Membrane Biochemistry: A Laboratory Manual on Transport and Bioenergetics [1 ed.] 978-3-540-09844-7, 978-3-642-67530-0

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Membrane Biochemistry A Laboratory Manual on Transport and Bioenergetics Edited by

E. Carafoli and G. Semenza With Contributions by H. Affolter· A. Azzi • R. Bachofen • A.D. Bangham W. Beyeler • A. Binder • E. Carafoli • J.B. Chappell • M. Chiesi M. Crompton • P. Gazzotti • K. W. Hanselmann • R. Hartmann A. Kepes • M. Kessler· K. Malmstrom· A. Martonosi • V. Niggli D. Oesterhelt • Ch. Pflugshaupt • Ch. Richter • P. Ronner H.-J. Schatzmann • E. Sigel • G. Toggenburger • M. Wikstrom

With 45 Figures

Springer-Vedag Berlin Heidelberg

New York

Ernesto Carafoli Laboratory of Biochemistry Swiss Federal Institute of Technology (ETH) Zurich, Switzerland

Giorgio Semenza Laboratory of Biochemistry Swiss Federal Institute of Technology (ETH) Zurich, Switzerland

The artwork on the cover is taken from an original drawing by Dr. Klaus Miiller.

ISBN-13: 978-3-540-09844-7 DOT: 10.1007/978-3-642-67530-0

e-ISBN-13: 978-3-642-67530-0

Library of Congress Cataloging in Publication Data. Main entry under title: Membrane biochemistry. Includes index. 1. Biological transport-Laboratory manuals. 2. Bioenergetics-Laboratory manuals. 3. Membranes (Biology)-Laboratory manuals. I. Carafoli, Emesto. II. Semenza, G., 1928- QH509.M43 574.8'75 79-26591 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks.

©

1979 by Springer-Verlag New York Inc.

The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

9 8 7 6 5 4 3 2 1

Preface

This manual collects in the form of laboratory protocols a series of experiments in the field of Membrane Transport and Membrane Bioenergetics. It represents the experience accumulated during four advanced courses held at the Department of Biochemistry of the Swiss Federal Institute of Technology on behalf of Federation of European Biochemical Societies (FEBS) in the years 1975 through 1978. The idea of collecting the experiments into a laboratory manual developed as a response to a demand from the students who took part in the courses. Further motivation came with the fmding that, in planning the laboratory sessions, the teaching staff had no organized, modern source of information in the literature. The experiments presented cover most areas of importance in the subject matter. Their presentation has been continuously modified in the course of the four years during which the manual took shape, to accommodate to experience and various suggestions. In their present form, all of the experiments described have been repeatedly practiced to optimize their execution. Efforts have been made to combine in the manual classical experiments, and techniques which require relatively unsophisticated instrumentation and can therefore be carried out in most laboratories, with more modern experiments and relatively newer technologies. In its present form, the manual should therefore provide a usefui tool in the hands of researchers and laboratory teachers at different levels of sophistication and instrumentation. That the field of Membrane Transport and Membrane Bioenergetics is undergoing an explosive growth is obvious. Thus, it is hoped that this manual will provide an answer to the predictably expanding request for experimental information in the area. This manual would not have been possible without the enthusiastic collaboration of the teacl•.;rs who planned and supervised the experiments and who tested and modified, sometimes substantially, the protocols of their experiments

vi

Preface

to optimize the results. It is a pleasure for the Editors to acknowledge this enthusiastic collaboration. Equal gratitude goes to the students, from about 30 different countries, who were at the receiving end of this effort and provided the feedback which was essential to it. Lastly, a very particular word of thanks goes to Kerstin Malmstrom, who undertook the task of doing most of the editing and of supervising the proofreading of the manual.

Zurich, 6th October 1979

Emesto Carafoli Giorgio Semenza

Contents

1. Nonelectrolyte Transport in Small Intestinal Membrane Vesicles. The Application of Filtration for Transport and Binding Studies Markus Kessler and Gerhard Toggenburger (With 5 Figures). . . . . . . . . . . 1 2. Transport of Sugars in Bacteria Adam Kepes (With 1 Figure) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3. Net Na+ and K+ Movements in Human Red Blood Cells Mter Cold Storage Hans-Jiirg Schatzmann, Peter Ronner, and Verena Niggli. . . . . . . . . . . . 36 4. Calcium Transport in Resealed Erythrocytes and the Use of a Calcium-Sensitive Electrode Peter Ronner (With 4 Figures) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 5. Calcium Transport in Sarcoplasmic Reticulum Vesicles Isolated from Rabbit Skeletal Muscle Michele Chiesi and Anthony Martonosi (With 2 Figures). . . . . . . . . . . . 51 6. Preparation and Assay of Animal Mitochondria and Submitochondrial Vesicles Paolo Gazzotti, Kerstin Malmstrom, and Martin Crompton (With 5 Figures) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 7. Measurement of Cytochrome Kinetics in Rat liver Mitochondria by Stopped Flow and Dual-Wavelength Spectrophotometry Angelo Azzi and Hubert Mfolter (With 2 Figures) . . . . . . . . . . . . . . . . 77

Contents

viii

8. Proton Translocation Catalyzed by Mitochondrial Cytochrome Oxidase Marten Wikstrom and Erwin Sigel (With 1 Figure). . . . . . . . . . . . . . . . 82 9. Determination of the Membrane Potential and pH Difference Across the Inner Mitochondrial Membrane J. Brian Chappell and Martin Crompton . . . . . . . . . . . . . . . . . . . . . . . 92 10. Anion Transport in Mitochondria Martin Crompton and J. Brian Chappell (With 4 Figures) . . ......... 98 11. Calcium Transport in Mitochondria Kerstin Malmstrom and Emesto Carafoli (With 3 Figures) . . . . . . . . . . 103 12. Redox Intermediates Between O2 and H2 0 Christoph Richter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 13. Photophosphorylation with Chromatophore Membranes from Rhodospirillum Rubrum Kurt W. Hanselmann, Walter Beye1er, Christian Pflugshaupt, and Reinhard Bachofen (With 11 Figures) . . . . . . . . . . . . . . . . . . . . 120 14. Oxygen Evolution and Uptake as a Measure of the Light-Induced Electron Transport in Spinach Chloroplasts Andres Binder and Reinhard Bachofen (With 3 Figures) . . . . . . . . . . . 144 15. The Function of the Purple Membrane in Halobacterium Halobium Dieter Oesterhe1t and Rainer Hartmann (With 2 Figures). . . . . . . . . . . 154 16. Characterization of Ionophores Using Artificial Lipid Membranes Michele Chiesi (With 2 Figures) . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 17. Characterization of Neutral and Charged Ionophores Using Vesicular Artificial Upid Membranes (Liposomes) Paolo Gazzotti and Alec D. Bangham . . . . . . . . . . . . . . . . . . . . . . . 171

List of Contributors

Affolter, H., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Azzi, A., Institute of Medical Chemistry, University of Berne, Berne, Switzerland Bachofen, R., Institute of Plant Biology, University of Zurich, Zurich, Switzerland Bangham, A. D., Agricultural Research Council, Institute of Animal Physiology, Babraham (Cambridge), England Beyeler, W., Institute of Plant Biology, University of Zurich, Zurich, Switzerland Binder, A., Institute of Plant Biology, University of Zurich, Zurich, Switzerland Carafoli, E., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Chappell, J. B., Department of Biochemistry, University of Bristol, England Chiesi, M., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Crompton, M., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Gazzotti, P., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Hanseimann, K. W., Institute of Plant Biology, University of Zurich, Zurich, Switzerland Hartmann, R., Department of Biochemistry, University ofWiirzburg, Wiirzburg, Federal Republic of Germany Kepes, A., Centre National de la Recherche Scientifique, Institut de Recherche en Biology Moleculaire, Universite de Parix VII, Paris, France

x

Contributors

Kessler, M., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Malmstrom, K., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Martonosi, A., Department of Biochemistry, St. Louis University, St. Louis, Missouri, USA Niggli, V., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Oesterhelt, D., Department of Biochemistry, University ofWurzburg, Wurzburg, Federal Republic of Germanv Pflugshaupt, Ch., hlstitute of Plant Biology, University of Zurich, Zurich, Switzerland Richter, Ch., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Ronner, P., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Schatzmann, H.-J., Institute of Veterinary-Pharmacology, University of Berne, Berne, Switzerland Sigel, E., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Toggenburger, G., Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland Wikstrom, M., Department of Medical Chemistry, University of Helsinki, Helsinki, Finland

1. Nonelectrolyte Transport in Small Intestinal Membrane Vesicles. The Application of Filtration for Transport and Binding Studies MARKUS KESSLER and GERHARD TOGGENBURGER

I. INTRODUCTION AND AIMS

Brush border membrane vesicles were used for the fIrst time in 1973 forinvestigating intestinal transport processes (Hopfer et al., 1973). This was an important step forward in two respects: (a) These vesicles allow to dissect clearly phenomena occurring in the brush border membrane from others occurring in the cell interior or in other membranes, as, for example, in the basal-lateral membrane. In these vesicles, which are virtually free of cytosolic constituents and metabolites, the composition of the internal and the external compartment can be decided upon (within limits) by the experimenter according to the experimental requirements. (b) Brush border vesicles are a suitable starting material for isolating membrane components. Working with vesicles, on the other hand, carries some inherent diffIculties. Due to an unfavorable ratio between a large membrane surface and a small vesicular volume, binding in some cases may interfere with measurement of transport into an intravesicular space. Because vesicles are formed from disrupted membranes, they may exhibit an increased permeability towards solutes compared with the original, native membranes. It might therefore be diffIcult to detect a low capacity transport process under the diffusional uptake. A third problem is encountered when analyzing transport processes kinetically: due to the small intravesicular space, unidirectional fluxes often can only be measured at very short incubation times. One goal of this article is to show ways' for overcoming these diffIculties. Experiments will be discussed allowing a discrimination between transport, binding, and diffusion, exemplifled on the Na+-dependent D-glucose transport. These tests, supplemented by further experiments (kinetic studies, inhibitor binding, /::,.1/1 - dependence of transport) will at the same time serve a second goal,

Markus Kessler and Gerhard Toggenburger

2

namely to illustrate some possibilities of characterizing a transport system by means of transport measurements. The following five topics will be discussed: A. Description of a procedure for isolating brush border membrane vesicles from small intestinal enterocytes. B. Performance of transport measurements, advantages and disadvantages of the filtration technique. C. Experimental possibilities to discriminate between transport, binding and diffusion. D. 1:!.tJ; - dependence of transport. KinetIC measurements. E. Use of the filtration technique for binding studies.

