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Glycophorin, the major glycoprotein of human erythrocytes, has been isolated and reincorporated into lipid vesicles. Freeze-fracture electron microscopy shows the reincorporated glycophorin to occur as small particles in vesicle fracture faces while the etch faces are smooth. The glycoprotein has a tendency to cluster into groups of several particles. Evidence is presented that, although lipids in immediate contact with glycopherin are likely somewhat immobilized, the entire lipid-protein complex has a tendency to occupy fluid regions of the bilayer. Reincorporated glycophorin assumes its proposed conformation in the intact erythrocyte in so far as it penetrates the hydrophobic membrane interior while its N-terminal end with attached carbohydrate residues is exposed to the aqueous compartment and is available as a specific recognition site.

Three classes of lipoidal nitroxide spin probes reversibly perturb erythrocyte membranes at low concentrations (10(-10)-10(-5) M). This is manifest in (a) decreased osmotic fragility and (b) alterations of surface topology. At bulk phase nitroxide concentrations providing maximal osmotic stabilization, the erythrocytes exhibit a classic echinocyte morphology. At nitroxide concentrations very slightly higher than those yielding minimal osmotic fragility (10(-5)-10(-4) M), the cellus undergo a sphering reaction and lyse. The morphologic sequence seen in intact cells is not observed in erythrocyte ghosts. We suggest that the spin probes initially concentrate in focal domains, which expand into echinocytic protrusions primarily due to localized weakening of membrane cohesion. We propose that cell lysis involves an irreversible breakdown in membrane domain structure.

Association of purified acetylcholine receptor from Torpedo californica electroplax with lipids from the same organism results in a vesicular membrane system in which the receptor protein is oriented so that all neurotoxin binding sites appear to be on the outer surface. The reconstituted system is chemically excitable by acetylcholine and carbamylcholine, as measured by (22)Na(+) efflux. This excitability is specifically blocked by the antagonist alpha-bungarotoxin. These results demonstrate that the purified receptor macromolecule contains not only the specific neurotransmitter binding site but also the molecular elements necessary for ion translocation in order to effect postsynaptic depolarization.

We propose that membranes whose proteins and polar lipids are distributed asymmetrically in the two halves of the membrane bilayer can act as bilayer couples, i.e., the two halves can respond differently to a perturbation. This hypothesis is applied to the interactions of amphipathic drugs with human erythrocytes. It is proposed that anionic drugs intercalate mainly into the lipid in the exterior half of the bilayer, expand that layer relative to the cytoplasmic half, and thereby induce the cell to crenate, while permeable cationic drugs do the opposite and cause the cell to form cup-shapes. This differential distribution of the drugs is attributed to interactions with the phosphatidylserine that is concentrated in the cytoplasmic half of the membrane. Impermeable amphipathic drugs intercalate only into the exterior half of the bilayer, and therefore are crenators of the intact cell. Several predictions of this hypothesis have been confirmed experimentally with erythrocytes and erythrocyte ghosts. The bilayer couple hypothesis may contribute to the explanation of many membrane-mediated phenomena in cell biology.

The electrical response of a pigmented lipid bilayer to a short laser pulse is measured by a tunable voltage clamp method. In this method, a variable access impedance permits "tuning" of the observed relaxations for optimal measurements. Analysis of the data so obtained leads to an equivalent circuit that contains a novel chemical capacitance charged by the specific photoreaction across a single membrane-water interface. This chemical capacitance is distinct from the ordinary membrane capacitance. The intrinsic chemical rate constant obtained from the equivalent circuit analysis is shown to be the pseudo-first-order rate constant of the reverse dark reaction of the reduced acceptor and the oxidized pigment. The tunable voltage clamp method of measurement and analysis allows unambiguous separation of this rate constant into resistive and capacitative elements, which are interpreted in molecular terms.

Phosphatidylcholines, sphingomyelins, cholesterol, and cholesterol esters were enriched with (13)C by chemical synthesis in specific positions of their hydrophilic groups and aliphatic chains. Their spin-lattice relaxation times were determined in organic solvents. The substances were organized as liposomes and recombined with total human high density apolipoproteins and the two separated main components, apolipoprotein A-I (apoLp-Gln-I) and apolipoprotein A-II (apoLp-Gln-II). These (13)C nuclear magnetic resonance data established that in reassembled high density lipoproteins the phospholipid molecules bind to the apoprotein moieties with their hydrophobic fatty acid chains and not with their hydrophilic zwitterionic groups. Apolipoprotein A-I preferentially binds phosphatidylcholine, although its lipid-binding capacity is smaller than that of apolipoprotein A-II. Apolipoprotein A-II avidly reassembles with sphingomyelin by hydrophobic interactions. A model of the molecular organization of the high density lipoportein particle has been derived.

