Transport by proteins: Proceedings of a symposium held at the University of Konstanz, West Germany, July 9 –15, 1978 9783111710013, 9783110076943

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Transport by Proteins FEBS Symposium 58

Transport by Proteins Proceedings of a Symposium held at the University of Konstanz, West Germany, July 9-15,1978 FEBS Symposium No. 58 Edited by Gideon Blauer and Horst Sund

W G DE

Walter de Gruyter • Berlin • New York 1978

Editors Gideon Blauer, Ph. D. Professor of Biophysical Chemistry Institute of Life Sciences The Hebrew University Jerusalem, Israel Horst Sund, Dr. rer. nat. Professor of Biochemistry Fachbereich Biologie Universität Konstanz Konstanz, West Germany

CIP-Kurztitelaufnahme

der Deutschen

Bibliothek

Transport by proteins : proceedings of a symposium, held at the Univ. of Konstanz, West Germany, July 9 - 1 5 , 1978 ; FEBS symposium no. 58 / ed. by Gideon Blauer and Horst Sund. — Berlin, New York : de Gruyter, 1978. ISBN 3-11-007694-2 NE: Blauer, Gideon [Hrsg.]; Federation of European Biochemical Societies

Library of Congress Cataloging in Publication Data Main entry under title: Transport by proteins. (FEBS symposium ; no. 58) Bibliography: p. Includes indexes. 1. Proteins—Congresses. 2. Biological transport—Congresses. 3. Protein binding—Congresses. I. Blauer, Gideon. II. Sund, Horst, 1926III. Series: Federation of European Biochemical Societies. FEBS symposium ; no. 58 QP551.T73 574.1'9245 78-26457 ISBN 3-11-007694-2

© Copyright 1978 by Walter de Gruyter & Co. Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm, or any other means - nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Color-Druck, Berlin. — Binding: Liideritz & Bauer, Buchgewerbe GmbH, Berlin. Printed in Germany.

PREFACE

This volume presents the proceedings of a symposium on "Transport by Proteins" held at the University of Konstanz, West Germany, on July 9-15, 1978. In addition to the invited lectures, records of the discussions are included. The role of proteins in the transport of various molecules, ions and electrons is known to be of great biological, medical and pharmacological

im-

portance. The topic of this Symposium covers a wide range of different systems of fundamental physiological significance, such as the transport of oxygen, metabolites, metal ions, drugs, hormones, etc., all mediated by proteins either in aqueous medium or through membraneous structures. Transport systems involving proteins also include multienzyme complexes and arrays of the oxidation-reduction chains of respiration and photosynthesis. Both reversible and irreversible thermodynamics provide fundamental concepts for the main theme of this Symposium. Therefore, introductory lectures demonstrating some relevant principles, have been included in the programme. With regard to newer methodology, chemical relaxation and nuclear magnetic resonance on whole cells demonstrate more advanced and sophisticated experimental approaches which can be applied to the study of transport systems. Another section has been devoted to short surveys of some selected transport proteins and protein systems of fundamental biological significance, bringing us to the present level of knowledge in these fields. Several important and developing membrane protein systems have been grouped together in a separate section. Some selected metaltransport and storage proteins have been reviewed in another section, which also includes recent advances in the study of their structure and function. Finally, typical examples have been presented for the role of proteins in physiologically and pharmacologically important transport processes, again with the emphasis on new results and concepts.

VI

Preface

Symposia or s c i e n t i f i c topics should both promote the exchange of relevant information and also stimulate future research by the a c q u i s i t i o n of new ideas and concepts. However, conferences are u s u a l l y either overpopulated or devoted to a highly s p e c i a l i z e d topic. The aim of the present Symposium has been d i f f e r e n t : a limited number of s c i e n t i s t s from various d i s c i p l i n e s in the natural sciences were invited to present and discuss various aspects of a more general topic relevant to t h e i r work. I t was hoped that, despite the d i v e r s i t y of systems and t h e i r functions presented, some common properties and p r i n c i p l e s underlying these transport processes would be recognized and developed further through i n t e r d i s c i p l i n a r y

interaction.

Both experimental and theoretical knowledge acquired for one system may be useful and complementary to another. In accordance with the purpose of t h i s Symposium, special emphasis was placed on the d i s c u s s i o n s , f o r which no time l i m i t was imposed. To f a c i l i t a t e the d i s c u s s i o n s , preprints of the invited lectures were sent to the p a r t i c i p a n t s some time before the Symposium. A complete report of the d i s c u s s i o n s i s added at the end of each lecture. These written records of a d i s c u s s i o n were composed d i r e c t l y by the p a r t i c i p a n t s , i n most cases immediately a f t e r a s e s s i o n . The invited papers have been reproduced d i r e c t l y from the t y p e s c r i p t s submitted by authors. The contents of both papers and d i s c u s s i o n remarks are presented with the personal

responsibi-

l i t y of the respective authors. Based on previous experience, the number of p a r t i c i p a n t s and lectures was considered optimal for the purpose indicated. I n e v i t a b l y , many relevant subjects could not be treated within the framework of the Symposium, nor could a l l workers in a given f i e l d p a r t i c i p a t e . The present volume should aid in transmitting the message of t h i s Symposium to a much wider audience. Our thanks are due to a l l p a r t i c i p a n t s who have cooperated in t h i s Symposium. The generous f i n a n c i a l

support provided by the Deutsche Forschungs-

gemeinschaft (DFG) and the Federation of the European Biochemical

Socie-

t i e s (FEBS) i s highly appreciated. Further support extended by the host U n i v e r s i t y of Konstanz, by the Gesellschaft der Freunde und Förderer der U n i v e r s i t ä t Konstanz and by the Byk Gulden Lomberg Chemische Fabrik GmbH, Konstanz, i s g r a t e f u l l y acknowledged.

VII

Preface

Thanks are due to the s t a f f of the Verlag Walter de Gruyter in B e r l i n for t h e i r effective cooperation in the preparation of t h i s volume. The highly s k i l l e d and devoted assistance of Mrs. H. Allen in a l l technical matters concerning the Symposium, i s g r e a t l y appreciated. Our gratitude i s also extended to Dr. Stephen Bayne for his help in editing the book. F i n a l l y , the e d i t o r s would l i k e to repeat the hope expressed in the wake of the symposium on "Protein-Ligand I n t e r a c t i o n s " held in Konstanz in 1974, that "meetings of a s i m i l a r kind on various biological and biophys i c a l topics of i n t e r e s t will be held in the future and w i l l be i n s t r u mental in the promotion of new ideas and approaches in the natural ces".

Konstanz, September 15, 1978.

G. Blauer H. Sund

scien-

CONTENTS

SECTION I .

GENERAL

Thermodynamics o f P r o t e i n - L i g a n d Discussion

I n t e r a c t i o n s byF.M.

C l a s s i f i c a t i o n o f T r a n s p o r t Mechanisms by Thermodynamics by O. Kedem Discussion

Pohl

Non-Equilibrium 15 25

Chemical R e l a x a t i o n S p e c t r o m e t r y : I n v e s t i g a t i o n o f M e c h a n i s m s I n v o l v e d in Membrane P r o c e s s e s by H. Ruf, I. Oberbäumer and E. Grell Discussion 13

C NMR S t u d i e s o f G l y c o l y s i s in S u s p e n s i o n s o f Escherichia Coli C e l l s by R.G. Shulman, T.R. Brown, J.A. den Hollander and K. Ugurbil Discussion

SECTION I I .

3 11*

27

i»7 52

SELECTED TRANSPORT PROTEINS

Serum A l b u m i n a s a T r a n s p o r t P r o t e i n by T. Peters, R.G. Reed Discussion L i g a n d i n and P r o t e i n A: I n t r a c e l l u l a r B i n d i n g B. Ketterer, T. Came and E. Tipping Discussion The Absence o f Heme-Heme I n t e r a c t i o n a t the Hemoglobin by R.G. Shulman Discussion

Jr.

and 57 7U

P r o t e i n s by 79 92

I r o n Atoms o f 95 100

M u l t i e n z y m e C o m p l e x e s . M o l e c u l a r O r g a n i z a t i o n and F u n c t i o n a l I n t e r a c t i o n o f A c t i v e S i t e s w i t h i n the F a t t y A c i d S y n t h e t a s e and F. Lynen M u l t i e n z y m e Complex o f Y e a s t by E. Schweizer Discussion

103 118

Modes o f R e d o x - T r a n s p o r t by P r o t e i n s : " H y d r i d e " and versus E l e c t r o n T r a n s p o r t by P. Hemmerich Discussion

123 1i»6

"Carbanion"

X

Contents

The Mechanism of Energy Transduction by M. Avron Discussion

SECTION III.

in Photophosphory1 at ion 151 167

MEMBRANES AND TRANSPORT BY PROTEINS

Proteins in Mitochondrial Calcium Transport by E. K, Schwerzmann, I. Roos and M. Crompton Discussion

Carafoli, 171 185

Conformational Changes and Cooperativity in the Mechanism of Proton Transfer in Bacteriorhodopsin by B. Hess, R. Korenstein and D. Kuschmitz Discussion

1B7 198

Proteins of Bacterial Active Transport Systems by W. Boos Discussion

199 212

The Dependence of Mediated Transport Systems on the Cholesterol Content of the Membrane by W. Wilbrandt and C. Becker Discussion

2 15 220

Ion Channels in Excitable Membranes by F. Hucho Discussion

221 236

SECTION IV.

METAL TRANSPORT BY PROTEINS

Ferritin Iron Deposition and Mobilisation: Molecular and Cellular Consequences by R.R. Crichton Discussion

Mechanisms 2^3 255

The Ferritin Molecule: A Sink and a Source by P.M. Harrison, S.H. Banyard, G.A. Clegg, D.K. Stammers and A. Treffry Discussion

259 272

Thermodynamic and Accessibility Factors in the Specific Binding of Iron to Human Transferrin by P. Aisen and A. Leibman Discussion

277 291

Heme Transport and Properties of Hemopexin by U. Muller-Eberhard Discussion A Genetic Defect in the Transport of Ceruloplasmin Copper from Blood to Bile as the Possible Pathogenesis of Wilson's Disease by I.H. Scheinberg Discussion

...

295 309

311 320

XI

Contents

SECTION V.

MOLECULAR ASPECTS OF TRANSPORT BY PROTEINS IN PHYSIOLOGY AND PHARMACOLOGY

Pharmacological Consequences of Plasma Protein and Tissue Binding of Drugs by M.H. Bickel D i scuss ion • The Effects of Tryptophan and Tyrosine Modification on the DrugBinding Properties of Human Serum Albumin by W.B. Müller, K.J. Fehske and U. Wollert Discussion

325 335

3*t1 352

Transfer of Ferriprotoporphyrin IX from Human Serum Albumin to Apomyoglobin in Aqueous Solution by G. Blauer and J. Silfen .... Discussion

355 366

The Role of Intracellular Proteins in the Transport and Metabolism of Lipophilic Compounds by E. Tipping and B. Ketterer Discussion

369 383

Binding Proteins in Plasma and Liver Cytosol, and Transport of Bilirubin by J.A.T.P. Meuwissen and K.P.M. Heirwegh Discussion

3fl7 401

Concluding Remarks by P. Aisen

£tD5

Index of Contributors

itOg

Subject Index

i+"|"|

LIST OF CONTRIBUTORS

P. Aisen, Department of Biophysics, Albert Einstein College of Medicine, Bronx, New York, USA M. Avron, Institute of Biochemistry, The Weizmann Institute of Science, Rehovot, Israel S.H. Banyard, Department of Biochemistry, The University, Sheffield, England C. Becker, Pharmakologische Abteilung der Universität, Bern, Switzerland R. Benz, Fachbereich Biologie der Universität, Konstanz, West Germany M.H. Bickel, Pharmakologisches Institut der Universität, Bern, Switzerland G. Blauer, Institute of Life Sciences, The Hebrew University, Jerusalem, Israel W. Boos, Fachbereich Biologie der Universität, Konstanz, West Germany E. Carafoli, Laboratorium für Biochemie, Eidgenössische Technisehe Hochschule, Zürich, Switzerland T. Carne, Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, London, England G.A. Clegg, Department of Biochemistry, The University, Sheffield, England R.R. Crichton, Unité de Biochimie, Université Catholique de Louvain, Louvain-la-Neuve, Belgium M. Crompton, Laboratorium für Biochemie, Eidgenössische Technische Hochschule, Zürich, Switzerland K.J. Fehske, Pharmakologisches Institut der Universität, Mainz, West Germany E. Grell, Max-Planck-Institut für Biophysik, Frankfurt, West Germany G. Gros, Physiologisches Institut der Universität, Regensburg, West Germany P.M. Harrison, Department of Biochemistry, The University, Sheffield, England K.P.M. Heirwegh, Hepatology Laboratory, Katholieke Universiteit, Leuven, Belgium P. Hemmerich, Fachbereich Biologie der Universität, Konstanz, West Germany B. Hess, Max-Planck-Institut für Ernährungsphysiologie, Dortmund, West Germany F. Hucho, Fachbereich Biologie der Universität, Konstanz, West Germany 0. Kedem, Weizmann Institute of Science, Rehovot, Israel

XIV

L i s t of Contributors

B. Ketterer, Courtauld I n s t i t u t e of Biochemistry, Middlesex Hospital Medical School, London, England R. Korenstein, M a x - P l a n c k - I n s t i t u t für Ernährungsphysiologie, Dortmund, West Germany P.W. Kühl, M a x - P l a n c k - I n s t i t u t für Immunbiologie, Freiburg, West Germany D. Kuschmitz, M a x - P l a n c k - I n s t i t u t für Ernährungsphysiologie, Dortmund, West Germany A. Leibman, Department of B i o p h y s i c s , Albert E i n s t e i n College of Medicine, Bronx, New York, USA F. Lynen, M a x - P l a n c k - I n s t i t u t für Biochemie, M a r t i n s r i e d , West Germany R. Marz, I n s t i t u t f ü r Molekularbiologie der österreichsichen Akademie der Wissenschaften, Salzburg, A u s t r i a J.A.T.P. Meuwissen, Hepatology Laboratory, Katholieke U n i v e r s i t e i t , Leuven, Belgium W.E. M ü l l e r , Pharmakologisches I n s t i t u t der U n i v e r s i t ä t , Mainz, West Germany U. Muller-Eberhard, Department of Biochemistry, Scripps C l i n i c & Research Foundation, La J o l l a , C a l i f o r n i a , USA I . Oberbäumer, M a x - P l a n c k - I n s t i t u t f ü r Biophysikalische Chemie, Göttingen, West Germany Ch.T. 0 ' K o n s k i , Department of Chemistry, U n i v e r s i t y of C a l i f o r n i a , Berkeley, C a l i f o r n i a , USA Th. Peters, J r . , The Mary Imogene Bassett H o s p i t a l , Cooperstown, New York, USA W. P f l e i d e r e r , Fachbereich Chemie der U n i v e r s i t ä t , Konstanz, West Germany F. Pohl, Fachbereich B i o l o g i e der U n i v e r s i t ä t , Konstanz, West Germany G.S. Rao, I n s t i t u t für K l i n i s c h e Biochemie der U n i v e r s i t ä t , Bonn, West Germany R.G. Reed, The Mary Imogene Bassett H o s p i t a l , Cooperstown, New York, USA H. Ruf, M a x - P l a n c k - I n s t i t u t f ü r Biophysik, Frankfurt, West Germany I . Roos, Laboratorium für Biochemie, Eidgenössische Technische Hochschule, Zürich, Switzerland R. Salomon, Department of Neurobiology, The Weizmann I n s t i t u t e of Science, Rehovot, Israel G. Sawatzki, Abteilung f ü r Transfusionsmedizin der U n i v e r s i t ä t , Ulm, West Germany A.L. Schade, Physiologisches I n s t i t u t der U n i v e r s i t ä t , Wien, A u s t r i a I.H. Scheinberg, Albert E i n s t e i n College, of Medicine, Bronx, New York, USA

L i s t of Contributors

E. Schweizer, I n s t i t u t f ü r Mikrobiologie und Biochemie der U n i v e r s i t ä t Erlangen-Nürnberg, Erlangen, West Germany K. Schwerzmann, Laboratorium für Biochemie, Eidgenössische Technische Hochschule, Zürich, Switzerland R.G. Shulman, Bell Laboratories, Murray H i l l , New Jersey, USA J. S i l f e n , I n s t i t u t e of L i f e Sciences, The Hebrew U n i v e r s i t y , Jerusalem, Israel D.K. Stammers, Department of Biochemistry, The U n i v e r s i t y , S h e f f i e l d , Engl and G. Strobel, I n s t i t u t für Toxikologie der U n i v e r s i t ä t , West Germany

Düsseldorf,

H. Sund, Fachbereich B i o l o g i e der U n i v e r s i t ä t , Konstanz, West Germany M. Szekerke, Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Budapest, Hungary A.L. Tärnoky, Department of C l i n i c a l Chemistry, Royal H o s p i t a l , Reading, England

Berkshire

E. Tipping, Courtauld I n s t i t u t e of Biochemistry, Middlesex Hospital Medical School, London, England A. T r e f f r y , Department of Biochemistry, The U n i v e r s i t y , S h e f f i e l d , England M. Tropschug, I n s t i t u t für Biochemie der München, West Germany

Ludwig-Maximilians-Universität,

W. Wilbrandt, Pharmakologische Abteilung der U n i v e r s i t ä t , Bern, Switzerland U. Wollert, Pharmakologisches I n s t i t u t der U n i v e r s i t ä t , Mainz, West Germany.

Section I.

General

THERMODYNAMICS

Fritz

M.

OF

PROTEIN-LIGAND

INTERACTIONS

Poh1

F a c h b e r e i c h B i o l o g i e der U n i v e r s i t ä t D-7750 Konstanz, Germany

Konstanz

Introduction

In

order

to

processes to

study

the

well

regulatory overview

of

of

The

recent

measurements

and

the

mational etc.,

can

recent

gives

an

binding

of

formalism

ligand

the

their

in

and

equilibrium

forces

with

the

the

and

of

conditions. data It

by

also

involved

protein

and

concentration

will

in

the

on

the

of

interaction.

problems

interpretation

is

effector

experimental

the

as

transport

requirements

ligands

1igand-protein

developments and

the

thermodynamics. on

such

proteins

the

molecule

factors, on

of

both

of of

of

under

analysis

treatment

macromo1ecu1es

interactions, with

of

one

An

involved be

in

given.

