Molecular Bioenergetics and Macromolecular Biochemistry: Meyerhof-Symposium Heidelberg, July 5–8, 1970 [1 ed.] 978-3-642-65311-7, 978-3-642-65309-4

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Molecular Bioenergetics and Macromolecular Biochemistry Meyerhof-Symposium Heidelberg, July 5- 8, 1970

With Lectures of M. Eigen . W. Hasselbach . K. C. Holmes . B. L. Horecker H. A. Krebs . F. Lipmann . F. Lynen . D. Nachmansohn S. Ochoa' L. Sachs' A. Weber' H. H. Weber' R. Winkler

Edited by H.H.Weber

Springer-Verlag Berlin Heidelberg New York 1972

Professor Dr. med. Dr. rer. nat. h. c. Dr. med. h. c. HANS H. WEBER Max-Planck-Institut fur medizinische Forschung, Heidelberg

With 122 Figures

ISBN-13: 978-3-642-65311-7 e-ISBN-13: 978-3-642-65309-4 DOLJ 0.1 007/93.803-fi42-65309-4

This work is subject to copyright. All rights are reserved. whether the whole or part of the material is coucerned specifically those of translation. reprinting. re-use of illustrations. broadcasting. reproduction by photocopying machine or similar means. and storage in data banks. Under § 54 of the German Copyright Law where copies are made for other than private use, a fee is payable to the publisher, the amount of the fee to be determined by agreement with the publisher. ® by Springer-Verlag Berlin· Heidelberg 1972.Library of Congress Catalog Card Number 75-189292' Softcover reprint of the hardcover 1st edition 1972 The reproduction in this book of registered trade-marks does not warrant the assumption, even without any special marking. that such names are to be considered free under the trade-mark law and may be used by anyone

Preface The Meyerhof Symposium on "Molecular Bioenergetics and Macromolecular Biochemistry" took place in Heidelberg from the 5th to the 8th of July, 1970. The timing was chosen to coincide with the creation of a new chair, in the Weizmann Institute of Science in Rehovot, in memory of OTTO MEYERHOF and the location was determined by the fact that so much of MEYERHOF'S scientific work was done in Heidelberg. The historical reason for the symposium was the urgent want of many leading biochemists and physiologists active in Molecular Biology to honour the memory of one of the greatest scientists in this field and also one of the greatest biologists of the 20th century. October 1971 is the twentieth anniversary of the death of OTTO MEYERHOF and 1972 marks 50 years since he was awarded the Nobel prize (1922). With regard to the age of some of his friends and pupils it was decided the symposium to be arranged in 1970, the first of the three commemorative years. Not only MEYERHOF'S former co-workers but also many other famous scientists feel like students of his who - like the biologists of the Weizmann Institute - never worked in MEYERHOF'S laboratory. For these scientists felt their work based on the work of OTTO MEYERHOF, too. Therefore among the chairmen, speakers and discussants of the symposium are the N obellaureates HANS ADOLF KREBS, FEODOR LYNEN, CARL CORl, MANFRED EIGEN, furthermore the Meyerhof Professor of the Weizmann Institute, LEO SACHS, and three scientific grandchildren of MEYERHOF. The other authors of this monograph were all former co-workers of MEYERHOF. It shows MEYERHOF'S influence and importance that, despite a coincidence of the symposium with the meeting of the Nobel laureates at Lindau, seven of the seventeen participants of this symposium were Nobel laureates. All participants of the symposium are very grateful to Springer-Verlag who, by publishing this work, made it possible for the admirers of MEYERHOF, who were not able to attend the symposium in person, to be present in spirit. The organizing committee of the symposium is indebted to the "Weizmann Institute of Science" and the "Gesellschaft fUr Biologische Chemie" for paying the travel costs of the active members. Without this help the symposium would not have been possible. Heidelberg, January 1972 HANS H. WEBER

Vorwort Das Meyerhof-Symposion iiber "Molekulare Bioenergetik und makromolekulare Biochemie" fand vom 5. -8. Juli 1970 in Heidelberg statt. Der Zeitpunkt ergab sich aus der Errichtung eines Lehrstuhles zum Gedachtnis an OTTO MEYERHOF im Weizmann Institute of Science in Rehovot und der Ort aus der Bedeutung von Heidelberg fUr die wissenschaftliche Tatigkeit von OTTO MEYERHOF. Die innere Ursache fUr das Symposion war das Bediirfnis zahlreicher und bedeutender, auf dem Gebiete der molekularen Biologie tatiger Biochemiker und Physiologen, das Andenken eines der groBten Wissenschaftler auf diesem Gebiet und damit auch einen der groBten Biologen des 20. Jahrhunderts zu ehren. 1m Oktober 1971 sind 20 Jahre seit dem Tode von OTTO MEYERHOF vergangen; und 1972 liegt das Jahr des MEYERHOFSchen Nobelpreises (1922) ein halbes Jahrhundert zuriick. Mit Riicksicht auf das Alter einiger Freunde und SchUler von OTTO MEYERHOF wurde das Symposion in das Jahr 1970, das erste der angefUhrten Gedenkjahre, gelegt. Ais Meyerhof-Schiiler fUhlten sich bei diesen Erwagungen nicht nur die ehemaligen Mitarbeiter MEYERHOFS, sondern auch viele andere weltberiihmte Gelehrte, die - ebenso wie die Biologen des Weizmann-Instituts - nie in MEYERHOFS Laboratorium gearbeitet haben. Denn auch diese Gelehrten empfanden ihr eigenes Lebenswerk durch MEYERHOFS Lebenswerk gepragt oder entscheidend beeinfluBt. Und so trugen als Vortragende, Chairmen und Diskussionsteilnehmer auch die Nobelpreistrager HANS ADOLF KREBS, FEODOR LYNEN, CARL CORI, MANFRED EIGEN, ferner der Meyerhof-Professor LEO SACHS des Weizmann-Institutes sowie drei wissenschaftliche Enkel MEYERHOFS zu dem Symposion bei. Die iibrigen Autoren dieses Bandes waren personliche SchUler und Mitarbeiter von OTTO MEYERHOF. Es ist bezeichnend fUr die Bedeutung und Wirkung von OTTO MEYERHOF, daB unter den siebzehn aktiven Teilnehmern des Symposions sieben Trager des Nobelpreises sind, obwohl das Symposion mit der Lindauer Tagung der Nobelpreistrager zusammenfiel. Alle Teilnehmer des Symposions sind dem Springer-Verlag dafUr dankbar, daB er durch dieses Buch dafUr gesorgt hat, daB auch die Gelehrten und Verehrer von MEYERHOF geistig an diesem Symposion teilnehmen konnen, die nicht personlich anwesend sein konnten oder nicht zugelassen werden konnten, weil die Teilnehmerzahl des Symposions recht eng begrenzt war. Das vorbereitende Komitee des Symposions ist dem Weizmann Institute of Science und der Gesellschaft fUr Biologische Chemie dadurch sehr verpflichtet, daB diese beiden Gesellschaften die Reisekosten fiir die aktiven Teilnehmer iibernommen haben. Ohne diese Hilfsbereitschaft hiitte das Symposion nicht stattfinden konnen. Heidelberg, Januar 1972 HANS H. WEBER

Contents First Day OTTO WIELAND: Address of Welcome I BegruBung . . . . . HANS H. WEBER: Otto Meyerhof - Werk und Personlichkeit HANS A. KREBS: Otto Meyerhof's Ancestry . . . . . . . . Second Day, a. m. CARL F. CORl: EinfUhrung . . . . . . . . . . . . . . . . . . . . . BERNARD L. HORECKER: Meyerhof's Aldolase - 35 Years Later . . . . FEODOR LYNEN: Fettsauresynthetase aus Hefe und verwandte Multi. enzymkomplexe . . . . . . . . . . . . . . . . . . . . . . . . SEVERO OCHOA: Role of Ribosomal Factors in Polypeptide Chain Initiation. . . . . . . . . . . . . . . . . . . . . . . FRITZ LIPMANN: Biosynthesis of Gramicidin S and Tyrocidine: Polypeptide Synthesis without Nucleic Acids . . . . . . . . . . . .

1

3 14

17 18 30 56 76

Second Day, p. m. PAUL OHLMEYER: EinfUhrung . . . . . . . . . . . . . . . . . . . 89 KENNETH C. HOLMES: Molecular Structure of the Actomyosin System in Cross-striated Muscle. . . . . . . . . . . . . . . . . . . . . . 90 ANNE MARIE WEBER: Physiological Regulation of the Activity of the Actomyosin System . . . . . . . . . . III LEO SACHS: The Mechanism of Carcinogenesis . . . . . . . . 118 Third Day HERMANN BLASCHKO: Einfiihrung . . . . . . . . . . . . . . RUTHILD WINKLER and MANFRED EIGEN: Alkali-ion Carriers: Dynamics and Selectivity . . . . . . . . . . . . . . . . . . . WILHELM HASSELBACH: The Sarcoplasmic Calcium Pump . . DAVID NACHMANSOHN: Bioenergetics and Properties and Function of Proteins in Excitable Membranes Associated with Bioelectrogenesis

129

130 149 172

Name and Subject Index . . . . . . . . . . . . . . . . . . . . . . 194

Authors Professor Dr. MANFRED EIGEN Max-Planck-Institut fUr biophysikalische Chemie, 34 Gottingen-Nikolausberg, Am Fassberg Professor Dr. WILHELM HASSELBACH Max-Planck-Institut fUr medizinische Forschung, 69 Heidelberg 1, Jahnstr. 29 Professor Dr. KENNETH C. HOLMES Max-Planck-Institut fur medizinische Forschung, 69 Heidelberg 1, Jahnstr. 29 Professor Dr. BERNARD L. HORECKER Department of Molecular Biology, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Avenue, Bronx, N.Y. 10461, U.S.A. Professor Dr. HANS A. KREBS Metabolic Research Laboratory, Nuffield Department of Clinical Medicine, Radcliffe Infirmary, Oxford, OX2 6HE, U.K. Professor Dr. FRITZ LIPMANN The Rockefeller University, New York, N.Y. 10021, U.S.A. Professor Dr. FEODOR LYNEN Max-Planck-Institut fur Zellchemie, 8 Munchen 2, Karlstr. 23 Professor Dr. DAVID NACHMANSOHN Departments of Neurology and Biochemistry, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, N.Y. 10032, U.S.A. Professor Dr. SEVERO OCHOA Department of Biochemistry, New York University Medical Center, School of Medicine, 550 First Avenue, New York, N.Y. 10016, U.S.A. Professor Dr. LEO SACHS, Meyerhof Professor of Biology Department of Genetics, The Weizmann Institute of Science, Rehovot, Israel Professor Dr. ANNEMARIE WEBER Department of Biochemistry, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, Mo. 63104, U.S.A. Professor Dr. HANS H. WEBER Max-Planck-Institut fUr medizinische Forschung, 69 Heidelberg 1, JaJmstr. 29 Dr. RUTHILD WINKLER Max-Planck-Institut fUr biophysikalische Chemie, 34 Gottingen-Nikolausberg, Am Fassberg

First Day

Address of Welcome OTTO WIELAND Chairman of the Gesellschaft fiir Biologische Chemie

Ladies and Gentlemen, as Chairman of the "Gesellschaft fUr Biologische Chemie" and on behalf of Professor SABIN, President of the Weizmann Institute in Rehovot, it is my pleasure to welcome you. The initiative for this symposium came originally from the Weizmann Institute, which has just established a Meyerhof Professorship in memory of the historical biologist. The institute feels it owes a special debt of gratitude despite the fact that not one of the people working there was a personal co-worker of OTTO MEYERHOF. The institution which I represent enthusiastically took up this suggestion of the Weizmann Institute. A Meyerhof symposium is by definition concerned with some of the topics of greatest interest in modern biology. The breadth of interest displayed in OTTO MEYERHOF'S research made it possible for him to lay the foundations for the wide range of research discussed here. We should regard it as a uniquely significant event that the program of this symposium is presented by a select body of speakers who have presided over the development of the principles laid down by MEYERHOF. Most of these speakers are indeed direct pupils of MEYERHOF, or else indirect pupils and admirers who are well aware how much they owe to him. The importance of this symposium is apparent from the fact that seven out of the seventeen participants are Nobel laureates. In the presence of so many distinguished persons, I refrain from mentioning individuals, with the single exception of Professor GoTTFRIED MEYERHOF, whom I particularly wish to thank for coming here as the representative of the three children of OTTO MEYERHOF, now settled in the United States. May I also express my thanks to all those taking part in the symposium whose enthusiasm has brought them together from five countries in all parts of the world, as well as to the large audience assembled to honour a great scholar of our time and to show their admiration of his scientific achievement.

BegriiBung OTTO WIELAND Vorsitzender der Gesellschaft fUr Biologische Chemie

Meine Damen und Herren, ieh freue mieh, Sie als V orsitzender der Gesellsehaft fUr Biologisehe Chemie, aueh im Namen des Prasidenten des Weizmann-Instituts Rehovot, Professor SABIN, begruBen zu konnen. Die Anregung zu diesem Symposion ging vom Weizmann-Institut aus, das gerade jetzt im Gedenken an einen der groBten Biologen des 20. Jahrhunderts eine Meyerhof-Professur in Rehovot eingeriehtet hat, urn zu zeigen, wie tief sieh das Weizmann-Institut dieser sakularen Personliehkeit der Biologie verpfliehtet fuhlt. Das bedeutet sehr viel, denn keiner der dortigen Gelehrten ist ein unmittelbarer SchUler von OTTO MEYERHOF. Die von mil' vertretene Gesellsehaft hat mit Begeisterung dieser WeizmannAnregung zugestimmt. Weil es sieh urn ein Meyerhof-Symposion handelt, werden wir es mit vielen del' aktuellsten Gebiete der modernen Biologie zu tun haben. Denn die Forsehungen OTTO MEYERHOFS waren von einer ganz seltenen Vielseitigkeit und dadureh die Grundlage fUr die Entwieklung der heute hier behandelten so versehiedenen Gebiete. Man darf es als ein Ereignis von seltenem Rang ansehen, daB das Programm dieses Symposions von einer Auswahl von Rednern getragen wird, die die von MEYERHOF gelegten Fundamente zur hoehsten Entfaltung gebraeht haben. Denn diese Redner sind zum groBen Teil unmittelbare Schuler von MEYERHOF oder abel' mittelbare Schuler und Verehrer, die sieh bewuBt sind, wieviel sie ihm verdanken. Die Bedeutung un seres Symposions geht schon daraus hervor, daB von insgesamt siebzehn TeiInehmern sieben Nobelpreistrager sind. Bei so viel Prominenz moehte ieh auf namentliche BegruBung einzelner Anwesender verziehten - mit einer Ausnahme: Es ist dies Herr Professor Dr. GOTTFRIED MEYERHOF, dem ieh besonders dafUr danken moehte, daB er als Vertreter der drei in Amerika ansassigen Kinder von OTTO MEYERHOF zu uns gekommen ist. Summariseh gilt mein Dank allen Mitgliedern des Symposions, die sieh aus fiinf Landern rings urn die Welt voller Begeisterung hier zusammengefunden haben und aueh den zahlreiehen Zuhorern, die in Verehrung eines epoehalen Gelehrten und in Bewunderung seiner wissenschaftliehen Werke hierher gekommen sind.

Otto Meyerhof - Werk und Personlichkeit HANS H. WEBER Max-Planck-Institut fur medizinische Forschung, Heidelberg

OTTO MEYERHOF war einer der groBten Biochemiker und Biologen unseres Jahrhunderts. Unser Jahrhundert aber wird wahrscheinlich einmal in der Kulturgeschichte als das J ahrhundert der Physik und der Biologie bezeichnet werden. Sein Leben war voll von Erfolgen, Ehrungen, Tragik - und endete in Weisheit. OTTO MEYERHOF wurde am 12. April 1884 in Hannover geboren. Er war ein SproB jener kulturgesiittigten Familien, die charakteristisch waren fUr das 19. Jahrhundert und die niitzlich waren fUr die Kultur des Abendlandes bis weit in das 20. Jahrhundert hinein. Denn in diesen kulturgesiittigten Familien verhalfen die Eltern den Kindern schon friih zu hohen MaBstiiben fiir das Denken, Fiihlen und Handeln. Diese MaBstiibe befiihigten die Nachkommen zu scharfer Selbstkritik und damit zur Weiterentwicklung der Kultur. So wundern wir uns nicht, daB OTTO MEYERHOF schon sehr friih sich intensiv mit Philosophie beschiiftigte. Er war und er bIieb sein Leben lang ein Philosoph der Erkenntniskritik und der Moralkritik von KANT, und zwar in der Spielart der Kantianer FRIES und NELSON. Er hat etwa ein Vierteljahrhundert in den philosophischen "Abhandlungen der Friesschen Schule" pubIiziert und war fiir lange Zeit einer der beiden Herausgeber dieser Zeitschrift. Und er wirkte aus moraIischem Verantwortungsgefiihl schon als Student mit an wissenschaftIichen Kursen fUr Arbeiter in Berlin. MEYERHOFS Urteil - besonders sein leidenschaftIiches moraIisches Urteil - blieb sein Leben lang von KANTischer Philosophie bestimmt; in MEYERHOFS beruflicher Tiitigkeit traten dagegen allmiihlich andere Interessen in den Vordergrund. Er studierte zuniichst Medizin und schloB dieses Stadium hier in Heidelberg 1909 durch das medizinische Doktora tab. 1910 erschienen seine ersten beiden Publikationen in den erwiihnten "Abhandlungen der Friesschen Schule", und zwar bezeichnenderweise noch als Publikationen mehr geisteswissenschaftlichen Inhalts (psychologisch und historisch). Er ging dann von 1910-1912 als Assistent zu LUDOLF KREHL an die Heidelberger Medizinische KIinik. Und dort vollzog sich die entscheidende Wendung seines Lebens. Denn er fand dort einen um 1 J ahr iilteren Kollegen, der ihm, dem so gefiihrlich vielseitig Begabten, den Weg wies zu der Tiitigkeit, die MEYERHOF zu einem der groBten Forscher in der Geschichte der Biologie machte. Der Name des neuen Freundes war OTTO W ARBURG. W ARBURG beeinfluBte MEYERHOF, SO wie ein Genic ein anderes Genie beeinfluBt: nicht durch Uberreden, sondern durch Beispiel. W ARBURG analysierte schon damals den molekularen Mechanismus der Zellatmung, der biologischen Oxydation; er war also molekularer Biologe, und

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HANS H. WEBER

er arbeitete mit einer so glanzenden gedanklichen und experimentellen Methodik, daB auch MEYERHOF sich sofort und fur immer fUr molekularbiologische Forschung entschied. So entstand hier in Heidelberg zwischen 1910 und 1912 eine Freundschaft zweier genialer Biologen, in der der kaum jungere den fast gleichaltrigen Freund fUr sein ganzes Leben nicht nur seinen Lehrer nannte, sondern ihn auch in seinem Herzen immer als Lehrer verehrte. Bereits 1912 erschienen 4 molekularbiologische Publikationen von MEYERHOF - eine da von zusammen mit W ARBURG. AIle 4 betrafen die Zellatmung! 1912 ging MEYERHOF als Assistent an das damals kleine Physiologische Institut der Universitat Kiel, zunachst unter BETHE, dann unter HClBER. Auch hier blieb er zunachst Gefolgsmann von OTTO W ARBURG und trug weiter zur Analyse des Mechanismus der Zellatmung bei. Dann aber fand er 1918 das Thema seines Lebens, ein fundament ales Forschungsgebiet, das seinen Stempel tragt und dauernd tragen wird: Die Frage nach der chemischen und energetischen Koppelung der einzelnen Reaktionen innerhalb einer biologischen Reaktionskette. Schon seit 400 Jahren - seit LEONARDOS bekanntem und detailliertem Vergleich des Lebens mit einer Kerzenflamme - wuBte die Menschheit eigentlich, daB das Leben materiell und energetisch ein "stationarer Zustand"' sei. Aber erst durch MEYERHOF wurde dieser Rahmen der Erkenntnis mit konkretem Inhalt erfUIlt, geschah der Schritt von der Betrachtung zur Forschung: Denn MEYERHOF zeigte zuerst, daB dieser stationare Zustand Ie bender Systeme auf chemischen Kreisprozessen und vor aIlem, daB er auf einer chemischen und energetischen Koppelung der Einzeischritte dieser Kreisprozesse beruht. Er hat dieses Feid zuerst allein bearbeitet und blieb bis zuletzt der fUhrende Geist auf diesem Gebiet der molekularen Biologie. Ais Objekt der neuen Forschungsrichtung verwendete MEYERHOF zunachst den Muskel. Er tat das nicht so sehr, weil er sich besonders fUr den Muskel interessierte, sondern aus experimentellen Grunden: Der Muskel ist oin bequem zugangliches Gewebe, das in groBen und damit sehr genau meBbaren Betragen chemische Energie in Warme und in mechanische Arbeit verwandelt. AuBerdem tut der Muskel das - je nach Situation - sowohl unter aeroben wie unter anaeroben Bedingungen. Gleich der erste Schritt auf dem neuen Weg revolutionierte aIle bisherigen Anschauungen uber den Zusammenhang der beiden wesentlichen Energiequellen des Lebens, uber den Zusammenhang von Atmung und Garung. Schon seit PASTEUR wuBte man, daB die Lebensenergie unter anaeroben Bedingungen durch Garungsvorgange und unter aeroben Bedingungen durch Atmung geliefert wird. Schon seit PASTEUR wuBte man, daB viele Lebewesen je nach der O2 - Versorgung bald Atmung, bald Garung als Energiequelle benutzen. Man nahm vor MEYERHOF an, daB unter anaeroben Bedingungen die Garung der erste und einzige Schritt der energetischen Verwertung der Kohlehydrate sei, an den die Verbrennung der Garungsprodukte anschlieBt, sobald Sauerstoff zuganglich ist. MEYERHOF stellte aber kalorimetrisch fest, daB die Spaltung nur etwa 5% der im Kohlehydrat vorhandenen Energie frei werden laBt, wahrend etwa 95% der Kohlehydratenergie erst bei der Verbrennung der Spaltprodukte - d. h.