II. EQUIPMENT AND SOLUTIONS Vacuum filtration set with vacuum flask and filter support. Filters. Nitrocellulose fIlters with pore size 0.45 to 0.6 J.1.m (e.g., Sartorius (GFR), Millipore, Mulpot (Switzerland». ')olutions. The preincubation- and incubation-media are prepared from the eollowing stock solutions:

1M 100mM 1M 1M 1M 1M 1 mM, 1M 1M 100 mM 100 mM 100 mM 1 mM 10 mM 1M 1M

mannitol HEPES, adjusted to pH 7.5 with Tris NaSCN NaCI Na2 S04 KCl D-glucose L-glucose D-galactose D-fructose L-methionine phlorizin ouabain choline SCN is prepared from KSCN and choline OW on an ion exchange column. choline2 S04 is prepared from H2 S04 and choline OW.

Stop Solution. 250 mM NaCl and ImM Tris/HCI pH 7.5. This solution is kept on ice.

Nonelectrolyte Transport in Small Intestinal Membrane Vesicles

3

m.EXPER]MENTALPROCEDURES A. Isolation of Brush Border Membrane Vesicles Among the different techniques available for the isolation of intestinal brush border membrane vesicles (Eichholz and Crane, 1965; Forstner et al., 1968; Schmitz et aI., 1973; Louvard et al., 1973; Murer et al., 1974; Murer et al., 1976; Kessler et al., 1978a) the method of Schmitz et al. as modified by Kessler et al. has proven to be most useful for performing transport studies, because the vesicles obtained appear to be well resealed. In addition, this technique is simple and fast (less than 3 h) and allows the processing of large amounts of material giving a correspondingly high yield of membrane vesicles. The method is described in detail in (Kessler et al., 1978a). Briefly: Intestinal mucosa (from 20 g of intestine) is suspended in 300 mM mannitol and 2 mM Tris/HCl pH 7.1 (300 ml) and vigorously homogenized in a Waring blender for 2 min., Upon addition of 10 mM CaCl2 most of the nonbrush border membranes aggregate and are spun down at low speed (3000 g, 15 min). Brush,border vesicles are collected from the supernatant at 27,000 g (30 min) and washed once by centrifugation in the buffer needed for the subsequent experiment. For transport studies the fmal pellet is resuspended in about 1 ml by repeated passage through a fme injection needle. A slightly better purification can be obtained by repeating the Ca2+ precipitation step and the subsequent differential centrifugation. The isolation technique can be applied to both frozen and fresh intestines. Intestines removed from a freshly killed animal should be washed with cold saline and then be either cut open or inverted on a rod. After laying the intestine on a cooled glass plate, the mucosa can be scraped off with glass slides. For our routine preparation intestines are obtained from a slaughterhouse, washed, inverted and collected in portions of about ｾ＠ kg and frozen on dry ice. Mter thawing, vigorous vibration is sufficient to release the mucosal cells into solution. The frozen intestines can be stored for several months (_20°C or better -80°C) without appreciable loss in quality, provided that they are tightly packed in plastic bags. When air has access to the frozen intestines, they yield nonresealing membranes already after a storage time of only a few weeks. The calcium precipitation method has been successfully applied with intestines of mammals, including guinea pig, ham(fresh and frozen ones) ｦｲｯｾｮｵｭ｢･＠ ster, rabbit, and man (LUcke al., 1977). One remarkable exception has to be mentioned: whereas fresh intesines from rats yield excellent vesicles, it has been repeatedly observed that membranes isolated from frozen rat intestines do not seal properly and are therefore less suited for transportation studies. A similar precipitation method (but using Mg2+ instead of Ca2+) has been worked out by Booth and Kenny (1974) for preparing brush border vesicles from kidney cortex. Twenty g of intestine yield at least about 20 mg vesicle protein. The diameter of the vesicles ranges between 0.05 and 0.3 fJ.ITl. The internal volume amounts to

4

Markus Kessler and Gerhard Toggenburger

1-1.5 pl/mg protein. How many of the membranes really enclose an internal aequeous phase cannot be answered safely, but several lines of evidence show that only a small fraction of the vesicles has to be considered to be "open" (Klip et al., 1979) (The terms "open" and "closed" should be used with precaution, since some vesicles might well be "open" for low molecular weight compounds and at the same time appear tightly sealed towards penetration of proteins). The vesicles, as far as they are "closed", are more than 90% in the right-side-out configuration, since addition of Triton X-100 does not yield any apparent activation of enzymes known to be confmed to the outside of the brushborder membrane (e.g., sucrase) (Kessler et al., 1978a). Normally, vesicles are prepared daily for transport experiments, because diffusional permeability of the membranes increases markedly with time. The tightness of the vesicles can be assessed by determining the extent of accumulation of D-glucose in the presence of a Na+ gradient (see below) or the amount of Dglucose or any other substrate taken up after long incubation time (equilibrium value). In general, accumulation and equilibrium value decrease by 50% within 5-15 h, when the vesicles are isolated from frozen intestines and kept at O°C. This stability is usually sufficient for transport studies. When better stability is required, the vesicles should be prepared from freshly killed animals.

B. Transport Measurements by the Filtration Method Separation of membranes from the incubation medium can be achieved in different ways such as centrifugation, gel filtration, or filtration. For erythrocytes, centrifugation may be the method of choice, because sedimentation is fast and filtration can induce lysis of cells. For transport studies with smaller membrane systems as, for example, with the brush border membrane vesicles described here, filtration is the preferred method because separation can be achieved within less than lOs whereas gel filtration and centrifugation require at least about 1 min. Standard Procedtp"e for Transport Measurements. The standard procedure as it is routinely used, for example, to check "tightness" of the vesicle preparation (see above) is described first. The following solutions are prepared in advance: Vesicle Suspension. (ca. 10-20 mg protein/rnl) in 300 mM mannitol, 10 mM HEPES/Tris pH 75. "Cocktail". The solution, which is added to start the incubation will be referred to as "cocktail" throughout this article. Standard composition of the cocktail: 300 mM mannitol, 10 mM HEPES/Tris pH 75, 200 mM NaSCN, 0.2 mM D[3H] glucose (ca 2 jlCi/50 pl cocktail) and 0.02% KN 3 . Since this cocktail will be mixed 1: 1 with the vesicles, the fmal concentrations of NaSCN, D-glucose and KN3 will be half of those listed (mannitol and HEPES/Tris are already present in the vesicle suspension, their level will be maintained at the indicated concentrations).

Nonelectrolyte Transport in Small Intestinal Membrane Vesicles

5

Stop Solution. 250 mM NaCl and 1 mM Tris/HCl pH 7.5, cooled to O°C in an ice/water mixture. Mannitol (D-glucose free) is kept at the high concentration of 300 mM to minimize osmotically induced volume changes during the incubation. HEPES (N-2hydroxyethylpiperazine-N' -2-ethanesulfonic acid) and Tris are used as standard buffers, but may be replaced by any suitable buffer system. KN3 serves as antimicrobial agent. Mannitol, HEPES/Tris and KN3 are routinely added at the same concentrations to all incubation and preincubation solutions. Therefore they will not be listed any more in the following sections. Fifty ｾ＠ cocktail are pipetted into the bottom of a plastic Eppendorf centrifuge tube. Fifty ｾ＠ of the vesicle suspension are then carefully placed close to the cocktail drop while keeping the Eppendorf tube in a horizontal position. Incubation is started by vigorously shaking the tube on a vortex mixer for about 5s. Twenty J,Ll aliquots are withdrawn and injected into 2 ml of icecold stop solution after ISs, 90s, 3 min and 90 min. The samples are immediately filtered through prewetted filters. The filters are quickly rinsed with 5 ml of the icecold stop solution and transferred into a counting vial for the determination of the radioactivity. Preloading of the Vesicles. When a composition of the internal aqueous phase different from the standard solution is required at the start of the incubation, the vesicles are preequfiibrated for 90 min at room temperature by mixing 25 ｾ＠ of vesicles with 25 ｾ＠ of a preincubation-cocktail containing all the additives in twice the concentration desired. In general, 90 min preincubation is sufficient (e.g., for Na\ K'", glucose), however, bulky and/or negatively charged molecules might require longer preincubation times. For computing "pmol substrate taken up/mg protein" the following additional values have to be determined: Background. 10 ｾ＠ of vesicle suspension and 10 ｾ＠ of cocktail are injected separately into 2 ml stop solution, f:tltered, and processed as usual. The background value determined in this way also accounts for trapping of substrate in the fJlter. Total Radioactivity. 10 ｾ＠ cocktail are pipetted directly into a counting vial. In addition, a wetted filter is added to match the degree of quenching (some quenching is introduced by the f:tlters; if, however, all vials have the same degree of quenching, tedious measurements of quench correction curves can be avoided. To be safe, check external standard ratios). From this "total radioactivity" value a relation between pmol substrate and the corresponding cpm's can easily be deduced. Spillover. In double-labeling incubations 3 H values have to be corrected for the 14 C counts spilling over into the 3 H channel. The fraction of 14 C appearing in the 3 H channel is constant and is determined by adding ca 0.1 J,LCi 14 C together with a wetted fJJ.ter into a counting vial.

6

Markus Kessler and Gerhard Toggenburger

Filters. Nitrocellulose fllters are used with a pore size of 0.45 to 0.6 J.lIll. Although the pore size as declared by the manufacturer is larger than the diameter of the vesicles, the membranes are quantitatively retained during flltration, if a proper stop solution is used (see below). At larger pore sizes retention drops, at smaller pore sizes flltration time increases drastically so that diffusion out of the vesicles leads to serious losses of the substrate taken up. Stop Solution. Cooling of the vesicles to O°C by itself strongly reduces the fluxes of substrates across the membrane. In addition, influx is further reduced by the 100-fold dilution with the stop solution. Efflux, however, cannot be totally blocked to zero during the 1O-l5s needed for flltration and fllter-washing. A rough estimation of the substrate loss can be obtained by arbitrarily prolonging the time between injection into the stop solution and the flltration and by extrapolating the losses thereby determined exponentially to the real time of stopping. The loss of D-glucose amounts to 5%-15%. Compounds for which the membrane has a high permeability such as SCN" or the lipophilic ions used for determining membrane potentials are lost by 50%-100% during flltration. Their uptake cannot be properly determined by the method described here. D-Glucose leaks out of the vesicles partially by "diffusion," partially also by way of the transport system (The losses of D-glucose are highest at high internal concentration of sodium). Whereas the diffusional component cannot be reduced any further, losses through the transport system could be reduced by adding inhibitors. However, well-known inhibitors such as phlorizin or Hg2+ are oflittle effect since their binding velocity at the temperature of O°C is very slow. The retention of the vesicles on the fllter has been found to depend on the composition of the stop solution. Optimal values are obtained with salt solutions of high ionic strength (250 mM to 500 roM NaCl, other salts may be used instead of NaCl). At very low ionic strength, even when the osmolarity is well balanced by a nonelectrolyte like mannitol, it may happen that 50%-90% of the membranes go through the fllter during flltration. Therefore it is advised to use the same stop solution for all incubations within an experiment, even if the various incubation mixtures have different osmolarities.

