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Freeze-fracture replicas have been prepared from two fast-acting vertebrate muscles (frog sartorius and rabbit sacrospinalis) and are described with particular reference to the distribution of intercalated particles in the plasma membrane, T-tubule and SR cisternae. Orthogonal assemblies of small particles are present on the A face plasma membrane in each instance, and their distribution (in sartorius) is found to be random with respect to the underlying myofibrillar sarcomere repeat. Such assemblies are not present on A or B faces of T-tubule or SR cisternae. Asymmetric particle distribution is described for fracture faces of the T-tubules and SR: the profuse particle packing of the SR A face is uniform from terminal cisternae to medial fenestrated collar. Intercalated particles are present on A and B faces of T-tubule fractures: more commonly on the latter. These results are compared with studies on insect muscles, and a comparative approach to further studies on the correlation between membrane structure and function is discussed.

The present paper deals with the calculation of structures, some elements of which do not admit compressive stresses. An inequality theory is formulated for the large displacement-calculation of these structures. It is proved that for the numerical calculation of this inequality-problem the algorithms of quadratic minimization must be used.

Spin lattice relaxation times (T1) and apparent spin-spin relaxation times (T2) derived from linewidth have been used to investigate model membranes composed of egg yolk phosphatidylcholine. T1 measurements appear to be largely dominated by segmental motion and as a consequence are not very sensitive to small changes in membrane structure. On the contrary, apparent T2 times are shown to be sensitive to such changes in the membrane and are thus suggested as a useful tool for further investigation of membrane structure.

SummaryThe effects of adenosine diphosphate (ADP) and Ca2+ on platelet morphology were studied in edetic acid (EDTA)-treated, washed platelets of rat by means of light scattering measurement at right angles. ADP caused a transient change of light scattering, which was independent of concentration above 5 μM and of pH between 4 and 10. When the platelets were pretreated with ADP or chilled persistently, Ca2+ also caused another transient change of light scattering. The effect of Ca2+ was abolished at pH below 6.5. The velocity of both ADP- and Ca2+-induced response was dependent on temperature, and the magnitude was reduced by metabolic inhibitors and SH-blocking agents. Adenosine triphosphate (ATP) competitively inhibited the ADP-induced response, but not the Ca2+-induced one. The response to Ca2+ of the control and chilled platelets was compared by scanning electron microgram. Apparent similarity of the effect of chilling on membrane structure to that of ADP was suggested.

The increase in chlorophyll a steady-state fluorescence, induced by high NaCl concentration in Porphyridium cruentum in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, is directly correlated with a significant decrease in thylakoid thickness. It does not appear affected either by alteration of light absorption due to configurational change or by electron transport processes. Oxygen evolution occurs only in intact structures. The interrelationship between membrane structure, oxygen evolution and chlorophyll a steady-state fluorescence is discussed.

Evidence is presented which indicates that radioiodine labelled diazotized di-iodosulfanilic acid is a useful nonpermeant probe for the localization of the protein components in the vesicles formed from fragmented skeletal muscle sarcoplasmic reticulum. The data obtained suggest that a high molecular weight (∼172,000 daltons) protein component or components and the (Ca ++ + Mg ++ )-ATPase are located on or near the external surface of these vesicles and that calsequestrin and related Ca-binding proteins are either buried within the membrane structure or located on the internal surface of the vesicle.

Human diploid embryonic lung fibroblasts were cultivated in Eagle's Minimum Essential Medium and labeled with3H-uridine. The release of soluble radioactive substances into the medium was used as an indicator of damage to the cell membrane. The assay method described is simple, sensitive and rapid and allows quantitative estimation of changes in membrane permeability before any morphological damage is observed microscopically. Crude commercial preparations of phospholipase C (E.C. 3.1.4.3.) (40 μg/ml) were highly active on the cell membrane but most of the membrane damaging activity was found to be due to contaminating theta-toxin. However, also highly purified phospholipase C caused a membrane damage as measured by release of isotope through the plasma membrane. The release could be increased by including an optimal concentration of calcium ions in the incubation buffer, by treating the cells in a hypotonic medium and by simultaneous treatment with sublytic concentrations of Triton X-100. To our knowledge this is the first report of membrane damage on a live, intact, metabolizing human diploid cell caused by a highly purified phospholipase C. The results are in agreement with a dynamic membrane structure with the polar groups of a part of the phospholipids accessible at the membrane surface.

