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Volume 74, number 1 FEBS LETTERS February 1977

ReviewLetter

TRANSMEMBRANE ELECTROCHEMICAL H+- AS A CONVERTIBLE ENERGY SOURCE FOR THE LIVING CELL

Vladimir P. SKULACHEV Department of Bioenergetics, Laboratory of Bioorganic , Moscow State University, Moscow II 7234, USSR

Received 1 November 1976 Revised version received 5 January 1977

1. Introduction oxidative phosphorylation. Lardy et al. [3] found that the antibiotic oligomycin prevents energy transfer In 1941 Lipmann [l] put forward the idea that between X - Y and ATP. In the presence of oligo- ATP occupies the point of intersection of biological mycin, X - Y produced by the second and third energy transformation pathways. In the following energy coupling sites of the respiratory chain was 3.5 years, comprehensive experimental proof of the shown to be utilized to support reverse electron- validity of this postulate was furnished by a great transfer via the first energy coupling site. ‘X - Y’ many independent lines of study. The impressive was postulated to be discharged by uncouplers of success of the ATP concept gave rise to the opinion oxidative phosphorylation added to mitochondria in that the system of high-energy compounds is the only vitro [3] or formed in vivo, e.g., in response to convertible and transportable form of energy in the exposure of warm-blooded animals to cold. living cell. The latter conclusion was, certainly, no Further investigations revealed that X - Y energy more than a speculation requiring deeper insight into can be used to actuate Ca”-uptake by mitochondria bioenergetic mechanisms to be confirmed or rejected. [5] and energy-linked reduction of NADP’ by NADH The study of one of these mechanisms, namely [6] . All these observations pointed to X - Y being a oxidative phosphorylation, resulted in several pieces polyfunctional energy source in mitochondria. of evidence being accumulated which indicated that the cell possesses another form of unified energy, 2.2. Electrochemical fl-potential (AjiH) besides ATP and related chemical substances. In 1961-1966 Mitchell [7,8] developed a new (‘chemiosmotic’) theory of biological energy coupling, postulating the existence of a of 2. ‘Non-phosphorylated intermediate’ and H+ potential electrochemical-potential of hydrogen (APH) across membranes competent in respiratory or photo- 2.1. ‘Non-phosphorylated intermediate’ of energy synthetic phosphorylation. coupling (X - Y) According to Mitchell the role of respiratory and T’he,first group of facts which were indicative of photosynthetic redox-chains in ATP-synthesis is the storage of oxidation-released energy in a form confined to the formation of AjIH. The latter, it is other than ATP, was obtained in experiments with suggested, is used to reverse the H’--ATPase reaction. mitochondria oxidizing substrates in the absence of For example, the mitochondrial respiratory-chain ADP and phosphate. Such studies prompted Slater would pump H’ from the mitochondrion to the cyto- [2] to suggest a ‘non-phosphorylated intermediate plasm, creating AjiH across the inner mitochondrial of oxidative phosphorylation’. He believed this to be membrane (negative and alkaline in the mitochondrial an unknown high-energy chemical compound (X - Y) matrix). Then H+-ions go back to the matrix down e.g., a thioester, functioning as an ATP-precursor in A,CiHthrough an H’-ATPase (ATP-synthetase) system