C. Experiments Designed to Differentiate Between Transport, Diffusion, and

Binding Association and dissociation of low molecular weight compounds to and from low affInity binding sites usually are fast processes, which are essentially complete within much less than one second. D-Glucose, if bound externally to vesicle proteins can, therefore, be expected to be completely lost during flltration and fllter-washing. This need not be the case, however, if such binding sites are located inside membrane vesicles, binding thus being in equilibrium with the free concentration of the internal compartment. Since the rate-limiting step

Nonelectrolyte Transport in Small Intestinal Membrane Vesicles

7

will be the penetration of the ligand into the internal environment, the time course of this binding will be relatively slow and not distinguishable from uptake into an internal aqueous compartment itself. If a solute is shown to be indeed taken up into an internal compartment, the further question remains whether crossing the membrane occurs through simple diffusion or by a mediated transport process. In this section the methodology will be described which should allow to distinguish between binding, diffusion, and transport, exemplified on the uptake of D-glucose into brush border vesicles.

1. Substrate Specificity (Transport vs. Passive Diffusion) Uptake showing strong discrimination between stereoisomers indicates the involvement of a specific recognition site, thus rendering diffusion unlikely (it should nevertheless be kept in mind, that also diffusion might exhibit some stereospecificity, since the building blocks of the membrane themselves are optically active). In experiments (1) and (2) the time course of D-glucose and L-glucose will be compared. This experiment can be designed in many different ways, two of which will be described in some detail, because they also provide information about the underlying transport process.

Experiment 1 The time course of D- and L-glucose is measured in vesicles containing 100 mM NaCl or 100 mM KCl at both sides of the membrane. ｾｩｮｵＮｑ｡Ａｬｧｊｳ＠ (a) 200 mM NaCl (b) 200 mM KCl (mannitol, HEPES/ Tris and KN3 are not listed any more because they are always present in all solutions at identical concentrations, see Sect. B). Cocktails. --(a) 100 mM NaCl 0.2 mM D-[3H] glucose 0.2 mM L_[14 C] glucose

(b) 100 mM KCl 0.2 mM D-[3H] glucose 0.2 mM L_[ 14 C] glucose Eighty p.l cocktail should contain 24 f..lCi of the tritiated and 1-2 f..lCi of the 14 C-labeled compound. Forty p.l vesicle suspension are preincubated for 90 min at room temperature with 40 f..ll preincubation-cocktail. Incubation is started by addition of 80 p.l cocktail. Time points to be taken: ISs, 90s,S min, 20 min and 180 min (20 p.l each).

Markus Kessler and Gerhard Toggenburger

8

Observations. (see Fig. 1) In the absence of sodium, the uptake of D-glucose is only insignificantly faster than that of L-glucose. In vesicles incubated with sodium, however, D-glucose reaches the fmal equilibrium value already after a few minutes, whereas L-glucose needs up to 2 h to come to the same level. (fhe half equilibration time ｴｾＬ＠ i.e., the time required to reach half the equilibrium level, is ca 10-30s for D-glucose and 5-10 min for L-glucose.) At equal substrate concentrations in the incubation medium, the substrates should reach the same equilibrium value. If this is not the case, this might be indicative of a component of binding, of a different degree of quenching in different vials or of bacterial growth. Similar experiments can be designed to measure efflux from vesicles preloaded with the two substrates.

pMoies mg

100

50

/

:......

•

:.. I

15

90s

60 min

Fig. 1. Time course of D-glucose uptake in vesicles equilibrated with 100 mM NaCl (-) or 100 mM KC1 (A.). The uptake of L-glucose (e) is the same in both incubations (for further details see Exp. 1) Experiment 2 Glucose and 100 mM NaCl are added simultaneously to the vesicles. Cocktail. --200 mMNaCl 0.2 mM D_[3H] glucose 0.2 mM L-P 4 C]glucose Eighty III vesicle suspension are mixed with 80 IJ.l cocktail. Time points are taken as in Experiment 1.

Nonelectrolyte Transport in Small Intestinal Membrane Vesicles

9

Observations. (Fig. 2) D-Glucose is rapidly taken up and reaches within 30s a value several times larger than the fmal value obtained after 90 min. L-Glucose uptake is identical to that in Experiment 1. nus "overshooting" of D-glucose beyond the equilibrium concentration can easily be interpreted in terms of the model originally put forward by Crane (1962), assuming a cotransport, i.e., a coupled translocation ofNa+ and D-glucose: The asymmetric distribution ofNa present at early incubation times can maintain an asymmetric distribution of the nonelectrolyte, therefore, D-glucose can be accumulated inside the vesicles to concentrations above the concentration in the medium, as long as the Na+ concentration inside has not reached the medium concentration. As the Na+ gradient diminishes due to influx of Nt and cr (by various routes), the "driving force" for the accumulation dissipates and D-glucose approaches the same equilibrium level as L-glucose (It should be noted, that the ratio "maximum uptake during overshoot"/"equilibrium" is not exactly the real accumulation, because the vesicles undergo some volume changes, albeit small at the mannitol concentrations ｵｾ･､Ｌ＠ during the incubation).

600

200 t

x

15

905

I

I

I

i

90 min

Fig. 2. Time course of D-glucose (e) and L-glucose (x) uptake in the presence of a gradient of NaCI (see Exp. 2)

10

Markus Kessler and Gerhard Toggenburger

2. Competition for a Limited Number of Recognition Sites: Saturation and Inhibitor Studies (Mediated Transport vs. Passive Diffusion) At flrst approximation diffusional fluxes should be proportional to the concentration (or rather activity) of a solute. Saturation and inhibition by substrate analogs are commonly taken as indication for a specific translocation process. Saturation means that the uptake velocity determined at early incubation times approaches a limiting value when increasing the substrate concentration. Again, some precaution has to be recommended: it has been shown that also diffusional systems may exhibit saturation phenomena. On the other hand, transport agencies with a large ｾ＠ for the substrate show a linear relationship between transport velocity and substrate concentration. A more detailed discussion of the validity of these criteria is found in the review of LeFevre (1975). It must be generally stated that it is not suffIcient to check only for one of the criteria described here. The evidence for the operation of a mediated transport process increases when several independent tests can be collected.

Experiment 3 Performance and interpretation of saturation studies will be discussed in Section D. In this experiment the effect of flve potential inhibitors on D-glucose uptake will be tested: D-galactose, D-fructose, L-methionine, phlorizin, and ouabain. Cocktails. 200mMNaCl 0.2 mM D- [3 H) glucose 0.2 mML_[l4C]glucose and one of the following compounds: 1-10 mM D-galactose 1-10 mM D-fructose 1-10 mM L-methionine 0.02-0.2 mM phlorizin 0.2-2 mM ouabain Fifty JLl vesicle suspension are mixed with 50,.11 cocktail. Time points: ISs, 90s, 3 min, 90 min. Observations. 5 mM galactose and 50 IlM phlorizin inhibit D-glucose uptake to about 90%, the others are of little effect. Only L-methionine may show a slight inhibition. The strong inhibition by galactose as well as the high-affInity inhibition of phlorizin point to a speciflc transport process. Care has to be taken with compounds like phlorizin: this glycoside at concentrations above 1 mM may act as a membrane perturb ant.

Nonelectrolyte Transport in Small Intestinal Membrane Vesicles

11

Phlorizin is known from measurements on intact intestines to inhibit Dglucose uptake competitively, D-galactose has been found to share the same transport system. D-Fructose is known to be taken up by a separate Na+-independent transport agency. Neutral amino acids have been found to share a separate, but also Na+-dependent carrier. The lack of inhibition of methionine on Dglucose uptake has an important implication for the measurements, which will be discussed in Section D. Finally, ouabain, which has been found to inhibit Dglucose uptake in intact intestines, is generally believed to exert its inhibitory action through blocking the Na-K-ATPase. This enzyme, whose function is vital for maintaining the electrochemical gradient of Na+ across the plasma membrane, is present, however, in the basal-lateral membrane and not in the brush border membrane. The lack of inlubition by ouabain in our vesicles is in agreement with this explanation. 3. Comparison Among Equilibrium Values of Different Substrates (Transport into an Osmotic Space vs. Binding) Criteria such as substrate specificity, saturation, and availability of specific inhibitors indicate the involvement of specific recognition sites, but they do not allow the distinction between binding and transport into an osmotic space. Whether "uptake" is indeed due to transport into an internal compartment can, however, be assessed by comparing the equilibrium values of D-glucose and L-glucose in the experiments discussed above. If no binding is involved, the equilibrium values only depend on the concentration of the solute and the internal volume of the vesicles; substrates offered at the same concentration must exhibit identical equilibria. Some deviations occur with charged substrates at low ionic strength, if the vesicles carry an excess of negative or positive charges. It must be stressed once again: discrimination between stereoisomers and saturation phenomena per se cannot be considered as conclusive evidence for a transport process unless it is shown that under all the conditions chosen, the same fmal equilibrium value is attained. This simple control has been neglected in many publications, which invalidates the authors' claim of having measured a transport process. If the uptake of a slowly permeating stereoisomer is too slow and the vesicles deteriorate during the long incubation, then the internal volume of the vesicles should be determined by comparing equilibria of different faster permeating substrates. 4. Variation of the Osmolality (Transport vs. Binding) At equilibrium, the amount of substrate taken up should directly depend upon the available intravesicular volume, if no binding is involved. Intravesicular space can be decreased by raising the osmolality of the outer medium with a solute of

Markus Kessler and Gerhard Toggenburger

12

low penneability. The intravesicular space should be inversely proportional to the medium osmolality, provided that the slowest penneating solute represents the main component of the medium. Extrapolation to infInite osmolality (zero space) must yield zero uptake. Experiment 4 ｾｯＮﾣ｡Ａｳ＠