During the early developmental stages of the toad, Bufo arenarum, Hensel. up to the stage of gill circulation (150 hr of development at 20–25°C) the total phospholipids content as well as that of phosphoglycerides of choline and of ethanolamine were found unchanged. The subfraction of both phosphoglycerides were separated according to the number of double bonds on silver-ion chromatography and were also found to be unchanged up to the tail bud stage. The distribution of non-polar side chains in the subfractions varied in both phosphoglycerides showing a structural heterogeneity. In the phosphatidylethanolamines predominate the polyenoic containing subfractions. In contrast with the constant concentration of polar lipids, during early embryogenesis a steady increase in 32P incorporation into phospholipids takes place when oocytes labeled during oogenesis are used. These changes were also correlated with the DNA content up to gill circulation stage. It is proposed that most of the nascent membrane polar lipids during early embryogenesis may be derived from a storage site through an active and specific intracellular redistribution process. At the arrival of the polar lipid to the nascent membrane a change in their covalent structure by introduction of a phosphorylbase from a highly labeled pool may explain the raise in specific activity. This change may be necessary to make possible the assembly of the lipid into the membrane structure.

Another publication (Rand, R. P., Pangborn, W., Purdon, A. D., and Tinker, D. O. (1975) Can. J. Biochem. 53, 189–195) has established that lysolecithin and cholesterol interact to form an equimolar complex. We have investigated this complex using the techniques of electron spin resonance (e.s.r.) and electron microscopy. By varying the cholesterol concentration with lysolecithin in both thin films and dispersions studied by these techniques, we have observed the interaction between lysolecithin and equimolar complex below 50 mol% cholesterol, and between crystalline cholesterol and equimolar complex above 50 mol% cholesterol. We have observed an interesting alteration in morphology by electron microscopy, and an isotropic to anisotropic spectral change using 3-doxylcholestane and 12-doxylstearic acid spin-labelled probes when the cholesterol concentration is increased from 20 to 33 mol%. The equimolar complex is stable in the presence of crystalline cholesterol, and exhibits no phase changes in the physiological temperature range. Implications for membrane structure are discussed.

Changes in the fluorescence of two N-arylaminonapthalenesulfonate dyes, ANS and TNS, were measured during the action potential and during voltage clamp steps in giant axons from the squid. We hoped to obtain information about alterations in membrane structure that occur during activity.

We have used spin-labels to detect prostaglan- din E induced changes in erythrocyte membranes. The ob- served changes in spin-label resonance spectra can be mim- icked in erythrocyte ghosts by loading them with CAMP or cGMP. These changes can also be observed by adding ei- ther of these cyclic nucleotides to intact cells. This entry of cyclic nucleotides into intact cells is blocked by an inhibitor The morphology, chemical composition, and dynamical properties of the plasma membranes of most biological cells are doubtlessly subject to continuous regulation. The pres- ent paper is concerned with the membrane of the mature human erythrocyte. Recent studies by Allen and Rasmus- sen (1971) and by Kury et al. (1974) have shown that the rheological properties of erythrocytes at high hematocrit are affected by physiological concentrations of prostaglan- dins and epinephrine. These studies suggest but do not prove that the flexibility of an isolated erythrocyte can be controlled by these substances. Studies by Allen and Valeri (1974) indicate further that erythrocyte morphology is reg- ulated by low concentrations of the prostaglandins PGE1,' or PGE2, which can decrease, or increase, respectively, the internal volume of the cell by a small amount (-3%). The paramagnetic resonance spectra of fatty acid spin-labels bound nonspecifically to these membranes show small but reproducible changes on the addition of low concentrations ( 10-'o-10-'2 M; of the order of few molecules per cell) of the prostaglandins PGEl and PGE2 (Kury et al., 1974), and also show reproducible changes on the addition of physio- logical concentrations of adrenaline and carbamyl choline (Huestis and McConnell, 1974). The spin-label concentra- tion and resulting spectra suggest that the associated changes in membrane structure arise from small changes of the anion channel. We suggest that the observed changes in paramagnetic resonance spectra are due to changes in lipid "fluidity" that are brought about by changes in the biochemical state of membrane-associated proteins (such as spectrin) and in the direct or indirect biophysical interac- tions of these proteins with membrane lipids. throughout much of the membrane, rather than large changes localized to small regions of the membrane. The resonance spectra are a measure of the flexibility of the spin-label fatty acid chains in the bilayer region of the membrane. It is interesting that substances that increase the apparent single cell flexibility (e.g., PGE,) also increase this fatty acid chain flexibility, and vice versa (PGE2 and epinephrine) (Kury et al., 1974). Recent studies of erythro- cyte ghosts using circular dichroism also show changes in the presence of prostaglandins (Meyers and Swislocki, 1974).