Developments

theoretical

1 igands

(A

the

other

Theoretical

way,

information

molecules

of

behaviour

macromolecule

thorough

established

interaction

such

the a

important

influence

the

quantitative

energetics

to

allows

gives

a

the

molecules This

understand

in

such

protein states be

as

or

the

handled

review

the

molecule,

the

is

binding

now

so

advanced

interaction the

equilibria

of

influence

more of

that than

dissociation

of

oligomeric

in

a

summary

with of

the the

© 1978 Walter de Gruyter & Co., Berlin • New York Transport by Proteins

complex one

ligand confor-

proteins

straightforward

literature present

between

different

mathematically

together

excellent

of

cited

state

there

(1)).

way.

4

F.M.

Most

of

the

molecules to

the

tion

formulae

in

logarithm

of

protein

P1

+

is

described the

K

=

the

and

such

as

by

where Hr

=

n the

Hoff

are

Both

of

free

becomes

more

Multiple N

simple

reac-

~

the

in

-AG°/RT

this on

(1)

are

niven

respect

is

external

concentration

temperature

reciprocal

of

by

the

variables,

other

dependence

mole-

is

given

T

1

~ r

(2) =

units

owing

to

the

such

to

also

also be

and

of

the

should

can

sites.

AS°/R

use

a

simple

complicated

if,

methods system,

for

gas of

on of

example,

R,

different

In

that

systems a

K which

free

exist but

change

constant

emphasize

based

concentrations Numerous

enthalpy

the

the

is

ligand for

dis-

and

the

deter-

situation

multiple

binding

occurs. equilibria:

sites

on

a

The

binding

m a c r o m o 1 e c u 1e

of

ligand

P according

molecules to

the

scheme

given n

H

equations

binding

P ^ ^ P is

A

reactants

dependence

in

binding

reaction

x

free

Sr

in

protein

In

change

related

K

by

expressed

binding of

to

-

the

the

proportional

plot

energetics

mination

X

r

entropy

units.

a

S

K,

defined

-

pressure,

complications

directly

to

its

of

Y P1T_^.P2

constant

thermodynamics

K and

are

notation

as

pj

of

they

potentials

concentration.

shorter

In

example,

=

AH°/R

cussion

of

of

of

For

K

avoiding of

-

chemical

X

constant,

p2

aim

van't

l

in

the

which

association

temperature,

etc.

the

free

concentrations

x.The

on

in

ligand

or

the

In

determination

cules

their

dissociation

In where

of

P with

by

based

solution

XF=^P2X

of

Pi

dilute

are

Pohl

J^-P

by =

P

3

N-1

.

X

yp

(3)

N

^

) _

i .L. i

/

(

1 + 1 . ,

L • ) i

(M

Thermodynamics of Protein-Ligand

where

n

is

the

average

Interaction

number

protein

molecule.

Using

binding

functions

L;

of

1 igand

intrinsic

are

given

5

molecules

binding

bound

constants

K^

per the

by

i =

L.

with

x

J | j=1

again

constants

the

those

of

free

ligand

which

have

constants

Kj

are

binding

data

In

this

(2).

in

equation

concentration. to

mu1tip1e-binding

binding

polynomial

(4a)

J

as

are

energetics

( K . x (N + 1 - j ) / j )

be

used

occasionally

case,

the

terms

are

given

by

The

intrinsic

discussing

interactions.

also

(4)

in

the

Statistical

used of

the

to

evaluate binding

i

= J~]

L.

K*

x

Ctb)

j=1 Similar action

equations energy

between

protein

(3)

X

according

and

Y

11

^ ^

Y

If

a

for type

have

P

21

p

ligand

(4)

^

derived

calculation

ligands

for

the

X

and

binding

of

the

Y

bound

of

two

interto

the

1 igands

P

(5)

]Y P

32

conformational

binding

"allosteric

the

to

22

concerted

allow

different

been

I *

12

upon

which

is

change

indicated,

enzymes"

can

be

the

of

the

m a c r o m o 1 e c u 1e

formalism

applied

for

developed

reactions

of

the

:

p J L p JL. p JL. „ 1 „2 .3

1 1 1

p

N

(6)

6

F.M.

Analytical reaction to

the

mechanisms

free

and/or to

numbers

are

can

be

Within

short

time.

As

sets

advantage

of of

an

where

of

led like

some

with such

in

molecules

for if

the

the

kind

binding moderate

calcula-

past

use of

to

the

of linearisation

graphical

¡s

using

of

curves

ligand

equation

presented

simulation

display

unnecessary.

binding

simulation

sites,

constants

numerical

bound

intrinsic

methods

theoretical the

complicated

circumstances.

render

binding

of

the

tedious,

has

computers

which

binding such

This

rather

expressions

are

formalisms,

example,

four

N

for

number and

These

sites

particular

generation

with

the

ligands

etc.,

years

available the

protein

different The

plots

few

become

relate of

manually.

the

last

available

constants.

simplified

Hill

now

binding

under

allow

a

which

achieved

have

to

of

or

They a

of

performed

application Scatchard

are

concentration

isomerisation

large

tions

expressions

Pohl

in

becomes

F i g . 1 : Simulation of the number of bound molecules tetramer as function of the free ligand concentration using egn. ( 4 ). Equivalent binding sites: a) = 10, = 1, c) K Non-equivalent sites : d ) Ki = K2 = 0.1, 4 = e ) K1 = K2 = 10, K3 = K4 = 1.

within binding

(k) Fig.

more

and 1. pro-

(n) per x by = 1

>

0.1,

T h e r m o d y n a m i c s of Protein-Ligand

nounced

when,

cules

is

total

protein

for

this

the

example,

are

by

case no

the

a

the

a

are

fitting

of

of

the

Deriving

even

for

with

but

should

corresponding

in

be

the

icated

Since

known

moleand

expressions

compl

the

computer. best

mechanisms

bound ligand

analytical

task

a

usually

of

total

moderately

trivial

obtained

which

concentration

function

is,

means

easily

parameters

studies,

as

concentration.

latter

mechanisms, curves

for

calculated

7

Interaction

these

are

experimental

done

at

this

data

as

level. The

degree

judged at

of

present

anism best

and

the set

large

the

1

of

of

in

of

in

make

to

improved termining between exists a

range

basis

which

mech-

gives

the

selection

the

a

This most

kind

of

appropriate

most

important

choice

difficult

of

recent

the

stoichiometry and

allows 1 -

and/or each

small

for

method

these

years

and

methods

and

can

energetics

be of

single

the

binding

M

complexed

studying

applicability

proper

permit

other.

ligands

m a c r o m o 1 e c u 1es . No

10^

which

for

the

(5)and

a

con-

parti-

task.

steadily

ligands

free

methods

and

accuracy, the

exist

supplement

macromo1ecu1es

over

of

often

sensitivity, the

methods

They

advances

from

provides

deciding

data.

of

sguared,

parameters

experimental

technological

which

for

meaningful

concentration

the

Differences

Owing

measured

experiments.

the

between

system

the

solution.

summarises

cular

the

and

deviations,

physicochemica1

interaction

venience

of

Methods

measurement

Table

simulated sum

reasonable

further

number

molecules

the

physically

allows

for

Experimental

as

most of

also

conditions

between

or

description

approach

A

fit

visually

measurement ' and

above.

of

have used

been for

de-

interaction general

method

constants

Equilibrium

dialysis

over

8

F.M.

Table I .

1:

Methods

"Di rect" a)

for

measuring

separation

dialysis,

of

adsorption

Selective

electrodes, molecules

enzymatic

assay

of

optical

gelfi1tration

of

free

ligand

:

conductivity

optical

properties

upon

binding

:

fluorescence

rotation,

circular

dichroism

es r

Raman b)

:

Methods

absorbance,

nmr,

ligand

partition

indicator

Change

bound

electrophoresis

determination

specific

a)

and

chromatography

extraction,

" I nd i r e c t "

free

ultrafiltration,

ultracentrifugation,

II.

interactions .

Methods

Physical

b)

p r o t e i n - 1 igand

Pohl

spectroscopy

Change

of

thermodynamic

sedimentation, light

or

viscosity,

macromo1 ecu 1 ar diffusion,

properties

:

electrophoresis

scattering

o s m o t ic

pressure

biological

activity

ca1 or imetry III.

"Kinetic"

Methods

relaxation association fluctuation

(a

"direct"

upon in

binding

the

Optical this

method) (an

majority methods

will

kinetics and

dissociation

and

the

"indirect" of are

probably

kinetics

analysis

detection method)

quantitative certainly continue

to

and

most be

of

have

optical

qualitative convenient

so

in

perturbation

previously

the

and

been

used

investigations general

future.

and

Therefore,

Thermodynamics of Protein-Ligand Interaction

to

study

should

1igand-protein

be

directly

a

is

which

be

Titrations

are

solution versa,

bound

with

molecules

order

to

adding

situ,

stirrer

obtain a

solution equal Fig.

a

indicator

previously

free

The

ligand

efforts which

molecules

deserved

due

with

atten-

concentration

concentration

the

of

spectroscopic

stepwise

via

non-absorbing

determination

is

in

shows

and the

and at

a

by

be beam

titration e.g.

by

reference

constant

result

and

of

a

normal

can

double

(7),

often two

and

and

an

ligand

free

requires large

amount

time

or

and/or

are and

the

values.

of

data,

necessary. mixing

automatic

in

data

col-

(6).

inserted

time

of

continuously

simplified

driven

differential

It

the

concentration

between

skill

adding

amount

accuracy

solution

by

known

the

fashion.

syringes

a

of

difference

reasonable

possible

sample

manually

determining

titrations

short to

performed

experimental

Using

amounts 2

signals

Titrations

small

procedure

becomes

instruments. to

using

not of

from

titrating

spectroscopic

within

a

a

care,

precision

simple

and

in

of

the

this

lection With

by

considerable

optical

macromo1ecu1es

obtain

considerable By

usually

mixing

determination In

for

velocity.

Differential

a

has

assay.

derived

Continuous

to

or

determination

enzymatic

can

initial

vice

interactions, search

binding

method

the

coupled

ligands of

the

detection.

"direct"

tion,

into

indicate

optical A

put

9

constant

speed

cuvette,

continuous

performed

with

curves

of a

cuvettes

high

such

an

allows

simultaneously

experiment.

one

precision

concentrated

speed.

and

commercial

spectrophotometer

adding

motor

ligand in

F.M. Pohl

10

ETHIDIUM - Br

(pM)

2 : Continuous Differential Titration (CDT) of poly Fig. (dG-dC) with ethidium bromide addition to two cuvettes. The difference of absorbance between two cuvettes of 1 cm light path upon the continuous addition of a 10 mM ethidium bromide solution over 30 min is shown as function of time or the total ligand concentration. The reference cuvette con306 \i.M poly (dG-dC) in l.o M-NaCl and 20 mM-Na-pho sphate tained buffer (pH6.8), while the sample cuvette was filled with the same amount of solution without polymer. (The arrow shows the of an air-bubble (0.4 \xl ) in one of the delivery tubes). effect

Continuous apparatus single

Differential but

differing

experiment,

dependence.

For

this

solution

but

are

presents

the

data

buffer

and

b)

Considering of

the

see

the

allows

the

obtained

when

contain of

advantage

the

automatic

digital

displays

data data in

the work

manually of

as

both

at

with

of

binding

purpose,

amount

advantage

graphic

Titration.

conditions,

maintained

both

information

easily

of

Temperature

a) same

obtain

and

its

the in

to

the

contain

obtain

this one

titrations. would

simulation,

optimal

same Fig.3 only

solution.

titrations,

way

same a

temperature

contain

cuvettes

polymer

this

Using study,

temperatures.

automatic in

acquisition to

both

necessary

such

as

cuvettes

different

performed

collected

order

well

the

kind can Full

require using

parameters

des-

T h e r m o d y n a m i c s of Protein-Ligand

Interaction

E c o 0-01 ui o z
C H 0 H / > C = 0 a n d

NAD(P)/NAD(P)H,

B) - b e t w e e n N A D ( P ) H / N A D ( P ) and F1 C) - b e t w e e n H „ F 1 ,/Fl and 2 red ox

ox

/H.F1 , 2 red

FeIIIS/FeIIS

In a d d i t i o n , f l a v i n - d e p e n d e n t d i o x y g e n a c t i v a t i o n m u s t be c o n s i d e r e d as side r e a c t i o n , i.e. the D) - b e t w e e n H„F1 2

,/Fl red

transport

and 0 o / H „ 0 „ . ox

2

2 2

For f l a v i n - d e p e n d e n t r e a c t i o n s , we can d e r i v e f r o m the thus d e f i n e d a c t i v i t i e s five m a i n classes of f l a v o p r o t e i n s as s h o w n in T a b l e

1. The f o l l o w i n g s e q u e n c e of

(4),

questions

m u s t t h e n be a n s w e r e d for each one of the a c t i v i t i e s A - C: 1) Is the t r a n s p o r t s t o c h i o m e t r y 2) If 2e

le

or 2e

?

, w h i c h is the 2e - c a r r i e r , h y d r i d e o r

3) If c a r b a n i o n , w h i c h is the g e o m e t r y of the a n i o n bond,

tt or a ?

carbanion?

flavin-carb-

P.

126

H O ci D O

in

ai rH p H o xi « c xi m 0 rH •P a) O c N •H

•H W 10 ai >H

< >

en 3 rH f-t •H M

a> G 0 G

Oí

i

ai CM

0) CM Q

i

ai CM

P-. IV tn tO 4-> ai .c •M c

G

ai 01 bO O •o •H O •a m G •H x: •H en >1 0 G +-> O « •p •H fd S H Uh

ai CM




>

tn ai E o tn 0

u

ai -H •H -H O XI 10 G X> IÖ O M ai 0 c N -rH

tn rH 4-" e (0 0 ß X) bO 3 ai ai e tn tn eu

•rH

XI ai

* X) ai

ai 3 rH Xi

ai 3 rH XI

a: 3 rH XI

ai Ol

0) CM

1

1

1

^

ai en o a 3 rH O

S 2: (M




XH + >CHO-5-dFl~

> HdFl" , red

+

[1]

>C = 0

L[2] J

This is the first chemical simulation of biological hydride transport. Since the intermediate adduct is essential

(fig.l)

and since step 2 is rate determining, we are confronted with a cyclic mechanism, involving a 1,3-fragmenting

shift of

hydrogen (8). This is in apparent contrast to the linear hydride transport assumed genases

in nicotinamide-dependent

dehydro-

(eq.[3] ): NAD(P) + +

H

>C NAD(P)H + >C=0 + H X + ,

[3]

where X denotes the auxiliary histidine or zinc center (9).

129

Redox Transport by Proteins

(d)Flox /NAD*

5-HFI

S c h e m e 1. Structural comparison between (5-deaza)flavin and NAD(P) and their radicals. Attention: the acceptor position in d F l o x w h i c h is a n a l o g o u s to the n i c o t i n a m i d e - C ( 4 ) , is n u m b e r e d C ( 5 ) .

H ( r0e d ) / N A D "

( blue)

1-HdFl

unstable

stable

H

"yo

CH,

CH,

X' H

V o

N

H

(d) F l r 2 d H 2 / N A D H

5 - RFlox CH3-CH Ç H . - Ç H ,

=

N(l)-blocked and N(5)-blocked models a n d s c h e m e 4- b e l o w ) .

1 - R F fO êX

RF1

(cf. a l s o t a b l e 2

P. Hemmerich

130 Table 2: E N Z Y M E A N D M O D E L ^KDOX P O T E N T I A L S FT

SYSTEM

> n • ox < — E2 (le )

Free F1 (le or 2e")

-

1-RF1 b) (2e"-onlv)

ox < — E

e)

+ 423

Flavodoxins (le"-only)

-

- 1 9 0 to - 1 3 0

NADP-reductase (le" or 2e")

-

-

c)

- 280

c)

+ 010

360

-

+ 80 + 10 + 40

irrev.

irrev.

+ 4 30

770

Nicotinamide (2e - o n l y )

NAD+

Dithionite ^ (le" or 2e~)

so2Ji»

Oxygen d) (le~, 2e or 4e~)

02

-

-

320

-

320

NAD' N A D + 850

-

660

-

330

+ 413

400

- 056 - 240

-

64

- 4 2 0 to - 3 7 7 + 240

- 051 - 220

5-Deazaflavin (2e"only)

-

not obtained

009

+ 206

-

167

- 046 - 200

Glucose p H 5.3 Oxidase p H 9.3 (2e"-only) 5-Thiaflavin (le~-only)

320

(mV)

Fl- — > F 1 , E < — red * 1,2 E1 (le")

- 199

231

not obtained

5-RF1 b) (le~-only)

red (2e")

(pH 7)

NADH

+ 130 NAD'

-900

NADH

+ 210

-1010

S0~ 2 S 0 2 < J S > ( SOj ) 2~ 6~

386

o2 F l " , (Scheme b b red

3). A n a l o g o u s r e s u l t s

b e e n o b t a i n e d by P o r t e r e t a l .

(13) w i t h D - a m i n o a c i d

a s e a n d n i t r o a l k a n e as a r t i f i c i a l mediate OjH-CHR-Fl

H

have

substrates.

The

is r a p i d l y f r a g m e n t i n g

oxid-

inter-

to y i e l d

trite, aldehyde and reduced enzyme. Again analogously and Ghisla

(26) h a v e b e e n d e s c r i b i n g w i t h l a c t a t e

the decay of the

intermediate

glyoxylate and reduced

~00C-CH(0H)-Fl~ed

to

yield

S u m m a r i z i n g , w e can a n s w e r the above f o u r questions 3) a , 4 ) C ( 4 a ) a c c e p t i n g

i n t e r a c t i o n as

or releasing

1) 2e

for

the

, 2) R ,

R~, N(5) doing the

same

R+.

