Ion currents and physiological functions in microorganisms.

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Ann. Rev. Microbiol. 1977. 31:181-203 Copyright © 1977 by Annual Reviews Inc. All rights reserved

ION CURRENTS AND

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Annu. Rev. Microbiol. 1977.31:181-203. Downloaded from www.annualreviews.org Access provided by University of Bristol on 01/25/15. For personal use only.

PHYSIOLOGICAL FUNCTIONS IN MICROORGANISMS F. M Harold Division of Molecular and Cellular Biology, National Jewish Hospital and Research Center, Denver, Colorado 80206 and Department of Microbiology and Immunology, University of Colorado Medical School, Denver, Colorado 80220

CONTENTS .. .. . ... .. . . .. . . . . . .... .. . . ........ ,""',.".,', .. ". Energy Transduction ,..,""", .........,.,"""", ......,"""""", ..............,""', ................,.., Ancillary 10/1 Movements "",.....,.,"""""""',',.....,"""""',"',.'..,"""',.."',.,,....,""""" In Search of Novel Functions .. ..... . . . "".. """........ ".".. "" .. ".........".. ,,"",,...... ION CURRENTS IN EUKARYOTIC CELLS . .. . . . . . .. . . Proton Circulation of Fungi and Algae """"....."".. """".,,",,.........,,""",, .....,,"'''' Action Potentials and Osmotic Regulation ""......"....""."."....."".."""."......"".. ,, Excitability. Calcium. and Locomotor Behavior " .. "..,", ........,...,"'"''"..........,''''''''' Electrical Control of Morphogenesis Of Ions and Clocks .............. "...............................................................,.................. THE IMPORTANCE OF BEING ELECTRIFIED ............. ""... "............."""".............,,

PUMPS, CHANNELS, CARRIERS, AND GATES

.

. . . ..

.

..... . . .. .

. . . ..

.. .

PROTONS AND THE METABOLIC ECONOMY OF BACTERIA

.

.

.

. .

.

. .. .

.

. .

..

..

...

... ...... .

..

....... ... .. ..

................... . . . . . . . . ........ .....

"

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181 184 184 187

188 189 189 192 192 195 198 199

It takes a membrane to make sense out of disorder in biology, . . To stay alive you have to be able to hold out against equilibrium, maintain imbalance, bank against entropy, and you can only transact this business with membranes in our kind of world. Lewis Thomas, The Lives of a Cell.

PUMPS, CARRIERS, CHANNELS, AND GATES

That living things can generate electrical currents and potentials has been known since Galvani's day. Most bioelectric phenomena arise from the movement of ions across membranes in such a way as to produce an imbalance of electrical charge; the quantitative relationships between ion movements and electrical parameters 181


1 82

HAROLD

have been thoroughly explored in systems as diverse as nerve axons, epithelial tissues, plant roots, and giant algae. As a result, electrophysiology is today one of the most sophisticated branches of biological science, but rather an arcane one, seemingly remote from the concerns of most biochemists or microbiologists. This comfortable apartheid has been shattered by developments in bioenergetics during the past decade: it has become clear that at the molecular level a central function of biological membranes is to generate gradients of ion concentration and electrical potential, and that many, though obviously not all, of the physiological roles that membranes play depend upon the controlled utilization of ion currents to perform diverse kinds of work. Electrification is not confined to squid axons and electric eels but is a fact of life for Paramecium, Neurospora, and even Escherichia coli. A voluminous and specialized literature has grown up around the role of pro ton-and of sodium currents in oxidative phosphorylation and active transport [for historical perspectives (see 1 6, 2 1 , 44, 84, 88, 92, 1 19, 1 24)]. But one comes to suspect that ion currents may be too important to be left entirely to students of "bioenergetics." The ubiquitous distribution of ion currents and their (presumptive) evolutionary antiquity encourages one to wonder about their involvement in other cellular activities that depend upon membranes-in sensory perception, inter- and intracellular communication, cell division, morphogenesis, differentiation, biologi cal clocks, and other mysteries. The present essay, therefore, attempts not to review the current literature in any systematic way but to articulate a point of view: that the circulation of ions across biological membranes is at the heart of many physio logical devices that allow cells and organisms to function as integrated units. In time, we may list ion currents along with membranes, genophores, and ribosomes among the essential attributes of cells. Let us at the outset distinguish three kinds of molecular mechanisms that translo cate ions across biological membranes. Simplest and best understood is passive diffusion through aqueous channels or through the lipid phase of the membrane, but ions of biological interest do not usually move in this fashion. We shall be mostly concerned with transport, processes that involve interaction of ions with specific membrane components and especially with macromolecules. Following Mitchell's terminology (86), I speak of two classes of transport, primary and secondary. Primary ion transport refers to movements integrally linked to some enzymic reaction; many, but not all, are electrogenic. Examp1j!s include the familiar Na+- and K+-stimulated ATPase of mammalian cell membranes and the translocation of protons by vectorial redox reactions. The energy required to move the ion counter to the electrochemical potential gradient is supplied by the metabolic reaction; thus, the colloquial "ion pump" properly applies to the primary transport processes that interconvert chemical, osmotic, and electrical forms of energy. Secondary transport refers to processes that involve exchange of secondary or ionic bonds but not a concurrent chemical reaction; they provide thermodynami cally passive pathways of greater or lesser specificity for the diffusion of ions in accordance with the concentration gradient, the electrical potential, or both. The driving force upon the ion in question and the physiological role of the translocation depend upon the molecular mechanism. Some secondary transport processes are


ION CURRENTS AND PHYSIOLOGICAL FUNCTIONS

1 83

electroneutral, others translocate one or more charges; in some cases the ion moves singly, in others its movement is coupled to that of another metabolite in the same direction (symport) or in the opposite one (antiport). Functions are equally varied, ranging from modulation of the transmembrane electrical potential by ion move ment to the accumulation of nutrients. The translocators usually found in biological membranes are macromolecules, and their mechanism of action is in no case well understood. For that reason, much attention has been devoted to the ionophore antibiotics that exemplify many features of secondary transport: for example, valino mycin and nigericin are carrier molecules that form specific complexes with K+, respectively charged and neutral; gramicidins and polyenes generate ion-conducting channels, respectively selective for cations and anions; and alamethicin or monazo mycin form conducting channels only when a voltage is applied across the mem brane and are said to exhibit "gating" (29, 48, 5 1 , 82, 95, 96, 1 39). Finally, ion current refers simply to the flow of an ion from a higher to a lower electrochemical potential, but in the present context, it usually involves the circula tion across the membrane between two transport processes separated by some significant distance (Figure 1 ). At this point, the reader may wonder why the distinction between primary and secondary ion transport has been so emphasized. The reason is that it corresponds to two kinds of physiological functions. The primary systems carry out reversible

Figure J

Energy transduction by the proton circulation in bacteria. The diagram illustrates

generation of a proton current by vectorial electrogenic H+ extrusion through the respiratory chain and completion of the current loop by several paths that perform useful work: proton translocating ATPase (oxidative phosphorylation); pyridine nucleotide transhydrogenase; the flagellar motor; and several kinds of proton-linked transport systems. Also shown is the generation of a secondary sodium current by Na+/H+ antiport and an example of sodium linked substrate transport. S, Substrate.