North-Holland Publishing Company - Amsterdam 1 Volume 74, number 1 FEBS LETTERS February 1977 localized in the same membrane and ATP is formed phores associated with a planar membrane, to be from ADP and Pi [7,8,8a,b]. measured. Maximal values of A$ found were 240 mV The electrochemical-potential of H+-ions consists for bacteriorhodopsin from Halobacterium halobium, of an electrical component (transmembrane electric 215 mV for the light-dependent system of cyclic potential difference, A$) and a chemical component electron-transport from Rhodospirillum rubrum, (gradient of H+-concentration, ApH). Both A$ and 100 mV for cytochrome oxidase from beef heart ApH have been demonstrated across coupling mem- mitochondria, 70 mV for R. rubum pyrophosphatase branes of many types (inner mltochondrial membranes, and about 40 mV for mitochondrial and R. rubrum chloroplast thylakoid membranes and bacterial mem- H’--ATPases (at currents of 1 X lo-” -1 X lo-l2 A). branes). A method of universal applicability for the It was shown in several laboratories that electro- detection of A$ in intact organelles (mitochondria phoretic movement of any penetrating across a and chloroplasts) and bacterial cells or their particles coupling membrane, down an electrical potential has been developed by our group [9,10]. It was gradient, results in the conversion of A$ to ApH (for suggested by Dr E. A. Liberman and consists of reviews, see refs. [8,21,22]). The pH outside the measuring the transmembrane distribution of synthetic vesicles was measured with a sensitive pH-electrode penetrating ions. or using pH-dependent changes in the light absorption It was found that penetrating cations and anions or fluorescence of some non-penetrating dyes. Pene- differing strongly ln their structures move through trating pH-indicators and detergent treatment were coupling membranes ln opposite directions. This used to determine the pH inside the vesicles. process, defined as transmembrane electrophoresis [ 111, was found to be supported by electron-transfer 2.3. Identity of ‘non-phosphorylated intermediate’ via any of four energy coupling sites of the respiratory and H’-po ten tial (X - Y = w) chain as well as by ATP-hydrolysis. Similar relation- In the first detailed presentation of the chemios- ships were revealed in experiments with chloroplast motic theory, Mitchell [8] considered the following and bacterial membrane systems. In chloroplasts and ‘energy transfer chain’: e- transfer via the redox-chain photosynthetic bacteria, A$ was detected also by measuring electrochromic shifts in absorption spectra coupling site + AiiH + X N Y + ATP. of carotenoids and chlorophyll [ 12-141. A light- dependent All/ was observed by means of a micro- However, the inclusion of X - Y in this pathway electrode inserted into a chloroplast in vivo [ 151. was no more than a tribute to the traditional view of To exclude any possibility of the electrical events the mechanism of energy coupling. Later Mitchell across mitochondrial, chloroplast and bacterial mem- [23] and Glagolev [24] put forward schemes not branes being a secondary consequence of the energiza- involving X - Y. Indeed, there are no experimental tion of a complex system like the cell or organelle, observations indicating that ‘non-phosphorylated we attempted to demonstrate the electrogenic activity intermediate’ is a high-energy chemical compound. of purified lipoproteins incorporated into an artificial Any attempts to identify X - Y with known high- membrane [ 16-201. The lipoprotein was incorporated energy substances failed. Moreover, several pieces of into spherical phospholipid membrane vesicles and the evidence were obtained suggesting ATP to be the resulting proteoliposomes were associated with a only covalent high-energy compound in the ‘energy planar phospholipid membrane through Ca’+-ions. transfer chain’ (for review, see refs [25,25(a)]). On Addition of an appropriate energy source utilizable the other hand, AiiH seems to meet requirements for by the lipoprotein was shown to induce generation of the component whose place had previously been both an electrical potential difference and a current occupied by X - Y, because: across the planar membrane. These were measured (1) &H can be formed by the redox-chain in the with a conventional electrometer and macro-electrodes absence of ADP and Pi, the process being resistant immersed in the electrolyte solutions on both sides to oligomycin. of the planar membrane. A similar procedure allowed (ii) Interconversion between AjiH and ATP is electrical generation, by intact bacterial chromato- sensitive to oligomycin.

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(iii) AjiH is a common product of the forward seems to be obvious that the level of AjiH, when it is electron-transfers via the four energy coupling sites mainly in the form of A$, requires some additional and can be responsible for energy-exchange between stabilizing mechanism when the activities of A,QH- these sites; it can be involved in the respiration-sup- producing and MH-consuming pathways are chang- ported reverse electron-transfer in an oligomycin- ing. In these conditions, AiiH-supported conversion resistant fashion. of Pi to PPi, which CXI be easily converted back to Pi me~~r~eIsI supports ion-transport through coupling and AjiH, might function as the A,%H-stabilizing mechanism. (v) AjiH runs down in the presence of protono- phorous uncouplers [22] . 4. Osmotic work by APH