The ability of various nonionic detergents to solubilize D-alanine carboxypeptidase and other membrane-bound enzymes was correlated to a physical property of the detergent, the HLB number. Only a fairly narrow range of detergents were effective solubilizing agents. Purified carboxypeptidase required either a detergent or detergent plus a lipid fraction for stability. Only those detergents effective in solubilizing the enzyme were effective in stabilizing it.

Electron spin resonance analysis of suspensions of animal cell plasma membranes consistently reveals four characteristic temperatures for lateral phase separations in the membrane lipids. Similar analysis of an aqueous dispersion of lipids extracted from these membranes reveals only two characteristic temperatures, indicating that some aspect of lipid organization in membranes is destroyed by the extraction procedure. The characteristic temperatures for surface membranes from two different species of homeothermic animals were nearly identical and were approximately 37 degrees , 31 degrees , 21 degrees , and 15 degrees . A treatment of the physical data revealed that these temperatures could identify independent phase transitions for two hydrocarbon compartments of approximately equal size with lower and upper characteristic temperatures of 21 degrees and 37 degrees , and of 15 degrees and 31 degrees . The analysis of the effects of temperature on a number of physiological parameters indicates that 21 degrees and 37 degrees are likely to define the boundaries for lateral phase separations in the inner monolayer and 15 degrees and 31 degrees the boundaries for lateral phase separations in the outer monolayer.

The growth of mouse myeloma XS 63.5 cells in cell culture is dependent on serum. Among the several growth factors present in serum, the lipid fraction is highly active. The growth factor(s) provided by the serum lipid fraction can be replaced by unsaturated fatty acids. If XS 63.5 cells are cultured in medium that is low in serum lipids, the cells become quiescent, with most of the cells in the G(1) (or G(0)) phase of the cell cycle. Addition of linoleic acid to such quiescent cells leads to reinitiation of DNA synthesis and growth.

A recently developed nuclear double resonance technique which permits sensitive detection, together with high resolution, of rare spins in solids or other dipolar-coupled nuclear systems [Pines, Gibby, and Waugh (1973) J. Chem. Phys. 59, 569] has been applied to the study of natural abundance (13)C-nuclear magnetic resonance in lipid mesophases and of selectively labeled carbon sites in bacterial membranes.Detailed microscopic information on the molecular organization and phase transitions of the lipid phases and their interaction with ions and other molecules can be obtained from the study of the chemical shift anisotropies and dynamical aspects of the (13)C NMR spectra of unsonicated lipid dispersions (liposomes). Experiments are reported which demonstrated the feasibility of quantitatively observing the (13)C-nuclear magnetic resonance of specifically labeled sites in unperturbed Escherichia coli membrane vesicles for the study of the physical state of the lipids with the aim of relating it to the known lipid-dependent functional properties of the membranes.

Studies on contact-inhibited mouse embryo fibroblast 3T3 cells and 3T3 cells transformed by oncogenic RNA and DNA viruses and by a chemical carcinogen have demonstrated differences in plasma membrane architecture. Spin-label and freeze-fracture ultrastructural studies have shown that contact-inhibited cells have ordered membrane lipids and aggregated intramembranous particles, whereas transformed cells have fluid membrane lipids and randomly distributed intramembranous particles. These findings suggest a model for how changes in the cell membrane may account for some of the characteristic differences observed between contact-inhibited and transformed cells.

This paper presents a simple theory for the calculation of micelle size and other properties of micelleforming systems. The theory is based on the separate estimation of the components of the free energy arising from hydrophobic attraction and from head group repulsion, each as a function of micelle size. The difficult problem of an a priori calculation of the repulsive factor is circumvented by using experimental values of this parameter, derived from pressure-area curves of monolayers at an interface between an aqueous solution and liquid hydrocarbon. Though some parameters of the theory are still somewhat uncertain, self-consistent and physically realistic values can be assigned that lead to almost exact prediction of experimental micelle sizes and critical micelle concentrations for two distinctly different ionic detergents.