Flavin-iron Interaction In the preceding shift

Hassey

oxidase

enzyme.

case of flavin-nicotinamide with

ni-

and the Role of the

chapter we have

seen that a

is r e g u l a t i n g t h e m i g r a t i o n a l 0H—

R-4a-Flred-5-H

2)

RdFL re(J H + dFL* x

RdFlredH f a s t

>

(carbanion t r a n s f e r ) (HdFL) 2 " + H + + R +

(comproportionation)

(HdFl)2 c o n s i s t s of a mixture of diastereomers which are i n t e r c o n v e r t i b l e . I t i s very d i f f i c u l t to obtain a pure specimen of either diastereomer. This prevents easy formation of mono-crystals for X-ray a n a l y s i s . The diastereomers are stable towards heat and l i g h t in the absence of oxygen. Autoxidation in the dark i s proceeding slowly with quantitative formation of d F l 0 X . Dehydrogenation and formation of d F l 0 X are also obtained t h e r m o l y t i c a l l y . The s t a b i l i t y i s independent of pH, i . e . the dimers can be obtained in s o l u t i o n in neutral, monoanionic and dianionic states. P f l e i d e r e r : What i s the reason of the r e l a t i v e l y low s t a b i l i t y of the central C-C bond connecting the two 5-deazaflavin moieties? Hemmerich: This i s a good question. I can only answer by a counter quest i o n : what i s the reason for the r e v e r s i b l e dimerisation of t r i p h e n y l methyl r a d i c a l ? Perhaps i t can be said that the interconnecting C-C bond which anyhow should be r e l a t i v e l y long, does not possess 100% a-character because of angular d i s t o r t i o n at the carbon centers in p o s i t i o n 5. For more data, please compare P. Hemmerich, V. Massey and H. Fenner. FEBS Lett. 84, 5-21 (1977), and H.J. Duchstein, H. Fenner, P. Hemmerich and W.-R. Knappe: (Photo) Chemistry of 5-Deazaflavin: A Clue to the Mechanism of Flavin-dependent (De)hydrogenation (submitted to Eur.J.Biochem.). Crichton: You have said that you would not be unhappy with formation of dioxetane in the reaction of oxygen with reduced f l a v i n s . Would you l i k e to comment on the recent proposal of Goddard from theoretical c a l c u l a t i o n s of the formation of a radical intermediate in t h i s reaction? Henmerich: I am well aware of the c a l c u l a t i o n s done by Goddard as reported recently in Chemical and Engineering News. These c a l c u l a t i o n s may be excellent, but a l l I can say i s t h i s : the author does not know the current status of experimental evidence concerning flavin-dependent a c t i v a t i o n of dioxygen. The c a l c u l a t i o n s may thus be f i n e , but nature prefers not to make use of what the computer i s proposing. The main experimental r e s u l t s in t h i s context have been obtained by the research groups of Massey (flavin-dependent oxygenases) and Hastings (flavin-dependent l u c i f e r a s e ) as quoted in the present lecture. 0 ' K o n s k i : The emission of l i g h t in bioluminescence from dioxetanes i s c u r r e n t l y an i n t e r e s t i n g quantum-chemical problem. Can you e x p l a i n , on the l a s t s l i d e , your cryptic mention of Dioxetane"? Hemmerich:

My mention of dioxetane means that I would not want to s t i c k

P. Hemmerich out my neck as far as the structure of the "very fine intermediate" f o l lowing attack of dioxygen at the d i h y d r o f l a v i n nucleus i s concerned. This extremely s h o r t - l i v e d intermediate may well be a iT,a-complex or a dioxetane, i . e . a a,a-complex involving a four-membered r i n g made up from the two oxygen atoms and the two bridge head carbons in p o s i t i o n 4a and 10a. 0 ' K o n s k i : You referred to a p o s s i b i l i t y of electron tunnelling mentioned by someone in r e l a t i o n to hypothetical mechanisms of electron transport in the synthetic dimers of nicotinamide and f l a v i n s . Whose suggestion was that? Might internal charge transfer be involved? Henmerich: I did not want to mention anybody in p a r t i c u l a r . But the problem of electron t u n n e l l i n g , and whether i t i s possible in enzymes or not, is widely discussed i n the l i t e r a t u r e . Up to now, nobody has been able to devise r e l i a b l e channels through which electrons could be tunnelled within a protein in order to reach a wel1-shielded active s i t e . In my opinion, in the active conformation, at least an edge of the active s i t e must reach the surface in order to make electron-transporting contact, as s h o r t - l i v e d as i t may be, of l a r g e l y a-character. Support for t h i s hypothesis can be derived from our model s t u d i e s , but of course no proof i s a v a i l a b l e . Schweizer: Regarding the f a t t y acid synthetase-catalyzed reductions, I would l i k e to mention that, with the exception of the yeast enzyme, the enzyme complex isolated from animal sources apparently contains no f l a v i n . So, t h i s would be an example where NADPH i s able to reduce also an enoylgroup, in addition to the usual carbonyl-group reductions you mentioned. Hemmerich: This i s indeed true. The fact that some bacterial f a t t y acid synthetases do not contain f l a v i n , as i t seems, i s a painful mystery. Since I myself have no experience with such synthetases, I do not know whether anybody has a clear idea of how the second reduction i s working in these enzyme complexes. Our model r e s u l t s would not exclude a d i r e c t transfer of 2e"-equivalents between nicotinamide and acyl-CoA. Our data would only exclude d i r e c t transfer of equivalents between nicotinamide and an iron center without mediation of f l a v i n . On the other hand, i f transhydrogenation between nicotinamide and acyl-CoA i s possible without f l a v i n , why then would nature make use of f l a v i n in most synthetases? All I can say i s : I know that I do not know. Sund: You described marvellous experimental r e s u l t s mainly with model compounds. However, after your lecture I have the f e e l i n g that you can perform the described redox-transport processes without the mediation by any proteins. Would t h i s be p o s s i b l e ? Hemmerich: This i s the standard question of a protein chemist in d i s c u s sions with coenzyme chemists: This question i s centered around the d e f i n i t i o n of the term "chemical model". A chemical model in the hands of a bioorganic chemist i s not meant to be a "bigger elephant than nature". I t was the aim of t h i s lecture to show that the various biological a c t i v i t i e s of protein-bound f l a v i n can be mimicked chemically in p r i n c i p l e . This i s a f i r s t step towards an understanding of an enzyme mechanism in terms of chemical structure. Of course, such simulated a c t i v i t i e s remain poor in comparison with the natural ones and, in p a r t i c u l a r , the natural coenzyme

Redox Transport by Proteins in combination with the apoprotein e x h i b i t s a l l a c t i v i t i e s at once, depending on structure and conformation of the apoprotein. The chemical models, however, are confined, quite generally, to one a c t i v i t y such as 5 - a l k y l f l a v i n s to l e " - t r a n s p o r t , and 1 - or 2a-alkylated f l a v i n s to dehydrogenation. In flavin-dependent dioxygen a c t i v a t i o n , on the other hand, the various types of a c t i v i t y , namely l e " - t r a n s p o r t via superoxide, 2e"-transport via hydrogen peroxide and 4e"-transport via oxene are governed by the pH and the p o l a r i t y of the microenvironment. Thus we know that l i p o p h i l i c environment of oxidized f l a v i n can be recognized, e.g., by the v i b r a t i o n a l substructure of the band at 450 nm, while water a c c e s s i b i l i t y at the f l a v o semiquinone can be s a f e l y judged by i t s blue c o l o r : chemically, the semiquinone has a strongly negative solvatochromic s h i f t which turns the r a d i cal green in the absence of water. Many flavoproteins are known where the oxidized from shows a l i p o p h i l i c environment, while the semiquinone remains blue, i . e . h y d r o p h i l i c ; all t h i s i s of course possible as a consequence of conformational changes accompanying electron uptake. Thus the free f l a v i n in unpolar s o l u t i o n i s a "model for an oxygenase" while in polar acid s o l u t i o n i t i s a model for an oxidase and in polar a l k a l i n e s o l u t i o n a model for a superoxide producing e~-transferase. I f we had to renounce the protein, we could only simulate the sequence of events by changing the reaction vessel twice in every s i n g l e enzyme-model turnover. Thus protein chemists may be reassured that requirement for proteins will persist.

THE MECHANISM OF ENERGY TRANSDUCTION

IN

PHOTOPHOSPHORYLATION

M. A v r o n D e p a r t m e n t of B i o c h e m i s t r y , W e i z m a n n R e h o v o t , Israel

Institute of

Science

Summary The r e l a t i o n b e t w e e n e l e c t r o n t r a n s p o r t , p r o t o n p r o t o n g r a d i e n t - f o r m a t i o n and A T P s y n t h e s i s and q u a n t i t a t i v e l y

investigated.

is

transport, qualitatively

Transmembranal

proton

con-

c e n t r a t i o n g r a d i e n t s have m a n y p r o p e r t i e s w h i c h are in a g r e e m e n t w i t h their being an i n t e r m e d i a r y e n e r g y pool

between

e l e c t r o n t r a n s p o r t and A T P f o r m a t i o n in the steady H o w e v e r , during the initial

s e c o n d or two following

light t r a n s i t i o n t r a n s m e m b r a n e a major energy transducing

electric potentials

device.

tons are r e q u i r e d to t r a n s v e r s e the membrane

state. a darkserve as

A p p r o x i m a t e l y three chloroplast-vesicle-

to p r o v i d e s u f f i c i e n t e n e r g y for the s y n t h e s i s of an

ATP molecule

in the steady state.

The system

fully r e v e r s i b l e t h r o u g h the e x p e r i m e n t a l

is s h o w n to be

demonstration

A T P d r i v e n p r o t o n g r a d i e n t s , reverse e l e c t r o n flow, l u m i n e s c e n c e , and p r o t o n g r a d i e n t d r i v e n reverse flow and 1.

pro-

of

and

electron

reverse-electron-flow-luminescence.

Proton Uptake and Proton

Chloroplast membranes

Gradients

isolated from h i g h e r p l a n t s or algae

show r a p i d and r e v e r s i b l e e l e c t r o n t r a n s p o r t d e p e n d e n t

proton

u p t a k e from the m e d i u m

(see

Ref. 1). natural

into their i n n e r v e s i c u l a r

The n u m b e r of p r o t o n s

space

taken up is a f u n c t i o n of the

internal buffering p o w e r , and c a n therefore

be

in-

c r e a s e d c o n s i d e r a b l y by the a d d i t i o n of c o m p o n e n t s w h i c h as internal b u f f e r s

(2,3).

In order to e v a l u a t e the

© 1978 Walter de Gruyter & Co., Berlin • New York Transport by Proteins

act

possible

152

Energy Transduction in

Photophosphorylation

Fig. 1: E q u i l i b r a t i o n of a m i n e s a c r o s s m e m brane v e s i c l e s .

JH^lin _ (RNH$)ing (R totollin ^ (H')oul (RNHJIoul (R total )ou)

ou|

c o n t r i b u t i o n of the p r o t o n c o n c e n t r a t i o n g r a d i e n t s

created

during

system

such p r o t o n u p t a k e to the e n e r g y c o n s e r v i n g

several m e t h o d s were d e v e l o p e d to m e a s u r e gradients.

M o s t of these m e t h o d s d e p e n d on the

of amines a c r o s s the c h l o r o p l a s t m e m b r a n e pH gradient

the size of

(Fig.

such

equilibration

in r e s p o n s e to the

1).

The n o n - p r o t o n a t e d

form of the a m i n e s

(RNH2)

is in all

a s s u m e d to freely p e r m e a t e the m e m b r a n e and t h e r e f o r e concentration

cases its

inside e q u a l s its c o n c e n t r a t i o n o u t s i d e .

w i t h a pK more than 1 p H u n i t s above that of the

Amines

reaction

m e d i u m will be m o s t l y in their p r o t o n a t e d form in the m e d i u m . W h e n a p H g r a d i e n t e x i s t s a c r o s s the v e s i c l e due to p r o t o n u p t a k e , the free amine inside will be p r o t o n a t e d b y the coming p r o t o n s p u l l i n g m o r e free amine in, until at state the following

r e l a t i o n will h o l d

(H+)in (H+)out

_

(RNHj)out

concentration concentration.

the t r a n s m e m b r a n e

C-labeled methylamine

u n d e r all e x p e r i m e n t a l

steady

(4):

(RNHj)in

The first m e t h o d d e v i s e d for m e a s u r i n g 14 gradient utilized

in-

pH

(pK=10.6),

where

c o n d i t i o n s e m p l o y e d the total

amine

is e s s e n t i a l l y equal to the p r o t o n a t e d C h l o r o p l a s t s were

illuminated

in the

amine presence

(1-IOm.M), a m o u n t s of1 4l a b e l l e d m e t h y l a m i n e , c e n t r i fuged in the light, and the C c o n t e n t of p e l l e t and s u p e r -

of trace

natant determined.

I n d e p e n d e n t l y , the o s m o t i c v o l u m e of the

153

M. Avron

14 C labelled

same c h l o r o p l a s t p r e p a r a t i o n was d e t e r m i n e d using

sorbitol w h i c h does not enter the c h l o r o p l a s t v e s i c l e therefore

labels o n l y the n o n - o s m o t i c c o m p a r t m e n t .

v a l u e s the m e t h y l a m i n e c o n c e n t r a t i o n v e s i c l e can be d e t e r m i n e d and hence V a l u e s of ApH a p p r o a c h i n g

and

From

inside and o u t s i d e (Equation 1) the

(4).

S i m i l a r t e c h n i q u e s using other a m i n e s have been

(5).

The m a i n d i s a d v a n t a g e

is the

the

ApH.

3 were d e t e r m i n e d w i t h this of such m e t h o d s

method employed

requirement

for rather h i g h c h l o r o p l a s t c o n c e n t r a t i o n s w h i c h r e s u l t s u n d e r s a t u r a t i o n w i t h light, and a c o n s e q u e n t of the p H

these

in

underestimation

gradient.

T h i s d i f f i c u l t y does not arise

in the following m e t h o d s ,

which

f o l l o w the p H g r a d i e n t c o n t i n u o u s l y in the r e a c t i o n m i x t u r e . Rottenberg

and G r u n w a l d

(4) a d d e d low c o n c e n t r a t i o n s of

nium and f o l l o w e d the a m m o n i u m c o n c e n t r a t i o n d i r e c t l y w i t h an a m m o n i u m - s e n s i t i v e

electrode.

e x c e l l e n t m e t h o d , but its u s e f u l n e s s

cent amine, strated

9 - a m i n o a c r i d i n e , since

in f l u o r e s c e n c e

vesicles.

From the

This

light conti-

is p r o b a b l y the m o s t w i d e l y u s e d

been a d e q u a t e l y e s t a b l i s h e d model systems

demontotally

and the o s m o t i c v o l u m e of the

m e t h o d and w i t h p r o p e r p r e c a u t i o n s

?

is

the light induced ApH can be a c c u r a t e l y and

nuously followed.

which

of a f l u o r e s -

it was p r e v i o u s l y

(7) that the f l u o r e s c e n c e of such amines

induced c h a n g e

is an

responds.

(6) f o l l o w e d the f l u o r e s c e n c e

q u e n c h e d inside the c h l o r o p l a s t vesicles

This

is l i m i t e d to m e d i a

c o n t a i n no o t h e r ions to w h i c h the e l e c t r o d e S c h u l d i n e r et al.

ammo-

in the m e d i u m

(8,9)

its r e l i a b i l i t y

in a v a r i e t y of b i o l o g i c a l

has

and

(10-17).

Prot on G r a d i e n t s and A T P

Formation

The r e l a t i o n of the m e a s u r e d ApH values to A T P f o r m a t i o n

in

c h l o r o p l a s t s was i n v e s t i g a t e d u s i n g several a p p r o a c h e s .

As

e x p e c t e d from the c h e m i o s m o t i c h y p o t h e s i s , w h i c h that ATP f o r m a t i o n is d r i v e n b y u t i l i z i n g

the

postulates

transmembrane

154

Energy Transduction in Photophosphorylation

9 i

?

0

2

3

'1 a.

J.

«

3.0

0

Tim« (min] Fig. 2. Time course of d e v e l o p m e n t of ApH and A T P f o r m a t i o n at low light i n t e n s i t i e s . The r e a c t i o n mixture c o n t a i n e d in 3.0 ml: t r i c i n e - m a l e a t e , pH 7.2, 30 mM; s o r b i t o l . 30 mM; M g C l , 1 mM; A D P , 1 mM; Pi, 1 m M (containing 3 x 1 0 7 cpm i 2 P ) . A T P , 100 uM, p y o c y a n i n e , 25 iiM; 9 - a m i n o a c r i d i n e , 0.5 uM; and c h l o r o p l a s t s c o n t a i n i n g 75 ug of c h l o r o p h y l l . In A light i n t e n s i t i e s were in ergs x cm~2 x sec"': 0 - 0 4.5 x 10^;a A 6 x 10 4 ; 4 x 1 0 4 ; O - O 1•5 x 1 0 4 . ( S e e Ref. 19.) electrochemical

potential

of p r o t o n s , the g r a d i e n t

observed

during p h o s p h o r y l a t i o n was s m a l l e r by about 0.5 pH units that o b s e r v e d in the absence of p h o s p h o r y l a t i o n

(18).

than

When

v e r y low light i n t e n s i t i e s were u s e d , the k i n e t i c s of the d e v e l o p m e n t of the pH g r a d i e n t and of ATP s y n t h e s i s c o u l d be simultaneously observed.

As can be seen in Fig.

g r a d i e n t d e v e l o p e d i m m e d i a t e l y upon turning a distinct point

On p l o t t i n g

of ApH at any

the

time

(Fig. 2), it is clear that no ATP f o r m a t i o n c o u l d be

o b s e r v e d before a t h r e s h o l d of ApH up.

the

the light on, but

lag in A T P f o r m a t i o n was o b s e r v e d .

rate of A T P s y n t h e s i s vs. the m a g n i t u d e

2 (19)

(about

2.3 units) was

B e y o n d that point the rate of ATP f o r m a t i o n was

and linearly d e p e n d e n t u p o n further A T P f o r m a t i o n and ApH are

increase

built

sharply

in ApH.

Clearly,

linked but their r e l a t i o n is not

linear. Toquantitate

the r e l a t i o n of p r o t o n m o v e m e n t across the m e m -

brane v e s i c l e to A T P f o r m a t i o n c h l o r o p l a s t s were p e r m i t t e d s y n t h e s i z e A T P at low light i n t e n s i t i e s until a steady was a c h i e v e d w h e r e no further ATP c o u l d be s y n t h e s i z e d . this point b o t h the m a g n i t u d e of the p r o t o n

At

concentration

g r a d i e n t and of the p h o s p h a t e p o t e n t i a l were m e a s u r e d .

to

state

In

155

M. Avron

3

tyi-H* Mri«d by light intmity -i 2 «IO* -» 2 > I09*r«t i em21 Me

Ab' A/Ih

2

200

Ì

220

A/i-H'.mllìwlt« Fig. 3. A n a l y s i s of ApH as a d r i v i n g force for A T P f o r m a t i o n in the s t e a d y s t a t e . R e a c t i o n m i x t u r e in 3 m l : K C 1 , 20 m M ; p h o s p h a t e , p H 8 . 0 , 2 m M ; M g C l 2 , 4 m M ; A D P , 0 . 2 m M ; p h e n a z i n e m e t h o s u l fate, 10 iiM; 9 - a m i n o a c r i d i n e , 1 uM; a n d c h l o r o p l a s t s c o n t a i n i n g 54 ^g of chlorophyll. It w a s i l l u m i n a t e d for 1 0 - 2 0 m i n u t e s u n t i l a steady state phosphate potential was attained. Phosphate p o t e n t i a l v a l u e s w e r e c a l c u l a t e d from A D P c o n t e n t d e t e r m i n ation with pyruvate kinase and phosphoenolpyruvate. AG® of 7.8 Kcalxmole at pH 8 . 0 , 10 m M M g + 2 , 2 5 o c , 0.1 i o n i c s t r e n g t h was used ( 2 0 ) . Au„+ w a s a s s u m e d to be c o m p o s e d of ApH o n l y . Fig.