20 mMNaCl 0.2 mM D- [3 H] glucose and one of the following mannitol concentrations (total concentration in cocktail): 300,500,700,900 mM Fifty pl vesicle suspension and 50 pl cocktail are mixed. D-Glucose taken up is determined after 0.5-2 h. NaCl is added at low concentration to speed up equilibration. The same stop solution is used for all incubations irrespective of the different osmolalities. The fmal mannitol concentrations during the incubation are 300, 400,500, and 600 mM. Osmotic volume changes are determined by the osmolalities (= Mol/kg solvent) and are most conveniently measured on an osmometer (at high concentrations the deviations from the osmolarity may become SignifIcant). In our experiment the osmolalities in the incubation media are: 320,430,550, 690 mosm. Inserting osmolarities instead in the plot may produce curves crossing the ordinate below the origin. Observations. The reciprocal value of the osmolalities in the outer medium is plotted on the abscissa, the pmol D-glucose taken up on the ordinate. The points fIt to a line extrapolating through the origin of the plot (Fig. 3). pMoles mg

100 50

0A5

Q73

1

Osmin

Osmaut

Fig. 3. Variation of the size of the internal volume by changing the osmolality of the incubation medium. D-Glucose uptake is shown after 30 min (e) and 60 min (A) incubation time. Initja1 mannitol concentration inside: 300 mM; mannitol concentrations outside during the incubations: 300, 450, or 600 mM (see Exp.4)

Nonelectrolyte Transport in Small Intestinal Membrane Vesicles

13

Comments. The osmolality test can be performed with other solutes as, for example, cellobiose or with salts. However, the main solute inside and outside the membrane must be identical, otherwise the osmotic volume changes can no longer be predicted in simple terms. If the same experiment is done with vesicles prepared in low mannitol concentrations (10-100 mM), a nonideal behavior is observed, i.e., shrinking at outer mannitol concentrations below 300 mM is smaller than expected. Obviously a certain minimum osmotic pressure must be applied to constrain vesicles to change their shape.

5. Counter Transport (Mediated Transport vs. Binding; Mediated Transport vs. Diffusion) If brush border vesicles have been preloaded with D-galactose (D-glucose and D-galactose share the same transport system) at concentrations high relative to the ｾ＠ and then are incubated with D-glucose as the substrate, D-glucose is accumulated inside the vesicles to a level significantly higher than in the absence of internal D-galactose. In this experimental set-up D-galactose is called "elicitor." The increased extent of accumulation is not primarily due to a change of D-glucose influx; it is effected by the competitive inhibition of D-glucose efflux (setting in after prolonged incubation time) due to the high concentration of the internal galactose. A fully analogous situation is found, when the vesicles are preloaded in a high concentration of unlabeled D-glucose ("elicitor") instead of D-galactose and diluted into a medium containing labeled D-glucose at low concentration. Also in this case, the accumulation of the tracer is strongly promoted. The increase in the "uptake" can hardly be reconciled with a binding or a diffusion process and therefore strongly favors transport as the underlying mechanism.

Experiment 5 The vesicles are preloaded with either 50 mM D-glucose or 50 mM L-glucose. At the start of the incubation the vesicles are diluted 50-fold. Final D-glucose concentration is 1 MM. ＡｊＢｾｵＮ｡ｑｬＹＱｧｳ＠ (a) 250 mM D-glucose (b) 250 mM L-glucose Cocktails. ---a: 100 mM NaCI b: 100 mM NaCI 1 mM D-glucose both cocktails contain in addition D_[3H] glucose (20 /.lCi/mI, fmal concentration oftracer: in) into the D-glucose gradient built up (in < out) only occurs if the flux of D-glucose is coupled-in an as yet not defInitely defmed way and stoichiometry-to a flux of Na+ ions. If the Na+ ions did not move along with D-glucose, their effect on D-glucose transport could only be catalytic, but not energetic, i.e., Na+ could only accelerate the equilibration of D-glucose without enabling an accumulation.

Markus Kessler and Gerhard Toggenburger

16

Since the fluxes of Na+ and glucose are coupled to one another, D-glucose flux must be accompanied by an electrical current, unless the movement of Na+ is electrically balanced by the movement of anoth'er ion through the same transport agency (possibly a It or a OW). If D-glucose flux generates a current, i.e., if it is "rheogenic" (from Greek "rheein"= to flow; also called "electrogenic"), then in turn the flux of D-glucose must also be influenced by an electrical potential t.1/I across the membrane.

Experiment 6 Asymmetrically distributed ions with differing permeabilities build up a diffusion potential. Assuming the validity of Goldman's approximation in vesicles incubated in the presence of Na+ and ｓｃｾＪ＠ as the only permeant ions, t.1/I is given by: 2.3 RT P SCN ' [SCNl in + PNa ' [Nalout

t.1/I

= - - log

F

=-----;=:-;-;---:-=----;:-:-;;-;--

PSCN ' [SCNl out + PNa ' [Nal in

If additional ions are involved, the equation has to be extended accordingly by introducing further additive terms. As long as no ions are present inside the vesicles (or only ions with very low permeability) the potential is primarily determined by the ratio Panion/PNi when a sodium salt is added together with D-glucose at t=O. Variation of the potential is therefore easily achieved by selecting different anions owing widely different permeabilities. In this way it was shown that the anions known to have a high membrane permeability such as SCN- or NO; produce a much higher accumulation than cr and this in turn was more effective than the little permeant ions cyclamate- or SO:' i.e., the more negative the potential inside, the faster is D-glucose uptake (Murer and Hopfer, 1974; Liedtke and Hopfer, 1977). These fmdings are indicative of a rheogenic transport mechanism. An alternative method to create membrane potentials is to select a highly permeant ion X and to vary the ratio of its concentrations inside and outside the vesicles, keeping the concentrations as high as possible. The diffusion potential is now roughly proportional to log Xout/Xin . This method has the advantage that it allows a continuous variation of the potential over a wide range and further, that the presence of additional ions with moderate permeabilities does not significantly affect the potential preestablished by the distribution of X. Possible *Lipid soluble (and chaotropic) anions such as SCN- and NO; have some inhibitory effect on the transporters of D-glucose and of other solutes. This effect is best detected by comparing tracer exchange fluxes across membrane vesicles preincubated in NaSCN, NaN0 3 , Na 2 SO 4 or NaCl (Kessler, unpublished observations).

Nonelectrolyte Transport in Small Intestinal Membrane Vesicles

17

candidates for X are: K'" together with the highly specific ionophore valinomycin or the anion SCN". The permeability of the thiocyanate anion was found to be about 4-IO-fold larger than that of Na+ (Kessler and Semenza, 1980). ｾ･ｪｰＮｦｵｑ｡Ａｮ＠

ｾｯＮｦ｡ｪＡｳ＠

20 mM, 50 mM, 100 mM,

200 mM cholineSCN

KSCN may be used instead of the commercially not available cholineSCN (if possible, Ie is avoided because it has been reported to exert some, albeit small, effects on D-glucose transport (Schultz and Curran, 1970). If desired ionic strength can be balanced in the preincubation cocktails by low-permeability salts such as, e.g., choline2 sulfate. Cocktails ---0.2 mM D_[3H] glucose 100 mM Na2 S04 enough cholineSCN to give a total fmal concentration of 100 mM in the incubation medium. Vesicles are preincubated by a 1: 1 dilution with the preincubation-cocktails. Incubation is started by mixing two equal volumes of preincubated vesicles and cocktail. The cholineSCN concentration outside is 100 mM, while the internal concentration is varied from 10 to 100 mM. Observations. D-glucose uptake correlates well with the ratio SCNout/SCNin. D-glucose flux is increased by a factor of 5 -1 0 when going from ｴＮＱＯｊｾ＠ (SCNin= SCNout=100 mM) to t.1/J

::>

C

C

30 TIME

min

TIME

min

Fig. 1. Experiment with branched time course. ia Strain 3300 in choline chloride; ib 300p Melibose in choline chloride Mel Melibose (inhibitor); Na (activator); PCMPS P-chloromercuriphenyl sulfonic acid

35

Transport of Sugars in Bacteria iil

TIME min

Fig. 1. ii Transient fluxes. Strain 3300 in incubation medium I. Preincubation at high bacterial density with ｃｾ＠ and ｾ＠ A Active uptake after recovery; B Transient uptake energized by efflux (overshoot); C Exit stimulated by uptake; D Control exit

REFERENCES Recommended Reading Bentaboulet M, Robin A, and Kepes A (1979) Artificially induced active transport of amino-acid driven by the efflux of a sugar via a heterologous transport system in de-energized Escherichia coli. Biochem J 178: 103-107 Cohen G N, Monod J (1957) Bacterial permeases. Bacteriol Rev 21:169-194 Flagg J L, Wilson T H (1977) A protonmotive force as the source of energy for galactoside transport in energy depleted Escherichia coli. J Membr BioI 31 :233-255 Hirata H, Altendorf K, Harold F M (1974) Energy coupling in membrane vesicles of E. coli. I. Accumulation of metabolites in response of an electrical potential. J BioI Chern 249:2939-2945 Kepes A (1971) The l3-galactoside permease of Escherichia coli. J Membr BioI 4:87-112 Lopilato J, Tsuchiya T, Wilson T H (1977) Role of Na+ and Lt in thiomethylgalactoside transport by the melibiose transport system of Escherichia coli. J Bacteriol 134: 147-156 Winkler H H, Wilson T H (1966) The role of energy coupling in the transport of l3-galactoside by Escherichia coli. J BioI Chern 241 :2200-2211

3. Net Na+ and K+ Movements in Human Red Blood Cells After Cold Storage HANS-JURG SCHATZMANN, PETER RONNER, and VERENA NIGGLI

I. INTRODUCTION AND AIMS During storage of whole blood at 4°C the red cell Na-K pump is slowed to a negligible rate (white cells may be disregarded). The cells gain Na and lose K through leak channels under these circumstances (these movements are not across the arrested pump because they are not sensitive to ouabain). Upon rewarming in the presence of glucose as substrate for glycolysis the alkali cations are pumped in the opposite direction, i.e., the cells tend toward the original steady state. The pump is obviously against a considerable gradient. Cardiac glycosides, like ouabain, block the Na-K pump. Therefore, the difference in Na and K movement between ouabain-poisoned and unpoisoned cells is equal to the movement through the pump mechanism. Maximal rate for the Na pump flux is about 3 mmo1/1 cell/h (when external K and internal Na concentrations are high enough to overcome the inhibition by external Na and internal K; see Garay and Garrahan, 1973). In the present experiment these conditions may not be satisfied and we expect to fmd a shift of Na of less than 10 mmo1/1 cell in 4 h. Reasonable precision in the measurements is therefore required.