1 84

HAROLD


interconversion between the free energy of chemical reactions and that of electrical potential or concentration gradients. These are Mitchell's chemiosmotic reactions (87,88,9 1 ) that have an intrinsic direction in space; they generate ion currents and also utilize them for chemosynthesis. In most cases, the current loop is closed by secondary transport systems,designed to couple the electrochemical potential of the ion gradient to the performance of other kinds of work. The remainder of this article explores some of the diverse ways in which living things, and particularly microor ganisms, exploit the interactions of primary and secondary ion transport.

PROTONS AND THE METABOLIC ECONOMY OF BACTERIA

Energy Transduction Electrophysiological principles emerged largely in the context of nerves and other electrically excitable cells, but it is the bacteria that most clearly illuminate the significance of ion currents to general physiology. The work of the past decade has established that metabolizing bacteria are continuously traversed by a strong cur rent of protons and that this is central to the conservation of metabolic energy and the performance of work. Our growing insight into the nature,genesis, and functions of the bacterial proton circulation stems almost entirely from Mitchell's chemiosmotic theory, formulated between 1956 and 1 966 (84, 85, 87, 9 1 ). The massive body of subsequent theoretical and experimental work has refined,extended, and supported the original conception but left its principles unchanged. The evidence is now so extensive that it is neither necessary nor possible to recapitulate it here. Major reviews consider in detail the chemiosmotic theory in general (39, 85, 87, 9 1 , 1 45, 146), the chemiosmotic inter pretation of bacterial physiology in particular (44, 45, 46) and specific topics such as transport (4 1 , 42, 47, 64, 1 26, 1 43), respiration and oxidative phosphorylation (40,9 1 , 146), and photosynthesis (22, 1 08, 1 14, 1 46). Here,I propose only to outline very briefly the kind of molecular machinery that is now envisaged (Figure I ). PROTON TRANSPORT BY VECTORIAL METABOLISM

The cytoplasmic mem brane of bacteria always contains one or several vectorial enzymic pathways so constructed as to catalyze electrogenic transport of protons outward.! These are the primary pumps that generate and maintain the imbalance of the electrochemical potential of protons across the membrane, making the cytoplasm electrically nega tive and alkaline,and thus they drive the circulation of protons. At present,we know four quite distinct kinds of primary pumps. (a) One is redox chains, both aerobic and anaerobic. Although the molecular details remain in dispute,the basic principle still appears to be that of the redox loop: a vectorial dehydrogenation carries hydrogen from a donor on the inner surface to an acceptor at the outer; the electrons lit is conventional to speak of proton translocation but one cannot distinguish movement of protons one way from that of hydroxyl ions the other way.



1 85

return to the cytoplasmic side; and protons are liberated at the junction of the two limbs [for recent adaptations of the principle (see 40, 90, 92, 113, 146)]. (b) Photo synthetic apparatus based on bacteriochlorophyll is another. Absorption of light generates the primary electric field, which redox reactions convert into an electro chemical proton gradient; this mechanism again depends on the positioning of primary acceptor and electron donor on opposite surfaces of the membrane. (c) Halobacteria produce a unique and chemically relatively simple proton pump com prising a single protein species and the chromophore retinal. These are so arranged that upon illumination the retinal is bleached, releasing to the outside one proton per photon absorbed; regeneration occurs with uptake of a proton from the cyto plasm. It is likely that the retinal gates a transmembrane channel. (d) A fourth kind is proton-translocating ATPase. The mechanism by which this sophisticated com plex functions remains highly controversial (9, 10, 60, 89, 91, 115). It clearly comprises two distinct regions: a headpiece (FI) that bears the catalytic site and a basepiece (Fo) that includes a specific proton channel spanning the membrane. The channel is normally so gated by the headpiece that protons can pass only in conjunc tion with the chemical reaction of ATP hydrolysis or synthesis. PERFORMANCE OF WORK BY THE PROTON CURRENT

The proton circulation serves as the driving force for the performance of chemical, mechanical, and osmotic work by bacteria. The molecular devices by which this is accomplished generally include some means of ion translocation (usually but not necessarily H+) cunningly articulated with elements of different specificity (Figure 1). We now recognize that a large number of transport systems couple the movement of a metabolite up its electrochemical gradient to the movement of protons down the gradient established by the proton pumps. Examples include electrogenic sym port of sugars and amino acids with one or more protons, electroneutral symport of anionic metabolites, and the extrusion of sodium and calcium by antiport with protons (41 , 42, 45, 46, 1 26, 143). From the proton's point of view, these are all secondary transport systems; little more than a beginning has been made towards understanding how they function at the molecular level (21, 129, 1 30, 1 36). Oxidative and photosynthetic phosphorylation exemplify the coupling of one primary transport to another via the proton current (Figure 1). The ATPase is thought to catalyze a reversible chemiosmotic reaction such that A TP hydrolysis drives protons outward, and conversely, movement of protons inward (down the potential gradient established by respiration) is coupled to ATP synthesis. Pyridine nucleotide transhydrogenase is probably another enzyme that links chemical synthe sis to the proton circulation. I hasten to add that this interpretation does not yet command universal assent; the molecular mechanism is particularly debatable. Finally, a word is in order concerning the role of the proton circulation in motility and chemotaxis, a kind of energy coupling that (unlike the preceding ones) appears to be confined to the bacteria. It is now generally agreed (2, 8) that bacteria swim by rotating their flagella around the point of insertion into the cytoplasmic mem brane. The energy source for the "motor" at the base of the flagellum is evidently not ATP but the proton circulation (2, 8 1 a). Indeed, it seems increasingly likely that