SUMMARY 1. The effects of various compounds on the fluorescence of microsome-bound 1-anilino-8-naphthalene sulphonate (ANS) have been studied. 2. Cationic drugs and divalent metal cations enhance the fluorescence of microsome-bound ANS whereas anionic drugs and primary aliphatic alcohols decrease it. 3. No changes were observed in the fluorescence lifetime or emission maximum of ANS and it was concluded that there were no significant changes in the quantum yield of ANS fluorescence. 4. The changes in fluorescence are shown to be related to changes in ANS binding. 5. The results suggest that drug-induced changes in the binding of ANS to the microsomal membrane reflect a change in the net charge on the membrane as a result of the binding of a charged drug and that changes in ANS fluorescence cannot be directly interpreted as changes in the structure of the membrane. 6. Primary aliphatic alcohols displace ANS from the microsomal membrane but cause no change in quantum yield. It is suggested that the alcohols change the net charge on the membrane either by exposure of negative charges or occlusion of positive charges. 7. The changes in ANS fluorescence can be used as a measure of the interaction of various drugs with the microsomal membrane. Apparent binding constants determined by this method for various drugs fall in the concentration ranges over which the drugs are reported to induce changes in membrane function in various in vitro systems.

The general chemical characteristics of the rare earth or lanthanide series of ele­ ments resemble those of the alkaline earth elements in many respects (I). Of the lanthanides, lanthanum has long been considered to have chemical properties most similar to the alkaline earths (2). Recent interest in the biological actions of lan­ thanum ion (LaH) is almost entirely based upon use of this rare earth ion as a substitute or antagonist for Ca2+ in a variety of cellular and subcellular reactions. The rapid and widespread employment of LaH in the last few years is a measure of the complex and varied functions of Ca2+ in membrane and coupling reactions. In this review article, I attempt to concentrate upon the cellular actions of LaH in muscle, nerve, and related tissues. A considerable literature has also accumulated concerning use of LaH to discern the presence or absence of anatomical barriers and to delineate the molecular nature and specificity of subcellular Ca2+ transport systems and membrane structure. References to these areas of research (as well as to the general pharmacology of LaH) will be limited to those studies most relevant to a consideration of the cellular basis of the actions of LaH. As long ago as 1910, Mines (3) examined the inhibitory effects of LaH upon contractile function in frog heart. However, the recent proliferation of LaH-related studies can quite clearly be traced to a prediction in 1964 by Lettvin and co-workers (4) that LaH, by virtue of an ionic radius similar to Ca2+ and a higher valence than Ca2+, will bind at superficially located Ca2+ sites in a less reversible manner than does Ca2+. The subsequent testing of this prediction (5) demonstrated that La3+ did indeed exert a blocking action in lobster axons and that it acted in a manner equivalent to an extremely high Ca2+ concentration. The high affinity of LaH for membrane binding sites was of particular interest when related to evidence that the distribution of LaH in many different preparations was confined to membrane areas contiguous with the extracellular space (6-12). Thus, use of LaH as a relatively specific Ca2+ substitute or antagonist at well-defined

Aromatic polyamide asymmetric “skin type” membranes have been prepared by the Loeb-Sourirajan technique. Two different structures were obtained, depending on the rate of precipitation. Low precipitation rates produced membranes with sponge-like structures. These membranes usually had high salt rejections and low water fluxes. High precipitation rates produced membranes with large finger-like pores. These membranes had low salt rejections and high water fluxes. Possible mechanisms producing these two structures are discussed. It is proposed that high concentrations of polymer in the casting solution, and hence high viscosities at the point of precipitation, or a thick viscous sublayer in advance of the precipitating polymer front both favour sponge-structured membrane formation. The suggested mechanisms are supported by data showing the effect of various preparation parameters such as polymer concentration and casting solution and precipitation bath composition on membrane structure.

After a general review of enzyme topology and the molecular architecture of endoplasmic reticulum the latest research results are discussed. Microsomal enzymes do not occur in specialized regions of the endoplasmic reticulum but are uniformly distributed over the entire membrane system. Functionally associated enzymes are also located in the direct vicinity of the membrane. It has been shown that the catalytic activity of integral enzyme proteins depends on phospholipid and membrane structure. These membrane-bound lipoproteid complexes exert specific effects as vectorial enzyme catalysts. The endoplasmic reticulum is active is active in the intracellular transport of metabolites.