3. A,UH-Supported chemical work Conversion of A,QH to concentration- of compounds penetrating membranes can also act as a This includes synthesis of high-energy phosphates A,QH-buffer. If a decrease in activity of AiiH-generat- (ATP and PPi) and reduction of compounds of negative ing enzymes takes place, efflux of penetrating cations redox-potential by means of the reverse electron- from mitochondria, accumulated earlier in the matrix transfer in the redox chains. It was shown that systems down an electrical gradient, should prevent AiiH from of ATP-production and reversal of electron-transfer being lowered for some time. However, this is hardly can utilize @H generated by (i) forward electron- the main function of the APH-supported ion-gradients. transfer and (ii) addition of penetrating ions and/or Osmotic work by AiiH is of great Importance for any changing pH outside the vesicles (for review, see ref. living cell being responsible for the unequal distribu- [26]). Non-specificity of the source of QH in the tion of many substances between intra- and extra- reaction of ATP-formation was clearly shown by cellular spaces (microorganisms) or between intra- Racker and Stoeckenius [26(a)] who demonstrated and extraorganelle compartments of protoplasm light-dependent ADP phosphorylation in proteolipo- (plant and animal cells). At present, all known pro- somes made of beef heart H’-ATPase, H. halobium cesses of uphill transport of ionized and non-ionized bacteriorhodopsin and soya-bean phospholipids. substances through mitochondrial and chloroplast It was observed [ 10,22,27] that NADPH oxidation membranes have been shown to be supported by by NAD’ in submitochondrial particles and bacterial A,UH or by one of its constituents, AJI or ApH. It is chromatophores produces A$ of the same direction clear now that &H is the driving force for many as the redox-chain and I-I+-ATPase, whereas NADH transport processes in bacteria (for review, see ref. oxidation by NADP+ forms A$ of the opposite direc- [28] ). Such a relationship seems to be specific for tion. This observation allowed the energy-linked coupling membranes. Other membrane structures (NADH + NADP’) transhydrogenase reaction to be utilize ATP-energy directly to perform osmotic work, explained as the AiiH-supported reverse electron- e.g., Ca2+-transport into sarcoplasmic reticulum, or transfer via an additional (fourth) energy coupling phosphorylation-linked transport of sugars or Na’/K’- site [8,22]. antiport across animal-cell outer membranes. In the Reversible Interconversion of AfiH and PPi energy latter case, a number of compounds can be accumu- is of especial interest. The rate of this process, e.g., lated in the cell down ApNa by symport with Na’ in chromatophores, is very high (a rather surprising [21,22]. fact since pathways of PPi energy utilization in meta- bolism are not numerous). Such a relationship may be accounted for if we assume that PPI behaves as a 5. @H-Supported motility of bacteria ‘AjiH-buffer’. Indeed, the electrical capacity of coupling membranes is too low to store a large amount It has been unambiguously established that of energy as A$. pH-Buffers can be used to store mechanical work performed by animal systems can AiiH-energy only if A$ is first converted to ApH. It be supported by contractile protein-mediated ATP-

3 Volume 74, number 1 FEBS LETTERS February 1977

cellular ATP level to < 0.3% of normal but had no effect on motility. On the other hand, addition of a protonophore, m-chlorocarbonylcyanide phenyl- hydrazone (CCCP), induced immediate and complete inhibition of motility, although the ATP-level remained unaffected [31]. These data were con- [ -[APH )-I 3 firmed by an observation on flagellum rotation: D- lactate oxidation was found to support rotation even in the presence of arsenate-induced ATP-exhaustion.