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HAROLD

the proton current turns the motor in some quite direct manner, but the molecular mechanism remains totally unknown. Chemotaxis requires modulation of flagellar rotation; evidence is beginning to accumulate that this most elementary instance of sensory perception and behavior also involves the proton circulation. Briefly, bacteria move in straight runs inter rupted by a tumble, after which the cell sets off in a new direction. Tumbling results from an abrupt reversal of the sense of rotation of the flagella, and it is the frequency of tumbling that is altered by environmental signals (attractants, for example, suppress tumbling with the result that cells tend to swim up the gradient). One way in which recognition of a signal might be communicated to the flagellar motor is via fluctuations in the membrane potential or in the flux of a particular ion. Several investigators have reported findings that point in this direction (8, 110, 1 1 1 , 1 53), but a compelling experiment has yet to be done. POWER TRANSMISSION BY THE PROTON CURRENT The amount of work that a proton current can perform depends on the proton flux (itself determined by the metabolic rate and molecular stoichiometry of proton transport) and on the electro chemical potential of protons across the membrane. The latter is usually expressed as the protonmotive force, IIp, analogous to the electromotive force of a battery: it is the amount of work done by a single proton going once around the circuit. The expression for IIp takes account of both forces that act upon protons, the electrical potential 1l1jI, and the pH gradient IlpH:

IIp

=

IlIjI

2.3 RT -

F-

-

IlpH :::: IlIjI

-

60llpH,

where IlIjI is the membrane potential, IlpH is the pH gradient and IIp is the protonmotive force, all in millivolts. R, T, and F have their usual meanings. Some representative data are collected (Table 1); more extensive compilations have been prepared by Rottenberg ( 1 28) and by Rosen & Kashket ( 126). In most cases,the magnitUde of IIp is sufficient to perform the work attributed to it,but some doubt remains with regard to ATP synthesis in oxidative phosphorylation. The contributions of IlIjI and IlpH to the total protonmotive force vary with external pH and potassium content, a matter that may prove very important in the formulation of molecular mechanism to link proton flux to work functions (4, 112, 120, 1 29). Less attention has been given to the flux of protons passing across the membrane, but some estimates are included (Table I) to facilitate comparison with eukaryotic cells. The figures imply a power output of the order of O.S W per g (dry weight) of Escherichia coli. For a membrane to sustain gradients of the order of 200 mY, it must be highly impermeable to protons and indeed to ions generally. The proton conductance is in fact of the order of 0.5 JLmho cm-2, comparable to that of artificial lipid bilayers. Any reagent that induces a non-physiological ion leak will short-circuit the proton circulation and dissociate metabolism from work. The ionophore antibiotics illus trate this effect, and it is at least plausible that some bacteriocins (e.g. colicins K, I, and E I ) exert their effects in this manner ( 1 Ia, 45, 69, 126).

ION CURRENTS AND PHYSIOLOGICAL FUNCTIONS Table

1

1 87

a Microbial proton circulations Proton motive Proton

force, mV J,leq Organism

g-l 1

flux peq

cm-2

1 sec-

Refs.

(48)

(120)

20,62

(28.6)

(72)

135

(4)

(10)

�pH

�y,

�p

sec-

-100

-137

-237

Prokaryotes


Escherichia coli Paracoccus denitrificans Streptococcus

-60

-150

-210

faecalis Halobacterium

-40

halobium Mitochondria

49; F.

M. Harold,

unpublished data

-100

-140

(4)

(10)

4; E.

Bakker,

unpublished data

18.7

93

-240

5-30

148, 151

-225

20-33

155

(1.2)

123

-84

-139

-223

-20

-220

-60

-165

7.5

(rat liver) Eukaryotes

Neurospora crassa Nitella translucens Chara corallina

-240

a Selected data from the literature, figures in parenthesis were recalculated. Proton nux + is given both as microequivalents of H per gram of protein per second and as picoequiv + alents of H per square centimeters of surface area. Where necessary, it was assumed that 2 l half the cell's dry weight is protein and that the surface area is 40 m g- of protein.

Ancillary Ion Movements The purpose of this section is not to discuss the many transport systems (142) that make inorganic ions available to cytoplasmic enzymes but to consider whether translocation of ions other than H+ has a dynam ic role in the operation of bacterial cells. This does appear to be the case, but movements of sodium, potassium, and calcium are ancillary in the sense of serving specialized functions that supplement the work of the proton circulation. SODIUM

The capacity to expel sodium from the cytoplasm is apparently ubiqui tous among bacteria. In no case is the mechanism well defined, but it seems that bacteria have no primary sodium pumps; sodium movement occurs by antiport with one or more protons and, thus, is secondary to the proton circulation (47, 73, 142, 1 65). A surprising number of bacterial transport systems require Na+; others are inhib ited by Na+ (45, 126). In view of the role of sodium symport in mammalian transport systems, attempts have been made to determine whether or not the bacterial trans-

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HAROLD

port systems also translocate sodium inwards, and if so, whether or not the electro chemical potential of Na+ contributes to the driving force upon the metabolite. Unequivocal positive results have so far been reported only for halobacteria, unique in other respects as well. According to Lanyi & MacDonald (72, 76), uptake of leucine responds both to 60/ and to the Na+ gradient; glutamate responds to the Na+ gradient alone. Sodium is expelled by electrogenic antiport for protons (73).