II. EQUIPMENT AND SOLUTIONS A. Equipment 3 Erlenmeyer flasks (25 ml) with stoppers shaking thermostated water bath refrigerated centrifuge for 10 ml conical centrifuge (3000 g)

Net Na+ and

K'" Movements in Human Red Blood Cells After Cold Storage

37

pipettes: 10 mI, 5 mI, 1 mI, 05 mI, 200 pl, 20 ｾ､＠ Eppendorf centrifuge mod. 5412, and tubes Vortex shaker spectrophotometer (546 nm), cuvettes emission flame photometer test tubes

B. Solutions heparin-solution (=llquemin R) NaCI : 155 mM HCS-solution : 200 mg potassium hexacyanoferrate (III) and 50 mg KCN are dissolved in 1 H2 0 isotonic MgCl 2 : 113 mM, (check osmolarity, titrate Mg2+ to ensure correct concentration) NaCI 1 mM KCI 1 mM

ID. EXPERIMENTAL PROCEDURES A. Cold Storage of Whole Blood Venous human blood is collected through a sterile needle into an Erlenmeyer flask containing 50 U USP heparin per 1 mI blood (=0.1 mI llquemin R). The flask is agitated in order to distribute the heparin evenly and is refrigerated immediately to 4°C. The stoppered flask is kept at 4°C under occasional shaking for 5 days.

B. Measurement of Na+ and K+ -Movements in Red Blood Cells After Cold

Storage

1. Incubation of Blood at 37°C in the Presence of Glucose, NaCl, and + or Ouabain 40 mg of powdered glucose is added to 20 mI of blood such as to make 11 mM and 2 portions of9 mI are formed in 25-mI Erlenmeyer flasks. To one flask 1 mI of ouabain solution is added to make 10-4 g ouabain/mI blood = 0.17 mM. To the other flask 1 mI of 155 mM NaCl solution is added. The flasks are stoppered and preincubated for 20 min in a gently shaking water bath at 37°C.

38

Hans-Jiirg Schatzmann, Peter Ronner, and Verena Niggli

After 20 min 0.5 m1 samples (precisely measured) are taken into conical centrifuge tubes at 0, 1, 2, 3, and 4 h. The Erlenmeyer flasks should be kept closed between sampling to avoid evaporation. If the cells sediment in the flasks, they should be mixed by hand (always before taking sample). At zero time an additional sample of 2 m1 is taken into a centrifuge tube which will serve to determine hemoglobin.

2. Hemoglobin Determination (in the Form ofCN-Hb) Five m1 of HCS solution is pipetted into test tubes. Twenty p.l of the 2 m1 blood sample taken at zero time is added to one test tube. The rest of this sample is centrifuged at 3000 g for 5 min. Ten p.l of the resulting packed cells are added in a similar way to 5 m1 of the HCS solution, rinsing the micropipette several times. The resulting samples are mixed vigorously, and their extinction is read after 20 min against pure HCS-solution at 546 nm. From these values the volume of the cell fraction in the blood can be calculated.

3. Na+ and K'" Determination in the Samples The tubes with the samples for Na and K-determination are cooled immediately on ice and centrifuged for 3 min at 3000 g in a refrigerated centrifuge. Two hundred p.l of the plasma is removed and put aside into Eppendorf centrifuge tubes. The cells are washed three times in the cold with 8 m1 icecold isotonic MgCl 2 solution (1l3 mM) by centrifugations as above. The cells are stirred after each refilling of the tubes on a Vortex shaker. The centrifugations should be done as quickly as possible. No cells must be lost during the washing procedure. For measurements of Na and K by emission flame photometry the following dilutions are made: plasma is diluted 1:20 and 1: 140 with water. The cells are diluted 1 :30 and an aliquot thereof 1:3 (fmal dilution 1 :90). From 1 mM NaCl and KCl solutions the following standards are made: 0.25, 0.50,0.75, and 1 mM. A calibration curve is taken on the emission flame photometer. The samples are read, whereby 1 mM standard should be checked frequently. The low plasma dilution (1 :20) should be used for K and the high plasma dilution (1: 140) for Na. The low cell dilution (1: 30) is used for Na and the high dilution (1 :90) for K. The results are read on the calibration curve.

IV. EVALUATION OF DATA A. The plasma concentrations are calculated from the dilution factor. B. The cellular concentrations are calculated per initial cell volume from the dilution and the hemoglobin result (by this procedure possible volume changes

Net Na+ and

r

Movements in Human Red Blood Cells After Cold Storage

39

during incubation cancel and concentration changes reflect mmol of Na and K having passed across the constant cell surface area). C. The concentration change is plotted as function of time for the cellular determination. The cellular concentration change is also calculated from the plasma determination. The cellular and plasma results can be averaged, or the result which shows less scattering may be retained. D. The concentration change in the ouabain sample is subtracted from that in the unpoisoned sample. This difference is plotted versus time, and the straight line of the best fit through the points is drawn. The ratio slope Na/slope K is formed. This gives the stoichiometry of the pump performance. It should be 15 (Le., 3 Na ions transported). It is recommended to average the results of several experiments in order to increase precision. In order to demonstrate statistical significance for the difference of the two slopes the following test may be applied:

x

(_1 +_1) Qx

I

Qx

2

slopes (regression coefficients) number of points Sy I Xl and ｾ＠

Qx

2 X2

= residual error. ...........Syx = ｾｸ＠

I

ｾｸ＠

I

1 2- n

2

1 2- n Ｈｾｸ＠

Ｈｾｸ＠

I

2

)2 )2

ordinate of regression at line at Xi The probability corresponding to t can be found in table t-distribution E. The Na and K concentration in the cell water are calculated for the original cells (assuming 0.63 rnl water in 1 rnl of cells), in order to assess the concentration gradient against which the transport took place.

REFERENCES Garay P R, Garrahan P J (1973) The interactions of sodium and potassium with the sodium pump in red cells. J Physio1231:297-325

40

Hans-Jiirg Schatzmann, Peter Ronner, and Verena Niggli

Recommended Reading Glynn 1M, Karlisch S J D (1975) The sodium pump. Annu Rev. Physiol37:l355

4. Calcium lransport in Resealed Erythrocytes and the Use of a Calcium-Sensitive Electrode PETER RONNER

I. INTRODUCTION AND AIMS Red blood cells contain a very low concentration of Ca 2 + (less than 1 IlM), whereas the surrounding plasma contains about 2.5 mM Ca2 +. The resulting gradient is maintained by the low permeability of the membrane toward Ca2 + and by an ATPase usually called (Ca2 + + Mg2 +)-ATPase or Ca,Mg-ATPase, which pumps Ca2 + in the outward direction. The pump capacity exceeds the passive leak flux by a factor of at least 100. An activator protein of the Ca,Mg-ATPase has been identified; however, the regulation of the enzyme's activity still remains to be elucidated. Apart from the fact that a raised intracellular Ca2 +-concentration disturbs the proper functioning of the cell membrane, a low intracellular Ca2 +concentration is demanded by the existence of several enzyme reactions which are regulated by Ca2 + in the IlM concentration range. Erythrocytes provide a unique tool in studying fluxes of Ca2+ and other ions, since they can easily be made permeable to a variety of normally impermeable substances, and then be resealed (Le., become impermeable again to substances like Ca2 + and ATP). Substances to be enclosed in the resealed cells are added to the hemolysis fluid. For a review on the properties of the Ca,Mg-ATPase see Schatzmann, 1975. The aim of the experiment is to demonstrate the ATP-dependent, outward pumping of Ca2 +. The experimental section shows how to set up an ion-selective liquid membrane electrode, and its application to the measurements of the activity of the calcium ions in solution. The experiment will demonstrate the usefulness and sensitivity of electrode measurements in viscous solutions of high optical density. The procedure for hemolyzing and resealing erythrocytes are also described.

42

Peter Ronner

ll. EQUIPMENT AND SOLUTIONS A. Equipment mY-meter with high impedance input 0 10 12 ohms; any modem pH-meter will be adequate), and if possible with 100 m V output recorder with a sensitivity up to 1 to 10 mV bucking voltage box (see below) reference electrode liquid-membrane ion-sensitive electrode shielded connecting wires thermostated water bath thermostated incubation vessel magnetic stirrer with a strong magnet (e.g., Pyromagstir from Cenco) magnetic stirring disc human erythrocyte sediment, as fresh as possible refrigerated centrifuge and rotors for 40 rn1 tubes (if'possible 1 ftxed angle + 1 swinging bucket rotor) 40-mI centrifuge tubes vacuum line to remove solutions balance (preparative) timer spectrophotometer (780 nm), cuvettes test tubes (phosphate-free) graduated cylinders, graduated tubes pipettes 0.5 mI, 1 mI, 2 mI, 5 mI, 10 rn1 Hamilton syringe 25 J.Ll variable automatic pipette 200 - 1000 J.d Eppendorf centrifuge mod.5412 and tubes Pasteur pipettes

B. Solutions incubation medium: 130 mM KCI, 1 mM CaCI2 , 10-4 gm ouabain/mI, 10 mM HEPES, pH 7.4 (at 37°C) calcium standard : 23 mM CaCl2 washing buffer: 140 mM KCI, 10 mM HEPES, pH 7.4 (at 20°C) hemolyzing buffer: a) 1 mM CaC12 , 4 mM MgCl2 , 5 mM HEPES, pH 7.4 (at 20°C) b) 1 mM CaCI2 , 4 mM MgCl2 ' 5 mM HEPES, 2 mM ATP, pH 7.4 (at 20°C)

43

Calcium Transport in Resealed Erythrocytes KCl: 3.6 M icecold medium (needed after resealing): 140 mM, 1 mM CaC1 2 , 10 mM HEPES, pH 7.4 (at O°C) perchloric acid: 14 %w/v LaC1 3 : 30mM phosphate standard: 1 mM sulfuric acid: 1.75 M ammonium molybdate: 25 %w/v reducing agent for Fiske-Subbarow phosphate determination (4-amino-3hydroxynaphthalene-l-sulfonic acid)

1lI.EXPER]MENTALPROCEDURES A. Preparation of the Electrode Set-Up Ion-selective electrodes must be used with a high impedance input (approximately 10 12 ohms) mY-meter, e.g., with the glass electrode terminal of a pH-meter. This is because the electrodes will be damaged even by small currents. An ionselective electrode may thus never be linked directly to a recorder. The reference electrode may be a calomel-KCl electrode, the reference terminal of a combined pH-glass electrode or a platinum wire. The following set-up is useful for measurements with ion-selective electrodes and will be used in the experiments. pH 0, mV meter

.