-I L...nL CCCP switched off the rotation (see [3 1 ] ). The UNCOUPLING ] authors concluded that ‘an intermediate of oxidative phosphorylation’, rather than ATP per se, is the energy source for bacterial motility [3 l] and the ‘energy-transducing ATPase’ is required for anaerobic Fig. 1. The central place of the transmembrane electrochemical- potential of hydrogen ions (ApH) in the system of the mem- but not for aerobic motility [32]. If ADH is the brane-linked energy conversions. oxidative phosphorylation intermediate and H’- ATPase is involved in ATP + AfiH energy transforma- tion, we can conclude that the bacterial motility hydrolysis. This was shown, in particular, for motility studied was supported by A,!iH. However, it remained of spermatozoa and unicellular animals. One might obscure whether this conclusion is of general impor- think that motility of bacteria by means of flagella tance or it is only true for the above mutants of two might be explained in the same way. However, the heterotrophic bacteria. structures of the bacterial flagellum and, for example, Recently, Dr A. N. Glagolev in this laboratory has that of the tail of the spermatozoon are quite studied the effects of inhibitors on the motility of different, the first being much smaller and simpler. Rhodospirillum rubrum wild strain. It was found that The bacterial flagellum is made of many identical R. rubrum can obtain energy for motility by the protein subunits (flagellin) demonstrating no ATPase four following means: (or any other enzymatic) activity. Mitchell [29,30] (i) Light-dependent (photosynthetic electron put forward a hypothesis that motility of bacteria by transfer) means of flagella is due to direct utilization of A,GH (ii) Rotenone-sensitive (respiration) generated across the bacterial membrane. Accordingly, (iii) Oligomycin-sensitive (glycolysis + H’--ATPase) H-ions, pumped out from bacteria by the redox-chain (iv) Fluoride-sensitive (glycolysis + H’-PPase). or I-I+--ATPase, return back to the cytoplasm via a The first energy source was studied in detail. This flagellum which is postulated to be giant ionophore. process was found to be resistant to oligomycin, Since H-ions are extruded through all the surface of rotenone and fluoride and very sensitive to CCCP. the bacterial cell and return down AiiH via a single Valinomycin (t K’) or a synthetic cationic penetrant, flagellum only, an ion-current appears along the cell tetraphenyl phosphonium (TPP’) decreased the rate surface which can, according to the hypothesis, cause of movement of the bacterium. Motility was com- movement of the bacterium along the long flagellum pletely inhibited by addition of a penetrating weak axis. acid, e.g., acetate, together with valinomycin t K’ or In 1974 Larsen et al. [31] and independently TPP’. In the absence of a penetrating cation, acetate Thipayathasana and Valentine [32] demonstrated was without effect. These facts suggest that R. rubrum that mutants of Escherichia coli and Salmonella motility can be supported by both constituents of typhimurium possessing no H’-ATPase and, hence, AiiH, namely A$ (which is discharged by cationic no oxidative phosphorylation, can support their penetrants) and ApH (which is discharged by penetrat- motility by respiration or nitrate reduction. Glyco- ing weak acids). lysis was not an energy source for motility in these In agreement with such a conclusion, it was found mutants. Additions of arsenate decreased the intra- that the rate of motility of R. rubrum cells closely

4 Volume 74, number 1 FEBS LETTERS February 1977 correlates with the level of membrane-potential. In treatments results in desorption of a portion of the these experiments, tetraphenyl ammonium accumula- cytochrome c pool from the inner membrane to the tion by the bacteria was used as the A$-probe, light intermediate space of liver mitochondria. This as the energy source and o-phenanthroline or anti- solubilized cytochrome c functions as a shuttle mycin A as the inhibitors of the light-dependent between the NADH-specific flavoprotein-cyto- electron transfer. No correlation of the motility-rate chrome b5 pathway in the outer membrane and and the ATP-level was found. It was also shown that cytochrome oxidase in the inner membrane. At the motility can be actuated by a rapid change in the same time skeletal-muscle mitochondria, having no extracellular pH (from 8.5-5.5) in the absence of cytochrome b5, respond by uncoupling of the phos- any usable energy source other than artificially formed phorylating pathway [4,37]. A similar mechanism ApH between the medium and the cytoplasm. The was revealed in brown-fat tissue which is specialized motility ceased in 1-2 min, most probably due to in thermoregulatory heat production [38] . The ApH-dissipation. mechanism of this phenomenon, called ‘thermoregula- It should be mentioned that the above data support tory uncoupling’ [4] , includes cold-induced release only that part of Mitchell’s hypothesis of bacterial of noradrenalin followed by lipolysis of fat both in motility which considers A,!iH as the driving force. brown-adipose tissue and muscles. This results in an However, the intimate mechanism by which A,QH increase in the concentration of unesterified fatty actuates motility remains obscure. It seems to me that acids in both cytosol and mitochondria [22,42]. an ion-flow along the bacterial membrane is hardly an Fatty acids enhance the H+-conductance of the mito- effective mechanism. It may be more probable that chondrial membrane, dissipating the ADH which is motility is a result of flagellum rotation Induced by a generated by the respiratory-chain. Nichols [39] rotating movement of the basal membranous protein obtained some indications that the H’conductance structure connected with the flagellum. In its turn, of brown-fat mitochondria is mediated by a special the rotation of the basal structure may be a result of mechanism which is controlled by the level of extra- the downhill H+-movement, via this structure, from mitochondrial purine nucleotides. There are nucleotide medium to cytoplasm. binding sites on the outer surface of the inner mem- It seems interesting to consider the possibility that brane of brown-fat mitochondria. Their saturation by, a A,iiH-supported type of biological movement is used e.g., GDP strongly decreases membrane H+-conduc- by systems other than bacteria, e.g., by Intracellular tance. organelles. It seems possible that warm-blooded animals are not the only group of organisms using ApH-dissipation to form heat. In some plants, anthesis is accompanied 6. AiiH and regulatory heat production by a strong increase of in the inflores- cence. For example, the temperature in the inflores- It is known that the regulatory heat production by cence of Eastern skunk cabbage (Symplocatpus warm-blooded animals, induced by their exposure to foetidus L.) was found to be +15”C when the air . cold, is due partly to actomyosin-mediated ATP temperature was -15°C [40] . Lowering of the hydrolysis (‘shivering thermogenesis’). Other mecha- ambient temperature induced an increased oxygen nisms of additional heat generation in cold conditions consumption by the inflorescences. This increase was are defined as ‘non-shivering thermogenesis’ which is the greater, the smaller the size of the inflorescence, inherent in animals adapted to low . in accord with Rubner’s law which was previously In some cases non-shivering thermogenesis occurs believed to apply only to warm-blooded organisms. by means of respiratory energy dissipation with no In Sauromatum spadix, this effect was found to be ATP involved. Beyer et al. [33] and Smith and associated with the loss of respiratory control in Fairhurst [34] have described some decrease in P/O mitochondria isolated from inflorescences during the ratio of liver mitochondria of rats acclimatized to day of anthesis [41]. A day before and a day after cold for several weeks. In our group [35] it was shown anthesis, coupling was at its usual high level. that 15 min cold exposure of rats adapted to cold The fact that in such diverse objects as muscle and