POTASSIUM

K+ is an essential nutrient for all cells and generally the most abun dant intracellular cation. Unfortunately, the mechanism of potassium transport is not well understood. What evidence we have suggests that K+ accumulation by Streptococcus faecalis is electrogenic (translocating positive charge inward) and requires both ATP and an electrical potential (interior negative). In E. coli, the trKA system has similar properties, whereas the Kdp system is e1ectroneutral (47, 1 22, 142) (E. P. Bakker and F. M. Harold, unpublished data). Potassium is a cofactor for many enzymes, and a high cytoplasmic K+ level is required for protein synthesis. A more critical role for K+ transport as such appears to be the control of the cytoplasmic pH. It has been shown repeatedly that K+ is required to establish a pH gradient (interior alkaline), presumably because K+ uptake compensates for the net extrusion of protons. The alkaline pH in turn appears to be involved in a number of functions, including regulation of metabolism, anion transport,and possibly osmotic adaptation (47). Indeed, Raven & Smith (121) argue that control of the cytoplasmic pH may have been the ancestral function of proton pumps in the early evolution of life. Thus, accumulation of potassium is ancillary to the proton circulation, both in terms of mechanism and of function. There is as yet no evidence for any process that depends upon the efHux of potassium from the cells. CALCIUM Extrusion of calcium from the cytoplasm appears to be another univer sal attribute of bacteria, except under special circumstances such as sporulation (141). No primary pump analogous to the mammalian calcium ATPase has been described in bacteria, but there are several reports of calcium/proton antiport in response to the proton circulation (141, 161). Eukaryotic cells of various kinds make good use of calcium gradients (see below), but the role of calcium movements in bacterial physiology remains to be discovered.

In Search of Novel Functions The continuous massive flux of protons out of and back into bacterial cells is a major feature of their physiology and may well have functions that remain to be recog nized. One that comes to mind is that the proton current may serve as an index of metabolic status or as a regulatory circuit complementary to energy charge and cAMP. It is intriguing in this connection that proton conductors stimulate the phosphotransferase system and perhaps the breakdown of intracellular energy re serves by a mechanism that is quite unknown (32, 53, 61, 144). One of the essential features of ion currents is their intrinsic direction in space. This has been emphasized in the context of transport and motility where the vector



1 89

is normal to the cell membrane, but one can also ask whether bacterial cells may have an overall electrical polarity as germinating Fucus eggs do (see below); this would arise if either primary or secondary ion transport systems were preferentially localized in a particular region of the cell, for instance at the ends of a rod. There appears to be no information on this point, which may prove as pertinent to studies on morphological differentiation in bacteria as it has for eukaryotic cells. Bacteria are conventionally depicted as having but a single compartment (Figure I), but it is evident that there are exceptions. Spores, Chlorobium vesicles, and the membraneous lamellae so conspicuous in some chemoautotrophs and in cyanobac teria are likely to be intracellular compartments, possibly endowed with ion circula tions of their own. Finally, one can ask whether proton circulation is directly involved in DNA replication, in cell division, or in assembling the structural framework of wall, membranes, and nucleoid of bacterial cells. Recent experiments (49a) strongly suggest that the answer here is no. S. jaecalis grows readily in the presence of ionophores under conditions so arranged that IIp, IlpH, 1l1jJ, and the K+ gradient are all zero and the membrane is freely permeable to H+, K+, and Na+. To be sure, such cells are crippled; they require a rich medium, an alkaline pH, and a high level of external K+; they are inhibited by Na+. Yet the fact that they grow and divide at a rate comparable to that of control cells and appear morphologically quite normal sets an upper limit to the biological importance of the proton circulation. From the findings of Kopecky et al (69), it is likely that this conclusion also applies to E. coli. ION CURRENTS IN EUKARYOTIC CELLS

The multiplicity of membranes and compartments is surely the most conspicuous feature of eukaryotic architecture. Nuclei, vacuoles, mitochondria, and chloroplasts define regions that differ in metabolic activity and in some cases at least segregate ion circulations that perform particular and localized tasks. It seems increasingly likely that, in principle if not in detail, chloroplasts and mitochondria operate on a proton circulation in the prokaryotic mode; the evidence has been reviewed at length by others (9, 10, 25, 34, 44, 60, 85,9 1 , 1 13, 1 1 9, 1 46). Whether or not other vesicular structures have private ion circuits is not clear; at least some nuclear membranes are permeable to small solutes and ions (3 1, 140). Interest, therefore, centers on the cytoplasmic membrane, which, released from its commitment to energy production, assumes new and extended duties. In what follows, I have selected examples from relatively simple eukaryotic microorganisms to illustrate how novel needs were met by ingenious variations on the prokaryotic theme of cation currents.

Proton Circulation of Fungi and Algae Electrical currents and potentials across the cytoplasmic membrane of bacteria must be inferred from indirect data, but those of the fungus Neurospora crassa can be measured directly by use of microelectrodes. In a study that remains a landmark,


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HAROLD

Slayman (147, 148) reported membrane potentials near -200 mV, comprised of two components. Part, amounting to some -30 to -60 mY, was sensitive to the concen tration of external monovalent cations and was therefore interpreted as an ionic diffusion potential; outward diffusion of K+ accounts for most of it. Addition of small amounts of calcium (0. 1-1 mM) largely abolished the effect of external K+ and Na+ on the membrane potential, presumably by modifying the K+ permeability. The major component of the membrane potential was evidently of metabolic origin as it was rapidly and reversibly abolished by cyanide, azide, or carbonylcyanide m-chlorophenylhydrazone. For example, upon addition of 1 mM sodium azide, the potential fell from -200 to - 1 5 mV within less than a minute and recovered when the inhibitor was removed. This potential could not be accounted for as an ionic diffusion potential and was attributed to electrogenic extrusion of protons across the plasma membrane. The proton flux so generated was estimated at 5-20 pmol cm-2 sec, in the same range as the flux in bacteria and mitochondria ( 1 47, 148), but is subject to complex modulation (37, 150). The energy source of the proton pump is almost certainly ATP; Slayman et al (149) estimated the stoichiometry to be about two protons per ATP hydrolyzed. Existence of the enzyme has now been confirmed by Scarborough ( 1 3 3),who showed that plasma membrane vesicles contain an ATPase capable of generating a potential gradient. Very little is yet known of the molecular characteristics of this proton pump, but its role in coupling transport to metabolism is well established. Slayman & Siayman (152) showed that uptake of glucose was accompanied by an inward current of protons sufficient to depolarize the membrane and inferred symport with one or two protons. The pattern is probably much the same in yeast. Eddy and his associates have provided convincing evidence that accumulation of certain sugars, amino acids, and inorganic phosphate occurs by electrogenic symport with protons (19, 137, 138). Misra & Hofer (83) observed energy-linked electrogenic proton extrusion by Rhodotorula, probably mediated by an ATPase sensitive to N,N'-dicyclohexylcar bodiimide. Data on K+ uptake ( 1 1 6, 127) are also consistent with the hypothesis that a primary proton pump generates a membrane potential, interior negative. To a first approximation then, we find dual proton circulations in fugal cells: a mito chondrial one, based on redox reactions, to produce ATP; and another across the plasma membrane, based on an ATPase, that functions in metabolite transport and the control of cytoplasmic pH (Figure 2). It now appears that a pattern of this kind may be a feature also of the economy of algal cells, albeit only recently recognized. The enormous body of information on algal electrophysiology and ionic relations has been summarized by Hope & Walker (56), MacRobbie (80), and Higinbotham (54). Suffice it to mention here that an electrogenic proton pump is evidently a major contributor to the electrical potential and ion flux pattern across the plasmalemma of the freshwater algae Nitella ( 1 55) and Chara ( 123). The electrical potential may go as high as -200 to -240 mY; proton flux is of the order of 20 pmol cm-2 sec, and it is likely that the current is generated by an ATPase sensitive to dicyclohexylcarbodiimide that extrudes two protons per ATP ( 1 55, 1 62). A proton current is also produced by the