ｉＭＫＺ［ｾ＠

ｾ＠

Iｾｬ＠

selective' electrode

u u

----

ｾｯｬｴ｡ｧ･＠

buck;n.

device

-:::fconle
'

\jj

Q.

t:l

I>'

"d

I>'

OQ

= ｾ＠ = .!"'"

'"d :;:!l

(1

....ｾ＠

C1I

\jj

'< C1I

ｾ＠

F

I>'

§"

C1I

CIl

::t: I>'

ｾ＠

ｾ＠

w

0\

-

K =s L = Ilmol

M=cpm

Y= cpm

C=ml

D=ml

For explanation of abbreviations, see p. 139.

N=ml

H =Ilmol 1= mg

F= cpm

300

G= cpm

cpm

X, (Y)

e.

::s

0

......

C')

i";'

go ...

300

B=ml

cpm

E

e.

::s .... ...

0

C')

(I)

ｾ＠

(I)

...0 S·

N

0

A=ml

S

Incubation time

300

300

300

300

300

...

...::s e.

0

C')

0

ｾ＠

-...

::r

0

0

......::s

C')

e.

ｾ＠

st

(I)

(I)

300

300

"d

ｾ＠

o·

IN -..l

-

'"

r::r ;;l ::s (I)

i3

(I)

:s::

(I)

...0

::r

0 '1:1

I\>

...i3

(3

i.l

::r ::r

::;:

::s :Ii!

I\>

....

...

'"

::r 0

0 '1:1

::r

0 0 '1:1

...::r

K. W. Hanselmann, W. Beyeler, C. Pflugshaupt, and R. Bachofen

138

Control. Add 25 JL1 molybdate reagent to a known aliquot (0) of the supernatant of the complete reaction mixture (A), dilute to 1 ml with solvent I and count directly to give the total cpm introduced (F).

H. Counting of P-32 We measure Cerenkov radiation of the P-32 decay in the liquid scintillation counter. The fmal volume of all samples in the scintillation vials is made up to 15 ml with water by visual comparison with a vial containing 15 ml of water (Asada et al., 1972).

I. Enzymatic Conversion of ATP into ADP Our assay contains: triethanolamine buffer 0.1 M, pH 7.6 H 2 0 dist.

Iml 0.450 ml O.2ml 0.3ml 0.050 ml Iml 3ml

MgCI 2 ,0.1 M Glucose (excess) 0.5 M hexokinase 0.2 mg/ml (27 units/ml) probe, neutralized Vtotal

Incubate at 25°C for 15 min, dilute with 3 ml Tris-CI 0.05 M, pH 8 and layer the complete volume onto a minicolumn. Elute and collect fractions as described under (F). G-6-P elutes in the fITst fraction.

IV. EVALUATION OF DATA 1. Calculation of the ATP formed as determined from the third fraction eluted from the minicolunms (experimental procedure (F)): X-V ATP = - - . A N

1 0

3600

-0

S

- -

[J.tmol

0

mg-1Bchl

0

hfl]

1 K 0

2. Calculation of the esterified phosphate, most of which can be found in ATP [experimental procedure (G)] : E-G = Pi C esterified

0

2.12

A 0

1

3600

U

I·K

-

B

[,umol

0

mg- l Bchl

0

hr- l ]

Specific activities can be calculated from the control vials of the appropriate experimental procedure:

Photophosphorylation with Chromatophore Membranes

M from procedure (F): specific activity S = [cpm L

139

ｾｯｲｬ｝＠

0

FoA

from procedure (G): specific activity U = - -

DoH

Calculations for procedure (I) are done accordingly. A = total volume of neutralized reaction mixture [ml] , usually 3 ml B = volume aliquot of A taken for mB-procedure [ml], usually 0.5 ml C = volume aliquot taken for counting from the remaining aqueous phase after the mB-procedure [ml], usually 1 ml D = volume aliquot of A taken for the determination of total cpm introduced [ml] , usually 0.1 ml E= cpmin C F=cpminD G = cpm in aliquot (same volume as C) of zero-time control H = Pi introduced !pmol] , usually 5 J.lmol 2.12 = B + 1.2 ml + xml to 2.5 ml + 0.8 ml + 0.02 ml- 1.2 ml= ｾ＠ of volumes for aqueous phase in mB-procedure [ml]. Mter three extractions with solvent II all the acetone (1.2 ml) may be assumed to be removed with the organic phase. 1= Bchl in total sample [mg] usually around 0.1 mg K = incubation time in seconds, usually 150s L = known amount of phosphate containing P-32 (JLmol) M= cpm from L N = volume aliquot loaded onto the column; 1 to 3 ml Sand U = specific activities as calculated above [cmp J.lmorl] X = cpm in ATP fraction eluted from the minicolumn Y = cpm in ATP fraction eluted from the minicolumn of the zero-time control probe 0

Average phosphorylation rates between 50 and 100 ｾｯｬ＠ ATP mg-1 Bchl. h- 1 can be expected with freshly prepared chromatophores under optimal conditions. The following two examples will demonstrate the kind of results that can be expected (Figures 7 and 8). 0

K.W. Hanselmann, W. Beyeler, C. Pflugshaupt, and R. Bachofen

140

100

-

I .r:. 80

:;: u

CD

1"'60 E

c.. ｾ＠

« (5

E

40

2: 20

0.2

0.6

1.4

1.0

Succinate [mM]

Fig. 7. Optimization of the redox conditions by varying succinate concentration 150

-....

" "\ \

\

\

I

.r:.

:;:

\

ｾ＠

I

\

\

100

\

'"

c.. ｾ＠

\

« (5

E

\

\ \

E

,

\

50

2:

\

\

\ \

0

II

0 10- 7

I

10- 6

"\

I

10- 5

ｾ＠

10- 4

••10-

3

CCCP[M]

Fig. 8. Inhibitory effect of CCCP on phosphorylation (experimental data by courtesy of M. Hofmann)

Photophosphorylation with Chromatophore Membranes

141

V.COMMENTS Specific inhibitors have proved to be very valuable tools in clarifying the mechanism of phosphorylation. Antimycin A, for example, is known to inhibit electron flow, thereby making it impossible for the electrochemical gradient to be established across the membrane (Baccarini-Melandri et al., 1977). Furthermore, the contribution of b.t/J and b. pH on phosphorylation has been evaluated by the use of suitable ionophones (for a short review see Gromet-Elhanan, 1977). Experiments with the energy-transfer inlubitor NEM indicate the involvement of a light dependent conformational change of the ATP-synthetase (BaccariniMelandri et al., 1975). Many of the data supporting Mitchell's chemiosmotic hypothesis of energy coupling stem from experiments with inhibitors and uncouplers of electron transport and phosphorylation and with molecules that disturb membrane integrity. This is true for mitochondria as well as for chloroplasts and the chromatophores of photosynthetic bacteria. Results that can be expected from the action of the different classes of inlubitory substances on photophosphorylation and on electron transport are summarized schematically in Figures 9 to 11.

c::

ｾ＠

Ｎｾ＠

I-

ｾ＠

-5

....,

"ii;"

ｾ＠

D-

-5

I-

"ii;" g.,"

g"

.," a;

D-

I-

«

....0

'0 2l

2l

a:'"

a:'"

Concentration of Uncoupler e.g., CCCP

« ....

" W

0

2l

....0

a:'"

ｾ＠

a:'"

Concentrati on of ET Inhibitor e.g., Antimycin A, HOQI\IO

Fig. 9. Effect of uncoupler on electron Fig. 10. Effect of inhibitor on ET in the transport (ET) and ATP-synthesis presence and absence of un coupler and (ATP) on ATP-synthesis

142

K. W. Hanselmann, W. Beyeler, C. Pflugshaupt, and R. Bachofen ET

+ uncoupler

ｾ＠

ｾ＠

I-

.'" 0

"

.;; c:

c:

ｾ＠

...!:!

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l-

ld

« ......

e;; ......

0

0

....'"

ｾ＠

.

ex:

ex:

Concentration of Energy Transfer Inhibitor e.g .• NEM

Fig. 11. Effect of energy transfer inhibitor on ET in the presence and absence of un coupler and on ATP-synthesis.

The experiments that are carried out with this phosphorylation assay may be complemented with measurements of pH gradients across chromatophore membranes, by following spectroscopically the changes in the electron carriers and by observing the behavior of coupling factor-depleted membranes. literature that might be useful for other experiments is included at the end of the chapter.

REFERENCES Asada K, Takahashi M A, Urano M (1972) Phosphorylation assay in liquid scintillation counter using Cerenkov radiation of 3:2 P: Application to photophosphorylation. Anal Biochem 48:311-315 Avron M (1960) Photophosphorylation by Swiss-Chard chloroplasts. Biochim Biophys Acta 40:257-272 Baccarini-Melandri A, Fabri E, Firstater E, Melandri B A (1975) Energy transduction in photosynthetic bacteria. VII Inhibition of the coupling ATPase by N-ethylmaleimide related to the energized state of the membrane. Biochem Biophys Acta 376:72-81 Baccarini-Melandri A, Casadio R, Melandri B A (1977) Thermodynamics and kinetics of photophosphorylation in bacterial chromatophores and their relation with the transmembrane electrochemical potential difference of protons. Eur J Biochem 78:389402

