Phospholipid Molecules Were Presumed to Be Separated

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23 Nicolson, M. O., and W. G. Flamm, Biochim. Biophys. Acta, 108, 266 (1965). 24 Hallinan, T., and H. N. Munro, Biochim. Biophys. Acta, 108, 285 (1965). 25 Nakada, D., J. Mol. Biol., 12, 695 (1965). 26 Lamfrom, H., and P. M. Knopf, J. Mol. Biol., 11, 589 (1965). 27 Moule, Y., in Cell Membranes in Development, ed. M. Locke (New York: Academic Press, 1964) P. 97. 28 Kern, M., E. Helmreich, and H. N. Eisen, these PROCEEDINGS, 45, 862 (1959). 29 Kuff, E. L., M. Potter, K. R. McIntire, and N. E. Roberts, Federation Proc., 21, 106 (1962). 30 Dalton, A. J., M. Potter, and R. M. Merwin, J. Natl. Cancer Inst., 26, 1221 (1961). 31 Kuff, E. L., W. C. Hymer, E. Shelton, and N. E. Roberts, J. Cell Biol., in press.

MEMBRANES AS EXPRESSIONS OF REPEATING UNITS* BY DAVID E. GREEN AND JAMES F. PERDUE INSTITUTE FOR ENZYME RESEARCH, UNIVERSITY OF WISCONSIN, MADISON Communicated March 24, 1966 The present communication is intended to serve as an introduction to a new interpretation of the ultrastructure of biological membrane systems. In the first part, the two classical theories of membrane structure are reviewed; in the second part, the limitations of these theories of membrane structure are set forth; and in the final section, the body of evidence is assembled for a more biochemically oriented interpretation of membrane structure. (1) Bimolecular Phospholipid Leaflets as the Backbone of Biological Membranes.- The Danielli-Davson paucimolecular model1 of the ultrastructure of the plasma membrane, first proposed in 1935 to explain the permeability of cells, has dominated thinking about the molecular organization of membranes for almost three decades. The notions implicit in this theory (see Fig. 1) are that: (1) one or more bimolecular leaflets of phospholipid are sandwiched between two layers of protein, considered to be globular; and (2) lipid and protein constitute essentially separate but continuous phases-the polar groups of the phospholipid being bonded electro- statically to protein. In the earlier versions of this model, the two layers of oriented phospholipid molecules were presumed to be separated by a space within which neu- tral lipid was contained; this space was eliminated in the later versions of the model,2 and the now standard phospholipid bilayer was suggested. Electron micro-

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scopic studies have since revealed the existence of numerous membrane systems distributed throughout the cell in addition to the plasma membrane. The morpho- logical similarity of all membranes, as observed with the electron microscope follow- ing fixation and positive staining, led Robertson to propose the unit membrane hypothesis'-' (cf. Fig. 2), an hypothesis which in some important respects was similar to that of Danielli and Davson, and in other respects different. The notion of phospholipid layers sandwiched between protein layers was retained in the Robertson model, but the number of bimolecular lipid leaflets was restricted to one layer. The membrane was considered to be asymmetric rather than symmetric as in the Danielli-Davson model, with mucopolysaccharide or mucoprotein on the out- side face and unconjugated protein on the inside face. The protein layers were visualized as extended polypeptide chains no more than 20 A thick. The thickness of the bimolecular leaflet was set at about 35-40 A. It was implicit in these two membrane models that the bonding between phospholipid and protein was primarily electrostatic and that lipid and protein were essentially separate phases. Supporting evidence for the models: The assumption that membranes are paucimolecular lipid-protein structures is borne out by an overwhelming body of experi- mental evidence. Lipid has been found to be an invariant component of all membranes examined thus far, accounting for at least 25 per cent of the total dry weight.7 Furthermore, electron micrographs of sections of cells show that the thickness of the constituent membranes, although variable, does not exceed 105 A.8 9 In a qualitative way electron micrography of sectioned cells which have been fixed and positively stained does, in fact, support the thesis that membranes are made up of three layers (two electron dense layers separated by an electron transparent zone). Much of the evidence for the unit membrane hypothesis is based on biophysical studies of myelin (polarization microscopy, X-ray diffraction, and electron microscopy). These studies of the myelin system generally support the idea that lipids are arranged in bilayers. X-ray examination by numerous investigators'0-12 of unfixed myelin sheaths has revealed spacings between repeating layers of 171 A. According to Finean,12 this spacing corresponds to two bimolecular leaflets of lipid. A unit membrane would