191

Figure 2 Dual proton circulation in Neurospora crassa. The diagram includes a mitochond rion, the ATP-driven proton pump in the cytoplasmic membrane, and a H+-symport carrier.

marine alga Acetabularia, but it appears that in this case an ATP-driven chloride pump, directed inwards, is the chief source of the electrical potential (35). There is also evidence that K+ transport requires ATP, but the coupling among K+, Na+, and H+ fluxes is in no case clearly established. Complexity is compounded by the presence in all these algae of a second major membrane system, the tonoplast, which bounds the vacuole and thus as much as 90% of the cell's volume. Small electrical potentials have been measured between cytoplasm and vacuole, but their genesis is none too clear. The physiological functions of algal ion currents, insofar as one can discern them now, are again related to m.;tauolite transport. In ChIarella, Komor et al (65-67) have carefully characterized a system that mediates secondary glucose-proton sym port. Concurrent measurements of /lpH and /lljs indicate that the system is an electrogenic one (68). One would expect,however,that,in algae such as Nitella and Chara that maintain cell turgor by accumulation of salt in the central vacuole, the critical function of the proton circulation would be in the transport of inorganic ions. It has indeed been proposed that both chloride and bicarbonate are taken up by exchange for cytoplasmic OH-,but the hypothesis remains in dispute (see 56, 80, 154). To sum up, a proton circulation evidently is part of the metabolic economy of fungal and algal cells; at least one of its functions is to drive the secondary transport of metabolites. I have been unable to find evidence that would warrant extension of this statement to protozoa. Turning to higher organisms, there is abundant evidence from mammalian systems that a circulation of Na+ is generated by the Na+, K+-ATPase and supports metabolite transport across cell membranes and epithelial tissues (e.g. 16,21,52). ATPases stimulated by Na+ and K+ have also been isolated from various plant plasmalemma preparations (55, 156), but their function is uncertain. The evidence from electrophysiology (54, 80) seems rather to point to a proton circulation as the dominant element in higher plants,and this is reinforced

1 92

HAROLD

by recent experiments ( 1 7, 1 8) that implicate some kind of proton pump in growth regulation by auxin.


Action Potentials and Osmotic Regulation It has been known for 30 years that some giant algal cells are at least mildly excitable: in response to a stimulus (usually but not necessarily electrical) that depolarizes the plasmalemma past a sharp threshold, one observes a marked further depolarization, followed by recovery of the resting potential. The duration of these action potentials is of the order of seconds (56). More recent work showed that action potentials are widespread in the plant world; they differ both in mechanism and in function from the better known and much faster action potentials of nervous tissue. In Chara and Nitella. the action potential results from a transient rise in the permeability to chloride; there ensues a substantial efflux of Cl- (0.5-1 nmol cm-2 in each action potential), accompanied by a smaller amount of K+. Calcium ions are probably involved in triggering the response. Part of the chloride flux is elec trically uncompensated, and it is this net movement of charge that accounts for depolarization of the membrane (56). A somewhat similar depolarization due to chloride efflux has been described for the single-celled alga Acetabularia (38, 97), but it now appears that the underlying mechanism is not a transient increase in chloride permeability but a change in the activity of the electrogenic chloride pump (36). In Neurospora, likewise, the sluggish action potentials appear to reflect modu lation of the proton pump ( 1 50). Quantitative studies with several organisms suggest that the function of action potentials has to do with osmotic regulation and apical growth patterns. In Acetabu laria, the electrical potential is about - 1 70 mY, yet the cells maintain a K+ concen tration corresponding to -90 mY. This is probably achieved by periodic release of K+: in a single action potential the discharge of K+ is approximately equivalent to the amount that leaks into the cell in 10 min; this fits well with a spontaneous firing rate of about six per hour (97). In embryos of the brown alga Pelvetia, turgor pressure is maintained by uptake of K+ and CI- from the medium. Periodic current pulses during growth (see below) were shown to be carried by efflux of CI- from the growing tip, accompanied by effiux of K+ elsewhere ( 1 04). Pulsing can be elicited by decreasing the external osmotic pressure,and the amounts of K+ and Cl- released thereby are approximately such as to restore the original turgor pressure ( 1 05). Nuccitelli & Jaffe ( 105) suggest that the growing tip serves as a pressure transducer that senses the turgor pressure and responds by triggering chloride release; here again, Ca2+ ions are somehow involved.

Excitability, Calcium, and Locomotor Behavior Paramecium is sldficiently robust to tolerate impalement by microelectrodes with little disturbance of cell function and behavior. The union of electrophysiology and genetics, exemplified in the work of Eckert (28) and of Kung and his associates (7 1 ), revealed that a current of calcium ions plays a key role in coupling sensory percep-