A theory is presented for the mechanics of plant cell wall extension and is based on the analogy of the cell wall with a membrane structure made of material capable of large non-linear deformations. These wall deformations may be elastic, elastic-plastic or visco-elastic. Mathematical analyses of such membrane structures show that there is, generally, a critical internal pressure at which dimensional instability occurs. This instability is characterized by a sudden drop in internal pressure accompanied by a large increase in the physical proportions of the membrane structure. The theory proposes that cell wall extension occurs when the cell turgor pressure reaches this critical instability value. The cell wall thus stretched is fixed by biochemical synthesis of wall material. Osmotic regulation re-establishes the turgor pressure and the instability cycle repeats itself as long as the critical instability pressure of the cell is below the osmotic pressure of the cell contents. Equalization of these pressures stops cell extension. The rate of cell extension depends on the frequency of the instability cycle and is thus dependent on the various rate processes associated with the instability cycle. The theory appears to be able to explain most of the known facts regarding cell extension such as the influence of temperature and the action of some growth substances.

Lateral diffusion of the fluorescent molecule, Pyrene, in phospholipid vesicles and mitochondrial membranes is demonstrated. Pyrene forms excited state dimers (excimers) in a diffusion limited reaction which depends upon membrane composition, temperature and pyrene concentration. The results obtained show that the classical theory of diffusion are not exactly applicable. The emission spectrum of the pyrene monomer in the membrane phase is highly structured and at 77 K a Shpol'skii spectrum is obtained (Shpol'skii, 1960). The formation of excimers in membranes affords evidence for the fluid mosaic model for membrane structure.

SummaryDibutyryl cyclic AMP inhibits growth of certain transformed mouse fibroblasts as does serum starvation. These treatments, however, fail to affect membrane structure as visualized by freeze fracture and electron microscopy. These observations suggest that such treatments do not restore the membranes of transformed cells to normal and that the organization and distribution of certain membrane proteins may be critical for the development and maintenance of the normal contact inhibited state.

An experimental study was made of the permeation of water, paradioxane, and water-paradioxane mixtures through nylon 6 membranes. Laminated films were utilized to determine actual liquid permeate concentration profiles within the membrane structure. Knowledge of these profiles permitted the investigators to measure the concentration-dependent diffusivities and to characterize the mechanism of mass transfer. Since water plasticizes nylon, the behavior of the water-dioxane mixture could not be accurately predicted from pure component performance. Thus the transfer of water was enhanced and the dioxane depressed for equimolar feed mixtures. Moreover, it was demonstrated that a simple, acid surface-treatment of the separative membrane can greatly enhance the transfer of both components. Concentration profiles measured during the unsteady state leading to equilibrium suggested that the nylon polymer matrix was in the process of undergoing structural changes.

Chloroplast membrane carboxyl groups were modified by carbodiimide activation followed by glycine methyl ester substitution, leaving the derivatized group uncharged. This charge alteration induced a number of effects similar to addition of salts to control chloroplasts suspended in a low salt medium. These include: ( a ) restacking or multiple membrane association in low salt-treated chloroplasts that lack grana stacks, ( b ) protection against polycation inhibition of photosystem I electron transfer, ( c ) reduction of the amount of polycations bound to the membranes, and ( d ) increased 90° light scattering due to membrane conformational changes. Carboxyl modification also altered acid-induced conformational changes. These effects are interpretated as the results of the reduction in the surface negative charge contributed by carboxyl groups. Membrane structure at both a local, polyelectrolyte level and at the level of membrane-membrane interaction (stacking) is controlled in part by these negative, charged groups.

The plasmodium of Physarum polycephalum reacts to various kinds of chemicals substances and moves towards or away from them. Threshold concentration of recognition of chemicals was examined in terms of membrane potential and of the averaged motive force of tactic movement by using a double-chamber method, i.e., a single plasmodium was placed between two compartments through a narrow ditch, and differences in membrane potential and in pressure between two compartments were measured. Results are summarized as follows: (a) By increasing the concentration of various substances in one compartment, the membrane potential started to change at a certain threshold concentration, C-th, for each chemical. Chemotactic movement of the plasmodium took place at the same threshold concentration. These results held both for attractants (glucose, galactose, phosphates, pyrophosphates, ATP, c-AMP, etc) and for repellents (various inorganic salts, sucrose, fructose, etc.). (b) The threshold concentration, Cth, for inorganic salts decreased remarkably with increase of the valences of cations, zeta, and was proportional to Z-6, I.E., THE Shultze-Hardy rule known in the field of colloid chemistry was found to be applicable. (c) The plasmodium distinguished the species of monovalent cations in the following order: H(Li(K(Na(Rb(Cs(NH-4 Plots of log Cth against the lyotropic number of anion fell on different straight lines for each monovalent cation species. (d) Plots of log Cth, against the reciprocal of the absolute tempe lines were almost the same and gave a value of 12 kcal/mol for the enthalpy change. These results suggest that the recognition of chemical substances appears as the result of a structural change of the membrane at the threshold point, and that the change in membrane structure is transmitted simultaneously to the motile system of the plasmodium.