account for about one half of the fundamental spacing observed in myelin; Robertson considered the X-ray data on myelin as ex- perimental support for his membrane model although the electron micrographs of myelin revealed spacings of 120 A in KMnO4-fixed, specimens and of some 100 A in osmium-fixed specimens.4 But, regardless of these dimensional and other un- certainties, the concept of a membrane as a paucimolecular lipid-protein structure now appears to be well established in fact and, in this respect, corresponds to the general prediction of the unit membrane thesis. (2) Limitations of the Unit Membrane Hypothesis. Chemical binding studies: Studies on the binding of phospholipid to proteins in various membranes has led to the conclusion that the predominant binding mode between lipids and protein is hydrophobic'3 rather than electrostatics (cf. S. Fleischer and his colleagues'5' 16 on the mitochondrial membranes, A. D. Brown'7 on the membrane of halotolerant bacteria, and A. Benson'8' 19 on the membranes of the chloroplast). The hydrophobic bonding established in these studies implies deep penetration of the hydrophobic region of protein by the hydrocarbon residues of the phospholipid molecules. This interaction is so tight that, when the disassociation of the lipoprotein complexes Downloaded by guest on September 27, 2021 VOL. 55, 1966 BIOCHEMISTRY: GREEN AND PERDUE 1297

comprising the mitochondrial electron transfer chain from one another is achieved with bile salts, the stoichiometry of lipid and protein within the separated complexes is not disturbed.20 21 Thus, in the case of beef heart mitochondria, binding studies do not support the thesis that lipids and proteins form separate phases joined by electrostatic interaction, as predicted by the unit membrane hypothesis. Benson and Singer19 have reached essentially the same conclusion in their study of the chloroplastic membrane system. The electron transfer complexes of the inner mitochondrial membrane lose the capacity for integrated electron transfer when lipid is removed, and regain this capacity when lipid is reintroduced. Essentiality of lipid for enzymic function can readily be rationalized with hydrophobic bonding between lipid and protein-a mode of bonding in which lipid can profoundly influence the conformation of the protein. Membrane spacings: Electron micrographs of negatively stained specimens of authentic bimolecular phospholipid leaflets show characteristic spacings and a lami- nar appearance.22 23 Such ultrastructural patterns are not observed in the membranes of mitochondria or in those of chloroplasts unless these have been damaged or modified by appropriate treatment. From mitochondria and other membrane systems phospholipid can be extracted almost quantitatively with a mixture consisting of 90 per cent of acetone and 10 per cent of water. Fleischer et al.22 have carried out an electron microscopic examination of such lipid-depleted mitochondria. The appearance of the cristae and the dimensions of the layers in the extracted membranes were virtually indistinguish- able from those in normal, unextracted mitochondria. If the protein layers were held apart by lipid bilayers, one might expect the structure to collapse when the lipid was removed; this collapse was not observed. Such a result indicates that (1) the basic framework of the membrane system must be protein and not phospholipid; and (2) that the characteristic spacings observed in electron micrographs are determined predominantly by the protein of the membrane. Thus, it must be questioned whether the clear space between densely stained layers in electron micrographs of positively stained membranes can be equated, as Robertson does, with the hydrocarbon chains of phospholipid bilayers. The special status of myelin: The myelin sheath which has provided most of the available evidence for the unit membrane hypothesis is now generally regarded as a specialized derivative of the plasma membrane.24 The enzymic activity and the turnover of the major components in myelin are essentially zero.2' The ratio of lipid to protein in myelin is 3:1 ;26 the analogous ratio in membranes is generally no higher than 1: 2, and usually less. Because of these special features of myelin it is our view that the preoccupation with the ultrastructure of myelin has been in the nature of a distraction, since this structured system appears to have little in common with cellular membranes either chemically or functionally.27 Extended protein chains in membranes: The extended protein configuration has been invoked in the unit membrane hypothesis but experiment has failed to verify this prediction for any membrane, including the myelin structure itself. Maddy and M'alcolm28 concluded from infrared absorption analysis that the pro- Downloaded by guest on September 27, 2021 1298 BIOCHEMISTRY: GREEN AND PERDUE Puoc. N. A. S.