Otto Meyerhof - Werk und Personlichkeit

5

im Fall des Muskels der Milchsaure - verfiigbar werden. Auf der anderen Seite fanden die Freunde HILL und MEYERHOF - HILL mit seiner glanzenden thermoelektrischen Methode, MEYERHOF durch Kalorimetrie -, daB der Muskel unter anaeroben Bedingungen nicht ca. 5% und bei nachtraglicher 02-Zugabe nicht ca. 95%, sondern anaerob und aerob etwa gleich viel Energie freisetzt. MEYERHOF loste diesen scheinbaren Widerspruch durch den Nachweis, daB im Muskel bei Zugabe von Sauerstoff iiberhaupt nicht die ganze anaerob gebildete Milchsaure verbrannt wird, sondern nur etwa ein Sechstel, wahrend fiinf Sechstel in Kohlehydrat zuriickverwandelt werden. Damit betragt die Energieabgabe durch Milchsaureverbrennung in der aeroben Phase nicht mehr das Zwanzigfache, sondern nur noch ungefahr das Dreifache der Energieabgabe der anaeroben Phase. In Wirklichkeit wird die Energieabgabe in der aeroben Phase noch etwas kleiner, weil ein Teil der Verbrennungsenergie des einen Sechstels Milchsaure zur Resynthese der anderen fUnf Sechstel der Milchsaure in das urspriingliche Kohlehydrat verbraucht wird. Dieses Verhaltnis zwischen aerob verschwundener Milchsaure und verbrannter Milchsaure ist der beriihmte M eyerhof -Quotient*. Es war zu vermuten und wurde spater teils durch MEiYERHOF, teils durch WARBURG bewiesen, daB in allen Lebewesen dieser Meyerhof-Quotient gleich ist. Oder mit anderen Worten: 1m ganzen Bereich des Lebens werden die Produkte des anaeroben Kohlehydratstoffwechsels aerob nur zu einem kleinen Teil verbrannt und im iibrigen in Kohlehydrat zuriickverwandelt. So fiihrte gleich der erste Schritt MEYERHOFS auf dem neuen Forschungsweg zur endgiiltigen Losung eines fundamentalen Problems, von dem andere Forscher nicht einmal erkannt hatten, daB es ein Problem war. Das beruhte ausschlieBlich auf jener Methodik MEYERHOFS, chemischeund thermodynamische Messungen sinnvoll zu vereinigen. Und diese Methode trug ihn weiter. Mit Hilfe des Meyerhof-Quotienten ware erklart, warum im Muskel bei der oxydativen Beseitigung der Milchsaure nicht zwanzigmal so viel Energie auftritt wie bei der anaeroben Entstehung der Milchsaure, sondern nur etwa dreimal so viel. Yom Muskel aber werden wahrend der anaeroben Phase und der aeroben Phase fast die gleichen Mengen an Energie freigesetzt. Also miissen im Muskel wahrend der Phase der Milchsaurebildung weitere unbekannte energieliefernde Vorgange stattfinden, die wahrend der Oxydationsphase der Milchsaurebeseitigung riickgangig gemacht werden. Denn nur durch solche weiteren unbekannten Vorgange konnte erklart werden, daB die Energieabgabe wahrend der anaeroben Phase groBer und wahrend der aeroben Phase kleiner ist, als sich aus Garung und Resynthese der Milchsaure zu Kohlehydrat ergeben hatte. Und so suchte MEYERHOF so lange nach diesen energetisch bedeutsamen weiteren Vorgangen, bis die anaerob und die aerob abgegebenen Energiemengen tatsachlich gleich waren. Als das schlieBlich erreicht war, konnten er und die Wissenschaft sicher sein, daB fUr weitere, die Energiebilanz beeinflussende Vorgange im Muskel kein Platz mehr sei. Schon 1922 wurde der erste neue energetisch bedeutsame ProzeB von OTTO MEYERHOF gefunden: Die Neutralisierung der anaerob gebildeten Milchsaure dureh Proteine. Diese Entdeckung war wieder von fundamentaler Bedeutung

* Er betragt also unter optimalen Bedingungen 6; er kann unter weniger guten Bedingungen aber auch Werte zwischen 3 und 6 annehmen.

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fiir die Biologie weit iiber den Muskel hinaus. Denn MEYERHOF fand , dal3 die Warmetonung durch " Neutralisierung" fiir alle Sauren und fiir alle Proteine sehr ahnlich ist; und dieser Befund wurde durch einen ehemaIigen Schiiler MEYERHOFS (H. H. WEBER) sehr bald zu einem Hauptargument fiir die Zwitterionennatur* der EiweiBkorper. Auch diese Erkenntnis betrifft wieder die ganze molekulare Biologie - wenn auch auf einem anderen Gebiet!

OTTO M E Y E RHOF

im Garten seines Freundes A. V. HILL in London, etwa 1925 (aufgenommen von A. V. HILL)

So wundern wir uns nicht, daB viele grol3e Gelehrte der Welt, besonders die groBen Muskelexperten in England, von MEYERHOFS neuen VerOffentlichungen geradezu fasziniert waren . DOROTHY NEEDHAM hat in ihrem Nachruf auf MEYERHOF beschrieben, wie HOPKINS und aIle seine Muskelexperten in aul3erster Spannung auf die Dbersetzung jeder neuen Arbeit von MEYERHOF warteten. Vor allem aber fiihrten die Muskelarbeiten von MEYERHOF dazn, dal3 sich seine wissenschaftIiche Verbindung mit A. V. HILL in lebenslangliche Zusammenarbeit verwandelte. So geniigten 4 Jahre einsamer Arbeit eines kleinen Assistenten in dem kleinen Kieler Institut, dam it dieser Assistent fiir das Jahr 1922 zusammen mit seinem Freund A. V. HILL den Nobelpreis fiir Medizin erhielt.

* Zwitterionennatur heiBt: Aile EiweiBkiirper aller Lebewesen sind unter physiologischen Bedingungen nicht elektrisch fast ungeladen - wie vorher angenommen -, sondern maximal gel aden mit vielen positiven und negativen Ladungen nebeneinander.

Otto Meyerhof - Werk und Personlichkeit

7

Dieser junge Ruhm hatte seinen Nahrboden nicht in begeistertem Verstandnis der deutschen Universitaten fur MEYERHOFS Ergebnisse. So wurde am Anfang des Nobeljahres 1922 an MEYERHOFS Universitat Kiel ein Lehrstuhl fUr physiologische Chemie erriehtet. Die Fakultat berief einen freundlichen Mann namens PUTTER; MEYERHOF blieb Assistent! Aueh fand sieh erst im Jahre 1922 der erste akademische Mitarbeiter bei MEYERHOF ein, ein junger Mann, der gerade

OTTO MEYERHOF in USA zur Zeit seines 65. Geburtstages 1949 (aufgenommen von L. JACOBI)

sein medizinisehes Staatsexamen hinter sieh hatte, der auch sonst ein wissenschaftlieh recht unbesehriebenes Blatt war und der bereits nach einem halben Jahr wieder verschwand, weil er eine bezahlte Stellung annehmen muBte! lch weiB das, weil dieser junge Mann H. H . WEBER hieB. Allerdings waren die Forscher aufJerhalb der Universitaten auch in Deutschland nieht so blind gegenuber dem Genie. So sagte der Chairman des Nobelkomitees, JOHANSSON, bei der Verleihung des medizinischen Nobelpreises mit besonderer Befriedigung, daB gerade das Urteil eines deutschen Gelehrten fUr die Verleihung des Preises an HILL und MEYERHOF den Ausschlag gegeben hatte. Geradezu groBartig aber war es, daB im Kaiser-Wilhelm-Institut fUr Biologie in Berlin-Dahlem Forscher wie CORRENS, GOLD SCHMID, HARTMANN und OTTO W ARBURG einen Teil ihrer Laboratorien abgaben, um sofort eine zusatzlie he Abteilung fur OTTO MEYERHOF zu schaffen. Nur so war es moglich, daB MEYERHOF schon 1924 in die Kaiser-Wilhelm-Gesellschaft berufen wurde. Denn

8

HANS H. WEBER

erst 5 Jahre spater wurde in Heidelberg das Kaiser-Wilhelm-Institut fUr Physiologie* fertig, dessen Leitung MEYERHOF 1929 ubernahm. In der Kaiser-Wilhelm-Gesellschaft hatte MEYERHOF nun endlich Raum und Geld, um zahlreiche Mitarbeiter aus der ganzen zivilisierten Welt um sich zu versammeln - von 1924-1929 in Berlin-Dahlem, von 1929-1938 in Heidelberg. Von diesen Mitarbeitern sind heute 10 aus USA, England, Frankreich und Deutschland in Heidelberg anwesend. Und so floB der Strom der Forschungen MEYERHOFS von 1924 bis zu seiner Flucht vor HITLER im Sommer 1938 machtig dahin. In diesen Jahren von 1924 bis 1938 wurden mit Hilfe mehr oder minder gereinigter Enzyme der Garung in MEYERHOFS Laboratorium in Wechselwirkung mit den Laboratorien von EMBDEN, PARNASS und WARBURG die 6 Reaktionsschritte geklart, die den energieliefernden Teil der Garungskette bilden. Es wurde ferner durch zahlreiche Untersuchungen der Meyerhof-Schule und auch OTTO W ARBURGS gezeigt, daB aIle diese Reaktionsschritte der Kohlehydratverwertung in allen Zellen und Geweben aller Pflanzen und Tiere identisch sind. Erst yom Pyruvat an gabelt sich der Weg fur die verschiedenen Garungsformen der verschiedenen Lebewesen (Milchsauregarung, alkoholische Garung usw.) und, wie spater von anderer Seite festgestellt wurde, auch fUr die Verbrennung der Kohlehydrate. Es ergab sich bei diesen Untersuchungen, daB aIle Zwischenprodukte dieser Reaktionskette phosphoryliert sind und daB die Zwischenreaktionen bis zum Pyruvat mit Umphosphorylierungen verbunden sind. Auch der Sinn dieser Phosphatverschiebungen wurde in der gleichen Periode MEYERHOFS geklart: Denn die Messung derEnergietonung dieser Schritte durchMEYERHOF und seine Schuler zeigte, daB durch diese Phosphatverschiebungen die Phosphatreste aus der anfanglichen "energiearmen" Bindung an das Kohlehydrat in "energiereiche" Bindungen an seine Abbauprodukte uberfuhrt werden**. Etwa gleichzeitig wurde mit anderen Methoden in MEYERHOFS Laboratorium festgestellt, daB diese Art von UmphosphoryIierungen Voraussetzung ist fUr den Ablauf der von MEYERHOF unermudlich gesuchten, unbekannten energieliefernden Vorgange der Kontraktionsphase (vgl. S. 9 f.). Denn diese Vorgange wurden ebenfalls bis etwa 1935 gefunden und ihre Verknupfung mit den Garungsvorgangen aufgeklart. Fur MEYERHOFS Suche nach den noch fehlenden Quellen der Energielieferung boten sich um 1930 drei Substanzen und ihre PhosphoryIierungsreaktionen an, deren Existenz vorher vollig unbekannt gewesen war. Diese Substanzen und ihre Reaktionen wurden teils in Wechselwirkung und teils unabhangig voneinander in den Laboratorien von EGGLETON und EGGLETON, von FISKE und SUBBAROW und im eigenen Labor von MEYERHOF entdeckt. Es handelte sich um 3 Substanzen, die von den Enzymen des Muskels dephosphoryliert werden: zwei Phosphoguanidine, Phosphokreatin im Vertebratenmuskel (EGGLETON und EGGLETON sowie FISKE und SUBBAROW) oder aber Phospho-

* 1m Rahmen des Kaiser-Wilhelm-lnstituts fiir Medizinische Forschung. ** DieBegriffe "energiearm"und "energiereich"wurden erst spater von demMeyerhofSchiiler LIPMANN in die Terminologie eingefiihrt, der die allgemeine energetische Bedeutung Bolcher Reaktionen ausgearbeitet hat.

Otto Meyerhof - Werk und Personlichkeit

9

arginin im avertebraten Muskel (MEYERHOF) und ferner Adenosintriphosphat = ATP in allen Muskeln (LOHMANN sowie FISKE und SUBBAROW). Nur in MEYERHOFS Laboratorium aber wurde gezeigt, daB die Dephosphorylierung dieser Substanzen hohe Energiebetrage freisetzt, d. h. daB auch diese 3 Substanzen energiereiche Phosphatverbindungen sind. Vor allem aber ergab sich aus LUNDSGAARDS Beobachtung der anaeroben alactociden Muskelkontraktion, daB die Energie der Dephosphorylierung der genannten Substanzen die Muskelkontraktion nicht nur begleitete, sondern fUr die Muskelkontraktion notwendig war. Denn LUNDSGAARD, der die eingehende Untersuchung seiner alactociden Kontraktion sofort von Kopenhagen nach Heidelberg in MEYERHOFS Laboratorium verlegt hatte, fand dort folgendes: (1) daB auch nach Vergiftung der Milchsauregarung durch Halogenazetate (Jodazetat) der Muskel sich vollig normal kontrahierte und (2) daB auBerdem die in der Kontraktion freigesetzte Energiemenge iibereinstimmt mit der Energiemenge, die sich aus der Dephosphorylierungsenergie und der umgesetzten Menge der fraglichen Substanzen rechnerisch ergab. Damit war zunachst gezeigt, daB diese Dephosphorylierungsenergie der Kontraktionsarbeit naherstand als die Energie der Milchsauregarung. Ferner ergab sich aber auch bald, daB mit dieser neuen Energiequelle endgiiltig die unbekannte energieliefernde Reaktion gefunden war, die fiir die normale, unter Milchsauregarung stattfindende Kontraktionsphase bis dahin gefehlt hatte. Dies ergab sich durch Arbeiten des groBen Meyerhof-Schiilers KARL LOHMANN, der den Zusammenhang der eben genannten neuen Reaktionen mit den Garungsreaktionen auffand. Denn LOHMANN fand, daB in Gegenwart von Muskelenzymen folgende chemische Reaktionen ablaufen:

+ Phosphoenolpyruvat --+ ATP + Pyruvat ADP* + Phosphokreatin ｾ＠ ATP + Kreatin

ADP*

(1) (2)

Wichtig ist dabei, daB direkte Phosphatiibertragung zwischen Phosphoenolpyruvat und Phosphokreatin nicht moglich war. Diese Befunde fiihren zu folgendem BUd: Wahrend der Kontraktionsphase wird zuerst das ATP dephosphoryliert und ohne Zeitverlust regeneriert durch Phosphatiibertragung yom immer anwesenden Phosphokreatin. Erst dann, wenn die Reaktionen der Garungskette zu energiereich phosphorylierten Umbauprodukten (Phosphoenolpyruvat und 1,3-Diphosphoglyzerinat) gefiihrt haben, wird die Phosphatiibertragung aus dem Phosphokreatin abgelost durch Phosphatiibertragung aus den genannten Garungsprodukten. Sobald durch die Garung der urspriingliche ATP-Spiegel wieder hergestellt ist, wird schlieBlich auch das Phosphokreatin durch Riickiibertragung von Phosphatresten aus dem ATP mehr oder minder regeneriert. Fiir eine vollstandige Regenerierung aber reicht die Zeit der anaeroben Phase nicht aus. Infolgedessen wird ein gewisser Teil des Phosphokreatin auch dann nicht in der anaeroben Phase regeneriert, wenn die Milchsauregarung nicht vergiftet ist. Die Phosphorylierungsenergie

* .ADP =

.Adenosindiphosphat

=

dephosphoryliertes .ATP.

10

HANS H. WEBER

dieses Anteiles des Phosphokreatin kommt zur Garungsenergie hinzu und ist der Energiebetrag, der bis dahin in der Energiebilanz des Muskels fehlte und der von MEYERHOF so lange gesucht wurde. Damit war jene Periode der Muskelenergetik 1935 abgeschlossen, die mit der Entdeckung des Meyerhof-Quotienten urn 1918 begonnen hatte. Die Ergebnisse MEYERHOFS wurden in dieser Periode auBerordentlich gef6rdert durch die Zusammenarbeit mit A. V. HILL. Denn die Mechanik der Muskelkontraktion wurde durch HILL viel genauer analysiert als durch MEYERHOF. Ferner wurden MEYERHOFS Warmedaten mit einer ganz anderen Methode bestatigt - vor aHem auch in ihrer Giiltigkeit fUr die normale Einzelzuckung des lebenden Muskels. Dnd schlieBlich wurden aIle Probleme zwischen den beiden Freunden intensiv und erfolgreich diskutiert. Mit den zuletzt genannten experimentellen Ergebnissen von MEYERHOF, LOHMANN und LUNDSGAARD aber wurde nicht nur eine Forschungsperiode der Muskelenergetik abgeschlossen, sondern auch eine neue Periode der Bioenergetik erOffnet, die alle Zellen und Gewebe aller Pflanzen und Tiere betrifft. Denn es ergab sich noch wahrend der Heidelberger Periode MEYERHOFS, daB nicht nur im Muskel die Milchsiiuregarung keinen anderen Zweck hat, als Energie und Phosphat zur Bildung und Restitution von ATP zu Iiefern, sondern daB das gleiche fUr alle Arten der Garung gilt (unabhiingig von den jeweiligen Endprodukten). Diese GesetzmaBigkeit ist ferner unabhangig davon, ob die betreffenden Zellen und Gewebe Phosphoguanidine enthalten oder nicht. Das bedeutet mit anderen Worten, daB die Reaktionen der Phosphoguanidine, die fUr die Energiebilanz des Muskels so wichtig waren, nur spezielle Bedeutung haben: Sie sind sofort wirksame Lieferanten von Energie und Phosphat in Geweben, deren explosiver Energie bedarf den ATP -Vorrat bereits verbraucht ha ben wiirde, ehe die ATP-Restitution durch die Garung beginnt. Infolgedessen kommen Phosphokreatin und Phosphoarginin nur in Geweben mit explosivem Energiebedarf vor (Nerv und Muskel). Dagegen bedeutet die Erkenntnis, daB alle die verschiedenen Garungsarten immer und ausschlieBlich der Formation von ATP dienen, ein universales Gesetz der Biologie. Dnd aus diesem Gesetz wurde sehr bald nach dem abrupten Ende von MEYERHOFS Heidelberger Periode - meist in angelsachsischen Laboratorien - ein noch viel weiter reichendes Gesetz: AIle Energie aller Nahrungs- und Betriebsstoffe wird zunachst im ATP gespeichert - unabhangig davon, ob diese Energie durch Garungsvorgange oder durch Oxydationsvorgange freigesetzt wurde. Diese Erkenntnis beherrscht seitdem die Bioenergetik. Die hier skizzierten epochalen Arbeiten und viele andere bedeutende Arbeiten der unerh6rt fruchtbaren Heidelberger Periode endeten im September 1938 durch MEYERHOFS Flucht vor HITLER. MEYERHOF entkam mit Hilfe seines ehemaligen Schiilers A. VON MURALT in die Schweiz. Er ging von dort nach Frankreich als "Directeur de Recherches" am "Institut de Biologie PhysicoChimique", wahrend MEYERHOFS letzter deutscher Schiiler OHLMEYER das wissenschaftliche Erbe in Heidelberg abwickelte. Schon vor der Flucht aus Heidelberg war alles fUr seine Aufnahme in Paris vorbereitet dank der Bemiihungen von RENE WURMSER, HENRI LAUGIER, JEAN PERRIN und DAVID NACH-

Otto Meyerhof - Werk und Personlichkeit

11

MANSOHN. Und so wurde MEYERHOF mit Begeisterung in Paris aufgenommen. Abel' bereits nach 20 Monaten muBte MEYERHOF VOl' dem Einmarsch del' deutschen Armeen weiterfliehen. 1940 gelangte er schlieBlich uber Spanien und Portugal in die Vereinigten Staaten. Auch hier fand MEYERHOF auf Grund seines Ruhmes und durch A. V. HILLS warmherzige und kluge Fursprache bald eine Position: Die Universitat Philadelphia und die Rockefeller Foundation richteten fUr ihn eine Stelle als "Research Professor" und ein Laboratorium im Department von WRIGHT WILSON ein. Hier hat er bis zu seinem fruhen Tod am 6. Oktober 1951 weitergearbeitet. NaturgemaB erreichte del' Strom seiner originalen Ergebnisse in seinen letzten Jahren - auch aus gesundheitlichen Grunden _. nie wieder die Machtigkeit seiner jungen Jahre und seiner Kaiser-Wilhelm-Periode*. Dafur abel' wurde sein Genius in diesen letzten Jahren in einem ganz besonderen Sinn zu einem international en geistigen Zentrum: Viele Gelehrte vieleI' Nationen spurt en die Wurde und Weisheit seiner Personlichkeit; viele bedeutende Forscher holten sich bei ihm Rat, er wurde zum "groBen altenMann" fUr viele Zweige del' Biologie. Mit del' Treffsicherheit des geborenen Forschers erkannte er immer die bedeutenden und zukunftsreichen Ansatze und setzte bis zu seinen letzten Tagen sein Ansehen fUr diese ein. So ist er in mehr als einem Sinne nicht eigentlich alt geworden. Diese unzulangliche Skizze des Lebenslaufes und des Werkes von OTTO MEYERHOF zeigt implicite schon manche Seiten seiner Personlichkeit. Abel' die Personlichkeit war so reich und groB, daB das Bild unbedingt del' Erganzung bedarf. AIle seine bedeutenden Schuler und fast aIle Mitarbeiter MEYERHOFS uberhaupt hingen und hangen ihm so an, daB mil' das Wort "Liebe" - nicht nur "Verehrung" - dafUr gerechtfertigt erscheint. lch beschranke mich auf zwei Beispiere: 1. Als MEYERHOF 1938 geflohen war, beschloB sein technischer Assistent W ALTER SCHULZ, das zuruckgelassene Eigentum seines ehemaligen Chefs zu retten. Er ermittelte zunachst auf sehr inoffizielIen Wegen, wann es als "judisches Eigentum" versteigert werden sollte. Schon das war gar nicht so einfach und ungefahrlich. Dann nahm er seine bescheidenen Ersparnisse und ersteigerte die Dinge, die nach seiner Meinung fUr MEYERHOF einen hohen GefUhlswert besaBen und leicht transport abel waren. Daruber hinaus aber hatte er daran gedacht, sich auf del' Versteigerung eine Adressenliste alIer Leute anzulegen, die das ubrige MEYERHOFSche Eigentum (z. B. MEYERHOFS MobiliaI') ersteigert hatten. So konnte er nach dem Krieg auch diese Dinge mit HiJfe des Wiedergutmachungsgesetzes zuruckholen! Dann lieB SCHULZ alles von einem Spediteur nach USA schicken. So viel Muhe und gleichzeitig so viel Umsicht beruhen auf Liebe, nicht nur auf Verehrung! Das gilt auch, wenn man beriicksichtigt, daB WALTER SCHULZ eine Personlichkeit von ungewohnlicher Herzenswarme ist.