3 (12) the r e s u l t s

The v a l u e

tons w h i c h m u s t ATP molecule at least

of s u c h an e x p e r i m e n t

of AG ' / Au[j+ r e p r e s e n t s traverse

under

the m e m b r a n e traverse

It is e v i d e n t

the m e m b r a n e p e r A T P

s y n t h e s i z e d u n d e r all the m e a s u r e d c o n d i t i o n s . tential

v a l u e s were not c o n s i d e r e d

it h a s b e e n a m p l y d e m o n s t r a t e d membrane

3.

potentials

exist

Proton Gradient

We have p r e v i o u s l y

(21,4,14)

in the s t e a d y

Membrane

that no state

in

shown that, under appropriate (22).

significant chloroplasts.

Thus

conditions, and ATP

(Figures

4, 5). A c c o r d i n g

intermediates

to the c h e m i o s m o t i c

s h o u l d be o b l i g a t o r y e n e r g y

in this p r o c e s s .

forma-

a d d i t i o n of A T P

c a u s e d the r e d u c t i o n of "Q" a n d o x i d a t i o n of c y t o c h r o m e proton gradients

po-

since

Flow

the c o u p l e d r e a c t i o n b e t w e e n e l e c t r o n t r a n s p o r t is r e v e r s i b l e

an

that

molecule

in the c a l c u l a t i o n ,

and R e v e r s e E l e c t r o n

t i o n in c h l o r o p l a s t s

plotted.

n u m b e r of p r o -

for the s y n t h e s i s of

the g i v e n c o n d i t i o n s .

3 protons must

are

the m i n i m a l

f

hypothesis transducing

Indeed, transmembrane

proton

156

Energy Transduction in Photophosphorylation

Fig. 4. An o v e r s i m p l i f i e d scheme of p h o t o s y n t h e s i s i n d i c a t i n g a site of c o u p l i n g b e t w e e n e l e c t r o n t r a n s p o r t and A T P f o r m a t i o n .

Fig. 5. A T P d r i v e n r e d u c t i o n of Q and o x i d a t i o n of c y t o chrome f. Q r e d u c t i o n was f o l l o w e d by the increase in c h l o r o phyll f l u o r e s c e n c e y i e l d w h i c h a c c o m p a n i e s it. Cytochrome f o x i d a t i o n in a dual w a v e l e n g t h s p e c t r o p h o t o m e t e r at 5 5 4 - 5 4 0 nm (see Ref. 22) . g r a d i e n t s were shown to be c r e a t e d d u r i n g the a c t i o n of the ATPase

(see Ref.

As o b l i g a t o r y

23).

i n t e r m e d i a t e s , the b u i l d u p of p r o t o n

gradients

w o u l d be e x p e c t e d to be k i n e t i c a l l y at least as fast as the i n d u c t i o n of reverse e l e c t r o n flow u n d e r all e x p e r i m e n t a l ditions.

T h u s , t r e a t m e n t s w h i c h slow down the d e v e l o p m e n t

the t r a n s m e m b r a n e p r o t o n g r a d i e n t w o u l d be e x p e c t e d to a corresponding

s l o w d o w n in the d e v e l o p m e n t

reverse e l e c t r o n flow.

of the

of

induce

ATP-driven

We c o n s t r u c t e d an a p p a r a t u s

e n a b l e d us to s i m u l t a n e o u s l y m o n i t o r the A T P - d r i v e n m e n t of ApH and r e d u c t i o n of Q under a v a r i e t y of

con-

which develop-

conditions,

157

I ' '

M. Avron i?

0* "--—02

V

0

Imdozole

V " V T '

Phenylened«n»n«____

^

30 20 10

Q

Pho»ho>c

0

——— •—2°0 12

t

.

-

.

V \

_

. . .

.

/ 30 I

Tai» offer ATP-oddition. KC Fig. 6. The effect of internal b u f f e r s on the k i n e t i c s of ATPd r i v e n b u i l d u p of ApH a n d r e d u c t i o n of Q. Buffers p r e - a d j u s t e d to pH 8.0 were a d d e d to the i n d i c a t e d final c o n c e n t r a t i o n s b e fore a c t i v a t i o n (24). a n d thus test the above p r e d i c t i o n the s i m u l t a n e o u s

(24). Figure 6

r e c o r d i n g of 9 - a m i n o a c r i d i n e and

f l u o r e s c e n c e , and the e f f e c t of adding increasing tion of three

illustrates chlorophyll concentra-

internal b u f f e r s on the A T P - i n d u c e d ApH b u i l d u p

and reverse e l e c t r o n flow.

All three c a u s e d a d e c r e a s e

rate and e x t e n t of not only the A T P - i n d u c e d ApH, as but also of the A T P - i n d u c e d reverse e l e c t r o n flow. results are e s s e n t i a l l y

in

expected, These

in a g r e e m e n t w i t h the p r e d i c t i o n s

the c h e m i o s m o t i c h y p o t h e s i s .

T h u s , slowing the b u i l d u p

ApH by internal b u f f e r s b r o u g h t about a c o r r e s p o n d i n g

of

of

change

in the rate and e x t e n t of the o b s e r v e d r e d u c t i o n of Q by reverse e l e c t r o n

flow.

If p r o t o n g r a d i e n t s serve as i n t e r m e d i a t e e l e c t r o n t r a n s p o r t a n d ATP f o r m a t i o n , demonstrate

also p r o t o n - g r a d i e n t

energy p o o l s

between

it s h o u l d be p o s s i b l e

d e p e n d e n t reverse

to

electron

flow.

Indeed

ratory

(Fig. 7). R a p i d a d d i t i o n of Tris to c h l o r o p l a s t

(25,26), such a r e a c t i o n w a s found in our

labosuspen-

sions p r e e q u i l i b r a t e d at pH 5.3 c r e a t e d a m o m e n t a r y pH grad i e n t w i t h the i n n e r v e s i c u l a r at pH 9.6.

space at p H 5.3 and the

medium

This m o m e n t a r y g r a d i e n t s e r v e d as a d r i v i n g

for reverse e l e c t r o n flow as i n d i c a t e d b y the t r a n s i e n t t i o n of Q

(Fig. 7).

since o x i d i z i n g

Reverse e l e c t r o n flow is c l e a r l y

force reduc-

occurring

the r e d u c e d e l e c t r o n c a r r i e r s b e t w e e n the two

p h o t o s y s t e m s by p r e i l l u m i n a t i o n w i t h f a r - r e d light

which

Energy Transduction in Photophosphorylation

158

DCMU

i

IO

| LIGHT

F i g . 7. P r o t o n g r a d i e n t d r i v e n r e d u c t i o n of Q. F q l e v e l i n d i c a t e s f u l l y o x i d i z e d Q a n d F,*, level ( o b s e r v e d on a d d i t i o n of D C M U a n d / o r d i t h i o n i t e ) f u l l y r e d u c e d Q. Base r e f e r s to a n i n j e c t i o n of 0 . 2 ml of 1 M T r i s , at a p r e d e t e r m i n e d pH to r e s u l t in a final p H of 9 . 6 , into a r e a c t i o n m i x t u r e w h i c h c o n t a i n s in 2.0 m l : m a l e i c a c i d , p H 5 . ? , 3 m M ; M g C ^ , 10 m M ; K C 1 , 30 m M ; a n d c h l o r o p l a s t s c o n t a i n i n g 40 ng of chlorophyll. W h e r e i n d i c a t e d 1.5 n M D C M U a n d a few g r a i n s of d i t h i o n i t e w e r e a d d e d (25) .

F i g . 8. E f f e c t of f a r - r e d (FR) a n d g r e e n p r e i l l u m i n a t i o n the p r o t o n g r a d i e n t d r i v e n r e d u c t i o n of Q. R e a c t i o n c o n d i t i o n s as d e s c r i b e d u n d e r Fig. 7 (25). excites

o n l y the p h o t o r e a c t i o n

electron transfer base

(see Fig.

sensitizing

4), severely

the P 7 0 0 to X

inhibits

i n d u c e d r e d u c t i o n of Q, a n d a f o l l o w i n g

with green

light

thus reducing reaction

(Fig.

(which e x c i t e s

also

the e l e c t r o n c a r r i e r s ) 8) .

on

the

acid-

preillumination

the s e c o n d

photoreaction,

fully restores

the

159

M. Avron

Time, min

Fig. 9. A T P - i n d u c e d s t i m u l a t i o n of l u m i n e s c e n c e . The light off arrow i n d i c a t e s the end of the 3 m i n lightactivation period. The a p p l i c a t i o n of flash pairs is i n d i c a t e d by zig-zag arrows. The b r o k e n lines r e p r e s e n t the decay of f l a s h - i n d u c e d l u m i n e s c e n c e w h e n no A T P was added. During illum i n a t i o n or flashing the p h o t o m u l t i p l i e r was t u r n e d off; m o n i toring of p o s t - i l l u m i n a t i o n luminescence was i n i t i a t e d about T e m p e r a t u r e , 10°C-(Ref. 28). S s after p r e i 1 l u m i n a t i o n . Recently, gradient the

c o n d i t ions

d r i v e n reverse

el e c t r o n flow we re e x t e n d e d to include

p h o t o s y s t e m (27 , 28) .

i n d u c e d the rimental and

were found under w h ich A T P or prot on T h u s , A T P or a cid-base

transi t ion

emis sion of li ght from the p h o t o s y s t e m .

p r o c e d u re and

Th e e x p e -

i l l u s t r a t e d in Figures 9

results are

10.

The c h l o r o p l a s t

s u s p e n s i o n was first

h e a t - f i l t e r e d white ATPase.

illuminate d with

light for 3 m i n to

a c t i v a t e the

From about 5 s after the a c t i v a t i n g

off, the decay of p o s t - i l l u m i n a t i o n

ligh t was

l u m i n e s c e n c e was

for 90 s, by w h i c h time it a p p r o a c h e d zero. flashes were g i v e n and the f l a s h - i n d u c e d

strong,

latent turned

monitored

Two s a t u r a t i n g

luminesc ence

was

r e c o r d e d for 90 s.

Two more flashes w e r e then gi v e n and A T P

was i n j e c t e d before

the f l a s h - i n d u c e d

luminescence had

c o m p l e t e l y . A T P i n d u c e d a m a r k e d burst of d e c a y e d w i t h a h a l f - t i m e of about lated l u m i n e s c e n c e by about illumination

12 s.

10-fold, as

lumines cence ATP

addi t i o n

stimu-

c o m p a r e d w i t h the

l u m i n e s c e n c e d u r i n g the same time pe r i o d

line in figure).

decayed which

At 90 s after the s e c o n d

post-

(dashed

flash p a i r , a third

160

Energy Transduction in Photophosphorylation

Fig. 10. P r o t o n g r a d i e n t d r i v e n r e v e r s e - e l e c t r o n flow luminescence. R e a c t i o n m i x t u r e i n c l u d e d in 2 ml: s u c c i n a t e , 3 m M ; KC1, 30 m M ; M g C l 2 , 10 m M a n d c h l o r o p l a s t s c o n t a i n i n g 40 ug of c h l o r o p h y l l . Final pH 5.4. 0.15 ml 0.5 M Tris and 0.1 ml 0.1 m M D C M U in 10 % m e t h a n o l were i n j e c t e d w h e r e i n d i c a t e d . Prei1lumination was w i t h g r e e n light 8x10^ ergs x cm~2 x sec"^ (27). flash pair was given and the l u m i n e s c e n c e m o n i t o r e d . d a s h e d lines in Fig. 9 i l l u s t r a t e l u m i n e s c e n c e w h e n no A T P was

the d e c a y s of

The

flash-induced

added.

This sequence d i s p l a y s the A T P - i n d u c e d l u m i n e s c e n c e

in two

ways: (i) The b u r s t of l u m i n e s c e n c e u p o n i n j e c t i o n of A T P . (ii)

The m u c h e n h a n c e d l u m i n e s c e n c e

following a

flash-couple,

w h e n A T P is p r e s e n t and h y d r o l y s e d by the A T P a s e

(compare

d a s h e d and s o l i d curves f o l l o w i n g the last flash

couple).

These e x p e r i m e n t s the ATPase

demonstrate

that after p r o p e r

activation,

s y s t e m can a f f e c t the e l e c t r o n - t r a n s p o r t

all the w a y to the p h o t o s y s t e m

II r e a c t i o n

system

center.

A s i m i l a r p r o c e d u r e was f o l l o w e d to o b s e r v e an a c i d - b a s e d u c e d reverse e l e c t r o n flow l u m i n e s c e n c e . i l l u m i n a t i o n p e r i o d , the c h l o r o p l a s t s d u r i n g w h i c h their n a t i v e l u m i n e s c e n c e c o m p l e t e l y , the s h u t t e r

are p l a c e d in the decays

reaction.

is

immedia-

(Fig. 10).

w h i c h b l o c k s e l e c t r o n flow b e t w e e n the site of c o u p l i n g Q fully i n h i b i t s the

dark

essentially

in front of the p h o t o m u l t i p l i e r

light e m i s s i o n is a p p a r e n t

in-

Following a pre-

t h e n o p e n e d to p e r m i t o b s e r v a t i o n , Tris i n j e c t e d a n d tely a t r a n s i e n t

the

DCMU, and

M. Avron

4.

Initial K i n e t i c s of

If t r a n s m e m b r a n e

Photophosphorylation

p r o t o n c o n c e n t r a t i o n g r a d i e n t s are the

i n t e r m e d i a r y energy t r a n s d u c i n g

devices

c o u l d be e x p e c t e d that on a d a r k - l i g h t

in c h l o r o p l a s t s , transition

e v e r , several

it

photophos-

p h o r y l a t i o n s h o u l d always p r o c e e d w i t h a m e a s u r a b l e as d e m o n s t r a t e d

only

lag

period,

in the low light e x p e r i m e n t s of Fig. 2.

laboratories

How-

r e p o r t e d r e c e n t l y that such lags

l e n g t h of w h i c h can be r o u g h l y c a l c u l a t e d by c o n s i d e r i n g rate of p r o t o n p u m p i n g and the internal b u f f e r content) not o b s e r v e d

(the

the are

(29-31).

We d e c i d e d , t h e r e f o r e ,

to compare p h o t o p h o s p h o r y l a t i o n

with

a m o d e l r e a c t i o n w h e r e one can be fairly c e r t a i n that ATP f o r m a t i o n is d r i v e n e x c l u s i v e l y by a t r a n s m e m b r a n e p r o t o n centration gradient.

v i o u s l y d e s c r i b e d in detail as p o s t - i l l u m i n a t i o n lation

(32).

In this r e a c t i o n c h l o r o p l a s t s are

phosphorypreilluminated,

in the a b s e n c e of A D P and p h o s p h a t e , and the latter are after the light has b e e n t u r n e d off. dark was c o n v i n c i n g l y

shown to be due only to the

Figure

transmembrane preillumin-

11 shows a c o m p a r i s o n of the early

n e t i c s of p h o t o p h o s p h o r y l a t i o n

added

The A T P f o r m e d in the

p r o t o n c o n c e n t r a t i o n g r a d i e n t built up during the ation period.

con-

L u c k i l y , such a r e a c t i o n has b e e n p r e -

and post-illumination

ki-

phospho-

rylation.

In a g r e e m e n t w i t h the p r e v i o u s reports we find no

detectable

lag in the p h o t o p h o s p h o r y l a t i o n r e a c t i o n , but a

clear lag of about 500 m i l i s e c o n d s reaction

(33).

Thus, a driving

in the

post-illumination

force of A T P

formation

to e x i s t during the first s e c o n d of i l l u m i n a t i o n , w h i c h a transmembrane proton concentration gradient.

An

seems is not

alternative

p o s s i b i l i t y a p p e a r e d to be that the initial f o r m a t i o n of A T P is d r i v e n by a t r a n s m e m b r a n e

electric potential.

dence e x i s t s for the t r a n s i e n t electric potential

following

f o r m a t i o n of a

illumination, although

steady state p h o t o p h o s p h o r y l a t i o n is p r e s e n t

(16).

Ample

evi-

transmembrane during

little or no s u c h p o t e n t i a l

162

Energy Transduction in Photophosphorylation

Fig. 11. Time course of ATP f o r m a t i o n in p h o t o p h o s p h o r y l a t i o n and p o s t - i l l u m i n a t i o n p h o s p h o r y l a t i o n in the p r e s e n c e a n d absence of p o t a s s i u m and v a l i n o m y c i n . R e a c t i o n m i x t u r e s included in 1.0 m l : N a - H E P E S , 33 m M , pH 6.8; M g C l 2 , 5 mM; KC1, 100 m M ; EDTA, 0.1 mM; m e t h y l v i o l o g e n , 1 m M ; and c h l o r o p l a s t m e m b r a n e s c o n t a i n i n g 73 ug c h l o r o p h y l l per ml. W h e r e i n d i c a t e d 4 ul of 0.5 mM e t h a n o l i c s o l u t i o n of v a l i n o m y cin was added. A r e a c t i o n mix c o n t a i n i n g the same c o n s t i t u e n t s w i t h 2 m M ADP and 4 m M 32pi (6x106 cpm/ mole) but no c h l o r o p l a s t s or v a l i n o m y c i n was rapidly injected prior to t u r n i n g on the light ( p h o t o p h o s p h o r y l a t i o n ) or i m m e d i a t e l y following light e x p o s u r e ( p o s t - i 1 l u m i n a t i o n p h o s p h o r y l a t i o n ) for the i n d i c a t e d length of time (33). To test this p o s s i b i l i t y we u t i l i z e d the ion a g e n t s v a l i n o m y c i n and n i g e r i c i n .

transporting

In the p r e s e n c e of p o t a s s i u m

ions, v a l i n o m y c i n p r o m o t e s the t r a n s p o r t of that ion membranes, annihilating where

a transmembrane

electric

it e x i s t s w i t h o u t a f f e c t i n g the p r o t o n

gradient.

a f f e c t i n g the t r a n s m e m b r a n e As can be seen in Fig.

electric

a pH gradient,

without

potential.