1 93

tion to behavior and promises to shed light on the molecular basis of excitability. Let us begin this brief and somewhat oversimplified summary with the classical avoidance reaction. When Paramecium encounters an obstacle, or a chemical stimulus such as Na+, it backs up briefly and then resumes forward motion in a slightly different direction. This results from a temporary reversal of the direction of ciliary beating over the entire cell surface. The mechanism of ciliary beating is beyond our scope here (see 94, 1 57) except to note that the energy source is ATP, usually derived from oxidative phosphorylation. The direction of the power stroke is a function of the calcium concentration in the basal region: organisms whose membranes have been disrupted by detergent extraction still swim when given ATP -forward at low calcium (10-8-10-7 M), backward at high calcium (above 10-6 M) (99). These and other findings suggested that calcium influx may be responsible for ciliary reversal, and electrophysiological studies implicated the membrane in con trolling the calcium flux (28). The electrical potential across the plasma membrane is normally around -20 to -40 mY; this is thought to be an ionic diffusion potential, determined primarily by the movement of K+ out of the cells and of Na+ inward, in accordance with the concentration gradients and the ionic conductance of the membrane (28). Paramecium, like other cells, accumulates K+ and extrudes both Na+ and Ca2+, but as yet, little is known of the transport systems that generate the gradients. Suffice it to state that the cells maintain a low cytoplasmic calcium level by continuously pumping the ion out, probably via a calcium ATPase (6, 1 3). Mechanical and electrical stimuli increase or reduce the membrane potential in a manner clearly related to motile behavior. Gentle tapping of the anterior of Paramecium with a small stylus causes it to back up and also generates an action potential: intracellular microelectrodes record transient depolarization of the mem brane, sometimes an overshoot to positive readings, followed by recovery. The action potential was traced to an influx of calcium ions in amounts sufficient to raise the cytoplasmic concentration to lQ-6 M or more. This influx is thermodynamically passive, down the gradient maintained by pumping calcium out. The sequence of events appears to be as follows: anterior stimulation causes localized depolarization of the membrane (receptor current) and increased calcium conductance; this spreads over the entire cell surface as calcium rushes in, further depolarizing the membrane; the cilia reverse in response to the elevated cytoplasmic Ca2+; within 50 msec or so the original low conductance recovers, Ca2+ is pumped out of the cell, and forward beating resumes (28, 79). Stimulation of the posterior end causes the organism to swim forward faster, as if to escape; this response is correlated with hyperpolarization of the membrane and an increase in the frequency of ciliary beating. The hyperpolarization was shown to reflect a transient increase in the K+ conductance (enhanced K+ efflux, hence a more negative potential) (98). The behavior of the organisms thus results from the inter play of physical and anatomical features: on the one hand, the electrical potential across the entire cell surface; on the other, the localization of responsive K+ chan nels at the rear, Ca2+ channels at the front (28, 1 09).


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One must add that the actual relationship between membrane conductance, cal cium flux, and ciliary beating is rather more complex than outlined here. Just how the calcium level determines the direction of beating remains unknown, as does the role of calcium-binding plaquesJocated at the base of the cilia (30, 118). Hyperpolar ization of the membrane (posterior stimulus), as mentioned above, accelerates for ward beating of the flagella; this also involves calcium. The mechanism depends on the rate of calcium influx in response to the potential gradient, rather than on the calcium level (77, 78), and again the molecular basis is unknown. These unsolved problems, however, should not obscure the fact that locomotor behavior of Paramecium is controlled by the current of calcium ions across the cytoplasmic membrane and particularly by the excitable transport system that mediates the entry of calcium. To be effective, an anterior stimulus must elicit the opening of the calcium channels whose conductance varies dramatically with the potential across the mem brane (that a "channel" mechanism is involved is suggested by the relatively low specificity of whatever mediates the entry of CaH). The molecular basis of "gating" has been approached by an ingenious combination of genetic, physiological, and biochemical techniques. Kung and his associates (7 1 ) isolated and characterized a series of mutants whose markedly altered behavior could be correlated with defects in their ion gates. Most dramatic is pawn, which lacks calcium action potentials altogether and in consequence is unable to back up; paranoiac has very prolonged depolarizations resulting from excessive influx of Na+ and exhibits excessive back ward swimming; and fast mutants have abnormalities in the K+ channels ( 1 4, 43, 7 1 , 131, 1 32). Nelson and his co-workers ( 1 2- 1 4) have begun to characterize the various transport pumps and channels by direct measurement of ion fluxes. At O°C, the pump that expels Ca2+ from the cytoplasm is shut down but the gated Ca2+ channel still operates: chemical or electrical stimuli that depolarize the membrane enhance 45Ca influx by tenfold and more ( 1 3, 14). The gate may also be opened by addition of certain local anaesthetics, which presumably interact with the lipids in the immediate vicinity of the channel ( 12). What molecular mechanisms could account for the gating of the CaH channel in such a way that depolarization of the membrane enhances its ion conductance? The same question arises with respect to the Na+ and K+ channels whose responses underlie the action potentials of nerve axons. The molecules that comprise the excitable channels are presumably proteins, as yet unidentified and not accessible to biochemical study. However, significant insight into the molecular basis of excit ability stems from the studies of Mueller and his associates (95, 96) on the iono phores, alamethicin and monazomycin. These antibiotics, like gramicidin, form cation-selective channels across lipid bilayer membranes but exhibit two unusual properties: the conductance depends markedly upon the electrical potential (in one experiment with alamethicin the conductance rose by three orders of magnitude when the voltage was raised from 40--80 mV); and channel formation is an exponen tial function of the antibiotic concentration, suggesting that many molecules cooper ate to construct each channel. Membranes doped with these ionophores generate action potentials that reproduce those of nervous tissue with remarkable fidelity. To



195

account for the findings, Baumann & Mueller (7) proposed a model in which formation of the channel depends on the voltage across the membrane: the iono phores under consideration are all lipophilic molecules of elongated form, bearing a hydrophilic group at one end and one or more cha.rges at the other. Under the influence of an applied voltage, the molecule would insert vertically into the lipid matrix, anchored at the surface by the hydrophilic group. Lateral diffusion would then generate channels containing three or more molecules arranged like the staves of a barrel. [For a somewhat different model see Gordon & Haydon (33)]. Models of this general kind can be constructed for macromolecular components as well (95, 9 6), and it is easy to conceive of situations in which abolition of a potential, rather than its imposition, is prerequisite to channel formation. Returning again to the biological world, one must ask how general is the use of a calcium current to couple reception of a stimulus to motor response. The data are fragmentary but highly suggestive. References to work with other ciliates will be found in the papers cited above; in Chlamydomonas also,calcium influx appears to link a light stimulus to reversal of the flagella (1 34),-and it is reasonable to look for an involvement of calcium in the remarkable photoresponses of Euglena (24). The photoreceptor here and in other systems is now thought to be riboflavin, which may function as an ion gate, perhaps across the membrane of an internal structure (23). Further afield are indications that calcium ions, possibly a flux of calcium across the plasma membrane, may be involved in chemotaxis and amoeboid movements of many kinds (26, 27, 50, 107, 160). Participation of calcium in aggregation and differentiation of Dictyostelium has been suggested (11, 81) but remains to be established. Finally, it is not a long step to the apparent involvement of Ca2+ in pinocytosis and phagocytosis,(3,70) phenomena,which may again be mediated via the electrical properties of the cytoplasmic membrane (26, 63) .