The responses in membrane lipid composition, structure, and function of four cultivars of wheat (Triticum aestivum L.) to growth at low temperature have been investigated. Marked growth temperature-dependent alterations in the fatty acid composition and unsaturation of the mitochondrial phospholipids correlate with changes in respiratory activity in all the varieties. Parameters such as the respiratory control ratio and the phosphorylative efficiency decrease in cold-adapted seedlings. Three temperature-dependent structural transitions were identified in the mitochondria by the spin-labeling method. The structural transitions occur at lower temperatures in the cold-grown material. The shift in one transition appears to be quantitatively greater in the winter hardy varieties. Cold-induced changes in all of the other measured parameters were indistinguishable in hardy and nonhardy varieties. The results indicate major involvement of the phospholipid matrix in cold acclimation. A link between cold acclimation and winter survival may exist involving the structural and functional modifications in membrane structure which occur during acclimation.

Artificial membranes bearing immobilized enzymes can be used to study some effects of membrane structure on enzyme kinetic behavior. The bienzyme system described is a mixture of beta-glucosidase and glucose oxidase. Gluconolactone, the product of thesecond enzyme, is an inhibitor of the first one. The resulting feedback effect has been compared using a mixed two-enzyme membrane, two separated one-enzyme membranes, and astirred bienzyme solution. The feedback effect is quicker and more efficient in the two-enzyme membrane than in solution; it is slower and less efficient in the case of the separated one-enzyme membranes. Effects of enzyme proximity in the structure are discussed. Conclusions are drawn concerning the efficiency of feedback mechanisms when enzymes are embedded within a single structure.

Summary The influence of divalent cations on the structure of the plasma membrane and the inner and outer acrosomal membranes of human ejaculated spermatozoa was investigated by electron microscopy. Calcium and strontium ions produced identical effects on the outer acrosomal membrane, namely an increase in thickness by about 20%, but did not change the thickness of the plasma membrane and the inner acrosomal membrane. Magnesium ions did not affect the thickness of any of the membranes. Averaged membrane profiles of the outer acrosomal membrane showed a marked difference between membranes fixed in the presence of calcium and the control. Electron microprobe analysis revealed an increase of the amount of calcium in the acrosomal region of the calcium-treated spermatozoa. It is concluded that the changes in membrane structure are due to the binding of calcium ions to the outer acrosomal membrane, indicating that the site of action of calcium on the acrosome reaction is located on the outer acrosomal membrane.

E. coli cells treated with the bifunctional crosslinking reagents dimethyl malonimidate, succinimidate, adipimidate, suberimidate, and sebacinimidate served for the isolation of rod-shaped “ghosts.” These ghosts proved to be crosslinked over their entire surface; i.e., a macromolecule (resistant to boiling 1% Na dodecyl sulfate) the size of the cell had been created. Also, ghosts could similarly be crosslinked. In both cases, the final “sacs” contained about 60-70% protein, and very little or no lipopolysaccharide. When ghosts from which phospholipid had been removed were crosslinked, the covalently closed ghosts were almost pure protein; 80-90% of their dry mass was accounted for by protein. Ammonolysis of the crosslinked material (whether stemming from crosslinked cells or ghosts) showed that the same four proteins (Na dodecyl sulfate gel bands) had been crosslinked that are found in normally prepared ghosts. These observations practically exclude the hypothesis that a fluid mosaic model of membrane structure can be applied to the outer membrane of the E. coli cell envelope; rather, extensive protein-protein interactions must exist over the whole surface of this membrane. These findings are consistent with the possibility that the ghost polypeptide chains are involved in the determination of cellular shape.

Proton nuclear magnetic resonance (PMR) spectra at 270 MHz of aqueous dispersions of nonsonicated egg lecithin, dipalmitoyl lecithin, egg lecithin-cholesterol (1 : 1) and dipalmitoyl lecithin-cholesterol (1 : 1), together with PMR spectra of mitochondrial membranes and their extracted lipids, have been obtained. Carbon-13 nuclear magnetic resonance (CMR) spectra at 25.2 MHz of egg lecithin, egg lecithin-cholesterol (1 : 1) and sphingomyelin, together with CMR spectra of chloroplast and mitochondrial membranes, and erythrocyte ghosts, have also been obtained. The results obtained using CMR appear very promising for further study of intact membrane structure. It is suggested, on the basis of CMR evidence, that the proteins in mitochondrial membranes may be considerably less mobile than the lipids .

Chloroplasts from spinach were fixed in glutaraldehyde and extracted with three different lipid solvents, after which the lipid composition was analyzed. Studies were also made with the electron microscope. In cold dry acetone, which removes 75 % of lipids, the basic structure of the membranes is unchanged. Acetone with 10 % water removes 89 % of the lipids and a mixture of chloroform with methanol removes 93 % of the lipids, both solvents leaving nearly unrecognizable membrane structures. The relationship between lipid composition and membrane structure is discussed.