tein of the erythrocyte membrane was not in the extended 3-configuration but was randomly coiled (15% a helix). Several electronmicroscopists, notably F. Sj6strand,29 E. De Robertis,30 D. Fawcett,31 and A. Frey-Wyssling,32 have expressed reservations about the appli- cability of the unit membrane hypothesis to biological membranes. However, until very recently the body of contrary evidence was insufficient for evaluation of the kind which is being attempted in the present communication. (3) Macromolecular Repeating Units in Membranes.-During the past 10 years a large body of evidence has accumulated, showing that the electron transfer system and certain other systems of the mitochondrion are built up from macromolecular complexes33-35 (molecular weights of the order of about 0.5-1.0 X 106). Similar complexes have also been isolated from chloroplasts36 37 and from microsomal membranes.38 The existence of these macromolecular functional units clearly argued for counterpart ultrastructural units (see Green and Oda for such an exposition in 1961).39 H. Fernaindez-Moran,4042 using a negative staining technique, was the first to observe repeating units in the inner membrane of the mitochondrion by electron microscopy; this particular observation has now been confirmed by several laboratories.43-48 Repeating units can be demonstrated in the electron micrographs of positively stained as well as those of negatively stained specimens.29 41 49 Frey- Wyssling and Steinmann50 were the first to observe globular units in positively stained specimens of chloroplastic membranes; others have demonstrated similar units in electron micrographs of negatively stained specimens.37' 51, 52 Repeating units have now been reported in each of a number of membranes of different types (microvilli,53 54 outer segments of the retinal rods,55 56 bacterial membranes,57-59 plasma membranes of liver cells,60 microsomal membranes,'8 61, 62 and in endo- plasmic reticula29). These repeating units are of variable shape and size, and cover a range of molecular weights ranging from 50,000 to well over a million. The verification of globular repeating units in mitochondria and other membrane systems by the freeze-etching technique introduced by Moor and Muhle- thaler63 has effectively disposed of the possibility of these repeating units being artifacts of the methods of staining and fixation.64 This technique, which involves sec- tioning of frozen specimens followed by surface replication, would be expected to minimize the danger of preparative artifacts. Blaisie et al.,56 employing low-angle X-ray diffraction and polarization microscopy, studied the membrane of the outer segments of the rods of the frog retina, and correlated these observations with those obtained by electron microscopy. The X-ray diffraction data indicate globular particles, 40 A in diameter, arranged in the membrane within unit cells, 70 A in width. Lipid extraction of the membranes failed to distort the diffraction pattern or the pattern observed with the electron microscope. Kreutz65 has similarly shown by X-ray diffraction that the chloroplastic lamella is also made up of repeating units with a diameter of 71 A. This repeating unit may be equated with a subunit of the "quantasome" reported by Park and Pon, N and Park 66 in electron micrographic studies and by Frey-Wyssling and Steinmann50 in yet earlier studies. It is highly probable that all membranes are built of repeating units. This conclusion brings the membrane into line with the whole of biochemical experience- Downloaded by guest on September 27, 2021 VOL. 55, 1966 BIOCHEMISTRY: GREEN AND PERDUE 1299

that complex macromolecular structures are invariably built up of smaller repeating units. Definition of membranes: We may now define membranes as vesicular or tubular systems, the continuum being made up of nesting, lipoprotein-repeating units (one layer thick). A diagrammatic representation of a tubular membrane is shown in Figure 3. According to this interpretation, these repeating units are the only structured elements in membranes. This conclusion is in accord with chemical studies of membranes. Essentially all the protein and lipid of membrane systems- for example, the mitochondrion-are structure-bound within the organelle.