Photophosphorylation with Chromatophore Membranes

143

Clayton R C (1963) Absorbtion spectra of photosynthetic bacteria and their chlorophylls. In: Gest H, San Pietro A, Vernon L P (eds) Bacterial Photosynthesis. The Antioch Press, Yellow Spring, Ohio, pp 495-500 Gromet-Elhanan Z (1977) Electrochemical gradients and energy coupling in photosynthetic bacteria. TIPS 2: 12, 274-277 Hanselmann K W (1977) Zur Struktur von Reaktionszentren in phototrophen Bakterien: eine Standortbestimmung. Ber Dtsch Bot Ges 90:459-476 Jones 0 T G (1977) Electron transport and ATP synthesis in the photosynthetic bacteria. In: Haddock B A, Hamilton W A (eds) Microbial energetics, 27th Symp Soc Gen Microbiol. University Press, Cambridge, pp 151-183 Recommended reading on general aspects of photosynthetic energy-transduction: Avron M (1975) The electron transport chain in chloroplasts. In: Govindjee (ed) Bioenergetics of photosynthesis. Academic Press, London New York, pp 373386 Hinkle P C, McCarty R E (1978) How cells make ATP. Sci Am 4:104-122 Jagendrf A T (1975) Mechanism of photophosphorylation. In: Govindjee (ed) Bioenergetics of photosynthesis. Academic Press, London New York, pp 413492 Newer summaries on many aspects of biological energy transduction written by six authorities in the field. Requires some background knowledge for profitable study. Boyer P D, Chance B, Ernster L, Mitchell P, Racker E, Slater E C (1977) Oxidative phosphorylation and photophosphorylation. Annu Rev Biochem 46:955-1026 The most comprehensive up to date bibliography on the phototrophic bacteria: 59 contributors summarized in 50 chapters, the present knowledge about: ecology, structure, biosynthetic abilities, physiology, and bioenergetics of the photosynthetic bacteria. In: Clayton R C, Sistrom W R (eds) (1978) The photosynthetic bacteria. Plenum Press, New York, 946 p You may consult the following literature for complementary experiments dealing with pH-gradient and coupling factor of chromatophores: Stedingk von L -V, Baltscheffsky H (1966) The light-induced, reversible pH change in chromatophores form Rhodospirillum rubrum. Arch Biochem Biophys 117:400-404 Stedingk von L -V (1967) Light-induced, reversible pH changes in chromatophores from Rhodospirillum rubrum. Arch Biochem Biophys 120:537-541 Gromet-Elhanan U, Briller S (1969) On the use of bromthymol blue as an indicator of internal pH changes in chromatophores from Rhodospirillum rubrum. Biochem Biophys Res Commun 37:261-271 Gromet-Elhanan Z (1974) Role of photophosphorylation coupling factor in energy conversion by depleted chromatophores of Rhodospirillum rubrum. J BioI Chern 249:2522-2527

14. Oxygen Evolution and Uptake as a Measure of the Light-Induced Electron Transport in Spinach Chloroplasts ANDRES BINDER and REINHARD BACHOFEN

I. INTRODUCTION AND AIMS

A. General The light-induced electron transport in thylakoid membranes of higher plant chloroplasts uses H2 0 as electron donor and is able to reduce components with a potential as low as -0.55 V while producing at least one molecule of ATP. Thus 1.65 V/electron are required for the process. One quantum of red light, however, has not more than 1.3 V available. This leads to the conclusion that the energy of at least two photons must be utilized in order to provide sufficient energy to drive an electron through the complete photosynthetic electron transport system. The manner by which this could be achieved was fIrst shown in the observation of the red drop phenomenon by Emerson (Emerson and Lewis, 1943). Today the proposed Z-scheme is still generally accepted (Hill and Bendall, 1960). According to this model, the electron goes through two photoreactions (pS II and PS I), which are linked together by an electron transport chain. This scheme and a possible arrangement in the thylakoids proposed by Trebst (1974) is summarized in Figure 1. In Figure 2 some possible electron donors, acceptors, and inlubitors are introduced which will be used in the experiments of this course. With these artiftcial components it is possible to elucidate the individual steps of the electron transport.

B. The Electron Transport Pathway 1. Oxygen Evolution In intact chloroplasts, where the excited electrons after PS I are used as reducing power in the intrinsic photosynthesis system and water functions as electron

145

Oxygen Evolution and Uptake OUT

hv

2W

hv

NADP + 2W

NADPH

Membrane

IN

Fig. I. Proposed arrangement of the photosynthetic electron transport in the thylakoid membranes. (Trebst, 1974) Natural components: Y water splitting system; P-680 reaction center of PS II; PS II photo system II; Q primary acceptor of PS II; PQ plastoquinone; PC plastocyanin; P-700 reaction center of PS I; PS I photosystem I; X primary acceptor of PS I; Fd ferredoxin; FAD ferredoxinNADP-Reductase. Artificial components: HQ hydroquinone (e-donor of PS II); DCMU 3-(3,4-dichlorphenyl)-I, l-dimethylurea (herbicide, e-transport inhibitor between PS II and PS I); DBMIB 2,S-dibromo-3-dimethyl-6-isopropyl-pbenzoquinone (e-transport inhibitor between PS II and PS I, e-acceptor of PS II); SiMo sllicomolybdate (e-acceptor of PS II); FeCy ferricyanide (e-acceptor, Hillreagent); DPIP 2,S-dichlorphenolindophenol (oxidized: e-acceptor, Hillreagent; reduced: e-donor of PS I); MV methylviologen (e-acceptor of PS I, e-donor to O2 ); BQ benzoquinone (e-acceptor of PS II).

Fig. 2. Site of action of artificial electron donors, acceptors and inhibitors in the photosynthetic electron transport. Abbreviations as for Figure 1.

Andres Binder and Reinhard Bachofen

146

donor of PS IT, one 02 per 4-electrons transported is released in the inside of the membrane vesicle. In isolated thylakoid membranes where the natural electron acceptor system after PS I is missing, oxygen evolution can only be measured when an artificial electron acceptor like ferricyanide (Jagendorf and Smith, 1962) or DPIP (Avron et al., 1958) is introduced (Hill reaction). The released oxygen can be measured with the oxygen electrode. This opens the possibility of testing the electron transport through both photo systems. Inhibitors acting at the oxidizing site of PS IT like hydroxylamine prevent oxygen evolution (Cheniae and Martin, 1970). Typical electron donors to PS IT like hydro quinone (Yamashita and Butler, 1969) or hydroxylamine itself (Cheniae and Martin, 1970) can overcome this block. Electrons will now flow again through both systems but no oxygen is evolved. Inhibitors which act between the two photosysterns like DCMU (Duysens and Sweers, 1963) or DBMIB (Trebst et al., 1970) will also inhibit oxygen evolution when a Hill reagent acts as electron acceptor. Silicomolybdate seems to be able to accept electrons directly from PS II before the inlubition site of DCMU (Giaquinta and Dilley, 1975). The reduced silicomolybdate is able to reduce ferricyanide and can restore oxygen evolution. Other e-acceptors of PS II are benzoquinone (Trebst, 1974) or DBMIB at high concentrations (Gould and Izawa, 1973). The latter is autooxidizable.

2. Oxygen Uptake The oxygen evolution of PS II is not the only possibility to measure electron transport activities with an oxygen electrode. When no electron acceptor is available at the reducing side of PS I, part of the free oxygen will be taken up and converted to H2 2 , Yet some artificial acceptors of PS I such as methylviologen in their reduced form are able to reduce oxygen to H2 0 2 more efficiently (Zweig and Avron, 1965). This type of reaction is called Mehler reaction (Mehler, 1951). If catalase is present and converts H2 2 to H2 and 2 , no net change in oxygen can be measured when H2 was the electron donor of PS II. This is called the pseudocyclic electron transport (Krall et aI., 1961). The catalase activity can be inlubited by adding Na-azide to the reaction mixture. In the Mehler reaction two electrons are used to take up one 2 , but when H2 functions as electron donor, one 02 released per four electrons in PS II has to be subtracted. The electron transport through PS I alone can be tested with the 02 uptake reaction by introducing an artificial electron donor to PS I such as DPIP reduced with ascorbate (Trebst, 1964). This reaction is not sensitive to DCMU or DBMIB. CCCP as an uncoupler accelerates the electron transport but acts at higher concentrations as an electron transport inhibitor near the water-splitting system (Katoh and San Pietro, 1967).

°

° °

° ° °

°

Oxygen Evolution and Uptake

147

ll. EQUIPMENT AND SOLUTIONS A. Preparation of Olloroplasts Two hundred g deveined washed spinach leaves were homogenized in 300 mI STN medium (0.4 M sucrose, 20 mM tricine-NaOH pH 7.8,10 mM NaCI) at 4°C for ISs in a Waring Blendor at top speed. The homogenate was strained through cheesecloth and glass fiber cloth (Miracloth). The flltrate was centrifuged for 90s at 200 g and the supernatant again for 7 min at 1500 g. The pellet was resuspended in about 5 mI of STN medium giving a concentration of about 1 mg chlorophyll per mI.

B. Determination of the Chlorophyll Concentration Zero point one mI of the chloroplast suspension was diluted with 20 mI 80% acetone. After shaking, the suspension was flltered and the clear green acetone solution with the extracted chlorophyll was measured at 652 nm in a 1 cm cell. (OD 652 ·5.8 = mg chlorophyll per m1 in the original suspension.)

C. Measurements with the Oxygen Electrode (Clark type, "Rank," see Fig. 3)

. ",

'.

11 10

7 1

9

6

Fig. 3. The Clark type oxygen electrode. (Explanations are given in the text.) The electrodes of the oxygen electrode (the negative one of platinum (1); the positive one of silver, coated with silver chloride (2); dip into a solution of concentrated KCL (3). The constant voltage between the electrodes is -0.65 V. The electrodes are covered with a thin Teflon membrane (4). The oxygen in solution can penetrate through the membrane and is reduced at the cathode. The current

148

Andres Binder and Reinhard Bachofen

which will flow by reducing 02 is recorded and is proportional to the oxygen concentration in the solution. The activity of the chloroplast membranes is given in: J.Lmo10 2 (evolved or taken up) per hour and mg chlorophyll or better, for comparison with other activities: J.Leq. electrons per hour and mg chlorophyll The system is calibrated by adding some dithionite in the reaction mixture in order to reduce all the oxygen. The recorded difference represents the oxygen tension of air-saturated water (at 25°C = 0.25 J.Lffiol per ml). Dithionite should only be added at the end of a set of experiments because this strong reductant may poison the subsequent reactions. The reaction vessel of the oxygen electrode is placed on a magnetic stirrer (5) with a flea as follower (6). The vessel contains 2-25 rn1 of the reaction mixture (7) which can be covered (8). The vessel has a water jacket (9) in order to control the reaction temperature. (The electrode is very temperature-sensitive.) The electrode dips into the reaction mixture and has to be frrrnly fIxed in a manner that the tip of the electrode is about 5 mm above the flea. The speed of the magnetic stirrer has to be constant during the reaction time, and no air bubble should be in the reaction mixture. The light of the reaction lamp (100-200 W) (10) should fIrst pass 5-10 cm of water containing CUS0 4 (11). The recorder should have a variable voltage range and is placed at 5 to 50 mV full scale depending on the slope. Two to 25 ml of the reaction mixture is placed in the vessel in the dark (cover or dim light) as specifIed under Experimental Procedures. Additional solutions (in Experimental Procedures given as add) are added with a syringe. The reaction is started by turning the light on for 2 to 5 min and stopped by turning the light off (in Experimental Procedures given as measure). The oxygen evolution or uptake is followed on the recorder. The calculations are based on the slopes: light minus dark.