Electrical Control of Morphogenesis Brown algae of the intertidal zone attain a length of several meters and hardly qualify as microorganisms, but their single-celled eggs deserve honorary member ship,if only because of the impressive researches of Jaffe and his associates (58, 106) on the developing eggs of Fucus and Pelvetia. These studies document that differen tiation into the future thallus and rhizoid is intimately related to the establishment of ion currents through and around the embryo. Moreover, they suggest possible mechanisms for the localization of morphogenetic processes and have far-reaching implications for higher plants and animals. A brief and readable review (58) summa rizes the basic observations and a current one is in press (106). During the first day following fertilization,the developing egg elongates and then divides into two unequal halves, one destined to become the rhizoid (holdfast), the other the thallus. At this stage,the rhizoid is the growing part as the embryo's first objective is to secure firm lodging. What makes this system so important is that, unlike animal eggs, the zygote of Fucus has no predetermined animal-vegetal axis or polarity: the site and direction of outgrowth are determined by one of a variety of external stimuli-light, an electric current,gradients of pH, or temperature (for example, the embryo grows away from the direction of incident light). The various


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stimuli are thought to produce a localized membrane change that confers polarity; once outgrowth has begun its direction is self-maintained. Studies on the mechanism that determines the site of growth initiation and then maintains its direction were guided by the hypothesis that localization is a function of an ion current: the plasma membrane in the growth region would be specifically permeable to a cation that is present outside the cell at a much higher electrochemi cal potential than in the cytoplasm (Caz+, Na+, or H+ are likely candidates). Movement of the ion across the membrane completes a current loop, localized in space by the positions of the pump and leak, and generates an electrical field across the cytoplasm with the positive pole at the site of cation entry (Figure 3). The field would be particularly marked for ions such as calcium, which bind strongly to anionic sites near the place of entry and thus set up a sharp gradient. The electrical field in turn may generate electrophoretic movement of negatively charged particles. Cell wall precursor vesicles, for example, would tend to be localized beneath the membrane region that admits Ca2+ and fuse with it, initiating localized growth. To maintain the direction, one must also envisage mechanisms to keep the growing region preferentially permeable to cations (58). Evidence in support of a scheme of this kind has accumulated rapidly in the past 2 years. Generation of an electric current by developing Fucus eggs was first demonstrated by use of an ingenious arrangement that, in effect, placed about a hundred embryos

Figure 3 Current pattern around a two-cell Pelvetia embryo as determined by current density measurements. From Nuccitelli & Jaffe (104) with permission.



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in series and thus amplified the minute voltage drop across each one (57). When the embryo begins to elongate, current begins to flow such that positive charge enters the growing (rhizoid) tip; the current persists for as long as elongation continues. Further progress became possible with the development of an ultrasensitive vibrat ing probe to measure the currents generated in the external fluid around a single egg. A map of the current pattern deduced from such measurements is shown (Figure 3) to illustrate that positive charges enter only at the growing tip but leave from the base of the rhizoid and the entire thallus cell. In actuality, the pattern of current flow is very much more complex because of fluctuations in time. Very briefly, there is a steady current of about 1-2 p,A cm-2 at the tip. Superimposed on this are larger current pulses ( 1 0-30 p,A cm-2), which occur spontaneously several times per hour. These current pulses look like action potentials but are not elicited by changes in membrane potential; it is possible that they arise from fusion of vesicles with the plasma membrane ( 102, 103). The overall pattern of current fl o w is established when the egg is first polarized, but the chemical composition of the current changes as development proceeds. While polarity is being established (6-12 hr after fertilization), much of the charge is carried by a flux of calcium that enters at the tip and leaves from the base of the embryo. Robinson & Jaffe ( 1 25) estimated a current density of 0.03 p,A cm-2 corresponding to 0. 1 5 pmol cm-2 of Ca2+; presumably, calcium is pumped out of the egg at the base and enters passively at the tip. Polarization of the egg by an external stimulus such as light may result from a redistribution of the transport systems, such that entry channels become localized away from the illuminated region and thereby determine the future growth point (58). During this stage, the orientation of development can be influenced by external electrical fields in a manner consistent with the proposal that the ion current localizes growth ( 1 1 7). Once the embryo has reached the two-cell stages and is pulsing actively, the composition of the current is quite different: calcium entry makes but a minor contribution and it is the efflux of chloride through the tip that carries most of the charge. In fact, the flux of chloride may be far larger than the current suggests since most of it is electrically balanced by efflux of K+, and calcium ions now play a role in triggering that efflux of KCl. Nuccitelli & Jaffe (104, 105) argue persuasively that these outward pulses of KCI regulate the turgor of the growing embryo by relieving excess osmotic pressure. Are localized and oriented electrical current a general feature of growing organ isms and, indeed, a determining factor in morphogenesis? Such an assertion would be premature but support is mounting rapidly. In a recent series of papers, Jaffe and his associates (59, 1 63, 164) reported that germinating pollen grains of the lily are also traversed by a strong positive charge current that enters the growing pollen tube and leaves from the grain. Most of the leaving current is carried by protons, whereas K+ ions are the main component of the entering current; calcium ions also enter the growing tip and tend to accumulate there, again pointing to a special relationship between calcium and localization of growth. In this connection, one should recall the meticulous' pioneering work of Lund (75) on the generation of bioelectrical

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potentials by growing plants and their relationship to the pattern of growth. Curi ously, in Avena coleoptiles and in roots, the charge current leaves the growing region rather than entering there as in the cases discussed above. The more recent literature records a growing number of observations on plants, enbryonic cells from amphibians to higher animals, sea urchin eggs, and transformed lymphocytes that implicate calcium and the electrical properties of the surface membrane in the control of differentiation (57, 58, 74). Microbiologists may wish to speculate on the role of ion currents in the apical growth of fungal hyphae (5); we may well be watching the first stirrings of a major development in biology.