DETACHABLE SECTORS OF REPEATING UNTS

BASEPIECES OF REPEATING UNITS (MEMBRANE FORMING SECTOR) FIG. 3.-Representation of a membrane FIG. 4.-Diagrammatic rep- as a fused continuum of repeating par- resentation of difierent pos- ticles. sible forms of repeating units. Modalities of repeating units: Each membrane appears to have its own specific repeating unit which is characteristic in respect to form or size or both. Different types of repeating units are shown diagrammatically in Figure 4. Repeating units are made up of a basepiece (the invariant or membrane-forming sector) and a detachable sector (this is not essential to the membrane continuum but is a projec- tion therefrom). The headpiece and stalk of the elementary particle of the mitochondrial inner membrane can be detached by sonication,46 or by exposure to bile salts, without disruption of the membrane continuum.20 The projecting elements in membrane-repeating units are thus intrinsic parts of the membrane but not essential for the maintenance of membrane structure. In any given membrane layer the repeating units are all complementary in form (this is mandatory for the formation of the membrane continuum) but not in com- position. In the inner membrane of the mitochondrion there may be as many as ten different species of elementary particles each chemically and enzymically unique.61 The repeating unit of the inner mitochondrial membrane has a molecular weight of about 1.2 X 106; this corresponds to some 40 molecules of protein and about 400 molecules of phospholipid. The molecular weights of repeating units in different membranes may vary over wide limits. Vesicularization of repeating units: The repeating units of membranes can be disassociated one from another by detergents such as bile salts to yield mono- merized lipoprotein complexes. When the bile salts are removed by appropriate means, the repeating units spontaneously reaggregate to form vesicular membranes (cf. Fig. 5). Brown'7 disaggregated the membrane of halotolerant bacteria by di- lution in salt-free water and then reconstituted the membrane by restoring the origi- nal high level of salt. Razin et al.59 depolymerized the membrane of Mycoplasma laidlawii with sodium dodecylsulfate and then reconstituted it by removal of the Downloaded by guest on September 27, 2021 1300 BIOCHEMISTRY: GREEN AND PERDUE PROC. N. A. S.

DETACHABLE Qo990999 detergent and addition of salt. In the case of mitochondrial membranes, the repeating units from which lipid has been extracted no longer SONICATIN G form membranous structures; reintroduction of lipid into the extracted particles restores the BILE SALTS Di~YSIS property of vesicular formation.68' 69 ACETONE REINSERTION EXTRACTION OF LIPID This capacity for vesicularization appears to °0°1= 1=°:1 be an intrinsic property of the invariant sector VESICULARIZATION ° of°the repeating units of all membranes. It is an FIG. 5.