A method of isolating the thick luminal membrane from homogenates of bladder epithelium is described, which entails pretreatment of the epithelium with fluorescein mercuric acetate and centrifugation of the homogenate on sucrose density gradients. A hexagonal array of hexamers is illustrated by negative contrast staining in whole mounts of the isolated thick membrane. Subunits are also shown in tangential sections of this thick membrane, in fixed, embedded bladder epithelium. The significance of the subunits is discussed in the context of membrane structure and permeability.

The two brain specific proteins S100 and 14.3.2 interact with lipid membranes inducing an increase in their permeability to cations in physiological conditions of pH and ionic strength. Such effect is regulated by Ca ++ and varies according to the membrane structure. Glucose permeability, in the same experimental conditions, is not affected by the two proteins.

To elucidate the organization of cytochrome oxidase in the mitochondrial membrane, a study has been made on the correlation between the fine structure of a cytochrome oxidase-rich submitochondrial membrane (green membrane) and purified cytochrome oxidase. The green membrane was obtained directly from beef heart mitochondria or from the mitochondrial electron-transfer particles by treatment with deoxycholate and potassium chloride, followed by centrifugation. Heme a content of the green membrane isolated from beef heart mitochondria and from the electron-transfer particles were about 2 nmoles per milligram protein and 3–4 nmoles per milligram protein, respectively. Electron micrographs of the green membrane revealed regular arrays of small particles on the surface of the membrane, each particle measuring approximately 50–60 A in diameter with a center-to-center distance of about 70 A. These particles were extracted from the green membranes and recovered in a purified cytochrome oxidase fraction. Membrane structure was re-formed with the purified cytochrome oxidase, and the surface of the reconstituted membrane exhibited a particulate structure similar to the original green membrane. These results indicate that there is a correlation between the particles on the green membrane and cytochrome oxidase.

The submicroscopic structure of the adrenal medulla was studied with the freeze-etching technique. In general the results are in good agreement with those obtained from chemically fixed specimens of adrenal medullas. Chromaffin granules show no interconnections; on the periphery of the cells they are closely aligned to the cell membrane. These features are discussed in relation to the secretion mechanism. The insertions of cristae mitochondriales appear as holes in the inner membrane. Examination of isolated chromaffin granules and of those in the intact cell showed that the fracturing planes run along either the surface or the inner part of the membranes. This is probably true also for other membranes. The exposed faces of the membranes vary from smooth to granular. The appearance of the membrane structure is altered after preincubation with glycerol in isotonic solution.

The limiting membrane of the equatorial segment of the acrosome has a five layered appearance due to a thin dense layer apposed to the inner aspect of the unit membrane. Shorter localized areas with the same appearance are seen in the inner membrane of the anterior segment of the acrosome, but most of this membrane and all of the outer membrane of this segment show the tri-laminar unit membrane structure. In longitudinal sections the postacrosomal sheath is seen to consist of two different portions separated by a circumferential groove in the cell membrane. The anterior part is composed of a homogeneous dense layer from which regularly arranged projections extend toward the cell membrane. The posterior part is composed of a dense granular or homogeneous material apposed to the inner aspect of the cell membrane. This layer is limited posteriorly by a close union of the cell membrane with the nuclear envelope via a dense homogeneous material. In the implantation fossa the two layers of the nuclear envelope are interconnected by regularly spaced linear structures. The centriole found in this region has a constant orientation with respect to the long axis of the flagellum, and a system of numbering the triplets is proposed. The coarse fibers of the flagellum are finely cross striated with a period of 40 A. Near the abaxial surface of the fibers every forth line is somewhat thicker than the others, thus demarcating a major period of 160 A with three intraperiod lines of which the middle one is slightly thicker than the others.—A somewhat regular mode of termination of the tubular elements of the flagellum in the end piece is described.

The functional activity of isolated mitochondria is known to be inhibited by sucrose. An investigation of the effect of sucrose on membrane structure as judged by lipid spin-label localization and mitochondrial function as judged by energizedproton translocation was undertaken. High sucrose concentrations inhibit oxygen-dependent hydrogen transport in mitochondrial and submitochondrial preparations and concomitantly change the partitioning of spin-label probes between polar and nonpolar membrane phases. It was concluded that sucrose probably interacts directly with the phospholipids of the membrane to cause disorder of the lipid structure. This conclusion is supported by using several types of nitroxide spin labels. Uncoupling agents such as fluorocarbonyl cyanide phenylhydrazone and 2,4-dinitrophenol influence lipid spin-label behavior in the same way as sucrose, only to a lesser degree. Changes in membrane structure are not brought about by sucrose in glutaraldehyde-fixed SMP which implies that the lipid domain is protein bounded.