Of Ions and Clocks Circadian rhythms are ubiquitous in the eukaryotic world from protists to primates, but the chemical and physiological basis of time-keeping has remained elusive. Most authorities invoke a biological clock; a minority prefers to endow organisms with sensitivity to subtle geophysical rhythms of the earth, but until recently both views lacked plausible biochemical expression. A clue, possibly a very major one, appeared with the discovery that valinomycin shifts the phase of the circadian rhythm in beans and in the marine dinoflagellate Gonyaulax ( 1 5, 1 58). Valinomycin, a highly selective K+ ionophore whose mode of action is intimately known, is the first reagent to affect the clock (as opposed to activities controlled by the clock) in a meaningful manner. Unfortunately, the meaning is anything but plain. Gonyaulax exhibits a number of circadian rhythms (stimulated and spontaneous bioluminescence, photosynthesis, cell division, and also K+ content), all apparently controlled by a single clock. Sweeney ( 1 58) found that exposure of the organisms to small amounts of ethanol shifts the phase of the free-running bioluminescence rhythm; the direction and magnitude of the shift depends on the time in the circadian cycle when ethanol is added. Presence of valinomycin in addition to the ethanol returns the phase of the rhythm to that of untreated control cells. An attempt to determine whether or not Gonyaulax undergoes oscillations of the membrane potential that might be affected by valinomycin gave ambiguous results ( 1 ) that were interpreted as indicating a rhythmic change in the structure or function of the cytoplasmic membrane. Sweeney ( 1 59) also showed that the number of intramembranous particles exposed in one fracture face of another membrane, that of the peripheral vesicle, varies as a function of circadian time. No clear interpretation of these results is possible but they do point to a link between clocks, membranes, and ion movements. Hastings and his associates ( 100, 101) have constructed an explicit and very appealing model along these lines. It is based on the premise that an ion (K+?) gradient across an unspecified membrane may oscillate in a manner determined by the interaction of pumps and leaks, modulated by ion gates sensitive to external stimuli such as light. The model accounts for many aspects of biological rhythms and has several testable features that should provoke skeptics to attempt its disproof.


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THE IMPORTANCE OF BEING ELECTRIFIED

What seems to emerge from all this is that both prokaryotic and eukaryotic cells are normally enveloped in a streaming cloud of ions, moving not at random but in defined currents. The pattern of currents varies from one cell to another, fluctuates in intensity and composition as a function of time or metabolic status or develop mental stage, but is rarely if ever quiescent. What makes sense and order out of the random motions of charged particles is, of course, an insulating membrane that forces the flow into loops: outward across the membrane by one path (typically but by no means always a primary electrogenic pump), back into the cytoplasm by another (often but not necessarily a secondary carrier or channel). In some organ isms, the various transport pathways may be scattered over the cell's surface so that there is no overall pattern of ion flow. But it is clear that in many cases, at least in eukaryotes, current sources and sinks have particular anatomical locations that may be spaced far apart and are related to the function of the current. The ion current is thus bent into a pattern that reflects anatomy and confers upon the cell an overarching chemical or electrical polarity. This is illustrated by the flow of ions around the embryo of Pelvetia (Figure 3) and by the bands of acidity and alkalinity along the surface of some giant algal cells (56). Ion currents serve two general kinds of physiological functions. One is energy transduction and the performance of work-osmotic, chemical, and even mechan ical, as is so clearly exemplified by the bacterial proton circulation (Figure I). The other is the processing and transmission of information, from environmental stimuli to the localization of growth. The special fitness of ion currents for such purposes is not far to seek. An aqueous medium is an efficient ion conductor and thus allows rapid power transmission between energy-generating and energy-consuming units that are not physically contiguous; for thermodynamic reasons, charged particles are superior to uncharged ones (87). Ion currents are intrinsically vectorial and lend themselves to modulation of intensity, coupling efficiency, and orientation in space. Finally, there may be an historical element: if it is true, as some have argued (46, 1 2 1 ), that ion pumps were among the necessary attributes of ancestral eobionts, they were available from the beginning for creative adaptation to the manifold purposes of life. If ion currents play a major role in such complex physiological functions as locomotor behavior, time keeping, development, and differentiation, one may expect external electrical fields and currents to affect these processes. There is indeed a vast literature, reaching back a century and more, that describes a multitude of effects of minute currents and fields on the growth of plants and animals, the regeneration of bones and nerves, the behavior of animals high and low, even commercial and medical application [this is beyond our scope here (see 74, 75, 106); also see forth coming papers from Jaffe's laboratory]. Among investigators with a molecular bent there has been some tendency to belittle such work, sometimes with reason; yet it may be that much of the early electrobiological research was simply premature in Stent's sense ( l 55a) of having been performed before the results could be logically

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connected to the general body of biological science. Now that the molecular basis of electrobiology is beginning to emerge, a more open-minded attitude may be in order.


ACKNOWLEDGMENTS

Thanks are due to my colleagues D. S. Cummings, M. Pato, and H. V. Rickenberg for constructive criticism; to Susan Walker and Nadia de Stackelberg for help in preparing the manuscript for publication; to Lewis Thomas and the Viking Press for permission to quote from The Lives of a Cell; and to Lionel Jaffe and the Journal of Cell Biology for figure 3. There remains a larger debt to Roger Eckert, Lionel Jaffe, Peter Mitchell, and Clifford Slayman, whose insights provided much of the substance of this review. Original research in this laboratory has been supported by grant AI 03568 from the National Institutes of Health. Literature Cited 1 . Adamich, M., Laris, P. c., Sweeney, B. M. 1976. Nature 261 :583-85 2. Adler, J. 1975. Ann. Rev. Biochem. 44:341-56 3. Allison, A. C., Davis, P. 1974. Soc. Exp. Bio!. Symp. 28:249-82 4. Bakker, E. P., Rottenberg, H., Caplan, S. R. 1976. Biochim. Biophys. Acta 440:557-72 5. Bartnicki-Garcia, S. 1973. Symp. Soc. General Microbiology 23:245-67 6. Baugh, L. c., Satir, P., Satir, B. 1976. J. Cell Bioi. 70:66a 7. Baumann, A., Mueller, P. 1974. J. Su pramol. Struct. 2:538-57 8. Berg, H. C. 1975. Ann. Rev. Biophys. Bioeng. 4: 1 19-36 9. Boyer, P. D., Chance, B., Ernster, L.,

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