-Vesicularization or mem- expression of the chemistry of these liproprotein brane formation by repeating units which predisposes them to align in a very spe- units. cific way to form a membrane continuum. This phenomenon is not unique but is, in fact, in line with the well-established principle of self-assembly as observed with viral coat proteins.70 Multiple membrane layers in organelles: Organelles are built up of two or more component membranes which have entirely different repeating units and functional attributes. Organelles are systems of membranes rather than individual membranes. Moreover, the relation of one membrane layer to the other within the system is highly precise and characteristic of the organelle. Membranes and metabolic sequences: A considerable number of integrated metabolic sequences have been shown to be localized within the repeating units of membranes.7" 72 Included in this list are the electron transfer chain,73 the system of enzymes catalyzing the citric cycle,74-76 the system of enzymes catalyzing the glycolytic sequence of reactions cycle,77 the mitochondrial system of enzymes for elongation of fatty acids,78 and the microsomal system for synthesis of proteins.79 It may be argued on the basis of presently available evidence that all integrated metabolic sequences are, perforce, localized in the repeating units of membranes, but this localization has still to be verified experimentally for some of the sequences. The study of the mitochondrial membranes has shown that enzymes constitute a large proportion of the total protein of both the outer and inner membranes. It is catalytic protein, rather than structural protein, that is the determinant of the capacity for membrane formation. The functionally active complexes can be divested of structural protein without losing the capability of forming membranes.80 Concluding Comment.-The Danielli-Davson theory, as well as the Robertson unit membrane hypothesis, although incorrect in detail, was a brilliant anticipation of membrane structure. If we substitute the macromolecular repeating units for the repeating structures of the Danielli-Davson or the Robertson membrane model, then we may make an orderly transition to the biochemically acceptable model. The names of Frey-Wyssling, Benson, Fernandez-Moran, Fleischer, Oda, Smith, Parsons, Stasny and Crane, Stoeckenius, and Sj6strand take places of honor in facilitating this transition. We owe a debt of gratitude to Dr. Mary Buell for assisting in the preparation of the manuscript, to Drs. Oscar Hechter, Edward Munn, and David Slautterback for their advice and suggestions, and to many members of our department for their comments. * This work was supported in part by U.S. Public Health graduate training grant GM-88 and Program-Project grant GM-12, 847 from the National Institute of General Sciences. Downloaded by guest on September 27, 2021 VOL. 55, 1966 BIOCHEMISTRY: GREEN AND PERDUE 1301

'Danielli, J. F., and H. A. Davson, J. Cellular Comp. Physiol., 5, 495 (1935). 2 Danielli, J. F., Proc. Symp. Colston Res. Soc., 7, 1 (1954). ' Robertson, J. D., J. Biophys. Biochem. Cytol., 4, 349 (1958). 4 Robertson, J. D., Progr. Biophys. Biophys. Chem., 10, 343 (1960). 5 Robertson, J. D., in Cellular Membranes in Development, ed. M. Locke (New York: Academic Press, Inc., 1964), p. 1. 6 Robertson, J. D., in Intracellular Membraneous Structure, ed. S. Seno and E. V. Cowdry (Okayama, Japan: Chugoku Press, Ltd., 1964), p. 379. 7 Bartley, W., in Metabolism and Physiological Significance of Lipids, ed. R. M. C. Dawson and D. N. Rhodes (London: John Wiley and Sons, Ltd., 1964), p. 369. 8 Yamamoto, T., J. Cell Biol., 17, 413 (1963). I Stoeckenius, W., Proc. Intern. Congr. of Biochem., 6th, New York, p. 603 (1964). 