5 Volume 74, number 1 FEBS LETTERS February 1977 ’ brown-adipose tissues of animals and flowers of the dria, if they are the usual form of this organelle, might above mentioned plants, the same type of mechanism be giant cells and among them the muscle-cell is of of urgent heat production is used, i.e., AiiH-dissipa- special interest. Inside the very large cells that form tion or a bypass of the AiiH-generating steps of muscle tissue, gradients of available energy should respiratory-chain, may suggest that this phenomenon exist, since : is of common biological significance. (i) Actomyosin fibrils being the main intracellular energy consumers fill the major part of the cell volume (ii) Energy sources come from the cell periphery 7. ApH as a transport form of energy in the ceII (from capillaries and intercellular spaces) (iii) The distance between the edge of the cell and Consideration of possible biological functions of its core is much greater than in cells of other tissues. membrane-potential suggested to me the idea that it To organize the A,GH-conducting pathways in may play the role of a unified transportable physical muscle-cell, ‘super-giant’ mitochondria would be form of energy in the cell, just as ATP (or more required. Indeed, structures of this type have been generally, - P) functions as a chemical energy carrier described in diaphragm [47,48] and skeletal-muscle [42,43] . In fact, if-P is used as the only transportable [49]. Mitochondrial material in the red-flbres of form of energy, the energy transport can be impeded these tissues was found to be organized as structures by intracellular membrane systems and the high visco- of three types: sity of cytoplasm. Membrane-potential spreading (i) Thin transverse tubules forming networks in rapidly along the membrane may create, together with isotropic regions of the muscle-cell, oriented parallel ATP-diffusion over short intermembrane distances, a to the% discs. united energy service in the cell. (ii) Longitudinal tubules crossing two or three 55 Without question, AiiH can spread along the mem- discs. brane of a single mitochondrion. One would think that (iii) Large spherical bodies, localized close to the if A,5H-transport is limited to a single mitochondrion, cell edges, with branches leading to the cell core. it cannot be used to transfer energy over distances It was suggested that all these structures are con- comparable with those existing in cells, since mito- nected, thereby organizing the united mitochondrial chondria are believed to be small, spherical corpuscules system of the muscle-cell [47] . dispersed in the cytoplasm. This assumption is based Reconstitution of a rat diaphragm-cell, by Dr L. E. on electron micrographs of random sections across cells Bakeeva and Dr Yu. S. Chentsov in this laboratory, and tissues. However, analysis of serial sections of confirmed such an idea [49(a)]. Connections between some unicellular organisms has given evidence of the networks, longitudinal tubules and clusters of the existence of quite another type of mitochondrion, a mitochondrial material in the cell periphery were giant branched membranous structure of a size compa- demonstrated. The system described, defined as mito- rable to that of the cell itself. In particular one of the chondrial reticulum [20], seems to be an association most fascinating recent publications presents a picture of several giant, branched mitochondria joined without of a single mitochondrion reconstituted from serial fusion of inner and outer membranes. Reconstitution sections of a flagellate (Polyfomella agilis) cell [44] . of one of these giant mitochondria revealed a struc- In one of the cells studied, the authors observed a ture crossing a muscle-cell from one edge to the other, single mitochondrion which looks like a hollow being parallel to asdisc. In this structure, continuity sphere with many apertures, arranged at a small of at least the outer mitochondrial membrane was distance from the outer cell membrane. revealed. Some branches of the mitochondrion in The view that giant mitochondria are monsters, question were found to form specially organized with which one may meet, only in quite peculiar contacts with adjacent giant mitochondria. At the forms of cells, was recently shaken by the discovery sites of the junction of two branches, four (two outer of such structures in a common yeast, Saccharomyces and two inner) mitochondrial membranes were situated cerevisiae [45] and in rat liver cells [46]. so close to each other that extramitochondrial and The best object in which to study giant mitochon- intermembrane spaces could not be seen. It is not