*

Etwa 50 Publikationen in USA gegeniiber 330 Publikationen in Deutschland.

12

HANS H. WEBER

2. Nach meiner Flucht aus Konigsberg begegnete mir in Tiibingen ein Professor GENEVOIS aus Bordeaux als franzosischer "Attache Scientifique" fiir die deutschen Universitaten der franzosischen Besatzungszone. Ganz zufallig ergab sich, daB er 1927 in Berlin und ich 1922 in Kiel mit MEYERHOF gearbeitet hatten. Von diesem Tage an kam Herr GENEVOIS nie mehr nach Tiibingen, ohne mich zu besuchen und ohne mir eine groBe Kiste mit Zigarren mitzubringen. Er hatte bemerkt, daB ich das Rauchen sehr entbehrte. Er hat Besuch und Zigarren nie vergessen! Kurz zuvor hatten er und sein Land unter deutscher Besatzung furchtbar gelitten. Dber mich wuBte er zunachst nichts, als daB auch ich ein Deutscher war. Aber Meyerhof-Schiiler fiihlten sich immer sofort als Freunde, auch wenn sie vor ihrer Begegnung sich nie gesehen hatten, weil sie zu ganz verschiedenen Zeiten und an verschiedenen Orten mit MEYERHOF zusammengearbeitet hatten! Die Wirkung MEYERHOFS auf die Menschen seiner Umgebung hatte beinahe etwas Magisches: Denn er war nicht eigentlich liebenswiirdig. Er war vielmehr vollig sachlich und iiberhaupt nicht auf Menschenfang aus. Aber unter der sproden Oberflache verbarg sich ein starkes Gefiihl: wen er gewogen und nicht zu leicht befunden hatte, dem blieb er treu. Er war universal begabt. Er philosophierte nicht nur, er war ein Literaturkenner und ein Kenner und Verehrer der bildenden Kunst. Er schrieb wunderschone Gedichte. Er nahm mit brennender moralischer Leidenschaft an der Politik teil. Da er auch immer bereit war, iiber diese Gebiete zu diskutieren, galt sicher vielfach: "Wer vieles bringt, wird manchem etwas bringen". Vor allem aber war MEYERHOF ohne Fehler und Launen. Ein Genie mag auf bestimmten Gebieten faszinieren. Ein Genie ohne Fehler und Launen ergreift das Gegeniiber ganz. Er hat 1914 die Mathematikerin und Malerin HEDWIG SCHALLENBERG aus Koln geheiratet. Der alteste Sohn aus dieser Ehe, GoTTFRIED MEYERHOF, ist heute hier. Die beiden anderen Kinder, BETTINA und WALTER, haben mir lange Briefe geschrieben aus tiefer Verehrung fiir ihren Vater. Dieser Vater aber hat iiber die 37 Jahre der Ehe hinweg Frau HEDWIG so geliebt, daB er ihr immer wieder - zuletzt noch in seinem Todesjahr 1951 - formvollendete Gedichte von liebender Weisheit gewidmet hat. Diese Gedichte wurden nach Frau HEDWIGS Tod gefunden. Liebesgedichte, die sich ein ganzes Leben lang an die gleiche Frau richten, sind in der Weltliteratur selten. Wer zum SchluB die beiden Photographien MEYERHOFS nachdenklich vergleicht, wird mit Dberraschung sehen, wie erstaunlich MEYERHOFS Geist nicht nur das Gesicht seiner Wissenschaft, sondern auch sein eigenes Gesicht im Laufe seines Lebens verandert hat. In seinen Mannesjahren ist das Gesicht nicht sehr auffallig: ein kluger Mann, kein schoner Mann, ein Mann mit ausgeglichenem Temperament, mit Humor und vielleicht sogar ein sehr kluger Mann. Gesichter dieser Art sind zwar nicht sehr haufig, aber keineswegs sehr selten. Das Altersbild 20 Jahre spater zieht unweigerlich den Blick auch eines fliichtigen Betrachters an: ein durch seine Vergeistigung geradezu schones Gesicht. Man glaubt das Genie. Und man ist iiberwaltigt, wenn man sich klarmacht, daB diese Schonheit erst im Laufe des Lebens dadurch entstand, daB in diesem Fall wirklich "sich der Geist den Korper baute".

13

Otto Meyerhof - Werk und Personlichkeit

Literatur Verzeichnisse aller Publikationen aus dem Laboratorium von OTTO MEYERHOF finden sich in den Nachrufen auf ihn: MURALT, A. v.: Ergebnisse der Physiologie 47, I (1952) (Schriftenverzeichnis zusammengestellt von D. NACHMANSOHN, S. VI). PETERS, R. A.: Orbituary Notices of Fellows of the Royal Society 9,175 (1954). Die Gesamtdarstellung der wissenschaftlichen Entwicklung der behandelten Periode (mit Weltliteratur) gibt das Buch NEEDHAM, D.: Machina Carnis; the Biochemistry of Muscle Contraction in its Historical Development. London: Cambridge University Press 1971. Eine gute historische Einordnung von MEYERHOFS Werk findet sich auch in KALCKAR, H. M.: Biological Phosphorylations, Development of Concepts. Cliffs, N.J.: Prentice-Hall 1969.

Englewood

Einige zusammenfassende Darstellungen aus MEYERHOFS Laboratorium sind: MEYERHOF, 0.: Thermodynamik des Lebensprozesses. In: Handbuch der Physik, Bd. II. Berlin: Springer 1926, S. 238. MEYERHOF, 0.: Die chemischen Vorgange im Muskel und ihr Zusammenhang mit Arbeitsleistung und Warmebildung (Monographien aus dem Gesamtgebiet der Physiologie der Pflanzen und der Tiere, Bd. 22). Berlin: Springer 1930. LOHMANN, K.: Der Stoffwechsel des Muskels. In: Handbuch der Biochemie, Erg.-Bd. III. Jena: Fischer 1935, S. 351. MEYERHOF, 0.: Uber die intermediaren Vorgange der enzymatischen Kohlehydratspaltung. Ergebnisse der Physiologie 39, 10 (1937).

Otto Meyerhof's Ancestry HANS A. KREBS Metabolic Research Laboratory, Nuffield Department of Clinical Medicine, Radcliffe Infirmary, Oxford

My connection with OTTO MEYERHOF does not stem from having worked in his laboratory or in his immediate field, though for almost four years, from 1926 to 1930, I worked under the same roof at the Kaiser-Wilhelm-Institut fUr Biologie at Berlin-Dahlem when I was in the laboratory of OTTO W ARBURG. The links between the two laboratories were very close and provided many occasions for personal contacts; so I got to know OTTO MEYERHOF quite well. But there are other links - family links. OTTO MEYERHOF was born in Hanover, and his father had come from Hildesheim, 30 km away, which is also my home town. The records of the MEYERHOF family in Hildesheim go back to about 1720, to OTTO'S great-great-great-grandfather. It was a well established and large family. One of the sons of this great-great-great-grandfather, ISAK MEYERHOF, born in 1753, established a family foundation to honour the memory of his father, for the benefit of any needy descendant. A consequence of this legacy was the keeping of a register of descendants who were entitled to benefit from the foundation. A large family tree, including many hundreds of names, was printed in 1932 and assembled at that time by another OTTO MEYERHoF. I knew many of the recent generations personally. It is, I think, of general interest to look into the background which has contributed to the shaping of personalities, through both genetics and environmental factors. OTTO MEYERHOF was not the only member of the family who became exceptionally distinguished. A second cousin of his, and personal friend, was MAX MEYERHOF (1874-1945). MAX was ten years older than OTTO and was primarily trained as an ophthalmologist. It so happened that in 1900 OTTO, then 16, was advised to go to Egypt to recover from a renal ailment and the family arranged that his cousin MAX should accompany him. MAX was apalled by the enormous amount of blindness which be saw in Egypt and after his return to Germany he decided in 1903 to settle in Egypt to practice as ophthalmologist. He was also attracted to Egypt by a deep interest in Egyptology which had been kindled by his first cousin WILHELM SPIEGELBERG (1870 to 1930), a professor of Egyptology at Strassburg. MAX later settled in Egypt where he lived, with the exception of the war years (1914-1923), until his death in 1945. He became distinguished both as an ophthalmologist and as an Arabist. An appreciation of his life's work appeared in the Bulletin of the History of Medicine 19,375 (1946), written by CLAUDIUS F. MAYER. His list of publications includes more than 279 items, about half on ophthalmology, the rest on history of medicine and Arabism.

Otto Meyerhof's Ancestry

15

Another relation of OTTO MEYERHOF is WALTER Dux, born in Hildesheim in 1889, an exceptionally able physical chemist whose doctoral work, carried out around 1912 under the supervision of MAX BODENSTEIN, represented a major achievement because it described the fundamental discovery of chain reactions. The research was concerned with the kinetics of .the reaction between chlorine gas and hydrogen gas leading to the formation of HCI. Dux did not pursue an academic career; he went into industry and joined the factory of his father-inlaw making adhesives. The importance of his work was recently fully recorded in connection with a memorial to MAX BODENSTEIN written by ERIKA CREMER in Chem. Ber. 100, XCV (1967) and Hanover University awarded him an honorary degree. Dux now lives in London. My own maternal family intermarried with the MEYERHOFS. A first cousin of OTTO MEYERHOF married a cousin of my maternal grandmother. The MEYERHOF family is also closely connected, through many intermarriages, with the family from which CARL NEUBERG, the distinguished biochemist, stemmed. CARL NEUBERG was born at Sarstedt, a small town 12 km from Hildesheim. These inter-connections between the families are not a matter of accident, but have to do with the smallness of the Jewish communities. Marriages between Jews and non-Jews were infrequent in those days, mainly for religious reasons. The Jewish population in the Hildesheim area was less than 1 % of the total population. This meant that within a radius of, say, 25 km there were only one or two thousand Jews. This smallness of course favoured some measure of inbreeding. OTTO'S family background, then, was one which held the pursuit of learning and of the arts in high esteem. It was an atmosphere where the young were encouraged to interest themselves in literature and music, fine arts and higher learning. It was an atmosphere of respect for matters of the mind. There was also a sense of self-respect and pride. I must conclude with a sad note. Of the large, prosperous and proud family not a single member was left, after the Hitler holocaust, in the area where the family had lived for hundreds of years. Many were deported and killed in concentration camps. These include the compiler of the family tree, the other OTTO MEYERHOF. Fortunately, many members survived but they are scattered all over the world. As you are aware the family tradition of academic pursuits continues. OTTO'S elder son GODFREY is now Professor of Engineering at Dalhousie University in Halifax (Nova Scotia). His younger son WALTER is a distinguished professor of nuclear physics at Stanford University.

Second Day, a. m.

Einfiihrung CARL F. CORI, Chairman Boston Es ist mir eine besondere Ehre, an dieser Gedenkfeier fur OTTO MEYERHOF mitwirken zu durfen, denn wir standen viele Jahre hindureh in freundsehaftMein letzter Besueh in Heidelberg war kurz vor seiner Ablieher ｂ･ｺｩｨｾｮｧＮ＠ reise ins Ausland, und es sehien mir tragiseh und ein groBer Verlust fur die Wissensehaft, die Tatigkeit eines so bedeutenden Forschers so plotzlich unterbrochen zu sehen. In der Tat war es MEYERHOF nicht moglich, in Philadelphia eine neue Forschungsstelle zu schaffen, die mit seiner hiesigen vergleiehbar war. Er arbeitete mit kleinen Mitteln ununterbroehen bis zu seinem Tode weiter und beklagte sieh nie. In dieser wie in vielen anderen Beziehungen war er ein nobler Mensch. Herr WEBER hat bereits in eindrucksvoller Weise uber den Lebenslauf von OTTO MEYERHOF gesprochen. Sein Werk lebt weiter in seinen Mitarbeitern, von denen viele hier versammelt sind und von denen wir horen werden. leh bitte also Herrn HORECKER, mit seinem Vortrag zu beginnen. Sein Thema "Meyerhof's Aldolase" bringt uns in Erinnerung, daB dieses kohlenstoff-verknupfende Enzym auf dem Hauptweg der Glykolyse von MEYERHOF schon vor 35 Jahren entdeckt wurde.

])Ieyerhof's Aldolase - 35 Years Later* BERNARD.L.HoRECKER Department of Molecular Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y.

Introduction MEYERHOF'S genius lay not only in his ability to integrate chemical and physiological observations into broad, often revolutionary, biological concepts, but also in his emphasis on the value of quantitative data and the development of methods for obtaining such data. His early interest in the problem of utilization of chemical energy for life processes led him to the discovery that the lactic acid which HOPKINS [1] had shown to be formed during muscle contraction arose from the breakdown of muscle glycogen, and that the resynthesis of glycogen from lactic acid was an endergonic process which depended on energy derived from the complete combustion of a portion of this lactic acid. These concepts of the breakdown and resynthesis of glycogen from lactic acid led him to the search for an enzyme which would catalyze the formation of carbon-carbon bonds and specifically for a reaction in which three-carbon precursors could give rise to six-carbon sugars. He correctly deduced that this step in the process, the coupling of two three-carbon compounds to form the six-carbon chain, would not be the energyrequiring step, since EMIL FISCHER [2] had earlier demonstrated the spontaneous formation of hexoses from trioses. EMBDEN [3] had already proposed, on theoretical grounds, that hexose diphosphate would be formed by the condensation of dihydroxyacetone phosphate and glyceraldehyde phosphate. In 1934 MEYERHOF and LOHMANN [4] succeeded in demonstrating that muscle extracts would catalyze the reversible condensation of 2 moles of dihydroxyacetone phosphate to form the Harden-Young ester, hexose diphosphate. They called this enzyme zymohexase. Two years later [5,6] they showed that these extracts would also catalyze condensation reactions between dihydroxyacetone phosphate and aldehydes, such as acetaldehyde, D- or L-glyceraldehyde, as well as other aldehydes, and named the enzyme aldolase, in recognition of its ability to catalyze reversible aldol condensations. Finally, in 1938 [7,8], MEYERHOF succeeded in trapping glyceraldehyde-3-phosphate as a product of the enzymatic cleavage of fructose diphosphate and identifying this labile product, which had been synthesized some years earlier by H. O. L. FISCHER and BAER [9]. Thus the overall process catalyzed by "zymohexase" (Fig. 1) involved two enzymes, aldolase, which catalyzed the cleavage of hexose diphosphate,

* The original work reported in this article was supported by a grant from the National Institute of General Medical Sciences, National Institutes of Health (GM 11301). This is Communication No. 207 from the Joan and Lester Avnet Institute of Molecular Biology.

19

Meyerhof's Aldolase - 35 Years Later

and an isomerase [10], which catalyzed the interconversion of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate [11]. Aldolase was crystallized from rat muscle by W ARBURG and CHRISTIAN [12], who also isolated the enzyme from yeast and showed that the yeast enzyme, unlike aldolase from muscle, required a metal ion for catalytic activity. RUTTER has recently confirmed this difference, and identified two classes of aldolases, Class I, present in animals and higher plants, and Class II, the metal aldolases, which were found in bacteria and molds [13]. Aldolase was crystallized from rabbit muscle by TAYLOR et al. [14], following a remarkably simple procedure which made large quantities of this enzyme readily available. Most of the information now available has been obtained with this preparati.on.

2T

H OH

c=o I

2-

H2COP0 3 Hexose Diphosphate

Q-Glyceraldehyde 3-Phosphate

Fig. 1. Zymohexase reaction (from

Dihydroxyacetone Phosphate MEYERHOF

[11])

In their early classical experiments MEYERHOF and his coworkers not only defined the overall zymohexase reaction, but also obtained important information bearing on the intimate mechanism of the reaction catalyzed by the enzyme, aldolase. They established that the condensation reaction was specific for dihydroxyacetone phosphate, which could not be replaced by free dihydroxyacetone nor by ex-glycerol phosphate. On the other hand, D-glyceraldehyde3-phosphate was replaced by a number of aldehydes, including D- or L-glyceraldehyde, acetaldehyde, formaldehyde, glyceraldehyde and propionaldehyde. It could be concluded from these observations that aldolase catalyzed a specific activation of dihydroxyacetone phosphate, a conclusion which was confirmed many years later when a number oflaboratories [15-17] reported that aldolase catalyzed the exchange of a proton of water with one of the two protons of the C-3 carbon atom of dihydroxyacetone phosphate. MEYERHOF and his coworkers also concluded that this activation of dihydroxyacetone phosphate was stereospecific. The condensation reaction, whether with D- or L-glyceraldehyde, always led to the same L-configuration in the new asymmetric carbon atom formed at the C-3 position, and to the Dconfiguration at the asymmetric C-4 carbon atom; D-fructose-l-phosphate was formed in the condensation with D-glyceraldehyde, but L-sorbose-l-phosphate was formed from dihydroxyacetone phosphate and L-glyceraldehyde. MEYERHOF correctly drew attention to the fact that in this respect the enzyme-catalyzed

20

BERNARD

L.

HORECKER

reaction differed from the alkali-catalyzed reactions in which both D-fructose and D-sorbose are formed from the condensation of dihydroxyacetone with Dglyceraldehyde (Fig. 2) [6, 18]. In this "spontaneous" condensation, neither of the cis-isomers could be demonstrated. This conclusion was again confirmed by the isotope-exchange reactions referred to above; the proton in dihydroxyacetone phosphate which is labilized by aldolase occupies the same position as the carbon atom which replaces it in the condensation product. Thus considering only the studies of MEYERHOF'S group on the properties of the aldolase reaction, one might postulate a structure for the active site which would account for these observations. This is illustrated in Fig. 3. The specificity for dihydroxyacetone phosphate suggests the presence of specific sites for interaction with the negatively-charged phosphate group (site X) and with the carbonyl group (site Y). Thus neither dihydroxyacetone, which lacks the negative charge, nor ex-glycerophosphate, which lacks the carbonyl group, will replace dihydroxyacetone phosphate in the condensation reaction. The stereospecific labilization of the proton at carbon atom 3 of diH2COH

H2 COH

H2COH

c=o

c=o

c=o

I

I

I

I

H2COH Dihydroxyacetone Phosphate HC=O

I

HCOH I H2COH Glyceraldehyde 3Phosphate

I

I

HOCH OW ｾ＠

I HCOH I HCOH I

H2COH

Q-Fructose

HCOH

+

I

HOCH

I

HCOH

I

H2COH

Q-Sorbose

Fig. 2. Products formed in the alkali-catalyzed condensation of trioses

ｾＢＧ＠

ｾｈＲｐＱ＠

Lr:1 ,LI1

1+ｾｈＲｃｏｐＰＳ＠

ｾ＠

Fig. 3. Structure of the active site of aldolase based on MEYERHOF'S studies on the specificity of the reaction

hydroxyacetone phosphate, and the specific orientation of the OR group of fructose diphosphate at this position, point to a functional group, R, which either interacts directly with the hydroxyl group, or more likely, participates in the labilization of the prochiral proton in dihydroxyacetone phosphate. Two possible explanations may be offered for the stereospecific orientation of the hydroxyl group at the 0-4 position. One is that the trans-configuration is obligatory, and that this configuration is imposed by the fact that the configuration of the 0-3 hydroxyl has already been specified. This conclusion, which is supported by the results of the alkali-catalyzed reaction, where only. the products with trans-configuration were found, was challenged by our observations with deoxyribose phosphate aldolase. In the reaction catalyzed by that enzyme, the new asymmetric carbon atoms always assume the cis-conformation, regardless of the configuration of hydroxyl groups on the neighboring carbon atom [19].