10 Schmitt, F. O., R. S. Bear, and K. J. Palmer, J. Cellular Comp. Physiol., 18, 31 (1941). 11 FernAndez-MorAn, H., and J. B. Finean, J. Biophys. Biochem. Cytol., 3, 725 (1957). 12 Finean, J. B., Intern. Rev. Cytol., 12, 303 (1961). 13 Green, D. E., and S. Fleischer, Biochim. Biophys. Acta, 70, 554 (1963). 14 There are several instances of electrostatic interactions between phospholipids and basic proteins which have functional significance. However, these are special interactions and do not affect the general conclusion that the paramount bonding modality in biological membranes is hydrophobic. 15 Fleischer, S., S. Richardson, A. Chapman, B. Fleischer, and H. 0. Hultin, Federation Proc., 22, 2186 (1963). 16 Richardson, S., H. 0. Hultin, and S. Fleischer, Arch. Biochem. Biophys., 105, 254 (1964). 17 Brown, A. D., J. Mol. Biol., 12, 491 (1965). 18 Benson, A. A., Ann. Rev. Plant Physiol., 15, 1 (1964). '9 Benson, A. A., and S. J. Singer, in Abstracts, 150th National Meeting, American Chemical Society, Atlantic City, September 1965, p. 8c. 20 Kopaczyk, K., J. F. Perdue, and D. E. Green, Arch. Biochem. Biophys., in press. 21 Fleischer, S., G. Brierley, H. Klouwen, and D. B. Slautterback, J. Biol. Chem., 237, 3264 (1962). 22 Phospholipid micelles can exist in a variety of forms; the bimolecular leaflet is a particular form of the micelle that is uniquely invoked in the Danielli-Davson and Robertson models. 23 Bangham, A. D., and R. W. Horne, J. Mol. Biol., 8, 660 (1964). 24 Shanes, A. M., in Biology of Myelin, ed. S. Korey (New York: Hoeber and Harper, 1959). 25Davison, A. N., in Metabolism and Physiological Significance of Lipids, ed. R. M. C. Dawson and D. N. Rhodes (New York: John Wiley and Sons, Ltd., 1964). 26 O'Brien, J. S., in Abstracts, 150th National Meeting, American Chemical Society, Atlantic City, September 1965, p. 6c. 27 W. L. G. Gent, N. A. Gregson, D. B. Gammack, and J. H. Raper [Nature, 204, 553 (1964)] have shown that there are lipid-protein-repeating units in myelin, and maintain that myelin is not merely a bimolecular micelle stabilized by associated protein. 28Maddy, A. H., and B. R. Malcolm, Science, 150, 1617 (1965). 29 Sjbstrand, F. S., in Intracellular Membraneous Structure, ed. S. Seno and E. V. Cowdry (Oka- yama, Japan: Chugoku Press, Ltd., 1964), p. 103. 30 De Robertis, D. R., W. W. Nowinski, and F. A. Saez, in General Cytology (Philadelphia: W. B. Saunders Co., 1960), p. 244. 31 Fawcett, D. W., in Intracellular Membraneous Structure, ed. S. Seno and E. V. Cowdry (Oka- yama, Japan: Chugoku Press, Ltd., 1964), p. 15. 32 Frey-Wyssling, A., Macromolecules in Cell Structure (Cambridge, Mass.: Harvard Univ. Press, 1957), p. 49. 33 Hatefi, Y., A. G. Haavik, and D. E. Griffiths, J. Biol. Chem., 237, 1676 (1962). 34 Ziegler, D. M., and K. A. Doeg, Arch. Biochem. Biophys., 97, 41 (1962). 35 Griffiths, D. E., and D. Wharton, J. Biol. Chem., 236, 1850 (1961). 36 Park, R. B., and N. G. Pon, J. Mol. Biol., 6, 105 (1953). 37 Oda, T., and H. Huzisige, Exptl. Cell Res., 37, 481 (1965). 38 MacLennan, D. H., A. Tzagoloff, and D. G. McConnell, in preparation. 3'9 Green, D. E., and T. Oda, J. Biochemistry (Tokyo), 49, 742 (1961). Downloaded by guest on September 27, 2021 1302 BIOCHEMISTRY: GREEN AND PERDUE PROC. N. A. S.