D. Solutions

Ascorbate

DPIP FeCy MY SiMo

HQ DCMU DBMIB

500 mM (use 10 mM) 2 mM (use 0.1 mM) 10 mM (use 05 mM) 25 mM (use 50 J.LM) 1 mM at pH 7.0 (use 20 J.LM) 250 mM (use 5 mM) 05 mM in methanol (use 10 J.LM) 200 J.LM in methanol (use 2 J.LM for inhibitor, 20 J.LM for acceptor)

Oxygen Evolution and Uptake Hydroxylamine Na-azide

ADP Phosphate

CCCP

149

500 mM (use 10 mM) 5 mM (use 100 pM) 100 mM (use 2 mM) 250 mM (use 5 mM) 100 pM in methanol (use 1 pM for uncoupler, 10 pM for e-transport inhibitor) 250 mM (use 5 mM)

m. EXPERIMENTAL PROCEDURES The following basic components are added in all experiments: 2 m1 20 mM Tricine-NaOH pH 7 .8 (except for Exps. 1 and 2) + Chloroplasts containing 2040 pg chlorophyll further additions are given in each experiment.

Experiment 1 Test of integrity of chloroplasts (fype A) in STN buffer add 2 m1 STN buffer and chloroplasts measure endogenous 02 evol.of Type A chloroplasts (weak activity with the given preparation). add 0.1 m1 FeCy measure accelerated 02 evol. (Hill reaction) of broken chloroplasts.

Experiment 2 PS II + PS I transport, PS II-I inhIbitor, PS II transport add 2 m1 20 mM Tricine-NaOH pH 7.0 and chloroplasts add 0.1 m1 FeCy measure 02 evol. by PS II + PS I = Hill reaction add 40 pI DCMU measure inhibition of 02 evol. add 40 pI SiMo measure restoration of 2 evol. by PS II.

°

Experiment 3 PS II + PS I transport, PS II-I inhIbitor, PS I donor measure 02 uptake minus 02 evol. by intrinsic PS II + PS I add 0.1 m1 DPIP measure 02 evol. by PS II + PS I = Hill reaction (DPIP ox.)

Andres Binder and Reinhard Bachofen

150

add 40 III DCMU measure inlubition of02 evol. add 40 III ascorbate + 40 III Na-azide + 40 III MY measure 02 uptake by PS I = Mehler reaction (DPIP red.).

Experiment 4 PS II + PS I transport, PS II-I inhibitor, PS I donor measure 02 uptake minus 02 evol. by intrinsic PS II + PS I add 40 pI MY + 40 III Na-azide measure 02 uptake minus 02 evol. by PS II + PS I add 40 III DCMU measure inhibition of 02 uptake and 02 evol. add 0.1 m1 DPIP + 40 III ascorbate . measure 02 uptake by PS I.

Experiment 5 PS II + PS I transport, hydroxylamine as inhibitor add 0.1 m1 DPIP measure 0) evol. by PS II + PS I = Hill reaction add 40 III hydroxylamine measure 02 evol. inhibited, e-transport from PS II donor.

Experiment 6 PS II + PS I transport, hydroxylamine as inlubitor and donor to PS II. add 40 III MY + 40 III Na-azide measure 02 uptake minus 02 evol. by PS II + PS I add 40 III hydroxylamine measure 02 uptake faster, 02 evol. inhibited add 40 III DCMU measure 02 uptake inlubited.

Experiment 7 DBMm as inhibitor and e-acceptor of PS II add 0.1 m1 FeCy measure 02 evol. by PS II + PS I (Hill reaction) add 20 III DBMm measure inhibition of 02 evol.

Oxygen Evolution and Uptake

151

add 200 J.Ll DBMIB measure 02 uptake (Mehler reaction) minus 02 evol. ofPS II add 40 J.Ll DCMU measure inhibition of 02 uptake and 02 evol.

Experiment 8 Uncouplers can accelerate electron transport at low concentration and inlubit at high concentration. add 0.1 ml PeCy measure 02 evo!. by PS II + PS I (Hill reaction) add 20 J.Ll CCCP measure faster 2 evo!. through uncoupling add 100 J.Ll CCCP 1J1easure 02 evol. inhibited.

°

Experiment 9 The electron transport which runs under phosphorylating conditions (Coupled electron transport) is more than twice as fast as under nonphosphorylating conditions. This is called the photosynthetic control. add 0.1 ml PeCy measure 2 evo!. by PS II + PS I = Hill reaction add 40 J.Ll MgC12 + 40 J.Ll Na-phosphate + 40 J.Ll ADP measure faster 02 evol. through phosphorylation.

°

IV. EVALUATION OF DATA Chlorophyll concentration OD652

•

5.8 = mg chlorophyll/ml

Electron transport activity

A= 11JJ. •

Y'T'f'6'104

JJ.eq electrons

C ·I1U

h' mg chl

6p. = voltage difference during the reaction (in mY/min) = volume of reaction mixture (in ml)

Y

Andres Binder and Reinhard Bachofen

152

= factor for conversion of Ilmol 02 in Ileq.e: = 4 for 02 evolution = 2 for 02 uptake. T = 02 -tension at given temperature (25°C =0.2Sllmol 02/ml) C = chlorophyll in reaction mixture (in Ilg) b.U = voltage difference of calibration = total 2 reduction with dithionite (in mY) f

°

For the same chlorophyll concentration and the same conditions of the electrode and the recorder, a factor F can be calculated which is constant for all experiments:

v . T • f· 6 • 104 F=-----c· b.U The activity for each experiment is then simply determined as:

A

= b.1l • F

REFERENCES Avron M, Krogmann D W, Jagendorf A T (1958) The relation of photosynthetic phosphorylation to the Hill reactions. Biochem Biophys Acta 30: 144-153 Cheniae G M, Martin I F (1970) Sites of function of manganese within photosystem II, roles in 02 evolution and system II. Biochim Biophys Acta 197:219-239 Duysens L N M, Sweers H E (1963) Mechanism of two photochemical reactions in algae as studied by means of fluorescence. In: Ashida J (ed) Studies on microalgae photosynthetic bacteria. University of Tokyo Press, Tokyo, pp 353-372 Emerson R, Lewis C M (1943) The dependence of the quantum yield of Chlorella photosynthesis on wavelength of light. Am J Bot 30: 165-178 Giaquinta R T, Dilley R A (1975) A practical reaction in photosystem II. Reduction of silico-molybdate prior to the site of dichlorophenyldimethylurea inhibition. Biochim Biophys Acta 387:288-307 Gould J M, Izawa S (1973) Photosystem - II. Electron transport and phosphorylation with dibromothymoquinone as the electron acceptor. Eur J Biochem 37:185-192 Hill R, Bendall F (1960) Function of the two cytochrome components in chloroplasts: A working hypothesis. Nature (London) 186: 136-137 Jagendorf A T, Smith M (1962) Uncoupling phosphorylation by absence of cations in spinach chloroplasts. Plant Physiol 37: 135 -141 Katoh S, San Pietro A (1967) Ascorbate supported NADP photoreduction by heated Euglene chloroplasts. Arch Biochem Biophys 122: 144-152 Krall A B, Good N E, Mayne B C (1961) Cyclic and non cyclic photophosphorylation ｾｩｮ＠ chloroplasts distinguished by use of labelled oxygen. Plant Physiol 36:4447

Oxygen Evolution and Uptake

153

Mehler A H (1951) Studies on reactions of illuminated chloroplasts I. Mechanism of the reduction of oxygen and other Hill reagents. Arch Biochem Biophys 33:65-77 Trebst A (1964) Ueber die photosynthetische NADP-reduktion mit Phenylendiaminen in isolierten Chloroplasten. Z Naturforsch 19B:4l842l Trebst A (1974) Energy conservation in photosynthetic electron transport of chloroplasts. Annu Rev Plant Physio125:423458 Trebst A, Harth E, Draber W (1970) On a new inhibitor of photosynthetic electron transport in isolated chloroplasts. Z Naturforsch 25B: 1157-1159 Yamashita T, Butler W L. (1969) Inhibition of the reaction by Tris. Restoration by electron donation to photosystem II. Plant Physio144:435438 Zweig G, Avron M (1965) On the oxidation-reduction potential of the photoproduced reductant of isolated chloroplasts. Biochem Biophys Res Commun 19:397400

Review

Encyclopedia of Plant Physiology, New Series. Photosynthesis I Trebst A, Avron M (eds) vol. V. Springer, Berlin Heidelberg New York.

15. The Function of the Purple Membrane in HaJobacterium HaJobium DIETER OESTERHELT and RAINER HARfMANN

I. INTRODUCTION AND AIMS

Halobacteria are Gram-negative rods of the approximate dimensions 0.5 f,l x 5 f,l, which require more than 15 % NaCl and smaller amounts of potassium and magnesium ions for optimal growth (see Bergey's manual of determinative bacteriology, 1974; Larsen, 1967; Dundas, 1977). If growth of Halobacterium halobium is limited by low oxygen supply the cells synthesize the retinal-protein-complex bacteriorhodopsin. TIlls chromoprotein forms, together with lipids, patches in the cell membrane. Because of their color these particles are called purple membrane. The purple membrane can be isolated by sedimentation and sucrose density centrifugation after lysis of the cells and fragmentation of the cell membrane by exposure to pure water. Upon illumination bacteriorhodpsin undergoes a photochemical cycle which is accompanied by the release and uptake of protons. The photochemical cycle is composed of several spectrally distinct intermediates of the bacteriorhodpsin chromophore called purple complex. The 412 nm chromophore is the intermediate with the longest life time (Fig. 1). The steady state concentration of any of the intermediates upon illumination of bacteriorhodopsin with light from a 150 W projector is too small to be detectable at room temperature by standard spectroscopy. If the photochemical cycle, however, is taking place in a salt-ether mixture the velocity of the cycle is slowed down. This is mainly due to a largely decreased rate of the backreaction from the 412 nm intermediate to the purple complex. Consequently the 412 nm complex accumulates under these conditions to a measurable (visible) steady state concentration. Bacteriorhodopsin is oriented in the cell membrane in such a way that protons are extruded into the medium and taken up from the cytoplasma during the

The Function of the Purple Membrane in Halobacterium Halobium ｾ＠

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The cycles per t,me Ivl are proportional to the steady slate concentrotlQn 01"'12' I Formahon 01 "412" ' E ' Y" J 2 Decoy 01 "412· : k2 ( slowest darl