6 Volume 74, number 1 FEBS LETTERS February 1977 excluded that there is electrical conductivity between (light or respiratory substrates) may be converted to the two mitochondria through the junction site and &H. This process is supported by reducing equivalent that the mitochondrial reticulum represents a united transfer down a redox potential gradient via energy electrical system which is equivalent to a single mito- coupling sites of the photosynthetic- and respiratory- chondrion, meaning the ability to transport energy in chains. In the case of bacteriorhodopsin, AiiH-forma- the form of A,QH. (Electrical conductivity through the tion is apparently coupled to the light-induced conform- junctions of the outer membranes of the adjacent ation change of retinal. animal-cells is well known.) However, if such conduc- tivity between mitochondria is low, the intermito- AiiH, when formed, can be used to support: chondrial energy-exchange may be organized as a (i) ATP-synthesis ‘mixed relay’ in which the energy is transmitted over (ii) PPi synthesis the major part of the distance as ApH (along the (iii) Uphill transport of ions and non-ionised com- membranes of giant mitochondria) whereas the short pounds distances at junctions of adjacent mitochondria are (iv) Cell motility (bacteria) overcome by ATP-diffusion: (v) Heat production in cold conditions Besides this, AfiH can be utilized to move reducing respiration + A,iiH + ATP + @H + ATP + equivalents against a redox-gradient (reverse electron- actomyosin contraction. transfer via coupling sites) which results in NAD’ and NADP’ reduction. An interesting possibility may arise from the co- existence of two types of mitochondria in muscle- To perform all these functions, energy in the form cells: small spherical ones, near the cell nuclei and of AiiH must be transported for some distance along giant ones, joined as a mitochondrial reticulum the coupling membrane. This distance is limited by penetrating the bulk of myofibrils. The small ones the size of the coupling membrane vesicle, e.g., the may give energy-support to biosyntheses and other mitochondrion. In cells possessing large mitochondria, usual cellular functions whereas the giant mitochondria A,5H might be transported for distances comparable may be specialized in providing energy for muscle- to the celI size. contraction. At present, discussions concerning membrane- Summarizing the discussion on the energy-transport potential and energy coupling problems are focused function of AiiH, I should like to stress that there is a mainly on the questions: precedent for the involvement of such a system in (1) How is AitH formed by the redox-chain? cellular energetics. This is photophosphorylation in (2) How is &H utilized by H’--ATP synthetase? H. halobium [SO-521. This bacterium converts light- (3) Is the &H-route the only possibility for energy into AfiH by means of bacteriorhodopsin, coupling electron-transfer and ADP phosphorylation? localized in a membrane region, which contains no In this paper I have tried to demonstrate another other proteins (purple-sheets). The light-generated aspect of the problem, namely that whatever the A,QH is utilized for ATP-synthesis by an ATP-synthe- results of the studies to answer the three questions tase which must be localized in a membrane region above, one can already conclude that the transmem- other than the purple-sheets. Connection between branous electrochemical-gradient of hydrogen ions is, sheets and ATP-synthetase-containing membrane areas as well as ATP, the convertible and transportable form can occur via &H-transfer along the membrane over of energy in the living cell. distances of thousands of A..