Meyerhof's Aldolase - 35 Years Later

21

The second, and more likely, explanation for the stereospecific orientation of the hydroxyl group at C-4 is the presence of a functional group (site S, Fig. 3), which interacts with this hydroxyl group. MEYERHOF'S observation that the rate of the condensation reaction with Dglyceraldehyde was only slightly faster than with the L-form [8] suggests that the configuration at the C-5 position of fructose diphosphate is of little importance. There is thus no reason to postulate the presence of a functional interacting group at this site. On the other hand, it has since been shown'that muscle aldolase reacts much more rapidly with fructose 1,6-diphosphate than with fructose I-phosphate [20-22], which prompts us to suggest the presence of a site T which interacts with the 6-phosphate group. The studies of MEHLER and CUSIC [23] have shown that this interaction does not alter the primary binding of the substrate, but rather enhances the ability of the enzyme to catalyze the dealdolization reaction. The presence of this group appears to increase V max' rather than decrease the value of Km' Subunit 8tructure. Fructose diphosphate aldolase has now been shown to have a molecular weight of 160000, and to contain four subunits of molecular weight 40000 [24, 25]. Although the four subunits may not be identical [26-28], the difference in their structure now appears to be relatively minor [29,30], and for the purposes of our present discussion we will assume that the enzyme contains four functionally equivalent active sites. 2ｾ＠

ｾ＠

I

CH 20!03 ｾ］ｎＭｌｙｓ＠

H

ＭｉＮｾ＠

H2 COH

Dinydroxyacetone Phosphate

Aldolase

Schiff Base

•

Fig. 4. Postulated interaction of dihydroxyacetone phosphate with a charged lysine group

at the active center of aldolase

Nature of the functional group8. The activation of dihydroxyacetone phosphate has been shown to involve the formation of a Schiff base intermediate with specific lysine residues in the protein [31-33]. Thus site Y has been identified as a lysine residue which combines with the carbonyl group of the substrate to form the covalently-linked Schiff base derivative. This structure has been established by reduction of the dihydroxyacetone phosphate-Schiff base intermediate with sodium borohydride and isolation ofN6-,B-(1,3-dihydroxypropyl)lysine after acid hydrolysis. The positively-charged group which interacts with the I-phosphate group of fructose diphosphate (or with the phosphate group of dihydroxyacetone phosphate) has not yet been characterized. This interaction is responsible for the primary binding of the substrate by the enzyme, as has been established by the binding studies of BARKER and his coworkers [34, 35] and of MEHLER and GINSBURG [36, 37]. It is possible that group X is the same lysine residue which

22

BERNARD

L.

HORECKER

forms the Schiff base intermediate (see Fig. 4). It is not known whether the phosphate groups in dihydroxyacetone phosphate and fructose diphosphate react as the doubly or singly charged ions. MEYERHOF and SURANYI [38] calculated the values for the secondary dissociation constants of the phosphate groups of fructose diphosphate as pK;a = 6.1 and pK;b = 6.5. . The first step in the reaction between fructose diphosphate and aldolase, formation of the Schiff base derivative, is followed rapidly by the elimination of glyceraldehyde-3-phosphate, leading to the formation of the bound dihydroxyacetone phosphate (Schiff base) carbanion (Fig. 5). 2-

H2yOP03 C=O

I

+

HOCH I

HCOH I

HCOH I 2H2COP0 3 Fructose Diphosphate

Schiff Base Carbanion

Aldolase

Glyceraldehyde 3Phosphate

Fig. 5. Schiff base intermediate formation between fructose diphosphate and aldolase

2-

ｾ＠ IC=N-Lys H+

H

H2COP0 3

I

HOCH

e

Ketimine

"

ｃｏｐｾ＠

ＲＭｾＧ＠

2/ H 3 C-N-Lys

II

HOCH

ｾ＠

ｾ＠

Eneamine

Fig. 6. Proposed structures of Schiff base intermediates

The existence of the carbanion has recently been confirmed by CHRISTEN and RIORDAN [39]. Stabilization of the ketimine carbanion is probably promoted by resonance with the eneamine form (Fig. 6). The Schiff base thus forms an electron sink, stabilizing the dihydroxyacetone phosphate carbanion by absorbing the negative charge. The carbanion adduct may be further stabilized by insulation from protons of water in an apolar microenvironment. In this case, the reaction mechanism would require the participation of groups which might effect the transfer of protons to and from this region of the protein. We have postulated that this is the function of site R at the active center (see Fig. 3), and that R is the imidazole group of a histidine residue at the active center. Photooxidation of the enzyme in the presence of the photosensitizing dye rose Bengal resulted in the progressive destruction of about half of the 40 histidine residues in the molecule, and a concomitant loss of catalytic activity [40]. However, this loss of catalytic activity was not complete, and the residual activity, which was about 10% of that of the native enzyme, could be largely

23

Meyerhof's Aldolase - 35 Years Later

restored by the addition of an aldehyde acceptor, such as acetaldehyde or erythrose-4-phosphate. The enzyme after photooxidation resembles transaldolase, which has an absolute requirement for an aldehyde acceptor (Fig. 7). The fact that photooxidized aldolase is capable of forming the Schiff base carbanion, as demonstrated by the ability to react with acetaldehyde or erythrose-4-phosphate, but has lost its capacity to produce free dihydroxyacetone phosphate, suggests that the function which has been lost is the ability to transport protons to the active center, and that this step is now rate-limiting. This hypothesis was confirmed by the observation that the proton exchange Fructose-6-P + Transaldolase

-LL. ｾ＠

Dihydroxyacetone-Transaldolase + Ga3P

Acetaldehyde

I

Ketotetrose-l-P Fructose-l,6-f2 +

\Erythrose-4-p Sedoheptulose-7-P

Photooxidized Aldolase, Aldolase ｾ］Ｚ＠ Dihydroxyacetone-P-Aldolase ,

ａ｣･ｴ｡ｬ､ｨｙｾ＠

ｾｅｲｹｴｨｯｳ･ＭＴｰ＠

Ketotetrose-l-P ｓ･､ｯｨｰｴｵｬｳＭＬＷｾＲ＠

Fig. 7. Comparison of the reactions catalyzed by transaldolase (TA) and by photo oxidized aldolase

!

H20

Dihydroxyacetone Phosphate

Fig. 8. Role of histidine residues in the transport of protein residues to the Schiff base carbanion

reaction, which is characteristic of the native enzyme, could not be detected with the photooxidized enzyme. Our conclusion was that the proton exchange reaction is mediated by one or more of the histidine residues destroyed during photooxidation (Fig. 8). Further support for this mechanism was provided by evidence that these histidine residues are indeed situated in a hydrophobic region of the protein. This was deduced from the observation that their sensitivity to photooxidation was atypical, in that they were just as susceptible to photooxidation at pH 5.5 as at pH 8.5. This was in contrast to imidazole or histidine in aqueous solution, which are resistant to photooxidation in the protonated form, below pH 6.5 [41].

24

BERNARD

L.

HORECKER

It may be concluded that the histidine residues which are responsible for proton transport cannot be protonated even at pH 5.5, possibly because they are situated in a hydrophobic microenvironment. The large number of histidine residues destroyed when rose Bengal is the photosensitizer precludes any possibility of identifying the specific histidine(s) involved in this part of the reaction mechanism. Recently, however, we have examined the photoinactivation of aldolase with pyridoxal phosphate as the photosensitizing agent [42]. Under these conditions the reaction is much more specific, and only one histidine per active center is destroyed [43], possibly because of the specific binding of pyridoxal phosphate at the active center (see below). Additional evidence for hydrophobic regions at the active center was derived from the effect of o-phenanthroline, which catalyzed the oxidation of a pair of sulfhydryl groups at the active center to form a disulfide bridge, in which form the enzyme was inactive [44]. Of particular interest was the fact that this oxidation was prevented by the presence of the substrate. The experiments with 0- phenenthroline, as well as with other sulfhydryl reagents, suggested that the enzyme contains at least one sulfhydryl residue

HCO

I

+

HCOH

I

HCOH

I

2-

H2COP0 3

Fig. 9. Reaction of erythrose-4-phosphate with aldolase

per active center, which is essential for catalytic activity. The role of these sulfhydryl groups was established by experiments with the second substrate, glyceraldehyde-3-phosphate, which was found to inactivate aldolase when it was incubated with the enzyme in the absence of dihydroxyacetone-P [45, 46]. Similar inactivation was observed with the L-isomer and with erythrose-4phosphate, analogues of this substrate. Experiments with radioactive erythrose4-phosphate established that inactivation was associated with the incorporation of one equivalent of erythrose-4-phosphate per active center, and the loss of one sulfhydryl group, which couIC!. no longer be titrated with sulfhydryl reagents. The properties of the covalent derivative formed suggest that it may be a hemithioacetal (Fig. 9). This places the essential sulfhydryl group at site S (see Fig. 3). The evidence for the identity of site T, which interacts with the 6-phosphate group of fructose diphosphate, is less direct and is based on the binding of pyridoxal phosphate mentioned earlier. This substance, which has been proposed as a specific reagent for phosphate binding sites at the active center [47], is a competitive inhibitor of aldolase, and can be shown to react with lysine residues, which are not the same as the lysine residues involved in Schiff base formation with the substrate [48]. This inhibitor can be irreversibly fixed to the

Meyerhof's Aldolase - 35 Years Later

25

enzyme by reduction with sodium borohydride and the expected N6- pyridoxyllysine derivative has been isolated and identified. We suggest that this lysine residue is at site T, which interacts with the 6-phosphate group of the substrate. Site T appears to be more complex, and there is evidence that it may interact with site S, and also contain the tyrosine residue which is found at the COOH-terminus of the peptide chain. This tyrosine residue has been shown to be required for full activity with fructose-I,6-diphosphate, but not for the cleavage of fructose-I-phosphate (Table 1). Furthermore, the enzyme after treatment with carboxypeptidase is no longer inactivated by glyceraldehyde-3phosphate or erythrose-4-phosphate. Conversely, the COOH-terminal tyrosines are protected from digestion by the presence of substrate, or in the enzyme inactivated with erythrose-4-phosphate [46]. Similar protection is obtained with the substrate, fructose diphosphate, or the substrate analogue, hexitol diphosphate. It is suggested that the COOH-terminal tyrosines are essential for the interaction of the enzyme with the 6-phosphate groups, and conversely, when this phosphate group is present at the active center there is a change in conformation which renders these tyrosine residues less accessible to carboxypeptidase. Table 1. Effeot of removal of COOH-terminal tyrosine residues on properties of rabbit muscle aldolase Properties

Native enzyme

Carboxypeptidase treated enzyme

Cleavage of fructose-I,6-P2 Cleavage of fructose-loP Inactivation by glyceraldehyde-3-P

100

5

2

3

+

Mechanism of the reaction Based on the information which has been summarized in the preceding sections, a mechanism i.s proposed for the cleavage of fructose-I,6-diphosphate to yield dihydroxyacetone phosphate and glyceraldehyde-3-phosphate (Fig. 10). In the first step the substrate is oriented at the active center through binding of the I-phosphate group, placing the carbonyl group in proper position for formation of the Schiff base. This is promoted by temporary withdrawal of the proton from the positively-charged lysine residue, which may then return to form the protonated ketimine. DealdoIization is favored not only by the Schiff base structure, but also by the formation of the charged sulfur group adjacent to the hydroxyl on the C-4 carbon atom. This charge on the cysteine sulfur is induced by the 6-phosphate group, which, in proximity to the second lysine residue, induces the shift of a proton from the sulfhydryl group to the lysine amino group. The carbanion which remains when the glyceraldehyde-3-phosphate (Ga3P) moiety is lost is neutralized by a proton transported by the histidine residue to the active center. Finally, the Schiff base is hydrolyzed to yield dihydroxyacetone phosphate (DHAP), regenerating the free enzyme, which can the'n catalyze a second cycle.

26

BERNARD

-DHAP

II

L.

HORECKER

ll-G03P

Fig. 10. Model for the cleavage of fructose diphosphate by muscle aldolase

This mechanism also accounts for the abortive reaction which occurs when only glyceraldehyde-3-phosphate is present (Fig. 11). As before, the thiolate ion is induced by the interaction of the phosphate group of glyceraldehyde-3phosphate. In the absence of the dihydroxyacetone phosphate carbanion, this results in a nucleophilic attack on the carbonyl group, leading to the formation

Fig. 11. Tentative mechanism for the reaction of muscle aldolase with glyceraldehyde-3phosphate

27

Meyerhof's Aldolase - 35 Years Later

of a thiohemiacetal, probably stabilized by the phosphate-lysine ion pair. The COOH-terminal tyrosine is shown as stabilizing the structure required for the specific interaction of the cysteine and lysine residues. In the absence of this tyrosine residue, we no longer observe the enhanced dealdolization of fructose diphosphate, which is now cleaved at the same slow rate as is fructose-6-phosphate. Similarly, in the absence of the COOH-terminal tyrosine residue, the enzyme no longer reacts with glyceraldehyde-3-phosphate and finally, as already mentioned, the presence of the phosphate group tightens the conformation so that the tyrosine residue is much less accessible to carboxypeptidase.

The primary structure of rabbit muscle aldolase Dr. LAI, in our laboratory, is making rapid progress toward the elucidation of the complete primary structure of the enzyme, and it may soon be possible to locate all of the functional groups which I have mentioned in this primary structure. Several years ago [33] we reported the sequence of a tryptic peptide from the active center, containing the lysine residue which forms the Schiff base derivative with the substrate. We have now determined the sequence of two cyanogen bromide peptides [49,50], one of which overlaps the active site peptide, and the other adjacent to it, so that a continuous sequence of 89 amino acids, including the active site, can now be written (Fig. 12). In addition, a 1

Pro-His ----------------------------------------------------------CNB\.158 160. 170 ------Glu-Asn-Ala-Asn-Val-Leu-Ala-Arg-Tyr-Ala-Ser-Ile-Cys-Gln-Glx-Asp-Gly-Pro180 190 Ile-Glu-Val-Pro-Glu-Ile-Leu-Pro-Asp-Gly-Asp-His-Asp-Leu-Lys-Arg-Cys-Gln-Tyr-ValT

f

200 210 Thr-Gln-Lys-Val-Leu-Ala-Ala-Val-Tyr-Lys-Ala-Leu-Ser-Asn-His-His-Ile-Tyr-Leu-GlnCNBr T 22, 141 (1941). WARBURG, 0., CHRISTIAN, W.: Biochem. Z. 314,149 (1943). RUTTER, W. J.: Federation Proc. 23, 1248 (1964). TAYWR, J. F., GREEN, A. A., CORI, G. T.: J. BioI. Chem. 173, 591 (1948). ROSE, I. A., RIEDER, S. V.: J. Amer. Chem. Soc. 77, 5764 (1955). BLOOM, B., TOPPER, Y. J.: Science 124, 982 (1956). RUTTER, W. J., LING, K. H.: Biochim. Biophys. Acta 30,71 (1958). FISCHER, H. O. L., BAER, E.: Helv. Chim. Acta 19, 519 (1936). PRICER, W. E., Jr., HORECKER, B. L.: J. BioI. Chem. 231>, 1292 (1960). TUNG, T.-C., LING, K.·H., BYRNE, W. L., LARDY, H. A.: Biochim. Biophys. Acta 14,

488 (1954). 21. DRESCHLER, E. R.. BOYER, P. D., KOWAT,SKY, A. G.: J. BioI. Chem. 234,2627 (1959). 22. RUTTER, W. J., RICHARDS, O. C., WOODFIN, B. M.: J. BioI. Chem. 236, 3193 (1961). 23. MEHLER, A. H., CUSIC, M. E., Jr.: Science 11>5, nOl (1967).

Meyerhof's Aldolase - 35 Years Later 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

29

KAWAHARA, K., TANFORD, C.: Biochemistry 5,1578 (1965). Su, C. L., HORECKER, B. L.: Arch. Biochem. Biophys. 123, 186 (1968). WINSTEAD, J. A., WOLD, F.: J. BioI. Chem. 239, 4212 (1964). CHAN, W., MORSE, D. E., HORECKER, B. L.: Proc. Nat. Acad. Sci. U.S. 57,1013 (1967). MORSE, D. E., CHAN, W., HORECKER, B. L.: Proc. Nat. Acad. Sci. U.S. 58, 628 (1967). KOIDA, M., LA!, C. Y., HORECKER, B. L.: Arch. Biochem. Biophys. 134, 623 (1969). LA!, C. Y., CHEN, C., HORECKER, B. L.: Biochem. Biophys. Res. Commun. 40, 461 (1970). GRAZI,E., CHENG, T., HORECKER,B.L.: Biochem. Biophys. Res. Commun. 7,250 (1962). HORECKER, B. L., ROWLEY, P. T., GRAZI, E., CHENG, T., TCHOLA, 0.: Biochem. Z. 338, 36 (1963). LAI, C. Y., HOFFEE, P., HORECKER, B. L.: Arch. Biochem. Biophys. 112, 567 (1965). HARTMAN, F. C., BARKER, R.: Biochemistry 4,1068 (1965). CASTELLINO, F. J., BARKER, R.: Biochem. Biophys. Res. Commun. 23, 182 (1966). GINSBURG, A., MEHLER, A. H.: Biochemistry 5, 2623 (1966). GINSBURG, A.: Arch. Biochem. Biophys. 117, 445 (1966). MEYERHOF, 0., SURANYI, J.: Biochem. Z. 286, 319 (1936). CHRISTEN, P., RIORDAN, J. F.: Federation Proc. 27, 291 (1968). HOFFEE, P., LA!, C. Y., PUGH, E. L., HORECKER, B. L.: Proc. Nat. Acad. Sci. U.S. 57, 107 (1967). WESTHEAD, E. W.: Biochemistry 4, 2139 (1965). RIPPA, M., PONTREMOLI, S.: Arch. Biochem. Biophys. 133, 112 (1969). DAVIS, L. C., BROX, L. W., GRACY, R. W., RIBEREAU-GAYON, G., HORECKER, B. L.: Arch. Biochem. Biophys.140, 215 (1970). . KOBASHI, K., HORECKER, B. L.: Arch. Biochem. Biophys. 121, 178 (1967). LAI, C. Y., MARTINEZ DE DRETZ, G., BACILA, M., MARINELLO, E., HORECKER, B. L.: Biochem. Biophys. Res. Commun. 30, 665 (1968). ADELMAN, R. C., MORSE, D. E., CHAN, W., HORECKER, B. L.: Arch. Biochem. Biophys. 126, 599 (1968). RIPPA, M., SPANIO, L., PONTREMOLI, S.: Arch. Biochem. Biophys. 118, 48 (1967). SHAPIRO, S., ENSER, M., PUGH, E., HORECKER, B. L.: Arch. Biochem. Biophys. 128, 554 (1961). LAI, C. Y.: Arch. Biochem. Biophys. 128, 202 (1968). LAI, C. Y., CHEN, C.: Arch. Biochem. Biophys. 128, 212 (1968).

Fettsauresynthetase aus Hefe und verwandte Multienzymkomplexe FEODOR LYNEN Max-Planck-Institut fUr Zellchemie, Miinchen

Zu den groBen Erfolgen, die OTTO MEYERHOF bei der AufkHirung der chemischen Reaktionskette der Glykolyse und ihrer Rolle im Energiehaushalt der Zelle erzielt hat, trug ganz wesentlich bei, daB es ihm erstmals gelang, das beteiligte Enzymsystem aus Muskeln zu extrahieren und in geloster Form zu studieren. Damit wurde die Moglichkeit erOffnet, die Methoden der Proteinfraktionierung einzusetzen und das beteiligte Multienzymsystem in seine einzelnen Komponenten aufzutrennen. Dieses Unterfangen hat schlieBlich zur Reindarstellung, ja sogar zur Kristallisation der an der Glykolyse beteiligten Enzyme gefUhrt, an denen dann die kinetischen Parameter und etwaige Regulationsphanomene studiert werden konnten. Untersuchungen dieser Art sind am Multienzymsystem der Glykolyse und an zahlreichen anderen Enzymsystemen gleicher Komplexitat weit vorangetrieben worden, und sie haben unsere Kenntnisse von den molekularen Prozessen im Bereich des Lebendigen ganz wesentlich bereichert. Gleichzeitig stellte sich damit jedoch die neue Aufgabe, der Verteilung solcher Enzymsysteme innerhalb der Zelle nachzugehen. In diesem Zusammenhang interessiert uns heute vor allem die Frage, ob solche Systeme innerhalb der Zelle in einer geordneten Struktur konzentriert sind oder ob sie ganz unregelmaBig verteilt im Raum des Oytoplasmas vorkommen. Tatsachlich haufen sich die Befunde, wonach ersteres zutrifft und dies sogar im Fall des Enzymsystems der Glykolyse.