11, a d d i t i o n of v a l i n o m y c i n a n d p o t a s -

sium i n d u c e d a lag in the p h o t o p h o s p h o r y l a t i o n

r e a c t i o n of a

length to that o b s e r v e d in its absence or p r e s e n c e

the p o s t - i l l u m i n a t i o n r e a c t i o n . riments

potential

concentration

N i g e r i c i n , on the other hand, p r o m o t e s a p r o t o n -

p o t a s s i u m e x c h a n g e , thus a n n i h i l a t i n g

similar

across

Figure

12 shows s i m i l a r

in

expe-

in the p r e s e n c e and absence of n i g e r i c i n and p o t a s s i u m .

Nigericin completely abolished post-illumination r y l a t i o n a n d steady state p h o t o p h o s p h o r y l a t i o n ,

photophosphoas

expected,

but d i d p e r m i t s i g n i f i c a n t ATP f o r m a t i o n to p r o c e e d d u r i n g first two seconds of p h o t o p h o s p h o r y l a t i o n .

Both

results

the

163

M. Avron

2

4

4

6

SECONDS OF ILLUMINATION

6

SECONDS OF PREILLUMINATION

Eig. 12. The e f f e c t of n i g e r i c i n on p h o t o p h o s p h o r y l a t i o n a n d post-illumination phosphorylation. R e a c t i o n c o n d i t i o n s as under Fig. 11, except that 20 ul of 10 uM n i g e r i c i n r e p l a c e d v a l i n o m y c i n (33). s u p p o r t the s u g g e s t i o n that a t r a n s m e m b r a n e is the m a j o r

initial d r i v i n g

two after a d a r k - l i g h t

c e n t r a t i o n g r a d i e n t as the m a j o r driving

5.

potential

force d u r i n g the first s e c o n d or

t r a n s i t i o n , changing

tion during steady state

electric

into a p r o t o n

force of A T P

con-

forma-

phosphorylation.

Conclus ion

I have tried to show d a t a from e x p e r i m e n t s w i t h

isolated

c h l o r o p l a s t s w h i c h d e m o n s t r a t e the intimate q u a l i t a t i v e quantitative

r e l a t i o n of p r o t o n g r a d i e n t s as an

e n e r g y pool b e t w e e n e l e c t r o n t r a n s p o r t and the A T P apparatus.

synthesizing

The system can be e x p e r i m e n t a l l y m a n i p u l a t e d

show light i n d u c e d e l e c t r o n t r a n s p o r t , gradient, proton gradient

light i n d u c e d

proton

formation, ATP

induced

reverse e l e c t r o n t r a n s p o r t , ATP i n d u c e d l u m i n e s c e n c e ,

proton

i n d u c e d reverse e l e c t r o n t r a n s p o r t and p r o t o n

gra-

dient induced reverse-electron-transport-luminescence. three p r o t o n s

About

seem to be n e c e s s a r y to drive the s y n t h e s i s

an A T P m o l e c u l e

in the steady state.

E v e n t h o u g h the

d i e n t as the m a j o r e n e r g y storage device transducing

electric potentials

intermediate

of

chloro-

p l a s t system uses the t r a n s m e m b r a n e p r o t o n c o n c e n t r a t i o n transmembrane

to

i n d u c e d ATP f o r m a t i o n , and in re-

v e r s e , A T P induced p r o t o n g r a d i e n t gradient

and

intermediary

in the steady

serve as an i m p o r t a n t

d u r i n g the first s e c o n d or two

gra-

state, energy fol-

Energy Transduction in Photophosphorylation

lowing a dark-light transition in strong actinic light.

All

this, and much more information which space and time do not permit to include here, provide us with much insight into the overall outline of coupled ATP synthesis in the chloroplast. However, the mechanism through which this feat is achieved still eludes us, and remains as a challenge for the future.

References 1.

Jagendorf, A.T.: Mechanism of Photophosphorylation. In: Bioenergetics of Photosynthesis. (Govindjee, Ed.), Academic Press, New York (1975), pp. 423-492.

2.

Nelson, N., Nelson, H., Nairn, V., Neumann, J.: Effect of pyridine on the light induced pH rise and post illumination ATP synthesis in chloroplasts. Arch. Biochem. Biophys. m , 263-267 (1 9 71 ).

3.

Avron, M.: The relation of light induced reactions in isolated chloroplasts to proton concentrations. In: 2nd International Congress on Photosynthesis. (G. Forti et al. , Eds.), N.V. Junk, The Hague, 1 972 , pp. 861 -871..

4.

Rottenberg, H., Grunwald, T.: Determination of a p H in chloronlasts. Eur. J. Biochem. 2J5, 71 -74 ( 1 972).

5.

Portis, A.R., McCarty, R.E.: Effect of adenosine nucleotides and of photophosphorylation on H + uptake and the magnitude of the H + gradient in illumination chloroplasts. J. Biol. Chem. 249, 62SO-62S4 (1974).

6.

Schuldiner, S., Rottenberg, H., Avron, M.: Determination of ApH in chloroplasts - fluorescent amines as a probe for the determination of ApH in chloroplasts. Eur. J. Biochem. 25, 64-70 (1972).

7.

Kraayenhof, R.: Quenching of uncoupler fluorescence in relation to the "energized state" in chloroplasts. FEBS Letters 6, 161-165 (1970).

8.

Fiolet, J.W.T., Bakker, E.P., Van Dam, K.: The fluorescence properties of acridines in the presence of chloroplasts or liposomes. Biochim.Biophys. Acta 568, 432445 (1974).

9.

Fiolet, J.W.T., Haar, L.V.D., Kraayenhof, R., Van Dam, K.: On the stimulation of the light induced proton uptake by uncoupling aminoacridine derivatives in spinach chloroplasts. Biochim. Biophys. Acta 387 , 320-334 (1 975).

165

M. Avron

10.

D e a m e r , D.W., P r i n c e , R.C., C r o f t s , A . R . : The response of f l u o r e s c e n t amines to pH g r a d i e n t s across liposome membranes. Biochim. B i o p h y s . A c t a 2 74, 323-335 (1972).

11.

C a s a d i o , R., B a c c a r i n i - M e l a n d r i , A., M e l a n d r i , Eur. J. B i o c h e m . 47, 121-128 (1974).

12.

A v r o n , M.: membranes. (Y. Hatefi Press, New

13.

G r a b e r , P., W i t t , H.T.: R e l a t i o n b e t w e e n the e l e c t r i c a l p o t e n t i a l , pH g r a d i e n t , p r o t o n flux and p h o t o p h o s p h o r y lation in the p h o t o s y n t h e t i c m e m b r a n e . Biochim. B i o p h y s . A c t a 4^3, 141-162 (1976).

14.

Chow, W.S., Hope, A.B.: Light induced pH g r a d i e n t s in isolated spinach chloroplasts. Aust. J. Plant Physiol. 141-152 (1976).

15.

C a s a d i o , R., M e l a n d r i , B.A.: The b e h a v i o r of 9 - a m i n o a c r i d i n e as an i n d i c a t o r of t r a n s m e m b r a n e pH d i f f e r e n c e in liposomes of natural bacterial p h o s p h o l i p i d s . J. Bioenerg. Biomembr. 9, 17-30 (1977).

16.

A v r o n , M.: Energy t r a n s d u c t i o n in c h l o r o p l a s t s . Biochem. 46, 143-155 (1977).

Ann.Rev.

17.

N i c h o l s , J . W . , D e a m e r , D.W.: P r o t o n and h y d r o x y l b i l i t y c o e f f i c i e n t for liposomes. In: A b s t r a c t s , tiers of B i o l o g i c a l E n e r g e t i c s , No. 143, J o h n s o n tion, U n i v e r s i t y of P e n n s y l v a n i a , 1978.

permeaFronFounda-

18.

Pick, U., R o t t e n b e r g , H., A v r o n , M.: Effect of p h o t o p h o s p h o r y l a t i o n on the size of the p r o t o n g r a d i e n t across chloroplast-membranes. FEBS Letters 3_2 , 91 -94 (1 973).

19.

Pick, U., R o t t e n b e r g , H., A v r o n , M.: Proton g r a d i e n t s , p r o t o n c o n c e n t r a t i o n s and p h o t o p h o s p h o r y l a t i o n . In: Proc. 3rd International C o n g r e s s on P h o t o s y n t h e s i s (M. A v r o n , E d . ) , E l s e v i e r , A m s t e r d a m , 1974, pp. 9 6 7 - 9 7 4 .

20.

R o s i n g , J., S l a t e r , E.C.: The value of A G 0 for h y d r o l y s i s of ATP. B i o c h i m . B i o p h y s . A c t a 2>67 , 275-290 (1 972).

21.

S c h r o d e r , H., M u h l e , H., R u m b e r g , B.: R e l a t i o n s h i p b e t w e e n ion t r a n s p o r t p h e n o m e n a and p h o s p h o r y l a t i o n on c h l o r o p l a s t s . In: Proc. 2nd Internat. C o n g r . P h o t o s y n t h e s i s (G. Forti et al. , Eds.) , W. J u n k , T h e H a g u e , 1971, pp. 919930.

22.

R i e n i t s , K.G., H a r d t , H., A v r o n , M.: E n e r g y d e p e n d e n t reverse e l e c t r o n flow in c h l o r o p l a s t s . Eur. J. Biochem. 291-298 (1974).

B.A.:

Energy t r a n s d u c t i o n in i s o l a t e d c h l o r o p l a s t In: The S t r u c t u r a l Basis of M e m b r a n e F u n c t i o n . and L. D j a v a n i - O h a n i a n c e , E d s . ) , A c a d e m i c York, 1976, pp. 227-238.

3,

43,

Energy Transduction in Photophosphorylation

166 23.

B a k k e r - G r u n w a l d , T . : A T P a s e . In: E n c y c l o p e d i a P l a n t Physiol. (Trebst, A. and A v r o n , M. , Eds.), S p r i n g e r V e r l a g , H e i d e l b e r g , 1977, V o l . 5, p p . 3 6 9 - 3 7 3 .

24.

A v r o n , M . , S c h r e i b e r , U . : P r o t o n g r a d i e n t s as p o s s i b l e intermediary energy transducers during ATP driven reverse e l e c t r o n f l o w in c h l o r o p l a s t s . F E B S L e t t e r s 7J7, 1-6 (1977) .

25.

Shahak, Y., H a r d t , H., A v r o n , M.: Acid-base driven rev e r s e e l e c t r o n flow in i s o l a t e d c h l o r o p l a s t s . FEBS L e t t e r s 54, 1 51-1 54 (1 9 7 5 ) .

26.

Shahak, Y., Pick, U., Avron, M.: Energy dependent rev e r s e e l e c t r o n f l o w in c h l o r o p l a s t s . In: P r o c . 1 0 t h FEBS Meeting, (Desnuel1e,P.and Michelson, A.M., Eds.), E l s e v i e r , A m s t e r d a m , 1976, V o l . 40, p p . 3 0 5 - 3 1 4 .

27.

Shahak, Y., Siderer, Y., Avron, M.: Reverse electron flow i n d u c e d l u m i n e s c e n c e t r i g g e r e d by a c i d b a s e t r a n s i t i o n of P l a n t a n d Cell P h y s i o l . , S p e c i a l Issue chloroplasts. No. 3 "Photosynthetic Organelles, Structure and Function" ( M i y a c h i , S . , K a t o h , S . , F u j i t a , Y. a n d S h i b a t a . K . , E d s . ) , J a p a n . Soc. P l a n t P h y s i o l . , T o k y o , 1977, p p . 115127.

28.

S c h r e i b e r , U., A v r o n , M.: ATP induced chlorophyll lumin e s c e n c e in i s o l a t e d s p i n a c h c h l o r o p l a s t s . FEBS Letters 82 , 1 59- 1 62 (1 977) .

29.

O r t , D . R . , D i l l e y , R . A . : P h o t o p h o s p h o r y l a t i o n as a f u n c t i o n of i l l u m i n a t i o n time. I. E f f e c t of p e r m e a n t c a t i o n s and permeant anions. Biochim. Biophys. Acta 449, 95107 ( 1 9 7 6 ) .

30.

Ort, D.R., Dilley, R.A., Good, N.E.: Photophosphorylation as a f u n c t i o n of i l l u m i n a t i o n t i m e . II. E f f e c t of p e r m e a n t buffers. Biochim. Biophys. Acta 449, 108-124 (1976).

31.

B e y e l e r , W. , B a c h o f e n , R. : I n i t i a l e v e n t s in l i g h t d e p e n d e n t A T P s y n t h e s i s in s p i n a c h c h l o r p l a s t p a r t i c l e s . Eur. J. B i o c h e m . , in p r e s s .

32.

G a l m i c h e , J . M : P o s t - I 1 l u m i n a t i o n A T P F o r m a t i o n . In: E n z y c l o p e d i a Plant Physiol. (Trebst, A. and A v r o n , M., E d s . ) , S p r i n g e r - V e r l a g , H e i d e l b e r g , 1 9 7 7 , V o l . 5, p p . 374-392.

33.

Vinkler, C., Avron, M., Boyer, P.D.: Initial formation of A T P in p h o t o p h o s p h o r y l a t i o n d o e s n o t r e q u i r e a p r o t o n gradient. F E B S L e t t e r s , in p r e s s .

Rece ived J u l y

14,

1978.

167

M. Avron

DISCUSSION Kuschmitz: I s i t necessary to activate the ATPase in order to measure reversed electron flow with ATP? Avron: Yes, of course. Since the native ATPase of chloroplasts i s latent, preactivation by illumination in the presence of d i t h i o t h r e i t o l and magnesium i s always done before ATP i s added to induce the reverse reactions. Kuschmitz: Were you able to confirm the H+/ATP = 3 stoichiometry in reversed electron flow? Avron: No, That i s not easy to do experimentally. We have attempted to estimate the ATP to electron r a t i o in the reverse reaction, but the numbers obtained thus far are rather high (above 10), indicating most probably poor coupling. Kuschmitz: What do the i r r e g u l a r i t i e s mean which were seen after ATP addition in the curves representing chlorophyll fluorescence and 9-aminoacridine fluorescence? Avron: The curves, as you noticed, are not monophasic. We have analyzed the chlorophyll fluorescence curve to c o n s i s t of at least three phases, and the acridine fluorescence quenching curve to c o n s i s t of at least two phases. However, the s i g n i f i c a n c e of these d i f f e r e n t k i n e t i c phases i s not clear at present. Marz: Can in your system a membrane potential replace the pH gradient for d r i v i n g ATP s y n t h e s i s ? Does t h i s t e l l us anything about the molecular mechanism of ATP s y n t h e s i s ? Avron: Yes, i t can p a r t i a l l y . That i s , you can replace part of the pH gradient by an a r t i f i c i a l l y imposed d i f f u s i o n potential and show that the c h l o r o p l a s t s use the potential to drive ATP s y n t h e s i s . At the present stage, a l l we can say i s that chloroplasts can use the total e l e c t r o chemical energy available through proton pumping to drive ATP s y n t h e s i s .

Section III.

Membranes and Transport by Proteins

PROTEINS IN MITOCHONDRIAL CALCIUM TRANSPORT

E. Carafoli, K. Schwerzmann, I. Roos and M. Crompton Laboratory of Biochemistry, Federal Institute of Technology (ETH), Universitatstrasse 16, CH - 8092 Zurich, Switzerland

Introduction The transfer of C a 2 + across the mitochondrial membrane appears to be catalyzed by two independent routes, one used for the influx of C a 2 + from the cytosol into the inner mitochondrial space, one used for the exit of C a 2 + from mitochondria

(1).

The influx route appears to be driven, at least in part, by the negative electrical potential generated by respiration at the matrix side of the membrane. The efflux route is probably different in different mitochondrial types. In heart, and a variety of other excitable tissues (2 - 3), it consists of a specific exchange between intramitochondrial C a 2 + and extramitochondrial Na + . In liver, and other non-excitable tissues, the N a + / C a 2 + exchange system is absent (3), but the efflux of Ca^

may also be mediated nevertheless by an independent path-

way (4), whose charge-compensating ion has not yet been identified. The possibility that the efflux pathway in non-excitable tissues is influenced by substances that act as natural "activators"

(5), as well as the existence of natural low-mo-

lecular weight components active as C a 2 + ionophores must also be considered.

Abbreviations used: EDTA = Ethylenediamine-tetraacetic acid, EGTA = ethyleneglycol-bis-( -amino-ethyl ether) N,N'-tetraacetic acid, PEP = Phosphoenolpyruvate, RR = Ruthenium red, SDS = Sodium dodecylsulfate, succ = Succinate.

© 1978 Walter de Gruyter & Co., Berlin • New York Transport by Proteins

172

The

E. C a r a f o l i e t

al.

Influx Pathway: Driving-Force, Inhibitors, Molecular

Components Most workers favour the idea that Ca^ + penetrates into mitochondria by an electrophoretic uniport process, occurring with a charge transfer of 2 (6). Recent proposals that the penetration would occur via a carrier that obligatorily cotransports Ca^ + and phosphate have not been supported by experimental evidence

(7). The Ca^ + -influx route is inhibited by extremely

low concentrations of ruthenium red (NH3)5 C 1 6 - 4 H 2 o J ( 8 , bition by La^

+

£(NH3)5RU-O-RU(NH3)4-0-Ru-

9) and lanthanides

(10).

Indeed, the inhi-

and ruthenium red has been widely taken as an

indication for the existence of a specific "carrier" responsible for the translocation of Ca^ + across the membrane. The problem, now, is that of understanding whether the influx route consists of one component only, or whether it is more complex, and composed of several. That the influx route may be very complex is suggested by experiments on the resolution of its molecular components, which have produced at least one purified component

( 1 1 ) , a glycoprotein, which binds C a 2 + with high af-

finity, but which apparently does not act as a trans-membrane carrier. Table I presents a list of the mitochondrial C a 2 + binding proteins that have been isolated so far. Those that have been analyzed for carbohydrates have yielded a positive response, a finding which agrees with the inhibition of the C a 2 + uptake reaction by ruthenium red, a reagent with a marked specificity for carbohydrate-containing components

(12), and

also with the inhibition by hexamine cobaltichloride, another reagent with carbohydrate specificity. One of the fractions listed in the Table, indeed, binds ruthenium red, although its affinity for the inhibitor has not yet been determined. Among the different protein fractions listed in the Table, one has been purified to homogeneity, and characterized fairly extensively by Sottocasa, Carafoli, and their coworkers

(11,

17).