Acknowledgements 8. Conclusion I wish to thank Professor E. A. Liberman, Drs The ways by which AjiH is formed and utilized are A. N. Glagolev, A. A. Konstantinov, I. A. Kozlov and summarized in fig. 1. Energy from external sources L. S. Yaguzhinsky for very useful advice and discussion,

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I am grateful to MS T. I. Kheifets for correcting the (a) Barsky, E. L., Dancshazy, Z., Drachev, L. A., Il’ina, M. D., English version of the paper and to MS N. M. Gorey- Jasaitis, A. A., Kondrashin, A. A., Samuilov, V. D. and shina for the help during the preparation of the Skulachev, V. P. (1976) J. Biol. Chem. 251. (b) Drachev, L. A., Jasaitis, A. A., Kaulen, A. D., manuscript. Kondrashin, A. A., La Van Chu, Semenov, A. Yu., Severina, I. I. and Skulachev, V. P. (1976) J. Biol. Chem. 251. References (c) Drachev, L. A., Jasaitis, A. A., Mikelsaar, H., NemeEek, I. B., Semenov, A. Yu., Semenova, E. G. and Skulachev, V. P. (1976) J. Biol. Chem. 251. [l] Lipmann, F. (1941) Adv. Enzymol. 1,99-162. [20] Skulachev, V. P. (1975) Proc. 10th FEBS Meet. Paris, [2] Slater, E. C. (1953) Nature 172,975-978. 225-238. [3] Lardy, H. A., Johnson, D. and McMurray, W. C. (1958) [21] Mitchell, P. (1970) Symp. Sci. Gen. Microbial. 20, Arch. Biochem. Biophys. 78,587-597. 121-166. [4] Skulachev, V. P. (1963) Proc. 5th Int. Cong. Biochem., [22] Skulachev, V. P. (1972) in: Energy Transformations in 1961, Moscow 5, 365. Biomembranes, Nauka, Moscow. [5] Rossi, C. S. and Lehninger, A. L. (1963) Biochem. Z. [23] Mitchell, P. (1972) J. Bioenerg. 3, 5-24. 338,698-713. [ 241 Glagolev, A. N. and Skulachev, V. P. (1974) Biokhimiya [ 61 Lee, C. P. and Ernster, L. (1964) Biochim. Biophys. 39,614-618. Acta 81, 187-190. [25] Kozlov, I. A. (1975) Boorg. Khim. 1,1545-1569. [7] Mitchell, P. (1961) Nature 191, 144-148. (a) Kozlov, I. A. and Skulachev, V. P. (1977) Biochim. [ 81 Mitchell, P. (1966) Chemiosmotic Coupling in Oxidative Biophys. Acta. and Photosynthetic Phosphorylation, Glynn Research, 1261 Skulachev, V. P. (1974) Usp. Sovrem. Biol. 77, 125-154. Bodmin. (a) Racker, E. and Stoeckenius, W. (1974) J. Biol. Chem. (aa9MM;ell, P. (1976) Biochem. Sot. Transactions 4, 249,662-663. ~271 Liberman, E. A. and Tsotina, L. M. (1969) Biofizika 14, (b) Mitchell, P. (1977) Sot. Gen. Microbial. Symp. 27, 256-264. 383-423. Harold, F. M. (1972) Bacterial. Rev. 36, 172-230. [9] Liberman, E. A. and Topali, V. P., Biofiiika (1969) 14, WI Mitchell, P. (1956) Proc. Roy. Phys. Sot. (Edinburgh) 452-461. 1291 25, 32-34. Grinius, L. L., Jasaitis, A. A., Kadziauskas, J. P., Mitchell, P. (1972) FEBS Lett. 28, l-5. Liberman, E. A., Skulachev, V. P., Topali, V. P., [301 Larsen, S. H., Adler, J., Gargus, J. J. and Hogg, R. W. Tsofma, L. M. and Vladimirova, M. A. (1970) Biochim. 