Fettsauresynthetase aus Hefe Diese Frage ist ganz eindeutig entschieden, wenn die geordnete Assoziation mehrerer, fUr die Katalyse der aufeinanderfolgenden Schritte einer Reaktionskette verantwortlicher Enzyme so fest ist, daB beim Aufbrechen der Zellen die Ordnung erhalten bleibt und eine anschlieBende Proteinfraktionierung schlieBlich zur Isolierung des intakten MuItienzymkomplexes fUhrt. Auf einen solchen Enzymkomplex sind wir in unseren Studien zur Fettsaureneubildung in Hefezellen gestoBen [1, 2]. Diese Synthese kommt in zwei Schritten zustande. 1m ersten Schritt wird der Baustein Acetyl-OoA zu Malonyl-OoA carboxyliert, wobei ATP als Energiequelle beteiligt ist (Gleichung 1). Diese Reaktion wird durch die zur Klasse der Biotinenzyme zahlenden Acetyl-OoA-Oarboxylase katalysiert. 00 2 + Acetyl-OoA + ATP ｾ＠ Malonyl-OoA + ADP + Po (1) Acetyl-OoA + 8 Malonyl-OoA + 16 TPNH + 16 H+ --+ Stearyl-OoA + 8002 + 8 OoA + 8 H 2 0 + 16 TPN+ (2)

Fettsauresynthetase aus Hefe und verwandte Multienzymkomplexe

31

Die folgenden Schritte, welche von Acetyl-CoA und Malonyl-CoA ausgehend zur Produktion von Palmityl-CoA und Stearyl-CoA gemiiB Gleichung 2 fUhren, werden von Fettsiiuresynthetase katalysiert. Diesel' Multienzymkomplex aus Hefe wurde in unserem Arbeitskreis wiihrend der letzten Jahre sehr eingehend studiert [1 - 4], und dies ermoglichte einen detaillierteren Einblick in seine Funktion und Architektur. In del' Fettsiiuresynthetase sind nicht weniger als 7 verschiedene Enzymaktivitiiten [3] zu einem stabilen Aggregat vereinigt. Das Molekulargewicht betriigt 2,3 Millionen, was sich zur Isolierung der Fettsiiuresynthetase aus Hefezellen ausniitzen lii13t. Dazu werden die frischen Hefezellen durch hochtouriges Schiitteln mit kleinen Glasperlen aufgebrochen, der rohe Zellextrakt mit Ammoniumsulfat fraktioniert und die aktive Fraktion an Calciumphosphat-Gel adsorbiert. Nach Elution mit Phosphatpuffer werden die hochmolekularen Anteile in der priiparativen Ultrazentrifuge quantitativ niedergeschlagen und dann anschlie13end durch Sedimentation im Rohrzuckerdichtegradienten fraktioniert. Mit diesem Reinigungsverfahren konnte die Enzymaktivitiit rund 250-fach angereichert werden. Die auf diese Weise gereinigte Fettsiiuresynthetase erwies sich sowohl in der analytischen Ultrazentrifuge als auch bei der Diskgel-Elektrophorese als einheitlich. Au13erdem gelang es OESTERHELT in unserem Laboratorium, das gereinigte Enzym aus ammonsulfathaltiger Losung [5] zu kristallisieren. Die Kristalle besitzen die Form hexagonaler Prismen (Abb. 1), wobei allerdings das Verhiiltnis von Kristalliinge zum Durchmesser durch den pH-Wert wiihrend der Kristallisation beeinflu13t werden kann. Bei Kristallisation im schwach alkalischen Gebiet liegt dieses Verhiiltnis bei 1: 5, es verschiebt sich zu 1: 1, wenn man die Kristallisation bei schwach saurem pH (pH 5,5) durchfiihrt.

Abb. l. Kristallisierte Fettsauresynthetase aus Hefe. Die Kristallisation wurde bei pH 5,5 durchgefiihrt

Beim Studium des chemischen Mechanismus der Fettsiiuresynthese unter Verwendung des gereinigten Hefeenzyms wurde in unserem Laboratorium zuerst gefunden, da13 die Zwischenprodukte der Synthese iiber Schwefelbrii.cken kovalent an Protein gebunden sind [1 , 6]. Es wurde ebenfalls festgestellt, da13 zwei verschiedene Arten von Sulfhydrylgruppen sich in diese Funktion teilen und

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durch ihre Reaktivitat gegeniiber SH-Giften unterschieden werden k6nnen [6]. Wir bezeichneten sie zunachst als "zentrale" und "periphere" Sulfhydrylgruppen und fanden spater, daB die "zentrale" Gruppe in Form von 4'-Phosphopantethein vorliegt [7], das an einer Proteinkomponente des Komplexes gebunden ist [8], die mit dem von VAGELOS [9] aus Colibakterien isolierten "acyl carrier protein" nahe verwandt ist. Die strukturellen Beziehungen zwischen "acyl carrier protein" und Coenzym A sind aus Abb. 2 zu ersehen. Im Coenzym A ist 4' -Phosphopantethein iiber eine Pyrophosphatbriicke an ein Adenosin-diphosphat gebunden, im "acyl carrier protein" aus Colibakterien jedoch iiber eine Phosphodiesterbriicke an die Hydroxylgruppe eines Serinrests im Polypeptid, das 77 Aminosauren enthalt und dessenSequenz im Laboratorium WAKILS [10] aufgeklart werden konnte. In den sogenannten "peripheren" Sulfhydrylgruppen des Multienzymkomplexes liegen polypeptidgebundene Cysteinreste vor [11]. Die Synthese der Kohlenstoffkette der Fettsauren geht stufenweise vor sich unter wiederholtem Anbau von C2-Einheiten aus Malonyl-CoA (Abb. 3). Dabei

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uptake. The assumption that it is the calcium gradient which gives rise to ATP formation is supported by the fact that on addition of oleate or ether which makes the vesicles permeable, the phosphorylation reaction is abolished. It must be emphasized that neither oleic acid nor ether do interfere with any interaction of ATP with the vesicular membranes: In the presence of oleic acid or ether the vesicular membranes are phosphorylated, the ADP-ATP exchange takes place and the calcium ATPase remains maximally active. A possible phosphorylation produced by mitochondrial contaminations of the vesicular preparation is excluded because the incorporation of inorganic phosphate is neither reduced by dinitrophenol nor by azide. On the other hand, prenylamine which is one of the most potent inhibitors of the sarcoplasmic calcium transport suppresses the incorporation to the same extent as it reduces the rate of active calcium transport. The results depicted in Fig. 17 imply that the coupling between calcium outward movement and ATP synthesis does not depend on the calcium load. Obviously the calcium ratio calcium inside/calcium outside does

169

The Sarcoplasmic Calcium Pump

not fall below a critical level until nearly all calcium is released. As a consequence of the calcium phosphate store inside the vesicles the decline of the ratio is only the result of the rising calcium concentration in the solution outside. The minimal energy which has to be provided by the calcium gradient under the experimental conditions that establish a very low ATP concentration by the hexokinase reaction can be estimated to be about 2000 to 3000 calories. Since the translocation of two calcium ions gives rise to the incorporation of one phosphate, a calcium ratio calcium inside/calcium outside of about 20 is sufficiently high to provide the required energy. However, for systems in which calcium is precipitated as phosphate and not as oxalate it is difficult to estimate ...;

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the existing calcium ratio. At the same free calcium concentration in the solution outside, the ratio in the phosphate system is presumably higher than in the oxalate system because the concentration of ionized calcium in equilibrium with calcium phosphate crystals is higher than the free calcium concentration in equilibrium with calcium oxalate crystals. Maximal value for the oxalate system has been estimated to be about. 3000. This gradient is even sufficiently high to drive the reaction when ATP is accumulated (",,0.005 mM) in the absence of hexokinase. These results show that the calcium translocation across the sarcoplasmic membranes is reversibly connected with the phosphoryl transfer reaction giving

170

WILHELM HASSELBACH

rise to a splitting of ATP when calcium moves inward and to a synthesis of ATP when calcium moves outward. This calcium gradient dependent ATP synthesis takes place with high efficiency under most simple conditions in the absence of any kind of inhibitor. Thereby the sarcoplasmic membrane system appearded to be superior to most other membranes used for the attempt to gain support for the conversion of osmotic into chemical energy (cf. LEW et aI., 1970; COCKWELL et aI., 1967; MITCHELL, 1961). The author is indebted to Deutsche Forschungsgemeinschaft for permanent support.

References AGOSTINI, B., DRABIKOWSKI, W.: J. submic. CytoI. 1, 207 (1969). AGOSTINI, B., HASSELBACH, W.: Naturwissenschaften 58,148 (1971). ASIILEY, C. C.: J. PhysioI. 208, 32 P (1969). BADER, H., SEN, A. K., POST, R. L.: Biochim. Biophys. Acta 118,106 (1966). BALZER, H., MAKINOSE, M., HASSELBACH, W.: Naunyn-Schmiedeb. Arch. Pharmak. u. expo Path. 260, 44 (1968). BARLOGIE, V., MAKINOSE, M., HASSELBACH, W.: FEBS Letters 12, 267 (1971). CALDWELL, P. C., HODGKIN, A. L., KEYNES, R. D., SHAW, T.I.: J. PhysioI.152, 591 (1960). CARVALHO, A. P., LEO, B.: J. Gen. PhysioI. 50,1327 (1967). CoCKWELL, R. S., HARRIs, E. J., PRESSMANN, B. C.: Nature 215,1487 (1967). COSTANTIN, L. L., FRANZINI-ARMSTRONG, C., PODOLSKY, R. J.: Science 147, 158 (1964). DEAMER, D. W., BASKIN, R. J.: J. Cell. BioI. 42, 296 (1969). DUGGAN, P. F., MARTONOSI, A.: J. Gen. PhysioI. 56, 147 (1970). EBASHI, S., ENDO, M.: Progr. in Biophys. and Mol. BioI. 18, 123 (1968). EBASHI, S., LIPMANN, F.: J. Cell. BioI. 14, 389 (1962). EIGEN, M., DE MAEYER, L.: In: Technique of Organic Chemistry (WEISSBERGER, A., Ed.), 2nd ed., vol. 8. New York: Interscience PubI. 1963, p. 895. FIEHN, W., HASSELBACH, W.: Eur. J. Biochem. 13, 510 (1970). FIEHN, W., MIGALA, A.: Eur. J. Biochem. 20, 245 (1971). FRIEDMAN, Z., MAKINOSE, M.: Pfliigers Arch. 816, R 71 (1970). GARRAHAN, P. J., GLYNN, I. M.: J. PhysioI.192, 237 (1967). HASSELBACH, W.: Federation Proc. 28, 909 (1964). HASSELBACH, W.: Biochemistry of Intracellular Structures. Warszawa: PWN 1969. HASSELBACH, W., ELFVIN, L.-G.: J. Ultrastruct. Res. 17, 598 (1967). HASSELBACH, W., MAKINOSE, M.: Biochem. Z. 888, 518 (1961). HASSELBACH, W., MAKINOSE, M.: Biochem. Biophys. Res. Commun. 7, 132 (1962). HASSELBACH, W., MAKINOSE, M.: Biochem. Z. 339, 94 (1963). HEIMBERG, F. W.: Thesis, Heidelberg 1970. HEINZ, E., PATLAH, G. S.: Biochim. Biophys. Acta 44,324 (1960). IKEMOTO, N., BHATNAGAR, G. M., GERGELY, J.: Biochem. Biophys. Res. Commun. 44,1510 (1971). INESI, G., GoODMAN, J., WATANABE, S.: J. bioI. Chern. 242, 4637 (1967). KIELLEY, W. W., MEYERHOF, 0.: J. bioI. Chern. 176, 591 (1948). KIRSCHNER, K., EIGEN, M., BITTMAN, R., VOIGT, B.: Proc. Nat. Acad. Sci. U.S. 56, 1661 (1966). LANT, A.I., FRIESLAND, R. M., WHITTAM, R.: J. PhysioI. 207, 291 (1970). LEW, V. L., GLYNN, I. M., ELLORY, J. C.: Nature 225, 865 (1970). MACLENNAN, D. H., WONG, P. T. S.: Proc. Nat. Acad. Sci. U.S. 68,1231 (1971). MAKINOSE, M.: Biochem. Z. 345, 80 (1966a). MAKINOSE, M.: 2. Int. Biophys. Congr., Vienna 1966b, p. 276. MAKINOSE, M.: Eur. J. Biochem. 10, 74 (1969). MAKINOSE, M.: FEBS Letters 12, 269 (1971). MAKINOSE, M., HASSELBACH, W.: Biochem. Z. 343, 360 (1965). MAKINOSE, M., THE, R.: Biochem. Z. 343, 383 (1965).

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MARTONOSI, A.: Federation Proc. 23, 913 (1964). DE MEIS, L.: J. bioI. Chern. 244, 3733 (1969). DE MEIS, L.: J. bioI. Chern. 246,4764 (1971). MITCHELL, P.: Nature 191,144 (1961). NAGAI, T., MAKINOSE, M., HASSELBACH, W.: Biochim. Biophys. Acta 43,223 (1960). PANET, R, SELINGER, Z.: Eur. J. Biochem.14, 440 (1970). PEACHEY, L. D.: J. Cell. BioI. 20, 209 (1965). PORTZEHL, H., CALDWELL, P. C., RUEGG, J. S.: Biochim. Biophys. Acta 179, 581 (1969). PUCELL, A., MARTONOSI, A.: J. bioI. Chern. 246, 3389 (1971). SANDOW, A.: Ann. Rev. PhysioI. 32, 87 (1970). SRETER, F., IKEMOTO, N., GERGELY, J.: Biochim. Biophys. Acta 203, 354 (1970). WEBER, A., HERZ, R: J. bioI. Chern. 238, 599 (1963). WEBER, A., HERZ, R, REISS, I.: J. Gen. Physiol. 46, 679 (1963). WEBER, A., HERZ, R, REISS, I.: Biochem. Z. 340, 329 (1,966). YAMAMOTO, T., TONOMURA, Y.: J. Biochem. (Tokyo) 62, 557 (1967). ZEBE, E., HASSELBACH, W.: Z. Naturforsch. 210, 1248 (1966).

Bioenergetics and Properties and Function of Proteins in Excitable Membranes Associated with Bioelectrogenesis* DAVID NACHMANSOHN Departments of Neurology and Biochemistry, College of Physicians and Surgeons, Columbia University, New York, N. Y.

Professor H. H. WEBER has outlined in his lecture some of the fundamental notions and concepts of cellular mechanisms which resulted from MEYERHOF'S work on muscle, and the impact which they had on biology as a whole. These notions have inspired and deeply influenced the investigations to be presented in this lecture, i.e., on the properties and function of proteins and enzymes of excitable membranes associated with the permeability changes during electrical activity and involved in the conduction of nerve impulses. The feelings of affection of the writer for his teacher and friend have been expressed on several occasions [1-3). Due to his remarkable personality, MEYERHOF attracted an unusual group of people. Even before joining MEYERHOF, the writer was fortunate to have worked with H. H. WEBER, who greatly advanced his knowledge of the physical chemistry of proteins. Among the contemporaries in MEYERHOF'S laboratory were SEVERO OCHOA, FRITZ LIPMANN, KARL LOHMANN, HERMANN BLASCHKO. The frequent and stimulating discussions between the members of this group in addition to those with MEYERHOF had an important influence on their thinking. Moreover, the inspiring atmosphere and the sharing of ideas favored the formation of strong bonds of friendship.

A. Excitable membranes (a) General features of cell membranes. During the last decade, the properties and function of cell membranes have been one of the most actively explored fields in biological sciences; much information has been obtained from electron microscopy combined with biochemical and biophysical analyses. The notion of a "unit membrane", which was based on the DanieIli-Davson model, proposed a structure about 80 A thick, formed by a bimolecular leaflet of phospholipids to which proteins are attached on the inside and outside by ionic forces [4). These views have been contested by powerful evidence. Membranes appear to be a mosaic of functional units formed by lipoprotein complexes. The proteins apparently form the core of the complexes; phospholipids are attached on the outside, probably by Van der Waals and Coulombic forces and by hydrophobic bonds [5). This idea has found much support (e.g. [6]); it does not exclude the

* This lecture is based on the Lead Article in Science 168, 1959 (1970). - This work has been supported in part by grants from the U.S. Public Health Service Nos. NS-03304 and NS-07743, by the National Science Foundation, Grant No. NSF-GB-25362, and by the New York Heart Association, Inc.

Bioenergetics and Properties and Function of Proteins in Excitable Membranes 173

possibility of modifications, for example lipid layers located between these complexes [7]. While the precise molecular organization of cell membranes is far from elucidated, the most important change in the last few years has been conceptual. It is now well established that cell membranes are highly organized and dynamic structures, in which many proteins and enzymes are located and form by their activity an essential part of the control mechanisms effected by membranes. An illustration of the character and intensity of the chemical reactions taking place in these structures is offered by the well-explored mitochondrial and other membranes [8-11]. The central role of proteins and enzymes in cell membranes accounts for their great diversity of function, their specificity, and their remarkable efficiency more readily than the previous notions based essentially on the physicochemical properties of phospholipids. In view of the crucial role of proteins in membrane function, SJOESTRAND and BARAJAS [12] have applied new procedures in preparing specimens for examination by electron microscopy aimed at preserving the conformation of proteins in their native state. Their pictures are quite different from those obtained with the standard procedures and offer evidence for the presence of globular structures. (b) Special features of excitable membranes. Nerve impulses are propagated along nerve and muscle fibers by electrical currents; bioelectricity is thus linked to one of the vital functions of the body. Since the turn of this century it has been widely accepted that ions are the carriers of these currents; the control of the ion movements was attributed to rapid and reversible permeability changes to ions of the excitable membranes surrounding the fibers. This special ability is a key problem for understanding nerve function. HODGKIN and HUXLEY assume that the ion movements are a simple diffusion process and account exclusively for conduction [13, 14]. This view is difficult to reconcile with present notions of membranes in general and a variety of experimental facts observed on excitable membranes. Drastic modifications of ion composition, e.g., both in the interior of the axon and its outer environment have, for a considerable length ot time, no effect on the electrical parameters contrary to the predictions of the theory [15]. Another serious difficulty arises from the measurements of heat production and absorption associated with electrical activity [16]. These authors find it hard to believe that the drastic changes of permeability in a material like the excitable membrane could occur without the intervention of work or chemical reaction. In a subsequent lecture, HILL [17], discussing various factors which possibly contribute to the heat production observed, again stressed as the only reasonable assumption that the early heat produced and absorbed is due to chemical reactions associated with the permeability cycle during electrical activity. In still more recent measurements the heat production coinciding with electrical activity was found to be diphasic: heat is produced during the rising phase of the action current and absorbed during its falling phase [18].