173

Proteins in Calcium Transport

Table 1 Mitochondrial Ca2+-binding fractions Author

Nature of Fraction

Affinity for Ca 2 +

Evtodienko et al. (12)

Protein

high

Sottocasa et al. (11)

Glycoprotein

high

Gómez-Puyou et al. (13)

Glycoprotein

high

Kimura et al. (14)

Glycoprotein

low

Tashmukhamedov et al. (15)

Glycoprotein

high

from (18) It was isolated from liver and other mitochondrial sources, and found to be present in both mitochondrial membranes and in the Table 2 A summary of the properties of the mitochondrial calcium binding glycoprotein Extraction

Osmotic shocks, additional extraction by sonication and by chaotropic agents.

Monomer MW

33,000

Carbohydrates

About 10 % (xylose, mannose, glucose, galactose, N-acetyl-glucosamine, N-acetyl-galactosamine, sialic acid).

Phospholipids

up to 3 3 %

Amino acids

About 1/3 glutamic and aspartic acid.

Tightly bound Ca 2 + and Mg 2 +

3-5

Ca

2+

binding

Inhibition of Ca 2 + binding Intramitochondrial location from (18)

moles/mole

2 - 3 sites/mole high affinity (Kd 0.1 jiM) . 20 - 30 sites/mole low affinity (Kd 10 jiM) . ruthenium red. Inner and outer membrane. Intermembrane space.

E. Carafoli et al intermembrane space, but not in the matrix. As seen in Table 2, where its properties are summarized, the protein is very acidic and has a molecular weight between 30,000 and 33,000 depending on the species. It contains phospholipids and carbohydrates, including sialic acid, and rather large amounts of bound Ca^ + and

part of which can be displaced by incubation with

lanthanides. It binds Ca^ + at two classes of sites, one of which has small capacity but very high affinity, in a reaction which is inhibited by La^ + and

by ruthenium red. The protein

is evidently weakly associated with the inner and outer mitochondrial membrane, since it can normally be found in the intermembrane space, and it can be solubilized by gentle osmotic 2+

shocks. It may thus function as a Ca

2+

-binding, or Ca

-con-

centrating, component in that compartment. Some of the protein, however, is bound to the membrane more tightly, since it is not removed from it by osmotic shocks, nor by sonication, but requires the treatment with high concentration of chaotropic agents like lithium di-iodosalycilate. Other observations on this glycoprotein which may be directly relevant to its function are the following. An antibody against it, prepared by Panfili et al. the uptake of Ca^

+

(19), inhibits almost completely

by mitochondria at concentrations where a

variety of other energy dependent and independent mitochondrial reactions are not affected

(Figure 1). The inhibition is

visible in intact mitochondria, but is naturally much more evident in mitoplasts, which lack the outer membrane; this certainly indicates that the glycoprotein is involved in the

2+

transport of Ca tipino et al.

in intact mitochondria. Experiments by Pres-

(20) on planar lipid bilayers reconstituted with

the purified glycoprotein also indicate

its participation in

the transport of Ca^ + by mitochondria: they show an increase in the electrical conductance of the bilayer, specifically in the presence of Ca 2 + . The increase in conductance is abolished

Proteins in Calcium Transport

175

Figure 1. Inhibition of the transport of Ca 2 + in liver mitochondria and mitoplasts by an antibody against the mitochondrial Ca2+-binding glycoprotein. The preparation of the antibody, the preparation of mitochondria and mitoplasts, and the incubation conditions, are also described in (19). From Panfili et al. (19). by ruthenium red, which shows that the Ca 2 + binding ability of the glycoprotein must be intact for the effect on the electrical conductance to be seen. Both the antibody and planar bilayer experiments could be interpreted in 2 ways. Either the glycoprotein functions as a trans2+

membrane "carrier", which mediates the transport of Ca

across

the membrane, or it may act as a superficial receptor, which concentrates Ca 2 + on the surface of the membrane, and forms the "gate" to the hypothetical transmembrane Ca 2 + carrier. Given the marked anionic character of the glycoprotein, its interaction with Ca 2 + might permit its closer association with the bilayer, and thus, ultimately, bring Ca 2 + in closer contact with the hypothetical transmembrane Ca'i+ channel (Figure 2) . It has indeed been shown that EDTA selectively extracts the glycoprotein from mitochondria (21), and that it is possible to transfer

E. C a r a f o l i e t al

176 Plus Calcium

No Calcium Ca" carrier

?

©

Ca Ca

Ca*

Ca

• Ca

Ca* Inner M.

Outer

M.

(GP

Outer M.

Inner M.

Figure 2. A model for the function of the Ca protein. 40

2+

-binding glyco-

n

no addition W////A

« 30

c is Ï T> iÇ 2 0 V

2 mM Ca"

o

% 10 5* *

2+

o 2+

Figure 3. Ca -induced solubilization of the Ca -binding glycoprotein in n-decane. 2 mg of phosphatidyl-choline were dispersed in 4.0 ml distilled H2O with the aid of a vortex-mixer (10 min.). 0.5 ml of an aqueous solution of the Ca^+-binding glycoprotein (0.5 mg) were then added. The protein was extracted from osmotically-treated, sonicated mitochondrial membranes, with 0.6 M lithium di-iodosalicylate (15 minutes, with gentle stirring, at room temperature). The insoluble membranous material was removed by centrifugation, and the supernatant was dialyzed 24 hours at 0° C. The protein was then purified by polyacrylamide gel electrophoresis (11). The tube was then stirred on the vortex-mixer for further 10 minutes. 1.5 ml n-decane were then layered on top of the aqueous medium; 0.5 ml of distilled H2O plus or minus Ca^ + were added, and the mixing resumed for further 15 minutes. The separation of the phases was aided by centrifugation, and the protein remaining in the aqueous phase was then determined.

177

Proteins in Calcium Transport the hydrophilic glycoprotein added to the system

(22)

into apolar solvents if C a ^ +

(Figure 3). It must also be

that the glycoprotein, even if it acts as a superficial tor, can easily account for the observations that are taken to support the idea of a C a ^

+

carrier:

recep-

usually

inhibition

and binding of, ruthenium red, saturation kinetics,

by,

competitive

inhibition by S r ^ + . Against the idea of the glycoprotein as a transmembrane, mobile or immobile, C a ^ ponent, stands its very pronounced

+

is

emphasized

acting

transferring

hydrophilic character,

comwhich

makes its deep association with, and possible free mobility within, the apolar regions of the membrane bilayer rather probable. Moreover, no

+

transfer was ever demonstrated

imun-

der a variety of experimental conditions across the membrane of liposomes into which the glycoprotein

had been

incorporated.

It thus appears likely that the glycoprotein acts only

super-

ficially. The increased electrical conductance of the

bilayers

is most conveniently explained with the C a ^ + - p r o m o t e d

associ-

ation of the glycoprotein with the bilayer: once in the er, the glycoprotein would charged

increase the transfer of

(other)

species present in the medium. One difficulty

this explanation mitochondria

bilay-

with

is that one should then expect that also

the glycoprotein would

a variety of charged

increase permeability +

ions, in addition to C a ^ . This need

in to not

be so, however, owing to the differences between the natural mitochondrial membrane, and the artificial lipid

bilayer.

One important question at this point is whether one needs to postulate other protein components, in addition to the ficial glycoprotein, to explain the transfer of C a ^ the apolar region of the bilayer. hydrophilic

+

super-

across

In principle, a non-specific

pore, within which non-protein,

weight ionophoric components would act

small

molecular

(23), could be

suffici-

ent. However, recent experiments carried out in this Laboratory have indicated

that a very hydrophobic protein, able to

178

E. Carafoli et al.

bind

and ruthenium red, can be isolated from liver mito-

chondria. Although the research is still in its preliminary stages, and the participation of this protein in the uptake of Ca^ + is at the moment only a possibility, the properties of the fraction are rather interesting, and justify a detailed description. The protein is isolated from liver mitochondria labeled with small amounts of ^^Ru-ruthenium red, and extracted with 4 % Triton X-100. The insoluble precipitate, which represents 0.5 % of the original mitochondrial protein, is enriched 2 0 - 4 0

times in radioactive ruthenium red. The preci-

pitate is insoluble in a variety of organic solvents, and in detergents like cholate or deoxycholate, but is soluble in SDS. On analytical polyacrylamide gels, the SDS-dissolved fraction yields a main protein band, which represents about 80 - 85 % of the total protein of the input, and 3 - 4

more faintly vi-

sible bands (Figure 4). The main protein band has a M.W. of 560nm

Figure 4. SDS-Polyacrylamide gel electrophoresis of the Triton-insoluble protein fraction of the inner mitochondrial membrane. General conditions for the electrophoresis according to Lämmli (24). Polyacrylamide concentrations: stacking gel, 4 %, running gel, 10 %, SDS concentration 0.1 %. The gel was stained with coomassie Blue R-250, and scanned in a Gilford 2400S spectrophotometer equipped with a gel-scanning device.

1.0

0.5

0 I

179

Proteins in Calcium Transport

about 35.000 - 40.000, contains low amounts of phospholipids ( the d e p e n d e n c e

of

the i n i t i a l v e l o c i t y of f e r r i t i n f o r m a t i o n from

apoferritin

and Fe II on the i r o n c o n c e n t r a t i o n m e a s u r e d by

stopped-flow

techniques

c o u l d best be l i n e a r i s e d by a s s u m i n g that

e n t e r e d into the rrte e q u a t i o n to the s e c o n d p o w e r

iron

(17).

This

w o u l d c e r t a i n l y be c o n s i s t e n t w i t h a m o d e l for a c t i v a t i o n of m o l e c u l a r o x y g e n in w h i c h two a d j a c e n t i r o n atoms b i n d a d i o x y g e n m o l e c u l e a n d are o x i d i s e d to the Fe III state

with

c o n c o m i t a n t r e d u c t i o n of o x y g e n to p e r o x o . At t h i s stage in our s t u d i e s we w e r e s t r u c k by an

anomaly

r e l a t i n g to i r o n r e l e a s e f r o m f e r r i t i n . We h a d o b s e r v e d

(18,

19) that o by sucrose density gradient c e n t r i f u g a t i o n (26) and iron and protein

266

P.M. Harrison et al.

measured in the fractions.

Fig.¿lb shows that O^ alone gives a broad

distribution of iron contents with relatively few molecules containing more than J000 Fe atoms,

whereas with KLO /Na^

contained 3000 or more Fe atoms.

'

most molecules

These distributions agree with the

qualitative patterns noted previously (29) and the finding that the presence of chemical oxidant gives a larger average crystallite size (10). Thus KIO^/Na^S^O^ accelerates crystal growth relative to nucleation giving fewer nuclei, larger crystals and more completely filled molecules than alone.

(b)

Kinetics of aerobic iron uptake by apoferritin with and without KIO-j/Na^S^O-j.

In Fig. kc we compare progress curves of aerobic

uptake by apoferritin with and without KIO^/Na^S^O^,.

A single addition

of 1330 Fe atoms/ molecule was made to each apoferritin solution.

It

can be seen that the added oxidant inhibits the initial stage of the reaction.

At pH 6.7 iron uptake is faster in the presence of KIO^

after about half the iron has been incorporated, even though present in solution. then at pH 6.7. or when more only confirm

At pH 7.^ iron oxidation is initially faster

It halts when

originally containing

is still

has been depleted from the solution

alone, but continues in presence of KIO^/Na^S^O^

is mixed with the solution (arrows).

These results not

that KIO^/Na^S^O^ is an effective oxidant for crystal

growth, but indicate that under these conditions the overall stoichiometry of the reaction with molecule

is approximately b Fe (II) oxidized/

(1.09 mM Fe (il)oxidized by approx 2^0 -|iM 0^) as has been

found recently by Melino et_ al_ (30).

(c)

Kinetics of iron uptake by apoferritin with and without 0^ and by a ferritin fraction without 0^.

Progress curves shown in Fig.'+d compare

the uptakes of a small amount of added Fe (II) (90 Fe atoms/molecule) by apoferritin in air and by apoferritin and a ferritin fraction with 0^ excluded, but with KIO^/Na^S^O^

added.

With apoferritin it can be seen

that although Fe (II)- oxidation does proceed with KIO^/Na^S^O^ alone, it is faster in air.

However, with a fraction containing about 500 Fe

atoms, oxidation is just as rapid with KIO^/Na^ S^O^ and no 0^, as with apoferritin and

These results emphasise the difference between

267

F e r r i t i n as Sink and Source for Iron

10

10

20 30 AO Zn CpMD

AF_

20

30

40

Fe atoms/molecule^

(10-2)

ph74

ÎAF

\•

a ö

10

15

Time(min.)

—o—•— 0

AF

002 20

40 60 80 Tim* (min.)

Pig. 4« Iron uptake by apoferritin and ferritin fractions a. Inhibition of Pe uptake by ZnSO. at constant protein concn.(0.25 mg^ ml) in 20 mM HEPES buffer, pH 7.0. 40|iM Fe(NH4) (SO ) added. Oxidant 0g alone. Apoferritin, AP, fraction containing 500 Fe atoms, P. b. Distribution of Pe in ferritin reconstituted in 20 mM Imidazole, HC1 buffer, pH 6.7. Fe(WH ) 2 (S0 ) 2 was added in 5 increments, 1 mM each. Both solutions were open to air; one contained KI0,/ïTa-S_0, (10 mM/40 mM). Reconstitution products were dialysed, concentrated and fractionated by density gradient centrifugation. c. Progress curves of aerobic Pe uptake at two pH values. Uptakes were monitored at 420 nm. Protein concn. 0.5 mg/ml, Pe(MH ) (SO.)„ 1.5 mM, buffer 20 mM HEPES. Uptakes with,«, and without 07 KlOyHa S 2 0, (4 mM/l6 mM). When the reaction appeared to have stopped, the cuvette was inverted 3 times (arrows), d. Progress curves of Pe uptake by apoferritin (AP) and a ferritin fraction (P). Protein concn. 0.99 mg/ml. Pe(BH.) (SO )„ 0.2 mM. 24 mM HEPES, pH 7.4. Solutions*, •»•were 0„ free. Solutions 0, • contained KI0,/Ha2S 0, (2 mH/ 8 mM). P contained 500 Pe atoms/ molecule. the two phases of iron oxidation. The initial oxidation/nucleation phase involving binding by apoferritin is relatively specific for Og. The second, crystal growth phase, involving oxidation at surface sites

268

P.M. Harrison et al

on the micelles, occurs readily with KIO^/Na^S^O^.

(3)

Release of iron from ferritin fractions.

Iron release from micelle surfaces gives rise to a dependence on Fe content such as that of Fig. 3b with thioglycollate as reductant.

A

similar dependence, with a maximum for molecules \ - -j full is found for 1,10-phenanthroline

(31), c y s t e i n e ( 3 2 ) , dihydroflavin

Spencer & C. Walsh, personal communication) and citrate personal communication).

(T.Jones,R. (D.C.Harris,

The question arises as to whether these and

other molecules are able to penetrate the protein shell for direct attack on the micelle surfaces, or whether they release iron by an indirect mechanism involving sites on apoferritin.

To try to answer this we next

look at evidence on transport of molecules or ions into the protein molecule.

Permeability of the Protein Shell In Table I we list some of the molecules and ions known to enter ferritin or apoferritin.

Table 1 shows no marked selectivity for cations,

anions or neutral molecules under 7-108

diameter.

However, much of the

evidence for entry is based on equilibrium rather than kinetic measurements. Not included in Table I are those molecules involved in iron release discussed below.

Kinetic measurements of iron uptake show rates of incorporation into iron cores of at least k6 Fe/molecule/sec. rate-limiting and could be much faster.

Channel penetration may not be Diffusion through the shell by

glucose and amino acids was too fast to measure by low angle scattering

lk

(< 1 sec), sucrose taking about 10 sees (35).

On the other hand [

C]-

glucose and other molecules had apparent permeation times of several days measured as protein-associated radioactivity after incubation with labelled molecules and removal of free diffusant by gel chromatography (l8). In the laboratory, iron can be removed rapidly from ferritin by dithionite and thioglycollate at low p H values and by dihydroflavins

at

Ferrition as Sink and Source for Iron

269

C

•H > 01

•H

ro

A fi -pfi fi ECD •H -P CD •H o PH

•H PH m eu » A rH O to CO CO ft CD r—i •H a) •H — • Ö 1000

5 5 5

text

process

ly w i l l

0 D 0

1 5 B B

distribution

The

Antipyrine

dialysis*

Parenchymal tissues Skeletal muscle Fat tissues

and

drugs

vitro.

vivo* Parenchymal tissues Skeletal muscle Fat tissues

basic

in

Phenylbutazone

Imi p r a m i ne in

of m o d e l

distribution dialysis

by

which

degree which

333

Protein and Tissue Binding of Drugs

Table

3.

Characteristics

of

"first-pass

drugs".

Drugs o rd in a ry Hepatic

extraction

low,

First-pass

binding

effe-ct

drugs,

their first stributed

by

has

been

(see

Table

high

volume

fused 39%

by

effect to

drug

is

of

drugs and

drugs".

of

such

for

both

uptake,

free

or the

others,

psychotropic have

the

being

"first-pass years

isolated

rat

drugs

with

produces

and

binding

binding. drugs,

take is

in

a strong from

blood

protein-bound

basic

opiates,

characteristics

the sink

competition

Many

up

decreased

binders

transport plasma

a

per-

rate

is n o t

imipramine

effect,"

livers

Uptake

uptake

di-

(10,15)

concentrations

(13).

binding

during

before

lipophilic

or other

intracellular as

i.e., This

and

flow

disappear

recent

passage rate,

hepatic blood

a nonrestrictive

other words,

philic

basic E.g.,

first

in

in

therapeutical

albumin

results

in f a v o r

blockers,

for

intracellular

available

liver,

circulation.

metabolism

Thus,

highly

pass

at

ca

largely

characterized

in t h e

than

In

the

distribution.

drug

which

tissue.

orally,

is t y p i c a l

presence

perfusate.

sytemic

imipramine

the

nonrestrictive yes

metabolic pacity

through

>0.70

low

no

given

well

of

higher

the

the

3),

with

of

much

when

passage

which

high,

restrictive

Hepatic clearance d e p e n d e n t on

Certain

163-170 (1974).

12.

Chen, R.F.: Removal of f a t t y acids from serum albumin by charcoal treatment. J. B i o l . Chem. 242, 173-181 (1967).

