197 34 19MB
English Pages 435 [436] Year 1978
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 ^