40 Green, D. E., Plenary Lecture, in 5th International Congress of Biochemistry, Moscow (1961). 41 Fernindez-MorAn, H., Circulation, 26, 1039 (1962). 42 Fernindez-MorAn, H., T. Oda, P. V. Blair, and D. E. Green, J. Cell Biol., 22, 63 (1964). 43 Smith, D. S., J. Cell Biol., 19, 115 (1963). 44 Stoeckenius, W., J. Cell Biol., 17, 443 (1963). 4 Sjostrand, F. S., Nature, 199, 1262 (1963). 46 Stasny, J. T., and F. L. Crane, J. Cell Biol., 22, 49 (1964). 47 Lehninger, A. L., in The Mitochondrion (New York: W. A. Benjamin, Inc., 1964), p. 205. 48 Parsons, D. F., Science, 140, 985 (1963). 49 Andre, J., J. Ultrastruct. Res. (Suppl.), 3, 1 (1962). 50 Frey-Wyssling, A., and E. Steinmann, Viertelsjahresschr. Naturforsch. Ges., Zuerich, 98, 20 (1953). 51 Murakami, S., S. Katoh, and A. Takamiya, in Intercellular Membraneous Structure, ed. S. Seno and E. V. Cowdry (Okayama, Japan: Chugoku Press, Ltd., 1964), p. 173. 52 Bronchart, R., Compt. Rend., 260, 456 (1965). 53 Oda, T., and R. Sato, in Symposium, 4th Annual Meeting of the American Society of Cell Biology, Cleveland (1964). 64 Overton, J., A. Eichholz, and R. K. Crane, J. Cell Biol., 26, 693 (1965). 65 McConnell, D. G., J. Cell Biol., 27, 459 (1965). 56 Blaisie, J. K., M. M. Dewey, A. E. Blaurock, and C. R. Worthington, J. Mol. Biol., 14, 143 (1965). 57 Birusova, V. I., M. A. Lukolanova, N. S. Gelman, and A. I. Oparin, Biochemistry (USSR), 156, 198 (1964). 58 Abram, D., J. Bacteriol., 89, 855 (1965). 59 Razin, S., H. J. Morowitz, and T. M. Terry, these PROCEEDINGS, 54, 219 (1965). 60 Benedetti, E. L., and P. Emmelot, J. Cell Biol., 26, 299 (1965). 61 MacLennan, D. H., A. Tzagoloff, and D. G. McConnell, manuscript in preparation. 62 Cunningham, W., J. Stiles, and F. Crane, Exptl. Cell Res., 40, 171 (1965). 63 Moor, H., and K. Muhlethaler, J. Cell Biol., 17, 609 (1963). 64 Frey-Wyssling, A., Proc. Intern. Botan. Congr., 10th, Edinburgh, p. 57 (1964). 65 Kreutz, W., Z. Naturforsch., 19, 441 (11963). M Park, R. B., J. Cell Biol., 27, 151 (1965). 67 In our earlier study we assumed that there was only one species of repeating unit in the inner membrane, containing the complete electron transfer chain. The notion of multiple species is a more recent development. 68 McConnell, D. G., A. Tzagoloff, D. MacLennan, and D. E. Green, J. Biol. Chem., in press. 69 The capacity of membrane formation by complexes of the electron transport chain has recently been confirmed in the laboratory of Dr. E. Racker (personal communication). 70 Caspar, D. L. D., and A. Klug, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 27 (1962), p. 1. 71 Green, D. E., Israel J. Med. Sci., 1, 1187 (1965). 72 Some sequences that are associated with the basepieces of membranes are integral parts of the membrane structure. Other sequences associated with the detachable sectors of repeating units are more readily separable from the membrane structure. 73 Crane, F. L., J. L. Glenn, and D. E. Green, Biochim. Biophys. Acta, 22, 475 (1956). 74 Bachmann, E., D. W. Allmann, and D. E. Green, Arch. Biochem. Biophys., in press. 75 Allmann, D. W., E. Bachmann, and D. E. Green, Arch. Biochem. Biophys., in press. 76 Green, D. E., E. Bachmann, 1). W. Allmann, and J. F. Perdue, Arch. Biochem. Biophys., in press. 77 Green, D. E., E. Murer, H. 0. Hultin, S. H. Richardson, B. Salmon, G. P. Brierley, and H. Baum, Arch. Biochem. Biophys., 112, 635 (1965). 78 Kennedy, E. P., Federation Proc., 20, 934 (1961). 79 Palade, G. E., J. Appl. Phys., 24, 1419 (1953). 80 A more extensive treatment of the repeating unit concept may be found in the review by D. E. Green and J. F. Perdue, [Correlation of Mitochondrial Structure and Function (New York: New York Acad. of Sciences, 1966), in press]. Downloaded by guest on September 27, 2021