13.

Teale, F.W.J.: Cleavage of the haem-protein l i n k by acid methylethylketone. Biochim. Biophys. Acta 35^, 543 (1959).

14.

Yonetani, T.: Studies on cytochrome c peroxidase. J. B i o l . Chem. 242, 5008-5013 (1967).

15.

Gibson, Q.H., Antonini, E.: Kinetic studies on the reaction between native globin and haem d e r i v a t i v e s . Biochem. J. 77_, 328-341 ( I 9 6 0 ) .

16.

Gibson, Q.H., Antonini, E.: Rates of reaction of native human globin with some hemes. J. B i o l . Chem. 238, 1384-1388 (1963).

17.

Blauer, G., Z v i l i c h o v s k y , B.: U l t r a c e n t r i f u g a t i o n studies on the aggregation of ferriprotoporphyrin IX by e l e c t r o l y t e s in aqueous a l k a l i n e medium. Arch. Biochem. Biophys. 127_, 749-755 (1968).

18.

Shack, J . , Clark, W.M.: Metalloporphyrins. VI. Cycles of changes in systems containing heme. J. B i o l . Chem. 171_, 143-187 (1947).

19.

Adams, P.A.: The k i n e t i c s and mechanism of the recombination reaction between apomyoglobin and haemin. Biochem. J. 159, 371-376 (1976).

20.

Chen, R.F.: Fluorescence stopped-flow study of relaxation processes in the binding of b i l i r u b i n to serum albumins. Arch. Biochem. Biophys. 160, 106-112 (1974).

21.

Faerch, T., Jacobsen, J . : Kinetics of the binding of b i l i r u b i n to human serum albumin studied by stopped-flow technique. Arch. Biochem. Biophys. J84, 282-289 (1977).

22.

Gibson, Q.H.: The combination of porphyrins with native human globin. J. B i o l . Chem. 239, 3282-3287 (1964).

Ferri heme Transfer from Albumin to Apomyoglobin

365

23.

Asakura, T., Yonetani, T.: Studies on cytochrome c peroxidase. XIII. Crystalline complexes of apoenzyme with porphyrins. J. Biol. Chem. 244, 537-544 (1969).

24.

Itagaki, E., Palmer, G., Hager, L.P.: Studies on cytochrome b ^ z Escherichia coli. J. Biol. Chem. 242, 2272-2277 (1967).

of

Received Apri 1 27, 1978.

Addendum A)

(G. Blauer and J. Silfen)

By evaluation of the stationary concentration of [FP]f, introduction

of this value into Eqn. 2 and rearrangement, one obtains: d[C] dt

k k

0Tt

[C] (1

kQn[P]f

[P] f 1 + k M [Apo]

)

[4]

([Apo] is the apomyoglobin concentration) The "retarding factor" on the right-hand side in the parentheses approaches unity in case of a very weak scavenger ( k M [ A p o ] « k fore the system approaches equilibrium

d rc i ("-jf

=

[P],) and there-

0)- Conversely, for k ^ A p o ]

>>k„ [ P U , the ratio k [P]-/k M [Apo] will determine the retarding effect on f on f M r on the rate of initial complex dissociation, which may approach zero for an efficient scavenger such as apomyoglobin. B)

By measuring

as a function of temperature in the range of 5° to

25°C, an activation energy of about 7.5 kcal/mole and an entropy of activation of about -50 e.u. were evaluated. This unusually large negative value for dissociation of the ferriheme-HSA complex indicates a considerably higher order of groups or molecules around the ferriheme in the transition state as compared with the ground state.

G. Blauer and J. S i l f e n

366 Discussion

Peters: Is there the p o s s i b i l i t y of d i r e c t transfer of heme between albumin and apoMb? This type of transfer occurs with enzyme complexes such as f a t t y acid synthetase described by E. Schweizer. Blauer: There i s no evidence from hydrodynamic measurements in the anal y t i c a l u l t r a c e n t r i f u g e that HSA and apomyoglobin form molecular complexes under the conditions of our work. A l s o , as reported, rates of transfer were v i r t u a l l y independent over a range of apomyoglobin concentrations. These observations make u n l i k e l y a d i r e c t t r a n s f e r of ferriheme between the two proteins under our conditions. Tipping: globin?

Do you know the k

on

value for the binding of haem by apomyo-

5 -1 -1 Blauer: A k -value of about 3 x 10 M sec has recently been reported for the interaction between monomeric ferriheme and apomyoglobin at pH 7.4 and 20°C [Adams, P.A.: Biochem.J. 159, 371-376 (1976)]. This value may be considerably higher for denatured apomyoglobin (see Discussion and compil a t i o n of other data in the reference cited above). Tipping:

What i s the o r i g i n of the lag time in F i g . 3 ?

Blauer: At l e a s t part of the time lag o r i g i n a t e s from the manual mixing of the reaction components at the s t a r t of a k i n e t i c experiment. Aisen: In view of the large negative entropy of a c t i v a t i o n in the " o f f " reaction, i t might be interesting to study the e f f e c t s of organic solvents on the reaction k i n e t i c s . Muller-Eberhard: Since i t i s impossible, at present, to accurately determine Kd which are below 10"® you could p o s s i b l y compare several apoproteins for their r e l a t i v e a f f i n i t y f o r heme under standard conditions. To obtain good comparative "on" and " o f f " rates may be of physiological or pathophys i o l o g i c a l importance. I r e a l i z e that you need, however, several independent c r i t e r i a to ensure the " n a t i v i t y " of those proteins from which you f i r s t must " s t r i p " the heme. Schade: Can these transfer reactions be followed in serum under physiol o g i c a l conditions of pH and CO2 concentrations? Blauer: In p r i n c i p l e , t h i s should be p o s s i b l e . However, an anticipated interference of various components of the serum with the transfer system may render a quantitative a n a l y s i s of the transfer reactions extremely difficult. Salomon: Related to the point, that the small molecule binds i n i t i a l l y not to the proper s i t e on the macromolecule and therefore the time r e quired for consolidation of the binding i s the time necessary f o r the move along the macromolecule, cannot such a move r e s u l t in d i s s o c i a t i o n and change the d i s s o c i a t i o n constant? I s a free energy change involved with

Ferriheme Transfer from Albumin to Apomyoglobin

367

this process? Since the later reaction is much faster than the initial rate-limiting step, does it have any effect at all on the overall kinetics? Blauer: The affinity of the apomyoglobin for ferriheme should be very high, even in a possible intermediate complex, so that the dissociation rate of the latter may be negligibly small, as pointed out before for myoglobin. Under these conditions, the overall kinetics will be determined mainly by the dissociation rate of the initial ferriheme-HSA complex.

THE ROLE OF INTRACELLULAR PROTEINS IN THE TRANSPORT AND METABOLISM OF LIPOPHILIC COMPOUNDS

E. Tipping and B. Ketterer Courtauld Institute of Biochemistry, Middlesex Hospital Medical School, London W1P 7PN, U.K.

The possibility that the intracellular binding proteins ligandin and *

aminoazodye-binding protein A

operate by allowing the facilitated

diffu-

sion of their ligands, in the same way as myoglobin does for oxygen

(1,2),

has been pointed out by Meuwissen et at.

(4,5).

(3) and by Ketterer et aZ.

Since nearly all the compounds which bind to ligandin and protein A do so because they have hydrophobic moieties components of membranes.

(6), they also bind to the lipid

Their intracellular transport may therefore in-

volve lateral diffusion in membranes as well as unbound aqueous diffusion and protein—bound aqueous diffusion, and the three processes must be considered together in assessing the role of the proteins in transport.

In

this paper we offer mathematical descriptions of diffusion in simple situations which resemble to some extent those in the cell-type in which ligandin and protein A are most abundant, the hepatocyte.

From these

descriptions we are able to get an indication of the contribution of protein-binding to intracellular transport in the liver, and its dependence on

the extents of binding to the proteins and membranes.

effect of diffusion-rate on the efficiencies of metabolism of

The lipophilic

compounds is also considered.

* Aminoazodye-binding protein A referred to hereinafter as protein A, is probably identical to Z-protein described by Arias and colleagues (cf. ref. 5).

© 1978 Walter de Gruyter & Co., Berlin • N e w York Transport by Proteins

370

Intracellular Proteins in Transport and Metabolism

Information

Proteins

The properties of ligandin and protein A are reviewed and dis-

cussed elsewhere in this volume (6).

Here we only wish to consider three

basic properties of the proteins as follows (a) their shared ability to bind reversibly small molecules with hydrophobic moieties, (b) their high concentrations in the hepatocyte,

(c) their rates of diffusion.

The data for the binding of small molecules by ligandin and protein A which are available at present

(cf. ref. 6) all refer to systems at equilibrium,

and so strictly speaking are not applicable to the dynamic situations w e are concerned with here.

However in the absence of rate constants for

association and dissociation, and also to make for simpler mathematics, we make the approximation that there is chemical equilibrium at every point in the systems we study, as did Wyman in his treatment of myoglobinmediated oxygen transport

(2).

To see how valid this approximation is let

us consider the interaction of an uncharged ligand and a small globular protein: the maximum association rate constant, k , for such an inter3 9 - 1 - 1 A 7 action is about 10 M sec (7). For equilibrium constants, K, of 10 - 1 0 M

1

(roughly the range of values found for purified ligandin and protein A

- cf. ref. 6), the maximum dissociation rate constants, k,(=k a /K), are 10"'2 - 1 2 -1 . . . 10 sec

.

For k^=10 sec

the time for 99% dissociation of the protein-

ligand complex to occur is about 45msec, in which time a molecule of diffusion coefficient 10 ^ cm^sec ^ (i.e. a small globular protein) would dif-4 fuse in one dimension a root-mean-square distance of about 3x10

cm.

This

is about one-third the radius of a hepatocyte, i.e. it is of similar m a g nitude to the distances in which we It therefore appears - 1 the . are . interested. . . . . that for k^ = 10 2 sec approximation of chemical equilibrium at all points begins to break down.

The expressions we derive are thus appli6 —1 • 3 —1 cable only to values of K of 10 M or less, i.e. k^(max) ^ 1 0 sec , even if association and dissociation rates are maximal.

The concentrations of ligandin and protein A in rat liver cytosol are both about 10 ^M (cf. ref. 6) and are taken as exactly this value here.

The

diffusion coefficient (D° ) of ligandin is 7.6x10 ^cm^sec ^ (8), w h i c h -7 2 -1 w e round off to 8x10 cm sec , and that of protein A is taken to be

E. Tipping and B. Ketterer -7 11x10

37 1

2 - 1

cm sec

by comparison with values for globular proteins of similar

molecular weight (source: ref. 9).

Since diffusion coefficients are in-

versely proportional to viscosity, the high protein concentration of the cytosol of the hepatocyte (~10% w/v) must mean that the cellular diffusion coefficient is lower than that in pure water.

However the diffusion coef-

ficients of globular proteins do not fall very much up to concentrations of 10% (10) and so any decreases are probably counteracted by the lower viscosity at 37°C compared to that at 20°C. Ligandin and protein A bind many ligands with similar affinities (6), and this allows us to consider protein-binding in terms of one association constant rather than two.

Since the molar concentrations of the proteins are

equal (see above) we have d l [l]k l + d a [a]k a = (PL + da)[p]k = dp[p]k

(i)

2 where D^ and D^ are the diffusion coefficients, [jJ and

the concentra-

tions, and K^ and K^ the association constants of ligandin and protein A respectively, and QlJ +

= [Y] = 2xl0~^M;

K.

The relationship

of v, the moles of ligand bound per mole of protein, to the unbound concentration , c, is given by the usual equation for a single binding site: V =

Intracellular Membranes

Kc irtte

....

(ll)

In the hepatocyte the intracellular membrane li-

pid is 90% phospholipid (11), of which there are 30mg per g liver wet weight (12).

Taking the density of a hepatocyte to be 1.067gcm . . . -3

the density of phospholipid bilayers to be l.Olgcm

(13), and

(14), we obtain a

value of 0.032 for the volume fraction of the cell which is phospholipid bilayer.

Since we are interested in the cytoplasm only, we should sub-

tract the minor contributions to this volume fraction from phospholipid in the plasma membrane and the nucleus, and we take a value of 0.03 for the volume fraction which is cytoplasmic phospholipid.

The aqueous phase of

the cytoplasm is 0.44 of the total cell volume (13), so the volume fraction of phospholipid bilayer in the system bilayer + aqueous phase is given by 0.03/(0.03+0.44)=0.064, and that of aqueous phase by 0.44/ (0.03+0.44)=0.936.

372

Intracellular Proteins in T r a n s p o r t and M e t a b o l i s m

Table. 1

Binding to ligandin, p r o t e i n A and lecithin

bilayers

The d a t a are taken from refs 5,15,16,17 and 18. For haem and b i l i r u b i n the results refer to 25°C, for the other compounds 4°C. T h e p a r t i t i o n c o e f f i c i e n t , k = | c o n c n . in m e m b r a n e / c o n c n . in water^.

Association M

Ligand

ligandin

-1

x

10

_A

N,N-dimethyl-4-aminoazobenzene oestrone testosterone bilirubin

Partition Coefficient

protein A

(k)

1

12 000

8

12

28 000

9

24

4 500

20

10

700

700

2 000

16 000

10

N-acetyl-2-aminofluorene

(K)

constant

bromosulphophthalein

1 100

70

147 000

haem

1 000

400

600 000

66

30

1 600

oestrone

sulphate

D i f f u s i o n c o e f f i c i e n t s of a v a r i e t y of small m o l e c u l e s in p h o s p h o l i p i d — — — 8 7 2 1 b i l a y e r s are in the range 10

-10

cm sec

(reviewed in r e f s . 19 and 20),

the v a r i a t i o n not being o b v i o u s l y related to the character of the d i f f u sant.

In the absence of d a t a for the p a r t i c u l a r small m o l e c u l e s w h i c h

b i n d to l i g a n d i n— and p r o t e i n A, w e have adopted a v a l u e of —8 2 1 D = 3x10 c m sec for all of them. M To calculate the d i s t r i b u t i o n s of d i f f u s a n t s b e t w e e n cytosol and the p h o s p h o l i p i d b i l a y e r s of intracellular m e m b r a n e s w e require d a t a for b i n d i n g of the d i f f u s a n t s to b i l a y e r s .

We have u s e d egg l e c i t h i n

somes to o b t a i n such d a t a for a n u m b e r of ligands of l i g a n d i h and A , and these are shown in Table binding.

the

lipoprotein

1 together w i t h p a r a m e t e r s for p r o t e i n -

For the u n c h a r g e d compounds the p a r t i t i o n c o e f f i c i e n t s

(k) are

c o n s t a n t up to a m o l a r ratio of l i g a n d / l e c i t h i n in the b i l a y e r of 0 . 1 . O n the other hand the b i n d i n g of o r g a n i c anions shows

concentration-

d e p e n d e n c e : for h a e m , oestrone sulphate and b r o m o s u l p h o p h t h a l e i n k falls to zero at a m o l a r ratio

(bound ligand/lecithin) of 0-1 - 0 - 3 ,

in practice m u c h smaller m o l a r r a t i o s are expected under these c o n d i t i o n s k can be taken as constant.

However

(0.01 or less) and T h e c o n c e n t r a t i o n of

E'. Tipping and B. Ketterer

373

diffusant in the membrane, c^, is related to the free aqueous concentration, c, by the equation c w = kc M Small Molecules

(iii)

The ligands shown in Table 1 have molecular weights

between 200 and 800 and so, by Stoke's Law, their diffusion coefficients — ~6 6 2 *"1 in water, D, vary from 5x10 to 3x10 cm sec , We have taken the ave— —6 2 1 rage value of 4x10

cm sec

for the purposes of calculation, but since

very little of any of the compounds is expected to be unbound in the aqueous phase, this choice is not at all critical.

Mathematical Formulations

In one-dimensional diffusion there are two basic arrangements of membranes and cytosol.

In the parallel mode the aqueous and membrane phases offer

alternative routes of transport, whereas in the series mode they cooperate in the same route.

These are illustrated schematically in Fig. 1, to-

gether with a mixed arrangement.

To evaluate the roles of soluble pro-

teins in transport in these media, we need expressions for the effective diffusion coefficients,

°f the "slabs of cytoplasm" of Fig. 1.

To

do this we arrange for a constant concentration difference across the slabs and derive expressions for the steady state fluxes in the x-direction.

The treatments which follow are based on that given by Wyman (2)

for myoglobin-mediated oxygen transport in aqueous solution.

PARALLEL

SEPT'-V x=0

x=h

x=0

x=p

x=h

MIXED x=ü

x=p

x=h

Fig. 1 Modes of diffusion. The stippled areas represent membranes, the open areas aqueous phase.

37it

Intracellular Proteins in Transport and Metabolism

The Parallel

Mode

For the arrangement shown in Fig. 1, with aqueous

phase and membrane occupying fractions A^ and A^ respectively of the total cross sectional area, (A^ + J

1), we have

= V

where J is the total flux, J

V

+

V

(IV)

V M

the unbound aqueous flux, J_ the bound -2

aqueous flux and J^ the membrane-bound flux, all in moles cm

-1

sec

.

The

steady state fluxes are given by Fick's first law: J u = -D(dc/dx);

J B = - D [p](dv/dx);

J M = -D^(dc^/dx)

(v)

Substitution of these expressions into eqn. (iv) and integration gives -Jx

= AA(DC

+ Dp[p]v)

+ AJJDJJCJ,

+

B

(vi)

The constant of integration, B, is given in terms of the expression for x=0: B = -Aa(Dco

+

I^[p]v o ) - A M D M c M ) 0

(vii)

In the limiting case in which chemical equilibrium exists at all points in the system (see above) we can substitute for v and c^ from eqns. (ii) and (iii) and for x=h we then have

hJ - A a j D ( c o - c h )

+

Dp[p]K

-

+

The effective diffusion coefficient, D

D

The Series

Mode

eff

=

VMk(Co"

C

h)

(viii)

is given by

hJ c - c, o h

,. .

(IX)

In the steady state the fluxes in the membrane and

aqueous phase of the series arrangement must be equal.