B. Role of acetylcholine in nerve activity (a) Neurohumoral transmission. In contrast to conduction, transmission across the junctions from nerve to nerve or from nerve to muscle was proposed to be effected by chemical mediators, in many cases specifically

174

DAVID NACHMANSOHN

by acetylcholine (AcCh). In view of basic similarities of the electrical properties of the membranes ofaxons and those at junctions, many neurobiologists questioned the transmitter theory, which was based on experiments in which classical methods of pharmacology were combined with those of electrophysiology (e.g. [19]). These methods, essential for the study of many aspects of biology and medicine, are inadequate for an analysis of the molecular events in excitable membranes. (b) Biochemical approach. Attending frequently the vigorous controversies between proponents and opponents of neurohumoral transmission at the meetings of the English Physiological Society in 1935 and 1936, the writer was struck by the total lack of any biochemical approach to the problem. Having been trained in biochemical laboratories in which proteins and enzymes played a central role in the ideas about cell function, it seemed to him imperative to investigate a variety of aspects of the proteins associated with the function of AcCh. Such information seemed likely to provide pertinent clues to the controversial problem of the role of AcCh. If one accepts the notion that the reactions in a living cell are chemically and energetically coupled, which - as pointed out by WEBER - was one of the most fundamental concepts resulting from MEYERHOF'S muscle work, it appeared necessary to integrate the formation and hydrolysis of AcCh into the intermediary metabolism of the nerve cell, to study the sequence of energy transformations during nerve activity and to correlate the biochemical data obtained with the physical events of conduction. The new approach initiated more than three decades ago was based on the notion of the central role of proteins and enzymes in cell function. Four factors were decisive in the progress achieved since this approach was initiated three decades ago: (I) the information about biomembranes mentioned above; (II) the development of highly refined methods and instruments for analysis of cellular function on the cellular, subcellular, and molecular levels; (III) the explosive growth of protein chemistry, culminating in the elucidation of the threedimensional structure of several proteins and enzymes; and (IV) the availability of an extraordinary and uniquely favorable material for the analysis of the proteins associated with excitable membranes and with bioelectricity: the electric organs of certain fish. The electroplax, the single cells of these organs, have electrical parameters similar to those of other excitable cells; but they are arranged in series as in a voltaic pile. This arrangement accounts for the powerful discharge of 600 volts in Electrophorus (the "electric eel" of the Amazon River), whose electric organ is formed by 5000 to 6000 cells. The most crucial feature of electric tissue for biochemical studies is its high degree of specialization for its main function, bioelectrogenesis. The metabolism of the cell is low except in the membrane. This aspect of the tissue provides a unique opportunity for the protein chemist because membranes form a small fraction of the cell mass and their study in other tissues is very difficult. The distribution and concentration of acetylcholinesterase (E. C. 3.1.1.7), the enzyme that hydrolyzed AcCh, was studied in a variety of excitable cells of many different species. A remarkable concentration was found, in 1937, in the electric

Bioenergetics and Properties and Function of Proteins in Excitable Membranes 175

organ of Torpedo marmorata and in the following year in that of Electrophorus; 1 kilogram of tissue hydrolyzes 3 to 4 kilograms of AcCh per hour in spite of the low protein (3%) and high water content (92%) of these organs. The use of this material in the next three decades was instrumental in the isolation, identification, and characterization of some of the proteins controlling the permeability changes of excitable membranes during electrical activity and in obtaining information about their function. (c) Chemical theory of the function of AcCh in excitable membranes. As a result of the biochemical approach a modification of the original theory became necessary. Acetylcholine is not a chemical mediator between two cells; it is never released from the cell (see below); its action is intracellular, taking place within the excitable membrane. It is the trigger which initiates and controls the permeability changes permitting the ion movements during electrical activity. Its function is similar in the excitable membranes offibers and in those at the junctions. This view is supported by a vast, and - in the last few years - rapidly increasing number of experimental data. The picture of the role of AcCh that best fits the available data is the following. Excitation leads to the release of AcCh from its bound form in resting condition. It acts as a signal recognized within the membrane by a specific AcCh-receptor protein. The reaction induces a conformational change of the protein, thereby possibly releasing by allosteric action Ca z+ ions bound to the protein. Calcium ions are involved in the excitability of nerve and muscle fibers, which become inexcitable in the absence of divalent cations; they are distinguished from other divalent cations by se"veral remarkable features and properties. Their release may induce further conformational changes of phospholipid8 and other polyelectrolytes. The end result of the sequence of chemical reactions is the ｣ｨ｡ｾｧ･＠ of the membrane permeability to ions, a change that permits the movements of many thousands of ions, possibly as many as 20000 to 40000 in each direction per molecule of AcCh released. These reactions thus act as typical amplifiers of the signal given by AcCh. Acetylcholinesterase rapidly hydrolyzes AcCh, thereby permitting the return of the receptor protein to its original ｣ｾｭｦｯｲ｡ｴｩｮ＠ and reestablishing the barrier for the ion movements. A schematic presentation of the postulated role of AcCh in the axonal membrane is given in Fig. l. Acetylcholine in its bound form and the two proteins reacting directly with the AcCh released, receptor and esterase, are presumably linked together structurally as well as functionally and form a protein assembly in the excitable membrane in a way comparable with other enzyme systems - for example, the electron transfer system in mitochondrial membranes. The structural organization of the system may account for the efficiency, the precision, and the speed of the events in the membrane during electrical activity. While an essential role of Ca 2 + ions in the permeability changes of excitable membranes appears likely, their release requires a specific control mechanism. Among the cell components, only proteins are known to have the ability to recognize specific ligands and thereby provide the proper control for initiating and terminating a specific cell function. The early data supporting these conclusions have been summarized in a monograph [20] and the more recent advances in several reviews [21-24].

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Three proteins, or at least three, are specifically associated with the action of AcCh: the two enzymes forming and hydrolyzing the ester, choline O-acetyltransferase and AcCh-esterase, and the receptor protein, the primary target of the action of AcCh. Some pertinent features of the properties of these three proteins may be briefly discussed.

AXON . . lonophore

Outside

RESTING MEMBRANE

ｾ＠

ｾ＠

0.0 Inside Outside

ACTIVE MEMBRANE

Inside

•

AcCh

@Ca" Fig. 1. Schematic presentation of the role of AcCh in excitable membranes: it is postulated to act as a signal initiating a series of reactions that result in increased permeability to ions. In the resting condition of the axonal membrane AcCh is bound to a storage form. On excitation (electric current, K+, HaO+ ions ?) AcCh is released and acts on the receptor protein inducing a conformational change. Ca ++ ions bound to carboxyl groups of the protein may thereby be released as a result of allosteric action; these ions act on the "iono· phore", inducing conformational changes of phospholipids or other polyelectrolytes and thus permitting accelerated ion movements. The end result is an amplification of the signal initiating a new electric circuit. The process is repeated at successive points of the membrane and the impulse is thus propagated along the axon The AcCh-receptor complex is in a dynamic equilibrium with the free ester and the receptor. The free ester will be susceptible to attack by AcCh-esterase. The rapid hydrolysis will permit the receptor to return to its resting condition. The barrier to the rapid ion movements is reestablished, but in the meantime the electric circuit generated has activated the adjacent point. The protein assembly effecting these processes must be structurally well organized within the membrane, as known for other assemblies and multi-enzyme systems. The structural organization permits the high speed, precision and efficiency

C. Enzymes hydrolyzing and forming AcCh I. AcCh-esterase The two enzymes have been shown to be present in various types of conducting fibers and in a great variety of species throughout the animal kingdom: in motor and sensory, cholinergic and adrenergic, and peripheral and central fibers and in muscles of both vertebrates and invertebrates.

Bioenergetics and Properties and Function of Proteins in Excitable Membranes 177

(a) Localisation. On the basis of a variety of biochemical data it was postulated since 30 years that AcCh-esterase is localized in or near the excitable membranes. This evidence was of necessity indirect. In the last eight years direct evidence for the exclusive localization of the enzyme in excitable membranes has been obtained by electron microscopy combined with histochemical techniques. This localization was found in a variety of different types of nerve and in muscle fibers. On examination of myelinated fibers the enzyme was, however, frequently absent in spite of its regular presence when the enzyme activity was determined in homogenized tissue. Even in a section 500 to 1000 A thick, structural barriers

Fig. 2. Large myelinated (MY) ventral root axon (AX) taken from a frog sciatic nerve. The fiber was treated with Triton 100 X, before the incubation for testing acetylcholinesterase activity. Dense end product is present on the axolemmal (plasma) membrane (arrow). Magn. X 32000. [Figure reprinted by permission of Editor, Proc. Nat. Acad. Sci. (Wash.) 56, 1560 (1966)]

may slow down or prevent the reaction between the membrane-bound enzyme and the added compounds required for its detection, especially in a tissue rich in lipids. Therefore, BRZIN [25] applied a detergent, Triton 100 X, to sections of a single isolated frog sciatic nerve fiber and found that acetylcholinesterase was located exclusively in the plasma membrane between the myelin and the axoplasm (Fig. 2). In the electroplax of Electrophorus the enzyme is present in the membranes of the nerve terminal and in the synaptic and conducting membranes [26). When CHANGEUX et al. [27] separated mechanically the excitable and inexcitable membranes, virtually all of the enzyme was found to be localized in the former, very little in the latter. When the isolated membranes were stained with histochemical procedures usually applied for the localization of AcCh-esterase and Triton 100 X was used, the enzyme was shown to be present in the excitable membrane (Fig. 3); in the inexcitable membrane no enzyme was detectable. The enzyme was, as in previous observations, uniformly distributed throughout the excitable membrane. Because virtually all of the enzyme is localized in the excitable membrane, a more correct value of the extraordinary

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enzyme activity in electric tissue is obtained when this activity is expressed as the amount of AcCh hydrolyzed per gram of membrane and not per gram of whole tissue. Since the membrane forms only 10- 4 or less of the whole cell mass, 1 gram of excitable electroplax membrane hydrolyzes 30 kilograms or more of AcCh per hour. (b) Purification and crystallization. Studies on the properties of acetylcholinesterase started in 1938 when this enzyme was first obtained in a highly active solution by extraction from electric tissue [28]. In the early 1940's a 500-fold purification was obtained, but the protein amounts avail-

Fig. 3. Electron micrograph of an isolated excitable membrane fragment of the electroplax of Electrophorus. The picture shows the striking uniformity of the distribution of AcChes terase at the innervated surface (MS) of the membrane The separation of the electroplax membranes has been achieved by differential centrifugation and by means of discontinuous saccharose gradients. In the usual cytochemical staining procedure for demonstrating AcCh-esterase activity, acetylthiocholine was used as the substrate. Magn. X 8860. No staining was found in the noninnervated (NI) membrane. (From CHANGEUX et aJ. [27J)

able were small [29]. The preparation was useful for kinetic studies and for analyzing many reactions of ligands with molecular groups in the active site and the mechanism in the hydrolytic process [20, 30, 31]. The effects of potent competitive inhibitors widely used in neuropharmacology and medicine were explained in terms of their reaction with the molecular groups in the active site of the enzyme. Of particular theoretical as well as practical interest were the studies with organophosphates, potent inhibitors of acetylcholinesterase and other ester-splitting enzymes. Some organophosphates are potential chemical warfare agents, and many are widely used as insecticides. The fatal effects are due to the reaction with AcCh-esterase. The phosphorus atom of organophosphates forms a covalent bond with an oxygen atom of the serine residue in the active site of ester-splitting enzymes. The resulting phosphorylated enzyme is much more stable than the acetyl enzyme. The reaction was first thought to be irreversible, but this notion has undergone considerable

Bioenergetics and Properties and Function of Proteins in Excitable Membranes 179

modifications [24,31]. The explanation of the reaction mechanism permitted the development of a highly efficient and widely used antidote against poisoning by organophosphate insecticides - namely, pyridine 2-aldoxime methiodide (PAM), which rapidly and quite specifically reactivates the enzyme by removing the phosphoryl groups from the serine in a displacement reaction [32]. PAM repairs the specific biochemical lesion. It is more efficient and less harmful than atropine, still widely used as antidote, although it protects only the receptor against excess AcCh without restoring enzyme activity. However, in combination with atropine PAM protects against 20-fold (or more) lethal doses of insecticides [33]. The analysis of protein properties requires considerable amounts of homogeneous and (for X-ray crystallography) crystallized protein. Adequate amounts of homogeneous acetylcholinesterase have been obtained by a large scale purification of electric tissue with chromatographic methods, and the crystallization of the enzyme has been achieved [34]. The crystals have the form of hexagonal prisms. Measured by equilibrium dialysis the enzyme has a molecular weight of 260000; it seems to be formed by two different polypeptide chains [35]. Affinity chromatography has been recently shown to be by far the most efficient and reliable procedure for purifying the enzyme; the yield is, moreover, many times as high as that obtained with the methods used previously. (c) Interdependence between electrical and acetylcholinesterase activity. The localization of the enzyme in various types of excitable membranes does not yet indicate its essential role in electrical activity. Other properties of the enzyme are prerequisites - for example, the high rate of its activity with a turnover time of 30 to 40 microseconds or less. Because nerve fibers may conduct 1000 impulses per second, the signal interacting with the receptor must be removed with a speed compatible with the function proposed. The postulated role requires, however, the demonstration that the enzyme and electrical activities are directly associated; potent inhibitors of acetylcholinesterase should effect and eventually block electrical activity. This effect has been demonstrated with reversible as well as irreversible types of inhibitors. However, this kind of experiment offers many pitfalls; some of them, such as the existence of structural barriers, were obvious from the beginning. Others became increasingly apparent when information accumulated about the many complex factors which may change the behavior of enzymes in an intact structure. Today it is well recognized that it is frequently impossible to extraP9late from observations in of enzymes solution to their behavior in intact cells because a variety of factors may modify the reactions. A few observations may be mentioned. Electrical activity of the frog sciatic nerve was found to be reversibly blocked by physostigmine, a potent competitive reversible inhibitor of acetylcholinesterase with an inhibition constant Ki of 10- 7 mole per liter; but the concentration required was 10- 2 mole per liter, and the time of exposure was 30 to 60 minutes. This preparation is formed by several thousand myelinated fibers surrounded by a sheath poorly permeable to many compounds. When physostigmine was applied to a single fiber of this preparation, in which the excitable membrane of the axon is poorly protected at the Ranvier nodes where myelin is absent, effects on the electrical activity

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were obtained at 10- 5 to 10- 6 mole per liter; within seconds a potentiation was observed followed by a depression and finally a block [36]. Several other types of complications were encountered with organophosphates. Exposure of various types of nerve fibers to organophosphates blocks electrical activity irreversibly, but in higher concentrations than expected. For example, in the squid giant axon, 5 X 10- 3 M diisopropylphosphofluoridate (DFP) and 30 to 40 minutes exposure are required for producing irreversible block. The actual concentration of DFP inside the axoplasm of the giant axon, at the time when conduction was irreversibly blocked, was small, of the order of 10-5 mole per liter [37,38]. It was later found that in the giant axon, as in many tissues, a phosphorylphosphatase exists which rapidly inactivates many organophosphates. In some types ofaxons, rapid and irreversible effects were obtained with relatively low concentrations. Pyridine 2-aldoxime methiodide, the potent and specific reactivator of phosphorylated acetylcholinesterase mentioned above, restored, in some preparations, irreversibly blocked electrical activity, although the compound is a quaternary ammonium derivative and does not readily penetrate the cell [39]. The successful demonstration that potent inhibitors of acetylcholinesterase rapidly block electrical activity in several types of conducting fibers is strong evidence for the essential role of the enzyme in this process. When organophosphates became available, in the 1940's, it was thought that this type of compound would permit the determination of the minimum acetylcholinesterase activity still compatible with unimpaired conduction. This value would indicate the excess of enzyme present. In the last few years it has become evident that there is at present no possibility to answer this question. The methods now available do not permit a quantitative evaluation of the enzyme activity in normal tissue. Simple homogenization turned out to be inadequate. Several chemical procedures may increase activities severalfold. After the tissues are exposed to organophosphates, the difficulties are compounded by many complicating factors; some ofthem became apparent only recently. Thus, for the time being there is no answer to the question of the minimum activity of acetylcholinesterase required for electrical activity, but the claims of a successful dissociation of electrical and enzyme activity after exposure to organophosphates have lost their validity [24].

II. Choline O.acetyltransferase (a) Energetic aspects of bioelectrogenesis. Perhaps no other aspect of th€ studies on the biochemical basis of bioelectricity, the bioenergetics of this process and the role of AcCh in nerve activity, was as strongly influenced by MEYERHOF'S work and ideas as the determination of the sequence of energy transformation during electrical activity and the discovery of enzyme acetylation. It was in MEYERHOF'S laboratory that the well-known notion of two different classes of phosphate compounds was first developed: one class, formed by a group of relatively stable phosphate esters, such as ex-glycerophosphate or glucose 6-phosphate, is hydrolyzed with a relatively small free-energy change, of the order of -1500 to -3000 cal. per mole. The second class of phosphate derivatives, mostly anhydrides of phosphoric

Bioenergetics and Properties and Function of Proteins in Excitable Membranes 181

acid, are less stable and hydrolyzed with a large free-energy change, with a LlF of -10000 to -12000 cal. per mole. The free energy available from oxidative reactions is usually trapped by ATP, one of the latter class of phosphate derivatives, and used for endergonic life processes. When phosphocreatine was discovered, MEYERHOF and SURANYI [40] measured the heat of its hydrolysis and found a high enthalpy in contrast to that of other phosphate esters. Later ATP, phosphoenolpyruvic acid, 1,3-diphosphoglyceric acid and other phosphate derivates were found to be hydrolyzed with a large free-energy change. MEYERHOF [41] reviewed the importance of the distinction between these two classes of phosphate derivatives for the intermediary metabolism in general and in particular for the implications for the problem of energy supply and the sequence of energy transformation in muscular contraction. MEYERHOF'S ideas were further extended and elaborated in the articles of LIP MANN [42] and KALCKAR [43]. The history of the development of these early concepts as well as those of more recent years may be found in the book "Biological Phosphorylations" edited by KALCKAR [44]. . When the writer first began to suspect, in 1940 and 1941, that the action of AcCh was not that of a neurohumoral transmitter, but an intracellular process associated with bioelectrogenesis, the problem of the energy supply and of the sequence of energy transformations during electrical activity appeared to him to be essential elements for obtaining a satisfactory insight into the function of AcCh and the chemical basis of nerve activity. The ionic concentration gradients between the outer environment and the interior of conducting fibers are most likely the primary source of energy of the ion movements carrying the electrical currents. If AcCh is the trigger responsible for making the potential source of energy, the ionic concentration gradients, effective by initiating the series of processes leading to increased ion permeability of excitable membranes, and if the ester is rapidly inactivated by hydrolysis permitting the return of the membrane to its resting condition and reestablishing the barrier to ion fluxes, the question arises: what is the reaction that provides the energy for the acetylation of choline in the recovery period 1 Were phosphate derivatives with a large ｦｩＺ･Ｍｮｾｧｹ＠ change involved 1 The electric organs being the most powerful bioelectric generators known and being highly specialized in their function seemed to offer a most suitable material for testing this question. The electric tissue of Electrophorus, although formed to 92% by water, has a remarkably high concentration of phosphocreatine, as high or highert han striated muscle; the concentration of ATP is also high, although slightly lower than in muscle. When the breakdown of phosphocreatine, roughly coinciding with electrical activity, was measured in the electric tissue of Electrophorus, it turned out to be more than adequate to account for the total, external and internal, electrical energy released [20,21,45]. It was assumed that the phosphocreatine breakdown was coupled as in muscle to the hydrolysis of ATP and that it was used for the rephosphorylation of ADP. To the energy released by the breakdown of phosphocreatine had to be added that of lactic acid formation. The computation of the chemical energy was based on the Gibbs free energy, LlF or LlG; the energy of ATP hydrolysis was assumed to be -12500 cal. per mole, that of

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lactic acid formed -25000 cal. per mole. A detailed description and analysis of the data may be found in the reviews quoted above. It appears likely that under the experimental conditions used part of the chemical energy was released subsequent to electrical activity and was, therefore, used for the restoration of the ionic concentration gradient for which the energy of ATP hydrolysis is used, as was shown many years later in several laboratories for many cells. The process is not specific for nerve and muscle fibers. A direct relationship between ATP hydrolysis and the elementary processes of bioelectrogenesis appeared for many reasons unlikely, especially in view of the low speed of the reaction. But it appeared possible that the energy of ATP hydrolysis would be used for the acetylation of choline, the first recovery process following the hydrolysis of AcCh. (b) Discovery of enzymic acetylation. When ATP was added to extracts of brain and electric tissue, AcCh formation was obtained, but it was disappointingly small. Only when NaFl was added for inhibiting ATPase, a strong and regular synthesis of AcCh was observed [46]. Soon afterwards, the rapid inactivation of the enzyme system on dialysis was observed and suggested the necessity of a coenzyme in the system which was subsequently demonstrated. These observations were the first enzymic acetylation achieved in a soluble system with ATP hydrolysis as the source of energy. It seemed at that time surprising and difficult to explain that ATP provided the energy for acetylation and not acetylphosphate described by LIPMANN. However, in the following decade the mechanism of acetylation in bacterial and animal cells and the structure and role of the coenzyme, referred to by LIPMANN as coenzyme A (CoA), have been fully elucidated in the laboratories of LIPMANN, OCHOA, LYNEN and many others and have become an integral part of biochemistry textbooks. Choline acetylase, now generally referred to as choline O-acetyltransferase, was redefined as the enzyme that transfers the acetyl group from acetyl-CoA to choline. The properties and function of choline O-acetyltransferase, its partial purification, etc. have been repeatedly reviewed.

D. Acetylcholine receptor protein I. Monocellular electroplax preparation A turning point in the studies of this protein was the development of a monocellular electroplax preparation from Electrophorus by SCHOFFENIELS [47, 48]. This preparation is unique for studying the properties of the receptor within the membrane because of (I) the large size of the cell; (II) the rectangular shape of the excitable membrane, containing both synaptic and conducting parts readily distinguishable by means of electrical parameters; and (III) the presence of a nonexcitable membrane which facilitates comparison between the two functionally different parts. As described above, the outstanding feature of the cell (that is, the electroplax of Electrophorus) is the prevalence of metabolic reactions in the membrane. Although the electroplax responds to externally applied AcCh, curare, and other quaternary nitrogen derivatives at the

Bioenergetics and Properties and Function of Proteins in Excitable Membranes 183

synaptic junctions only, both AcCh-receptor and acetylcholinesterase are present and functional in the conducting parts of the excitable membrane. During the last decade several refinements have permitted more sensitive analyses of the reactions between the receptor protein and specific ligands by means of electrical parameters comparable with the precision of other methods applied in protein studies. The preparation permits precise and reproducible titration of the dose-response curves and the evaluation of dissociation constants between ligands and the protein. Acetylcholine and related compounds that affect electrical activity and simultaneously depolarize the membrane by inducing conformational changes are referred to as receptor activators. Other compounds closely related in structure to AcCh act as "antimetabolites"; they prevent the specific signal from interacting with the receptor and are referred to as receptor inhibitors.