Light microscopy studies have established the retina of Coleonyx as containing only rod-type visual cells of three classes, Single rods, and two classes of Double rods. Three additional classes of rods were found in this electron microscopic study which included a Twin rod and two classes of Triplet rods. A new visual cell class nomenclature was presented and defined, and was based on the general morphology of the receptor cell and the relationship of the receptor members to each other. The terminology presented was: Single, Doublets (D 1 and D 2 ), Twin, Triplet 1 and Triplet 2 . A Quintuplet rod was described, but only one was ever found, and this class may not be of physiological significance. The receptor outer segment disks had a five-layered membrane structure when the tissue was fixed in osmium tetroxide and the sections were stained with saturated uranyl acetate at 60°C. The ellipsoid body was comprised of tightly packed mitochondria which fell into two size groups: small mitochondria (3.0–5.5 m μ ) and large mitochondria (11–18 m μ , with a few up to 35 m μ ). The distribution of the small mitochondria formed a hemisphere about the base and sides of the ellipsoids with the large mitochondria in the ellipsoid center. Two types of radially symmetrical paraboloids were described, a granular type which had a central granular mass surrounded by a system of membrane lamellae, and a membranous type which lacked the central granular mass and consisted only of radially symmetrically arranged paired membrane lamellae between which granules were located. In cross sections of the vitread paraboloid and myoid, radial plasma membrane extensions from the visual cells, the lateral fins, were seen to interdigitate freely with Muller cell cytoplasmic extentions. A function of the inner segment-paraboloid-myoid structures in relation to a possible metabolic pathway was presented and discussed.

An attempt has been made to rationalize the variables in the preparation procedure of Loeb-Sourirajan-type reverse-osmosis membranes. The quaternary phase diagram of the system cellulose acetate–acetone–formamide–water was determined and has proved a useful tool in the discussion of membrane structures and properties. A mechanism based on differences in the precipitation rate of the polymer during the membrane formation process has been suggested to explain the observed asymmetry in the membrane structure. The porosity of the membrane has been ascribed to the relative rates of water entering and solvent leaving the cast film. The effects of the casting solution composition, the evaporation time, the wash bath temperature, and the annealing procedure have been studied. X-Ray diffraction and electron microscopy were used to supplement flux and retention data of membranes made from a cellulose acetate–formamide–acetone casting solution.

The spectra of non-covalent spin labels have been examined in the presence of erythrocyte membranes and their separated lipid and protein components. Evidence has been obtained for the perturbations induced in the membrane structure by a range of extraneous molecules. The perturbations are detected either as changes in the spectra of the bound spin label, or as changes in the partition of the spin label into the membrane. A comparison of the perturbations detected by different spin labels provides preliminary evidence for the localisation of the perturbing agents in the membrane. With steroid spin labels it is possible to detect the onset of irreversible structural breakdown in the membrane at high concentrations of perturbing agents. This results in the exposure of new protein binding sites for the steroid spin labels which are not accessible in the intact membrane. The interpretation of the spin label experiments is supported in detail by directly comparable studies of the nuclear magnetic relaxation of the same perturbing agents in erythrocyte membranes.

The relationships of phospholipid to membrane structure and function were examined in hepatic microsomes. Findings indicate that normal microsomal membrane structure is dependent on lipid-protein interactions and that it correlates closely with glucose-6-phosphatase activity. Modification of most phospholipid with phospholipase-C is associated with widening of the membrane which can be reversed following readdition of phospholipid.

The combined x-ray diffraction and electron microscopic examination of myelin has provided reasonable, but not conclusive, support for its structure as a basically bimolecular leaflet of phospholipid that is partially interspersed with protein. But there is very little basis for extending this concept to biological membranes in general. There is no adequate experimental support for the specific orientation of phospholipids as proposed in the unit membrane theory or for the proposed polar nature of protein-lipid bonds, even in myelin. Membranes differ widely in chemical composition, metabolism, function, enzymatic composition, and even in their electron microscopic image. The only similarity is their general resemblance in electron micrographs, but, until more is known about the chemistry of electron microscopy, this evidence cannot be interpreted with confidence. One positive conclusion to which I have come is that much more chemical evidence must, and can, be obtained. Techniques for the isolation of membranes are improving and protein and lipid chemistry are now highly refined arts. Quantitative analysis of many different membranes is possible and the data can be related in some instances, notably bacterial plasma membranes, to calculations of surface area. Chemical and physical changes induced in membranes of widely different lipid composition by the preparatory procedures of electron microscopy can be determined directly and correlated with the electron microscopic image. Model systems can be assembled whose compositions closely resemble those of biological membranes. Membranes can be disassociated into subunits whose properties can be studied. In particular, x-ray diffraction analysis and electron microscopy by negative staining of reaggregates of lipoproteins isolated from membranes would be very informative. Perhaps most important, the problem of membrane structure must be considered in relation to the problems of membrane function and membrane biosynthesis.