With c^ as the

unbound aqueous concentration at x=p we have J = ° M k ( c - c ) = D (c - c j 0 p h — h=5 p

+

D

p H K " T ^ T

j Cp _ Ch I ¡ÜKT ÜK^j

(x)

375

E. Tipping and B. Ketterer T o c a l c u l a t e J w e n e e d to e x p r e s s c^ in terms of the o t h e r A f t e r r e a r r a n g e m e n t of eqn. ac Where

a =

DkK M

2 P

(x) we

+ 6c

D„kc M o p

Mode

(xi)

_D_(1-Kch) h-p

+

Dc, h h-p

-

+

h-p

I i

l+KcL

h

D

TpIKc pL J h (h-p)(l+Kch)

c o n s i s t of k n o w n p a r a m e t e r s , c^ c a n be c o m p u t e d f r o m

(xi) a n d t h e n J f r o m e q n .

Mixed

0

h-p

6 - V(l-Kco) p

The

+ Y =

+ DK

P

Since a, 6 and y

P

parameters.

obtain

(x).

D ^

is a g a i n o b t a i n e d f r o m eqn.

For the a r r a n g e m e n t

s h o w n in F i g . 1 w e c a n d e r i v e

e x p r e s s i o n for the flux t h r o u g h the slab b y a p r o c e d u r e

s i m i l a r to

for the s e r i e s m o d e , e x c e p t that the f i r s t p a r t of the slab t r e a t e d as for the p a r a l l e l m o d e . eqn.

an that

(up to x = p )

We o b t a i n a n e q u a t i o n a n a l o g o u s

is

to

(xi): a ' C 2 + g'C + y' P P

where

eqn.

(ix).

a

,

=

A^DK

A ^ k K

+

P B' =

Y

i

c - ~~ _ o

A

A

D

K Pp LWJ

((l1 --

D_[p]K /

o

1+Kc q /

H

d -( lK- Kcc oJ) ^WT M

++

_ Kc.

1

h-P

V

l

j

w

\

(xii)

h-p

M A ^ (£ ( l 1--KK Cc„J) +

+

0

^

+

P

_D_ (1-Kc^) h-p

=

m

+

kcJ

-

W h-p

d

+

d

_ p W M 1+Kc /

+

376

Intracellular Proteins in T r a n s p o r t and M e t a b o l i s m

C a l c u l a t i o n of Effective D i f f u s i o n

Equations

(viii),

(xi) and

on a n u m b e r of p a r a m e t e r s .

Coefficients

(xii) allow us to assess the d e p e n d e n c e of D In p a r t i c u l a r w e w i s h to k n o w how D

^

varies

ef f

w i t h K , the p r o t e i n - d i f f u s a n t a s s o c i a t i o n c o n s t a n t , k, the m e m b r a n e p a r t i t i o n c o e f f i c i e n t , and the c o n c e n t r a t i o n s at either end of the slab, c^ and c^.

All

the other p a r a m e t e r s in the equations have had v a l u e s

assigned to them

(see Information) except for A ^ , A ^ and p.

In the p a r a l -

lel m o d e the f r a c t i o n s of the total v o l u m e of the slab due to aqueous phase and m e m b r a n e should be the same as those in vivo A a = 0.936 and A ^ = 0.064

(see I n f o r m a t i o n ) .

and so w e m u s t put

In the series m o d e w e have not

a t t e m p t e d to relate the a q u e o u s and m e m b r a n e v o l u m e s to those in b e c a u s e the a r r a n g e m e n t is complete ly a r t i f i c i a l , and w e h a v e , set p=h/2.

vivo

arbitrarily,

T h i s v a l u e has also b e e n c h o s e n for the m i x e d a r r a n g e m e n t ,

in

w h i c h v a l u e s of A ^ and A ^ of 0.88 and 0.12 respectively make the v o l u m e fractions-of

the slab as a w h o l e the same as the in vivo

values.

We h a v e c a l c u l a t e d v a l u e s of D

in the three d i f f u s i o n m o d e s d e p i c t e d in £f-i u F i g . 1 for v a l u e s of K up to 10 M , and of k up to 10 , a p p r o x i m a t e l y the e

ranges found e x p e r i m e n t a l l y

(Table 1).

A s d i s c u s s e d in the

s e c t i o n , our f o r m u l a t i o n s are not a p p l i c a b l e for K > 1 0 ^ M of the c a l c u l a t i o n s

Information The

results

(Table 2) reveal a number of i n t e r e s t i n g p o i n t s .

all three m o d e s it is clear that the h i g h e r is the a s s o c i a t i o n the greater is the effect of the p r o t e i n s , except w h e r e the at b o t h ends of the slab are very high

(column five).

In

constant

concentrations

The proteins have

m o s t influence at low s a t u r a t i o n , so that a l t h o u g h columns

three

= 10_6M, = 0) and five (c = 1 0 ~ 5 M , c, = 9 x l O ~ 6 M ) refer to the o h o h same c o n c e n t r a t i o n d i f f e r e n c e , the v a l u e s of D g £ £ are always greater in (c

c o l u m n three. v a r i a t i o n of D

C o l u m n s three and four illustrate the same p o i n t . err

diffusion mode.

The

w i t h the relative v a l u e s of K and k d e p e n d s on the In the p a r a l l e l case the p r o t e i n s h a v e their g r e a t e s t

effect at h i g h K/k r a t i o s .

In the series case the r e v e r s e is true since

for h i g h v a l u e s of k the aqueous p h a s e is a d i f f u s i o n a l b l o c k , the

effect

of w h i c h can b e r e l i e v e d by p r o t e i n - m e d i a t e d f a c i l i t a t e d d i f f u s i o n , w h e r e as at low v a l u e s of k d i f f u s i o n in the m e m b r a n e b e c o m e s r a t e - l i m i t i n g no amount of f a c i l i t a t i o n in the a q u e o u s phase can enhance the

overall

and

377

E. Tipping and B. Ketterer

Table 2

2 Effective diffusion coefficients (cm sec

A

6 X 10") for the

parallel, series and mixed modes» CONCENTRATIONS

c =10 _ 6 M

AT ENDS OF SLAB

co

PAR TITION COEFF'T

SERIES

PARALLEL

ASSOCIATION^^ CONSTANT (M KO

P Ca

rate.

0

h=° 10 2

c =10~ 5 M o c h =0

c =10~ 5 M 0 c, =9xl0" 6 M h

10 4

10 4

10 4

3.7

3.9

22.9

22.9

22.9

5.5

5.7

24.7

24.6

24.4

5

19.9

20.1

29.1

31.8

27.6

K=10 6

92.7

92.9

111.9

39.1

24.6

K=10 4 K=10

K=0

0

3.4

7.4

7.9

7.8

K=10 4

0

4.0

11.8

11.3

10.2

K=10 5

0

5.3

40.0

26.2

17.4

K=10 6

0

5.9

165.4

41.8

11.4

K=0

3.7

3.9

7.3

7.3

7.3

K=10

4

5.5

5.7

10.3

10.1

9.8

K=10 5

20.0

20.1

31.0

19.4

15.0

K=10 6

95.7

99.1

115.0

36.7

10.1

Thus it turns out that in the mixed case the extent of influence

of the proteins depends little on k, because at low values transport does not depend on the membrane and at high values the proteins relieve the diffusional block of the aqueous phase, as in the series mode.

Since the

mixed mode bears most resemblance to the situation in vivo we conclude tentatively that in the hepatocyte ligandin and protein A might enhance the rate of transport of their ligands 4 to 5-fold for an association constant of 10 5 M _ 1 and 15 to 25-fold for K = 10 6 M

1

.

Transport and Metabolism

We now ask whether this increased rate of transport could have any signi-

Intracellular Proteins in Transport and Metabolism

378

ficant effect on the efficiency of the hepatocyte in the overall process of metabolism and excretion of the diffusant.

Consider the situation in

Fig. 2 which is a schematic representation of a liver cell of width 2q with two sinusoids at opposite ends.

Let us suppose that the diffusant

passes across the sinusoidal membrane easily so that its uptake rate is governed by diffusion and metabolism in the cytoplasm.

Also let us sup-

pose that the diffusant is not excretable into the bile without first undergoing metabolism.

If the rate of the metabolism is given by Rc,

where R is a constant and c is the unbound aqueous concentration as before, then the conservation equation is

dc _ d2 c = D r r -3—7 dt eff dx2

-5—

In the steady state (dc/dt)

-

_ Rc

..... (xm)

= 0 and so

d2c d^2 =

R eff

•

, . ,

c

Referring to Fig. 2 we see that the boundary conditions which the solution of eqn. (xiv) needs to meet are (a) c=Cq at x=q and (b) (dc/

tot Gel

filtration

Gel filtration on Sephadex G-100 was performed using two columns (100 cm x 2.5 cm) simultaneously. 10 ml fractions were collected and elution profiles were monitored at 280 and 450 nm. Protein and bilirubin were determined chemically on each fraction.

Sedimentation studies

Mixtures of liver cytosol and plasma were centrifuged using a movingboundary technique (Beckman L2-65B preparative ultracentrifuge with SW 40

Binding Proteins and Transport of B i l i r u b i n swinging-bucket r o t o r ) . Centrifuge tubes were f i l l e d at constant composition throughout but provided with a D£0 density gradient (5-45%) in order to maintain convectional s t a b i l i t y . After centrifugation for 40 hrs at 39,500 rev/min the contents of the tubes were displaced through the top and analysed continuously as absorbance (470 nm) versus displaced volume. The recordings were sampled and fed to a minicomputer. Data reduction consisted of smoothing the data points followed by numerical

differentia-

t i o n and integration. Output from the computer was printed and o p t i o n a l l y plotted. Relative k i n e t i c measurements The rate of exchange of b i l i r u b i n between binding protein and b i l i r u b i n Sephadex G-10 complex was studied adopting e s s e n t i a l l y the same experimental set-up as used for the competitive binding studies. The time course of the exchange process was followed. The experimental data were f i t t e d by a non-linear least-squares regression technique (18) with a sum of exponent i a l s as the f i t t i n g function. B i l i r u b i n d i s t r i b u t i o n between an aqueous phase and microsomal membranes Smooth and rough microsomes were prepared by isopycnic centrifugation (19). Smooth membrane fragments were collected at density 1.180 and rough f r a g ments at density 1.223. Lipids were extracted for phospholipid phosphorus determination. Glucose-6-phosphatase and 5 1 -nucleotidase were used as marker enzymes. Microsomal suspensions (13.5 mg protein) were f o r t i f i e d with b i l i r u b i n a t e and gently mixed for 30 min. To vary t h e i r densities separate suspensions were diluted 1:1 with buffers of d i f f e r e n t D 2 0 content. The suspensions were layered on top of each other in order of decreasing density and centrifuged for 1 hr at 40,000 rev/min in the SW 40 rotor. B i l i r u b i n was determined in both the supernatant and the resuspended sediment. B i l i r u b i n associated with the microsomal p e l l e t was assumed to be in equilibrium with i t s free concentration in the supernatant. The p a r t i t i o n c o e f f i c i e n t (K p ) was defined as the molar concentration r a t i o of b i l i r u b i n i n the membranous and aqueous phases r e s p e c t i v e l y . The total volume of the membrane phase, and the phospholipid and protein volume

J.A.T.P. Meuwissen and K.P.M. Heirwegh

392

f r a c t i o n s were calculated by correlating the isopycnic density of the microsomal f r a c t i o n with i t s p r o t e i n / l i p i d composition and the s p e c i f i c densities of phospholipid and protein respectively. Miscellaneous 2 All experiments were carried out at 4°C. Male Wistar and Gunn rats

were

used as the experimental animals. Low concentrations of b i l i r u b i n in aqueous systems were measured spectrophotometrically

(A^q" 1

: 58,700)

after extraction into a small volume of CHC1^• Otherwise the ¿ - i o d o a n i l i n e method was used (20). Albumin and l i g a n d i n concentrations were measured with s i n g l e radial immunodiffusion. Reduced glutathione (GSH) and d i t h i o threitol

(DTT) were measured according to Ellman (21). Data reduction and

a n a l y s i s were performed with a Hewlett-Packard HP 2100A minicomputer with graphical c a p a b i l i t i e s in a disc-operating environment.

Results and Discussion Binding studies at equilibrium and the effect of mercaptans B i l i r u b i n binding of rat l i v e r cytosol diminishes when the cytosol i s aged or fractionated. A small molecule in whole cytosol may be required to maintain the binding a c t i v i t y (22,23). Ligandin possesses glutathionetransferase a c t i v i t y (7) and binds GSH weakly (22). Therefore the effect of mercaptans on the binding a c t i v i t y for b i l i r u b i n has been investigated in a l l three methods used here. 1) Binding method (Sephadex G-10). In the presence of mercaptans the binding a c t i v i t y of cytosol i s much higher than i n t h e i r absence ( F i g . 1). Either GSH (2 mM) or DTT (1 mM) had nearly identical e f f e c t s . GSH i s 2

The Gunn rat i s a recessive mutant of the Wistar s t r a i n with a d e f i c i e n cy of b i l i r u b i n UDP-glucuronyltransferase a c t i v i t y . The animal converts b i l i r u b i n to poorly characterized products. This process i s much l e s s e f f e c t i v e than i s glucuronidation. Therefore t h i s animal has a l i f e - l o n g b i l i r u b i n level about 100 times higher than that of normal r a t s .

Binding Proteins and Transport of Bilirubin

393

therefore routinely used in binding studies of cytosol with the Sephadex method. The mercaptans had no influence on the binding of plasma. Representative binding parameters of plasma and cytosol are given in Table I. With this method the binding activity of cytosol is higher than that of plasma although the values are of the same order of magnitude. In otherwise identical

conditions,

parallel assays were run on mixtures of cytosol and plasma and on each

C b : MxlO 6

preparation separately. Within Fig. 1. Bilirubin binding by vat liver cytosol in the absence (• J and in the pvesence (O) of GSH. Scatchavd type of plot.

experimental error the binding was additive. This indicates that both preparations do not interfere with each others binding activity when mixed.

2) Gel filtration (Sephadex G-100). Jaundiced cytosol from Gunn rats was fractionated in parallel on two columns. Apart from the presence of GSH (2mM) in the elution buffer and in the applied sample of one of the columns, separation conditions were otherwise identical. In the absence of GSH bilirubin was recovered in at least three fractions (Fig. 2a).

Table I: Bilirubin-binding parameters of vat plasma and liver cytosol. Capacity values were recalculated to in_ vivo concentrations. K

Bc

app M" 1 x 10~ 8

tot

M x IO 4

Ba

tot

x IO" 5

Plasma

2.2

4.8

1.1

Cytosol

7.5

2.3

1.7

J.A.T.P. Meuwissen and K.P.M. Heirwegh

39U

Fig. 2.

Influence of GSH on gel filtration profiles of Gunn rat liver oytosol and of its mixture with Wistar rat plasma. The arrows indicate: (I): albumin; (II): ligandin; (III): ): 280 nm; (////): bilirubin. small protein (A/Z). (

The pigment that eluted with the void volume was colloidal as indicated by its visual spectrum and therefore not genuinly bound. Furthermore, recovery of diazo-positive material was only 50%. In addition a high 450 nm absorption relative to bilirubin concentration was observed in both the void volume and ligandin regions. All this indicates that during separation ligandin progressively lost its binding activity with concomitant formation of colloidal bilirubin and diazo-negative breakdown products. In contrast, after fractionation in the presence of GSH recovery of bilirubin was almost complete, and 75% of the pigment was found in the ligandin fraction (Fig. 2b). When the same experiments were repeated with mixtures of Gunn rat cytosol and Wistar rat plasma (albumin/ligandin molar ratio approximatively 0.85) similar effects were observed. In the absence of GSH a sizable fraction of the ligand was found with albumin (25%) (Fig. 2c). Again ligandin carried the smallest fraction of the pigment (20%) but, in this case, no bilirubin was detected at the void volume and its recovery was complete. The latter effects were probably due to the presence of albumin. GSH again shifted the distribution in favour of ligandin at the expense of both other proteins (Fig. 2d). Comparison of the elution profiles indicates that GSH has a protective effect on the binding activity of ligandin, permitting it to

395

Binding Proteins and Transport of Bilirubin

DISTANCE FROM AXIS OF ROTATION : CM

Fig. 3.

Moving-boundary sedimentation of a mixture of Wistar rat liver cytosol with Gunn rat plasma. First derivative plot. The numbering of the arrows has the same significance as in Fig. 2. ( ): bilirubin (470 ran); ( ): albumin (radial immunodiffusion).

carry bilirubin in preference to the small protein and to albumin. Also, when GSH is present, the binding activity of ligandin is maintained at a level sufficient to prevent formation of colloidal complexes and loss of bilirubin. DTT offered qualitatively similar, although slightly less pronounced protection. 3) Moving-boundary sedimentation. Jaundiced plasma from Gunn rats was mixed in different proportions with bilirubin-free cytosol from Wistar rats and the bilirubin distribution analysed after centrifugation. Both the small protein and ligandin took up the pigment from albumin and the exchange was virtually complete at a molar ratio of ligandin to albumin of approximately 0.4 (15). A typical experiment is shown in Fig. 3 where the bilirubin exchange between the cytosol proteins and albumin is about two to one (ligandin/albumin ratio approximatively 0.25). Addition of GSH to the mixtures had no measurable effect in these experiments. The results clearly indicate that both cellular proteins have a binding affinity higher than that of albumin. They bind bilirubin some 5 to 8 times more strongly. It thus is clear that mercaptans protect the bilirubin-binding

properties

396

J.A.T.P. Meuwissen and K.P.M. Heirwegh of ligandin in certain environments. However, this protection is only partial. The highest binding affinity is observed in the moving-boundary

sedim-

entation method where GSH is not required. A loss of affinity, with partial prevention by GSH and DTT, has been observed only in procedures where ligandin comes into contact with a solid phase. The mercaptans

probably

act by retarding conformational

chan-

ges of the protein. Our results also indicate that caution should be exercised when assessing binding activities of these proteins by comparing Fig. 4. Time course of desorption of bilirubin bilirubin-Sephadex G-10 (9): liver cytosol;

the from complex. plasma.

their relative ligand content, particularly after fractionation.

Comparative kinetic studies

Desorption of bilirubin from bilirubin-Sephadex G-10 complex by cytosol

and

plasma preparations was studied as a function of time in the presence of GSH (2mM) (Fig. 4). The curves are best described by a sum of two exponentials. At comparable binding capacities cytosol takes up bilirubin 3 to 4 times faster than does plasma. This is in line with the results of the equilibrium binding studies which show a higher binding activity of the former preparation. The adsorptive properties of Sephadex G-10 for bilirubin have been used to advantage here because, due to the competitive effect of the adsorbent, the time-scale of exchange is increased considerably. This made it possible to study the process in a realistic timedomain. However, true rate constants could not be derived as the association-dissociation rates of bilirubin with Sephadex G-10 could not be measured ( t ^