1311 (1974) Proc. Natl. Acad. Sci. USA 71, 1239-1243. Biophys. Acta 216, 1-12. Thipayathasana, P. and Valentine, R. C. (1974) Skulachev, V. P. (1970) FEBS Lett. 11,301-308. [3x1 1111 Biochim. Biophys. Acta 347,464-468. Jackson, J. B. and Crofts, A. R. (1969) FEBS Lett. 4, 1121 Panagos, S., Beyer, R. E. and Masaro, E. J. (1958) 185-189. [331 Biochim. Biophys. Acta 29, 204-205. ]131 Witt, H. T. (1972) J. Bioenergetics 3,47-54. Smith, R. E. and Fairhurst, A. S. (1958) Proc. Natl. 1141 Barsky, E. L. and Samuilov, V. D. (1973) Biochim. [341 Acad. Sci. USA 44,705-711. Biophys. Acta 454-462. [351 Zhigacheva, I. V., Mokhova, E. N. and Skulachev, V. P. [I51 Bulychev, A. A., Andrianov, W. K., Kurella, G. A. and (1976) Dokl. Akad. Nauk SSSR 227,235-237. Litvin, F. F. (1971) Fiziol. Rast. 18,248-256. Skulachev, V. P. and Maslov, S. P. (1960) Biokhimiya 1161 Drachev, L. A., Kaulen, A. D., Ostroumov, S. A. and [361 25,1058-1064. Skulachev, V. P. (1974) FEBS Lett. 39,43-45. (a) Drachev, L. A., Jasaitis, A. A., Kaulen, A. D., [371 Lindberg, O., de Pierre, J., Rylander, E. and Afzelius, Kondrashin, A. A., Liberman, E. A., Nemdek, I. B., B. A. (1967) J. Cell Biol. 34,293-310. Ostroumov, S. A., Semenov, A. Yu. and Skulachev, V. P. 1381 Smith, R. E. and Horwitz, B. A. (1969) Physiol. Rev. (1974) Nature 249,321-324. 49,330-425. [17] Drachev, L. A., Kondrashin, A. A., Samuilov, V. D. and 1391 Nichols, D. G. (1974) Europ. J. Biochem. 49,573-583. Skulachev, V. P. (1975) FEBS Lett. 50, 219-222. 1401 Knutson, R. M. (1974) Science 186, 746-748. [ 181 Drachev, L. A., Frolov, V. N., Kaulen, A. D., Kondrashin, [41] Wilson, R. H. and Smith, B. N. (1971) Z. Pflanzen- physiol. 65, 124-129. A. A., Samuilov, V. D., Semenov, A. Yu. and Skulachev, [42] Skulachev, V. P. (1969) in: Energy Accumulation in V. P. (1976) Biochim. Biophys. Acta 440,637-660. (a) Skulachev, V. P. (1974) Biochem. Sot. Spec. Publ. 4, the Cell, Nauka, Moscow. 175-184. [43] Skulachev, V. P. (1971) Current Topics in Bioenerg. 4, [ 191 Drachev, L. A., Frolov, V. N., Kaulen, A. D., Liberman, 127-190. E. A., Ostroumov, S. A., Plakunova, V. G., Semenov, [44] Burton, M. D. and Moore, J. (1974) J. Ultrastruct. Res. A. Yu. and Skulachev, V. P. (1976) J. Biol. Chem. 251. 48,414-419.

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[45] Hoffman, H.-P. and Avers, C. J. (1973) Science 181, [49] Gauthier, G. F. (1969) Z. ZeIlforsch. 95,462-482. 749-751. (a) Bakeeva, L. E., Chentsov, Yu. S. and Skulachev, V. P. [46] Brandt, J. T., Martin, A. P., Lucas, F. V. and Vorbeck, (1977) J. Cell Biol. in press. M. L. (1974) Biochim. Biophys. Res. Commun. 59, [ 501 Oesterhelt, D. and Stoeckenius, W. (1973) Proc. Natl. 1097-1103. Acad. Sci. USA 70,2853-2857. [47] Bubenzer, H.-J. (1966) Z. ZeIlforsch. 69,520-550. (511 Dannon, A. and Stoeckenius, W. (1974) Proc. Natl. [48] Gauthier, G. F. and Padykula, H. A. (1966) J. Cell Biol. Acad. Sci. USA 71,1234-1238. 28,333-354. [52] Belyakova, T. N., Kadziauskas, J. P., Skulachev, V. P., Smirnova, I. A., Chekulaeva, L. N. and Jasaitis, A. A. (1975) Dokl. Akad. Nauk SSSR 223,483-486.