II. Reactions with the receptor protein (a) Active site. The molecular groups in the active site of the receptor have been analyzed with a great variety of compounds and compared with the reactions of the enzyme in solution. The functional importance of the anionic group in the active site of both proteins has been demonstrated. Quaternary ammonium derivatives tested on the receptor are a few hundred up to a thousand times more potent than their tertiary analogs. There is no esteratic site in the receptor protomer, as might have been expected from the different function of the two proteins. A vast amount of data has accumulated supporting the assumption of the protein nature of the receptor. (b) Role of receptor in bioelectricity. The essential role of the receptor in bioelectricity has been demonstrated with a group of competitive inhibitors, the so-called "local anesthetics". These substances are closely related in structure to AcCh and act as typical antimetabolites at synaptic and conducting parts ofthe membrane. The transformation of the molecular structure of AcCh from a receptor activator, which acts on the junction only, to a receptor inhibitor, which acts on both parts of the membrane, was analyzed by a series of replacements of molecular groups [49, 50]. Benzoylcholine was found to be structurally and functionally the intermediate form; it may act as a receptor activator or inhibitor, depending on the experimental conditions. With the addition of a para-amino group to the phenyl ring, the compound becomes a typical local anesthetic. Small modifications of either the acyl or of the quaternary nitrogen group may greatly increase its potency as a local anesthetic. These compounds block all electrical activity in all excitable membranes; thus, the data support the postulated role of the receptor. (c) Oxygen, sulfur, and selenium isologs. Pertinent information a bout the active site of receptor and esterase resulted from experiments with a large series of Sand Se isologs of AcCh and its congeners [51]. Striking differences in the biological activity of these isologs, tested by the effects on the receptor of the electroplax, and in their reactions with the enzyme in solution have been interpreted in part as being due to differences of electron distribution and in part as being the result of different configurations (gauche and trans) of the isologs, as determined by X-ray analysis.

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(d) Oooperativity and allosteric sites. Observations of CHANGEUX, PODLESKI, and their associates [52] suggest certain similarities between the behavior of regulatory enzymes and the reactions of the electroplax with ligands acting on the receptor. The dose-response curve of the receptor to AcCh and other activators has a sigmoid shape, thus deviating from the usual Langmuir isotherm. The S-shaped curve is considered a characteristic property of allosteric systems and of cooperative action. The Hill coefficient (nH) of the electroplax response is about 2. After exposure to sulfhydrylblocking and disulfide-reducing compounds the nH becomes about 1 [53]. In the presence of two different activators, the dose-response curve of the electroplax becomes a hyperbola, with an nH of 1; this is considered an indication that the interaction between both classes of ligands may be indirect or allosteric. At saturating concentrations of activators the maximum response to different activators differs, independent of the ionic environment. (e) Affinity labeling. Compounds forming a covalent bond with the molecular group in the active site of an enzyme have long been important tools in the analysesofthese sites. One type of covalent bonding is referred to as "affinity labeling"; the reagent, because of its steric complementarity to the active site, first combines specifically and reversibly with the site with which it forms a complex. Then a small and reactive group reacts with one or more amino acid residues to form irreversible covalent bonds. The first affinity labeling of the AcCh-receptor was described with p-(trimethylammonium) benzenediazonium fluoroborate (TDF); the compound blocks irreversibly the electroplax response at a concentration of 10- 4 mole per liter [54]. Recent observations of MAUTNER and BARTELS [55] indicate, however, that it is the positively charged diazonium group that is attracted to the negative subsite in the receptor. Affinity labeling has been achieved with a two-step procedure, when the labeling compounds are applied after reduction of disulfide bridges of the electroplax by dithiothreitol (DTT). N-ethylmaleimide presumably forms a covalent bond with the exposed sulfhydryl groups. Substitution of the ethyl by a phenyltrimethylammonium group, a potent receptor activator, increased the potency by several hundredfold; 4(N-maleimido)phenyltrimethylammonium iodide (MPTA) blocks the receptor at a concentration of 10-8 mole per liter. The tertiary analog is not more potent than N-ethylmaleimide [56]. Other potent affinity-labeling compounds that act on the reduced cell were designed by SILMAN; one of them is bromoacetylcholine, which forms a covalent bond between the nucleophilic sulfhydryl groups and the carbon of the carbonyl group. In contrast to MPTA, which is a receptor, inhibitor, this compound depolarizes the electroplax [57, 58]. The use of affinity-labeling compounds may be useful in experiments aimed at the isolation of the receptor. Drastic changes of biological effects may result from the exposure of the electroplax to DTT. Hexamethonium, a bisquaternary reversible inhibitor, becomes a receptor activator after reduction. The compound TDF, an irreversible inhibitor of the receptor at a concentration of 10- 4 mole per liter, becomes after reduction a reversible activator at 10- 6 mole per liter. Apparently opposite biological actions leading to excitation or inhibition may thus be due to relatively small changes in the state of the receptor and other factors in the

Bioenergetics and Properties and Function of Proteins in Excitable Membranes 185 membrane, and not - as is widely assumed - to different "excitatory" or "inhibitory" transmitters. An explanation of the basis of such differences requires an analysis of the molecular reactions, as exemplified by the dependence of the action of adenosine triphosphate on the concentration of Ca2+ ions in inducing either muscular contraction or relaxation.

III. Isolated excitable membranes ("vesicles") A new development was initiated by CHANGEUX and his associates [2] by using excitable membranes of electroplax prepared in a way that they form very small "vesicles" or "microsacs". Excitable membranes from electric tissue were prepared by homogenization and sonication of the tissue in sucrose, followed by differential centrifugation in a discontinuous sucrose gradient. Electron micrographs of a particulate fraction which contained AcCh-esterase showed vesicular structures devoid of any cytoplasm. These vesicles were isolated on the basis of their content of AcCh-esterase and then incubated overnight in a Na 22 Cl solution. After dilution in a cold salt solution the efflux of Na 22 from the vesicles was measured [59]. When these vesicles were exposed to carbamylcholine, the efflux of Na 22 was accelerated. Similar effects were obtained with decamethonium. When d-tubocurarine was added combined with carbamylcholine, the increased efflux of Na22 was blocked. No such effects were observed with vesicles obtained from the noninnervated parts of the membrane. The most significant of these observations, however, is the complete parallelism found with receptor activators and inhibitors when the effects on Na 22 efflux were compared to the effects of the same compounds on the membrane potential of the intact electroplax. The same affinities of the activators controlling these two processes are observed in either system. Even the sigmoid shape of the dose-response curve observed in the experiments with the vesicles was essentially similar to that found in the electroplax, indicating cooperativity and allosteric effects. The Hill coefficient for carbamylcholine was found to be virtually the same in vivo (intact cell) and in vitro (vesicles formed by isolated membranes). This preparation of isolated excitable membranes offers a new assay of the properties of excitable membranes and especially the reactions with the membrane proteins, permitting to compare their behavior (I) in solution, (II) in the subcellular structure and (III) in the intact cell.

IV. Characterization, identification and isolation of the receptor protein by toxins (a) cx-bungarotoxin. Recently, CHANGEUX et al. [60] introduced a new compound for the characterization and identification of the receptor, ex-bungarotoxin, a polypeptide of molecular weight of 8000 purified from the venom of the snake Bungaru8 multicinctu8, which had been studied by LEE and his associates [61]. The toxin produces irreversible neuromuscular block; d-tubocurarine protects against the action of cx- bungarotoxin. From these findings, LEE and CHANG [62] concluded that the toxin combines irreversibly with the cholinergic receptor at the motor end plate.

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The toxin was applied by CHANGEUX, KASAl, and LEE [60] to various preparations derived from the electric organ of Electrophorus, i.e., to the isolated electroplax and to isolated excitable membranes, "vesicles", and to proteins obtained by fractionation of the electric tissue. The toxin, in 1 mg/mI, irreversibly blocks the electroplax response to carbamylcholine; d-tubocurarine protects against the effect of the toxin in agreement with the observations of LEE and his associates. The same effects were observed with isolated membranes of the electroplax. Finally, the authors tested iX-bungarotoxin on the binding of radioactive decamethonium to a protein isolated from electric tissue and which presents in solution some properties characteristic of the cholinergic receptor protein. The toxin blocks, in equilibrium dialysis assay, the binding of decamethonium to the protein; it has no effect on AcCh-esterase. Thus, the observations suggest specific binding to the physiological receptor of AcCh without affecting the catalytic sites of the enzyme. (b) Separation of the AcCh-receptor protein from AcCh-esterase. In a still more recent development CHANGEUX and his associates [63] have succeeded in separating the receptor protein from the enzyme by using the iX toxin of Naja nigricollis. The authors prepared from electric tissue of Electrophorus an extract which contained both the receptor protein and the enzyme. The iX-toxin of Naja nigricollis was covalently coupled to Sepharose granules. The granules of Sepharose toxin were left in contact with the extract for 30 minutes. After this period of exposure the extract had lost 75 to lOO% of the AcCh-receptor protein, whereas 85 to lOO% of the enzyme remained in the supernatant. Thus, for the first time the two proteins have been unequivocally separated. The number of active sites of the receptor were found to be about equal to the number of active sites of the enzyme.

E. Similarities and differences between axonal and junctional membranes I. Reevaluation of the basis of the transmitter theory A criterion for the validity of a new concept is its ability to provide more satisfactory explanations for the experimental data than that provided by the preceding concept. In view of the progress achieved in the understanding of chemical reactions in membranes, a reevaluation of the theory of neurohumoral transmission appears imperative. (a) Effects of AcCh and curare; permeability barriers surrounding axonal membranes. The powerful pharmacological action of AcCh on junctions in contrast to its complete failure to act on axonal conduction has been emphasized as evidence for a mechanism of synaptic transmission basically different from that of axonal conduction. Many drugs act on junctions only, as has been frequently observed since CLAUDE BERNARD first demonstrated that curare blocks the transmission from nerve to muscle, but does not affect conduction. Curare, a receptor inhibitor with a higher affinity to the receptor than AcCh, prevents the depolarization by the ester. The compound is a quaternary nitrogen derivative and is insoluble in lipids as are AcCh, neostigmine, and other receptor activators or inhibitors. The conducting membranes ofaxons are surrounded

Bioenergetics and Properties and Function of Proteins in Excitable Membranes 187

by Schwann cells, which act as structural barriers for this type of compound. CLAUDE BERNARD applied curare to the frog sciatic nerve fiber, formed by several thousands of myelinated axons and surrounded by a sheath that is impervious to many chemical compounds. When a single axon of this fiber is exposed to curare, electrical activity at the Ranvier nodes is rapidly and reversibly blocked, just as at junctions where barriers do not exist or are more pervious [64]. Even in unmyelinated axons the Schwann cells, which contain phospholipids, usually prevent AcCh, curare, and related quaternary structures from reaching the receptor in the excitable membrane, although in some axons with apparently incomplete barriers, for example, those of the walking leg of lobster, a direct action of these compounds on electrical activity has been obtained [65]. However, chemical treatment may reduce the outside barriers and permit AcCh and its congeners to reach the receptor in the excitable membrane. In the giant axon of the squid the excitable membrane is surrounded by a Schwann cell about 4000 A thick. When this axon is exposed to AcCh, curare, or neostigmine at high concentrations, no effect is observed. In contrast, physostigmine, a tertiary nitrogen derivative and an inhibitor of acetylcholinesterase equal in potency to neostigmine, blocks electrical activity. The tertiary analog of neostigmine, although a much weaker inhibitor than the quaternary form, has the same effect on conduction. When the axoplasm of the exposed axons was extruded, physostigmine had penetrated, whereas the quaternary compounds had not [66]. After the squid giant axon has been exposed to phospholipase A for a short period of time in low concentrations, electrical activity is blocked by AcCh and curare [67]; when, under these conditions, the two compounds labeled with radioactive isotopes were used, they had penetrated into the interior [68]. Examination by electron microscopy revealed marked structural changes in the Schwann cell, but none in the plasma membrane [69]. The effect of phospholipase A is fully accounted for by the formation of lysolecithin [70]. Even synaptic junctions may not react to AcCh and curare, for example, the neuromuscular junction of lobster, although the concentrations of acetylcholinesterase and choline O-acetyltransferase in these junctions is very high. Lipid soluble compounds, such as physostigmine and DFP, affect these junctions. Thus, the system is present and functional but protected against quaternary derivatives. (b) Release at AcCh. The second observation considered as evidence for the theory of neurohumoral transmission is the appearance of AcCh in the extracellular perfusion fluid of junctions after stimulation. However, no trace of AcCh appears in the absence of physostigmine even after prolonged stimulation, as was emphatically stressed by DALE et al. [7l]. This failure to detect any extracellular AcCh unless acetylcholinesterase, the highly effective inactivation mechanism, is blocked, suggests that the appearance is an artifact attributable to the incomplete hydrolysis. The detection of an efflux of AcCh from the axonal membrane would be prevented by the structural barriers except where they are incomplete, as in the axons of the lobster walking leg. There an efflux was found, provided that the axons were kept in physostigmine; various ions affect this efflux in a way similar to that described for junctions [72]. Even in the presence of potent enzyme inhibitors, the extracellular amounts found at

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junctions are far from those necessary for the ester to exercise a transmitter function. Such a sensitive preparation as the electroplax does not respond to 10-6 M AcCh in the absence of physostigmine; in its presence 10-6 mole per liter is required. The amounts released are ten orders of magnitude smaller. This large discrepancy raises a serious difficulty in attributing the postulated intercellular function to AcCh.

. II. Theory based on biochemical data; role of AcCh at junctions An alternative explanation accounts more readily for the data. Acetylcholine-receptor and acetylcholinesterase are both localized in the two membranes of the junction, those of the nerve terminal and of the postsynaptic JUNCTION

Fig. 4. Schematic presentation of the role of AcCh at junctions postulated to be essentially similar to that in the axon (see Fig. 1; symbols used are the same) As in the axonal membrane, AcCh is released by the current arriving at the terminal; it acts as the signal that initiates the reactions which amplify the signal and lead to increased permeability of ions in the terminal membrane. K + ions coming out from the nerve terminal act as transmitters of the impulse from cell to cell: many millions of them cross the nonconducting gap per 1000 molecules of AcCh released. However, these ion movements require the reactions within the terminal membrane initiated by AcCh. The flow of K+ ions acting across the gap leads, directly or indirectly, to a release of AcCh in the postsynaptic membrane which then initiates the same series of reactions there

membrane. Both proteins are functional since AcCh, curare, neostigmine, and related compounds act on both membranes of the junction [73-75]. Thus, it seems reasonable to assume that the amplifier process takes place in the membranes of the junctions in a way similar to that of the axons; AcCh released in the terminal membrane acts on the receptor there and triggers the sequence of the reactions resulting in the influx of sodium and the efflux of potassium ions. When a strong efflux of potassium ions was found after stimulation at junctions as well as in axons [76, 77], ECCLES [78] attributed the transmitter function to potassium ions. But release of the ions requires the amplification process triggered by AcCh; many millions of potassium ions would cross the gap for each 1000 molecules of AcCh released and effect the release of AcCh in the postsynaptic membrane. Chemical reactions in living cells are chemically and

Bioenergetics and Properties and Function of Proteins in Excitable Membranes 189

thermodynamically coupled; most of them are structurally organized. It appears more plausible that the specific signal in one of the fastest cellular mechanisms is recognized by the target protein within the membrane where it is released, rather than by a protein in another cell. Fig. 4 is a schematic presentation of the postulated role of AcCh in the two junctional membranes. For many years, the failure to detect flow of current from nerve terminals was considered as a support for chemical transmission. The absence of current flow seemed particularly conspicuous in the giant synapse of squid, in which fractions of the pre- and postsynaptic axons are located side by side and permit monitoring of current flow by the insertion of microelectrodes into both axons. Recently, current flow has been observed from nerve terminals; in the squid synapse current flows in both directions [79]. The pharmacological action on junctions mimicking nerve stimulation must be reinterpreted, on the basis of the data resulting from the analysis of membrane proteins, as mimicking the signal within the two membranes of the junction and not acting between them. Such a view integrates the observations which seemed to suggest neurohumoral transmission with the biochemical data and with the concept of the fundamental similarity of the function of AcCh in the conducting and junctional membranes.

III. Special features of junctions The specific chemical forces underlying cellular mechanisms such as motility, energy supply, vision, and genetic control, are remarkably similar throughout the animal kingdom. It has become apparent that this similarity also applies to the specific chemical forces underlying bioelectricity, that is, to the specific proteins controlling the changes of ion permeability in excitable membranes. The great diversity of bioelectric phenomena and the variations of pharmacological effects may be explained by differences of cellular structure, shape and environment between junctions and axons. These factors are bound to modify the effects of chemical reactions in the membranes and the actions of compounds applied externally. Electrical parameters in axons differ in various types of nerves; conduction velocity, for example, varies from 0.1 to 100 meters per second. These differences are generally attributed to dissimilarities of structure and not to differing basic mechanisms of conduction. Knowledge of the chemical composition and molecular organization of membranes is much too limited to permit speculation about possible differences. The answer to these questions requires much more information on a molecular level.

F. Concluding remarks: Concepts and axioms in science In a symposium devoted to the memory of OTTO MEYERHOF it appears appropriate to make a few comments on the decisive importance of concepts and basic notions for the progress of science: they determine the thinking, the methods of approach, the design of experiments, and the interpretation of the results by the investigator. However, as history has shown time and again,

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concepts once accepted prevail for a long time and it usually takes several decades until a new concept, no matter how sound and convincing, is accepted and the preceding one abandoned. In his article on "Theoretical Concepts in Biological Sciences", KREBS [80] quotes several interesting and illuminating examples ofthe rejection of new concepts, the hostility, vituperation and vitriolic comments with which other colleagues met the new ideas. This response seems to be common and may reflect a natural human reaction. As mentioned by WEBER, it took decades until some of the basic notions by MEYERHOF were fully accepted and became an integral part of biological thinking. Actually, this may not be as surprising as it may seem to those who think of science in terms of a human endeavor essentially based on objective data and experimental observations. When one realizes, however, to what extent basic notions and methods of approach affect our reasoning, conflicts and opposition to new ideas appear almost unavoidable. The biochemist, or the molecular biologist, analyzing e.g. a problem on cellular, subcellular and molecular level will use different criteria and notions from those used by a physiologist who studies overall manifestations of complex preparations or organs characterized by a nearly infinite diversity of structure and organization. Even a subcellular structure, such as e.g. a cell membrane, is extremely complex and biochemists have become increasingly aware during the last decade of the many big loop-holes in our knowledge of chemical composition, ultrastructure, reaction mechanisms, etc. Nevertheless, it can hardly be questioned that many theoretical concepts of biology have been profoundly deepened and clarified by the biochemical approach and in particular by the analysis of proteins and enzymes associated with the cellular mechanism under investigation. As KREBS writes in the article quoted, even in physical sciences there are certain basic concepts, although supported by a vast body of evidence, which cannot be rigidly proved or disproved, but are nevertheless real and essential; he quotes MAX PLANCK: "Auch in der Physik gilt der Satz, dass man nicht selig werden kann ohne den Glauben". Biological "axioms" applied in the biochemical approach differ in many respects in a fundamental way from those of classical physiology and pharmacology. In summary, the notions of bioenergetics and of protein and enzyme chemistry, as developed by MEYERHOF in his work on muscular contraction, were applied to the problem of another cellular function, namely nerve conduction. The approach has provided much information about the properties and function of the proteins in excitable membranes associated with AcCh; it has resulted in a concept of the role of AcCh quite different from that assumed in the theory of neurohumoral transmission.

Addendum in proof: In the recent issue of the J. Membrane Biology (6, 1-88, 1971) CHANGEUX and his associates compare various parameters ofNa+ and K+ fluxes from the microsacs as a result of chemical stimulation by carbamylcholine with those across the axonal membranes by electrical stimulation of squid giant axons. A striking similarity is found between the response to chemical stimulation of the isolated membrane fragments, with no EMF involved, and the response to electrical stimulation of the axons, when the fluxes, cP (molesjcm 2 j

Bioenergetics and Properties and Function of Proteins in Excitable Membranes 191

sec), the permeabilities, p (cm/sec) and conductances, g (mho/cm 2 ) of Na+ and K + ions are compared:

Na* K* pNa pK gNa gK (JJ (JJ

Microsacs

Giant axons

4 2 2 1 1 9

3.7 8.0 3.3 7.4 3.3 2.4

X 10-13 X 10-12 X 10- 9 X 10- 8

X 10- 6 X 10- 6

X 10- 12 X 10- 11 X 10- 9

X 10- 7 X 10- 6 X 10- 4

* In the case of microsacs the figures represent effluxes ofNa and K, in the case ofaxons the figure for N a represents the influx, that for K the efflux. Only the average data are given.