1. The relation between osmotic gradient and rate of osmotic water flow has been measured in rabbit gall-bladder by a gravimetric procedure and by a rapid method based on streaming potentials. Streaming potentials were directly proportional to gravimetrically measured water fluxes. 2. As in many other tissues, water flow was found to vary with gradient in a markedly non-linear fashion. There was no consistent relation between the water permeability and either the direction or the rate of water flow. 3. Water flow in response to a given gradient decreased at higher osmolarities. The resistance to water flow increased linearly with osmolarity over the range 186-825 m-os M. 4. The resistance to water flow was the same when the gall-bladder separated any two bathing solutions with the same average osmolarity, regardless of the magnitude of the gradient. In other words, the rate of water flow is given by the expression (Om — Os)/[Ro′ + ½k′ (Om + Os)], where Ro′ and k′ are constants and Om and Os are the bathing solution osmolarities. 5. Of the theories advanced to explain non-linear osmosis in other tissues, flow-induced membrane deformations, unstirred layers, asymmetrical series-membrane effects, and non-osmotic effects of solutes could not explain the results. However, experimental measurements of water permeability as a function of osmolarity permitted quantitative reconstruction of the observed water flow—osmotic gradient curves. Hence non-linear osmosis in rabbit gall-bladder is due to a decrease in water permeability with increasing osmolarity. 6. The results suggest that aqueous channels in the cell membrane behave as osmometers, shrinking in concentrated solutions of impermeant molecules and thereby increasing membrane resistance to water flow. A mathematical formulation of such a membrane structure is offered.

A study was made of the effect of mitochondrial size on the electrical properties of the membrane and the internal conductivity of mitochondria. The dielectric constant and electrical conductivity of suspensions of guinea pig heart mitochondria were examined in the frequency range 5 x 105 to 2.5 x 108 C.P.S. Membrane capacity was calculated to be 1.1 to 1.3 µf./cm.2 and was virtually the same in mitochondria whose surface area was made to vary by a factor of 4 by osmotic means. This finding suggested that some mechanism must exist for the transfer of mitochondrial material into membrane structure during fluctuations in mitochondrial size. The electrical capacity of the membrane was unaffected by a 33-fold change in potassium chloride concentration. The internal conductance of swollen mitochondria was 2 to 3 times lower than that of the external medium. In shrunken mitochondria the internal conductance was virtually independent of the conductivity of the external medium. These results were discussed in relation to current concepts of mitochondrial structure.

Exposures of intact rat liver microsomes to various sulfhydryl reagents or to concentrations of taurocholate below 1 mM inhibited glucose 6-phosphate phosphohydrolase activity. The inhibitions were abolished by subsequent disruption of membrane integrity by higher concentrations of taurocholate or 1M NH 4 OH. Also the reagents were ineffective when tested directly with disrupted microsomes. Thus, a requirement for an intact membrane structure in the action of these reagents is indicated. Moreover, the involvement of a second component of the endoplasmic reticulum in the catalysis of glucose 6-phosphate hydrolysis is suggested.

Two of the fractions obtained by density gradient centrifugation of Tris-disrupted brush borders from hamster intestinal mucosa have been identified as the microvillus cores and their surrounding membranous coats, respectively. This identification has the following morphological basis. In shadowed preparations one fraction (cores) appears as rounded, compact rods, and the other fraction (coats) appears as flattened sheets. Both rods and sheets have dimensions appropriate to the identities assigned to them. In addition, negative staining shows that the rods are composed of aligned particles of roughly 60 A, consistent with the appearance of the core in tissue section, where 60-A fibrils are characteristic. The sheets are covered by non-aligned particles of approximately the same size. Sectioned preparations show that the core fraction contains predominantly fibrous material with some membranous contamination and that the coat fraction is apparently composed exclusively of elongated sacs with a unit membrane structure. Some details of the structure of the core are evident in cases where the compact rod appears to be loosened, revealing a doubled strand. The strand is approximately 350 A wide; the compact rod is roughly twice this width. With negative staining the strand shows a dense central region. The morphological identification presented here is consistent with the distribution of enzymic activity among the density gradient fractions described in the preceding paper.