Since in both membranes the same protein assemblies are present and respond to specific ligands reacting with these proteins in a similar way, the similarity of the parameters represents one of the most striking supports of the writer's theory, i. e., that the same signal activates the same protein assemblies which are in control of ion permeability in synaptic and conducting membranes.

References 1. NACHMANSOHN, D.: In: Metabolism and Function (NACHMANSOHN, D., Ed.). Amsterdam: Elsevier 1950, p. 1. 2. NACHMANSOHN, D.: In: 2" Congres International de Biochimie. Paris: Masson 1953, p. 34. 3. NACHMANSOHN, D., OCHOA, S., LIPMANN, F. A.: Science 115, 365 (1952). 4. ROBERTSON, J. D.: Progr. Biophys. Biophys. Chem. 10, 343 (1960). 5. BENSON, A. A.: J. Amer. Oil Chem. Soc. 43, 265 (1966). 6. KENNEDY, E. P.: In: The Neurosciences (QUABTON, G. C., MELNECHUK, T., SCHMITT, F. 0., Eds.). New York: Rockefeller Univ. Press 1967, p. 271. 7. ROTHFIELD, L., FINKELSTEIN, A.: Ann. Rev. Biochem. 37, 463 (1968). 8. RACKER, E.: Mechanisms in Bioenergetics. New York: Academic Press 1965. 9. RACKER, E.: Federation Proc. 26, 1335 (1967). 10. GREEN, D. E., PERDUE, J. F.: Ann. N.Y. Acad. Sci. 137, 667 (1966). 11. New York Heart Association: Membrane Proteins. Boston: Little Brown 1969. 12. SJOESTRAND, F. S., BABAJAs, L.: J. Ultrastruct. Res. 5, 121 (1968). 13. HODGKIN, A. L.: Biol. Rev. (Cambridge) 26,338 (1951). 14. HODGKIN, A. L.: The Conduction of the Nervous Impulse. Springfield, Ill.: Thomas 1964. 15. TASAKI, I.: Nerve Excitation. Springfield, Ill.: Thomas 1968. 16. ABBOTT, B. C., HILL, A. V., HOWABTH, J. V.: Proc. Roy. Soc., Ser. B, 148,129 (1958). 17. HILL, A. V.: In: Symposium on Molecular Biology (NACHMANSOHN, D., Ed.). New York: Academic Press, p. 153. 18. HOWARTH, J. V., KEYNES, R. D., RITCHIE, J. M.: J. Physio1. (Lond.) 194, 745 (1968). 19. ERLANGER, J.: J. Neurophysiol. 2,370 (1939). 20. NACHMANSOHN, D.: Chemical and Molecular Basis of Nerve Activity. New York: Academic Press 1959. 21. NACHMANSOHN, D.: In: Handbuch der experimentellen Pharmakologie, vol. XV (KOELLE, G. B., Ed.). Berlin-Giittingen-Heidelberg: Springer 1963, p. 701; ibid., p. 40. 22. NACHMANSOHN, D.: Proc. Nat. Acad. Sci. (Wash.) 61, 1034 (1968). 23. NACHMANSOHN, D.: J. Gen. Physiol. 54,187 (1969). 24. NACHMANSOHN, D.: In: Handbook of Sensory Physiology, vol. I (LOEWENSTEIN, W. R., Ed.), Berlin-Heidelberg-New York: Springer 1970, p. 18.

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25. BRZIN, M.: Proc. Nat. Acad. Sci. (Wash.) 56, 1560 (1966). 26. BLOOM, F. E., BARRNETT, R. J.: J. Cell. BioI. 29, 475 (1966). 27. CHANGEUX, J.-P., GAUTRON, J., ISRAEL, M., PODLESKI, T. R.: C. R. Acad. Sci. Paris 269, 1788 (1969). 28. NACHMANSOHN, D., LEDERER, E.: Bull. Soc. Chim. BioI. (Paris) 21, 797 (1939). 29. ROTHENBERG, M. A., NACHMANSOHN, D.: J. BioI. Chem. 168, 223 (1947). 30. NACHMANSOHN, D., WILSON,1. B.: Adv. Enzymol. 12,259 (1951). 31. Handbuch der experimentellen Pharmakologie, vol. XV (KOELLE, G. B., Ed.). BerlinGiittingen-Heidelberg: Springer 1963. 32. WILSON,1. B., GINSBURG, S.: Biochim. Biophys. Acta 18, 168 (1955). 33. KEWITZ, H., WILSON, 1. B., NACHMANSOHN, D.: Arch. Biochem. Biophys. 64, 456 (1956). 34. LEUZINGER, W., BAKER, A. L., CAUVIN, E.: Proc. Nat. Acad. Sci. (Wash.) 59, 620 (1968). 35. LEUZINGER, W., GOLDBERG, M., CAUVIN, E.: J. Mol. BioI. 40, 217 (1969). 36. DETTBARN, W.-D.: Biochim. Biophys. Acta 41,337 (1960). 37. FELD, E. A., GRUNDFEST, H., NACHMANSOHN, D., ROTHENBERG, M. A.: J. Neurophysiol. 11, 125 (1948). 38. ROSENBERG, P., HOSKIN, F. C. G.: J. Gen. Physiol. 46, 1065 (1963). 39. ROSENBERG, P., DETTBARN, W.-D.: In: Animal Toxins (New York Heart Association, Ed.). Oxford-New York.: Pergamon Press, p. 379. 40. MEYERHOF, 0., SURANYI, J.: Biochem. Z. 191, 106 (1927). 41. MEYERHOF, 0.: Erg. Physiol. 39, 10 (1937). 42. LIPMANN, F.: Adv. Enzymol. 1, 99 (1941). 43. KALCKAR, H. M.: BioI. Rev. (Cambridge) 17,28 (1942). 44. KALCKAR, H. M.: Biological Phosphorylations, Development of Concepts. Englewood Cliffs, N. J: Prentice-Hall 1969. 45. NACHMANSOHN, D., Cox, R. T., COATES, C. W., MACHADO, A. L.: J. Neurophysiol. 6, 383 (1943). 46. NACHMANSOHN, D., MACHADO, A. L.: J. Neurophysiol. 6, 397 (1943). 47. SCHOFFENIELS, E., NACHMANSOHN, D.: Biochim. Biophys. Acta 26, 1 (1957). 48. SCHOFFENIELS, E.: Biochim. Biophys. Acta 26, 585 (1957). 49. BARTELS, E.: Biochim. Biophys. Acta 109, 194 (1965). 50. BARTELS, E., NACHMANSOHN, D.: Biochem. Z. 342,359 (1965). 51. MAUTNER, H. G.: Pharmacol. Rev. 19, 211 (1969). 52. CHANGEUX, J.-P., PODLESKI, T. R.: Proc. Nat. Acad. Sci. (Wash.) 59, 944 (1968). 53. KARLIN, A.: J. Theor. BioI. 16, 306 (1967). 54. CHANGEUX, J.-P., PODLESKI, T. R., WOFSY, L.: Proc. Nat. Acad. Sci. (Wash.) 58, 2063 (1967). 55. MAUTNER, H. G., BARTELS, E.: Proc. Nat. Acad. Sci. (Wash.) 67, 74 (1970). 56. KARLIN, A., WINNIK, M.: Proc. Nat. Acad. Sci. (Wash.) 60, 668 (1968). 57. SILMAN, L., KARLIN, A.: Science 164, 1420 (1969). 58. SILMAN, 1.: FEBS Symposium 21, 337 (1970). 59. KASAl, lVI:., CHANGEUX, J.-P.: C. R. Acad. Sci. Paris, Ser. D, 270, 1400 (1970). 60. CHANGEUX, J.-P., KASAl, M., LEE, C. Y.: Proc. Nat. Acad. Sci. (Wash.) 67, 1241 (1970). 61. LEE, C. Y., TSENG, L. F., CHIN, T. H.: Nature (Lond.) 215, 1177 (1967). 62. LEE, C. Y., CHANG, C. C.: Mem. Inst. Butantan, Sim. Internac. 33, 555 (1966). 63. MEUNIER, J.-C., HUCHET, M., BOQUET, P., CHANGEUX, J.-P.: C. R. Acad. Sci. Paris, Ser. D, 272,117 (1971). 64. DETTBARN, W.-D.: Nature (Lond.) 186,891 (1960). 65. DETTBARN, W.-D., DAVIS, F. A.: Biochim. Biophys. Acta 66, 397 (1963). 66. BULLOCK, T. H., NACHMANSOHN, D., ROTHENBERG, M. A.: J. Neurophysiol. 9, 9 (1946). 67. ROSENBERG, P.: Toxicon 3,125 (1965). 68. HOSKIN, F. C. G., ROSENBERG, P.: J. Gen. Physiol. 47, 1117 (1964). 69. MARTIN, R., ROSENBERG, P.: J. Cell. BioI. 36, 241 (1968). 70. CONDREA, E., ROSENBERG, P.: Biochim. Biophys. Acta 150, 271 (1968). 71. DALE, H. H., FELDBERG, W., VOGT, M.: J. Physiol. 86, 353 (1936). 72. DETTBARN, W.-D., ROSENBERG, P.: J. Gen. Physiol. 50, 447 (1966). 73. MASLAND, R. L., WIGTON, R. S.: J. Neurophysiol. 3, 269 (1940).

Bioenergetics and Properties and Function of Proteins in Excitable Membranes 193 74. RIKER, W. F., Jr., WERNER, G., ROBERTS, J., KUPERMAN, A. S.: Ann. N. Y. Acad. Sci. 81, 328 (1959). 75. WERNER, G., KUPERMAN, A. S.: In: Handbuch der experimentellen Pharmakologie, vol. XV (KOELLE, G. B., Ed.). Berlin-Gottingen-Heidelberg: Springer 1963, p. 570. 76. CoWAN, S. L.: Proc. Roy. Soc. London, Ser. B, 115, 216 (1934). 77. FELDBERG, W., VARTIAINEN, A.: J. Physiol. 83, 103 (1934). 78. ECCLES, J. C.: J. Physiol. 84, 50P (1935). 79. GAGE, P. W., MOORE, J. W.: Science 166, 510 (1969). 80. KREBS, H. A.: In: Current Aspects of Biochemical Energetics (KAPLAN, N. 0., KENNEDY, E. P., Eds.). New York-London: Academic Press 1966, p. 83.

Name and Subject Index Abhandlungen der Friesschen Sehule 3 acetylation 182 acetylcholine (AcCh) 174-176 in excitable membranes 175 - receptor protein 175, 182-186 - release 187 -, role at junctions 188 - synthesis 182 acetylcholinesterase 175-180, 183, 186 Acetyl-CoA 31 acetylphosphate 157, 166 actin 93 -, decorated 97 -, F- 94 -, monomeric 116 -, purified 111 -, spin-labelled 116 active center 27 actomyosin 90, 111 -, pure 113 acyl carrier protein 33 acylphosphates 157 ADP 157, 160, 161, 166, 168 ADP-ATP exchange 168 affinity chromatography 179 - labeling 184 aldolase 18 alkali-ion carrier 130 - - complexes 136,144 allosteric action 175 - systems 184 ex-bungarotoxin 185 ex-toxin of Naja nigricollis 186 amino acid sequences 27 aminoacyl adenylates 79 - thioesters 80, 83 ammonium derivatives, quaternary 183 amplifiers of the signal 175 anaerobe alactocide Muskelkontraktion 9 antibiotic polypeptides 79 antibiotics 130 artifact 187 Athylester der Triacetsaure (TAE) 52 Atmung 4 ATP 9, 90, 157, 160, 161, 165, 168 - ,high concentration 181 - hydrolysis 160, 182 -, Restitution 10 - splitting 159, 161 - synthesis 168 ATPase 155, 162-164

axonal membrane 186 azide 168 Binding specificity 146 biochemical approach 174 - lesion 179 bioelectricity 183, 189 bioenergetics 180 biologische Reaktionskette 4 bromoacetylcholine 184 Calcium 114 accumulation 157 binding 157 efflux 165-168 influx 156 ions 175 oxalate 150, 155, 156, 165 permeability 163, 165 phosphate 150, 155 pump 149 release 165, 168 transport 156, 161, 162 carbamylcholine 185 carbamylphosphate 157 carcinogenesis, mechanism 118 carrier 130, 142, 143, 145, 146, 167 - action, selective 131 --complex formation 144 - selectivity 142, 145 - specificity 142 cell membranes, concept 173 - -, properties and function 172 - surface membrane, structure 119 cellular structure 189 chain growth 82, 83 chain initiation complex 56 - - factors 56 chaotropic agents 165 chelate 132, 145 chelation 133, 134 chemical mediators 173 - warfare agents 178 chemische und energetische Koppelung 4 chlorpromazine 162 cholesterol 151, 153 choline O-acetyltransferase 176, 182 chromosome analysis 125 CoenzymA 33 complex formation 132, 137 - -, rate constants 135, 136, 139, 144, 146 - -, rate-limiting step 135

Name and Subject Index complex stability 131, 132, 136 - -, constants 134, 139 - -, size specificity 132, 134 Concanavalin A 119 concepts and basic notions in science 189 conduction velocity 189 configurations 183 conformation 145 - change 146, 175 contraction 112 cooperative action 184 coordination sphere 132, 144 CORRENS 7 covalent bond with oxygen of the serine 178 cross bridges 92, 93 - -, movement 98 - -, orientation 102 - -, power stroke 108 cross-striated muscle 90 curare 186, 187 current flow 189 cyclic mechanism 109 cyclization 84, 85 cysternae 150 Dianemycin 131 diazonium group, charged 184 diffraction pattern from frog sartorius muscle 102, 104, 105 diffusion control 139/140, 144, 146 dihydroxyacetone phosphate 19 dinactin 141 dinitrophenol 168 1,3-Diphosphoglyzerinat 9 dithiothreitol 184 dodecylsulfate 150, 152, 154, 165 dose-response curves, titration 183 Dux, WALTER 15 EGGLETON und EGGLETON 8 EGTA 155, 166, 167, 169 electric organs 174 - tissue 174, 181 electrical activity 179, 180 electron distribution 183 electrophilic groups 145 Electrophorus 174 electroplax 174 - preparation, monocellular 182 Energieabgabe 5 energy transformations 174 enniatins 130, 131 enniatins A and B 132 enthalpy 181 - of reaction 142 enzyme activity 179, 180 -, crystallization 179

195

enzyme localisation in excitable membrane

177 -, number of active sites 186 enzyme-bound thioesters 80-83 enzymic acetylation 182 equatorial reflections 106 - X-ray diffraction diagrams 106 erythrocytes 149 Escherichia coli initiation factors 59 - - ribosomes 59 excitable membranes 173, 185 excitatory transmitters 185 extra-ATPase 158, 159 Fatty acids 151, 152, 155, 163, 164 - -, free 153 - -, unsaturated 164 Fettsauresynthese 33 Fettsauresynthetase 30 field effect 137 - - relaxation 140, 144 filament, I 93 - model, sliding 90-92 -, thick 94 fine-focus rotating anode X-ray tube 99, 100 FISKE 8 0 flight muscle 90, 100 free-energy change 180, 181 frog 90 fructose diphosphate 18 D-fructose-l-phosphate 19 Garung 4 glass mirrors, bent 100 glucose-6-phosphate 169 glyceraldehyde-3-phosphate 19 GOLDSCHMID 7 gramicidin S 77-84,130,131 growth control 118 HARTMANN 7 heat production and absorption 173 Hefe 30 hexokinase 169 hexose diphosphate 18 HILL, A_ V. 5,10 Hill coefficient 184 hydrolysis, incomplete 187 hydrophobic interactions 155 Image reconstruction 98 inhibitors, potent compctitive 178 inhibitory transmitters 185 initiation compex with natural mRNA, 30 S and 70 S 67 -69 insecticides 178 intracellular action within the excitable membrane 175

196

Name and Subject Index

ion movements, diffusion process 173 - permeability in synaptic and conducting membranes 191 ionic strength 155 isolated membranes 177 isologs, oxygen, sulfur, and selenium 183 ITP 166

murexide 137 -139 myelinated fibers 177, 179 myofibril 149 myosin 113 - filaments, lattice 103 - filaments, synthetic 95 -, purified III

Junctional membrane 188

N-formylmethionyl-tRNAf 56 - binding 57 -, ribosomal binding 67 4-(N -maleimido )phenyltrimethylammonium iodide 184 Na+ and K+ fluxes, parameters 190 Na+-monactin complex 137, 140 Na+-murexide 142-144 Na 22 efflux, accelerated or blocked 185 NEM 160 nerve activity, chemical basis 181 NEUBERG, CARL 15 neurohumoral transmission 173 neuromuscular block, irreversible 185 nigericin 131 nonactin 132, 141 NTP 157,158 - hydrolysis 157

KANT 3 Kompartimentierung 136 Kontraktionsphase 9 KREHL, LUDOLF 3 Lactic acid formation 181 Lethoceru8 maximu8 90, 100 leukemias 126 ligand binding 133 lobster, walking leg 187 local anesthetics 183 LOHMANN, KARL 9,10 LUNDSGAARD 9, 10 lysolecithin 150, 152, 163-165 lysophosphatidylcholin 153 Macrocyclic compounds 130 macrotetrolides 131, 141 magnesium 166 main group metal ion complexes 132 malignancy, genetic basis 123 malignant phenotype, reversion 122 Malonyl-CoA 31 membrane potential, effects on 185 - vesicles 146, 185 mercuri-phenyl-azo-ferritin 161 meromyosin, heavy and light 94 messenger initiation sequences 71 - start signals 71 methanol 136, 139 methionyl-tRNA 56 6-Methylsalicylsaure 44 6-Methylsalicylsa uresynthetase 44 MEYERHOF, GODFREY 15 -, MAX 14 -, OTTO 3-13, 18, 30 -, WALTER 15 MEYERHOF'S ancestry 14 Mayerhof-Quotient 5 microsacs 185, 190 mitochondrial membranes 173 mollusc 112, 113 monactin 132,141-144 monazomycin 131 monensin 131 multidentate complex 131 Multienzymkomplexe 30

Oleate 168 oleic acid 151, 155, 165 oligodepsipeptides 130 oligopeptides 130 organophosphates 178, 180 oubain 150 oxalate 161 Palmityl-CoA 31 pancreatic lipase 152, 164 pantetheine 84, 85 Penicillium patulum 44 peptidyl thioesters 83 - transferase 58 permeability barriers surrounding axonal membranes 186 - changes 173, 175 phenotypic markers 118 Philosophie 3 phosphate 166 compounds, two classes 180 - exchange 160 - liberation 157-159,161,162 phosphatidylcholin 151, 153 phosphatidylethanolamin 153 phosphatidylserin 153 Phosphoarginin 8/9 phosphocreatine breakdown 181 -, high concentration 181 Phosphoenolpyruvat 9

Name and Subject Index Phosphoguanidine 8,10 Phosphokreatin 8, 9 Phospholipase A 151, 152, 163, 165, 187 phospholipase C 151, 152, 163 phospholipids 151, 163, 165 phosphoprotein 164 phosphoryl transfer 160-164 phosphorylated enzyme 178 - intermediate 160 Phosphorylierungsenergie 9 phosphorylphosphatase 180 photooxidation 22 physostigmine 179, 187 plant agglutinins 120 Polyacetatregel 44 polypeptide chain initiation 56 prenylamine 162, 164, 168 proteins and enzymes 173, 174 psoas muscle 90 pyridine 2-aldoxime methiodide 179 pyridoxal phosphate 28 Quartz crystal, curved 100 Rabbit 90 Ranvier nodes 179, 187 rapid reactions, techniques for the study 135 receptor activators and inhibitors 183, 185 -, active site 183, 186 -, negative subsite 184 -, role in bioelectricity 183 relaxation Ill, 149 - amplitude 142, 143 - spectrometry 135, 146 ribosomal factors 56 ribosome cycle 72 - dissociation factor (DF) 65 rigor mortis III 81 and S2 portions 94, 95 S-Acetacetyl-N-acetyl-cysteamin (AAC) 52 sarcoplasmic reticulum 149 - tubules 150 sartorius muscle 90 Schiff base 21 Schwann cell 187 SH-groups and reagents 161

197

solvation 133, 134, 136 - energy 135, 145 - sphere 135 solvent coordination shell 132 - molecules 132, 144, 145 sound absorption 137, 143 spectrophotometric titration 138 sphyngomyelin 153 squid axon 149, 187, 190 Stearyl-CoA 31 stimulation, chemical and electrical 190 SUBBAROW 8 substitution 132, 135, 136, 144, 145 Target protein within the membrane 18\} temperature jump (T-jump) 136 - amplitudes 142 transformed properties, expression and suppression 125 transmitter theory 174 transport ratio 158, 159 - system, efficiency 159 trigger 175 triglyceride 153 p-(trimethylammonium) benzenediazonium fluoroborate 184 trinactin 141 tropomyosin 112 -, spin-labelled 116 troponin 112, 113 d-tubocurarine 185 turnover time 179 tyrocidine 84, 85 tyrocidin A 131 Umphosphorylierungen 8 unit membrane 172 uridine triphosphate 157 Valinomycin 130-132 vesicles 146, 185 VVARBURG,OTTO 3,4,7,14

Zellatmung 3 Zwitterionennatur der Eiweil3korper 6 zymohexase 18