Changes in slope of Arrhenius plots for transport can, in some instances, be detected at two different temperatures for cells that have a relatively simple fatty-acid composition in the membrane lipids. These characteristic temperatures correlate with the characteristic temperatures that define changes of state in membrane phospholipids as revealed by the paramagnetic resonance of the spin label TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl). The higher of these characteristic temperatures is that at which the formation of solid patches of membrane lipids is first detected. The lower is the end point of the course of lateral phase separations, at which all the membrane lipids are in a solid phase. For cells enriched for elaidic acid, the rate of transport increase by as much as 2-fold as the temperature is decreased by less than 1 degrees , at the higher characteristic temperature. At this characteristic temperature, lateral phase separations begin in the membrane phospholipids. This is also the temperature where one predicts a striking increase in the lateral compressibility of the membrane lipids. These data are thus interpreted to indicate that a component of the transport system vertically penetrates one or both monolayer faces of the membrane during transport, or that some other event involving the lateral compression of the phospholipids is important for transport.

A series of helical structures for gramicidin A, with alternating L and D residues, are characterized as to number of residues per turn, atoms in hydrogenbonded rings, and dihedral angles. Because of alternating peptide C-O directions, these helices are capable of forming head-to-head hydrogen-bonded dimers with the capacity of functioning as transmembrane channels. The dimers are characterized as to channel length, pore size, and expected ion selectivity. In a test of the proposed head-to-head association for channel formation, the malonyl dimer [N,N'-(dideformyl gramicidin A)-malonamide] was synthesized. The chemical and conformational integrity of the product was verified by nuclear magnetic resonance; in lipid bilayer studies, the dimer was found to be a potent mediator of ion conductance with the predicted concentration dependence.Thus, the results on malonyl gramicidin A prove head-to-head association in formation of the transmembrane channel, and the results are consistent with the specific geometrical configuration involved in head-to-head dimerization of pi((L,D)) helices. At this stage, the action of gramicidin A on membranes with lipid-layer thicknesses of 30 A or less can best be understood in terms of the pi((L,D)) helix with 6.3 residues per turn.

Experimental data obtained on bilayer membranes treated with either of two antibiotics, monazomycin or alamethicin, are presented showing the marked difference in the time course of the rise and subsequent decay of the conductance in response to a positive and negative step of potential established by means of a voltage-clamp feedback circuit. The variation of the conductance with time in these model systems qualitatively mimics the behavior of the "potassium conductance" of squid giant axons and other excitable biological systems; namely, the rise of the conductance to the steady state requires a longer time than its decay to the resting state. For the alamethicin system, the decay time becomes very brief-as short as 50 musec-as either the salt or the alamethicin concentration is reduced, while the rise time remains several seconds. This marked brevity of the decay time versus the rise time may have implications for the mechanism underlying the formation of conducting channels in such membranes.

Cultures of a yeast mutant, deficient in the synthesis of unsaturated fatty acids, were supplemented with either stearolic acid or with any one of three octadecanoic acids having a cis double bond 6, 9, or 11 carbons away from the acyl group. The resulting cells, with lipid alkyl chains well defined with respect to the position and nature of unsaturated sites, were then studied with spin-labeled stearic acids having a N-oxyloxazolidine ring located at 4, 6, 9, or 12 carbons away from the acyl group, and added in vitro to the cellular preparations. Differences in the molecular motion of each spin label were observed, as a function of the unsaturated site, in the intact yeast cells. Characteristic order to disorder phase transitions are inferred from data of temperature dependence. The results also indicate that triple bonds and cis double bonds inhibit a relatively ordered packing of lipid alkyl chains in the region between unsaturated sites and terminal methyl groups, leaving the hydrophobic region bounded by acyl groups and unsaturated sites unaffected.

Liposomes containing trapped glucose marker were prepared from the chloroform-soluble fraction of sheep erythrocyte membranes. These liposomes release glucose when incubated with rabbit anti-sheep erythrocyte serum and a source of complement. Experiments with purified human complement components show that loss of marker is absolutely dependent on the presence of components 2 and 8. An absolute requirement for component 9 cannot be demonstrated, although it stimulates glucose release from the liposomes. These results establish a parallelism between the response of biological membranes and liposomal membranes to antibody and complement.

The effects of temperature and of the action of a purified phospholipase C enzyme preparation on human red blood cell membranes has been investigated by chemical analyses, circular dichroism, and proton magnetic resonance measurements. The results indicate that a substantial fraction of the phospholipids and the proteins of the membranes can change structure independently of one another, suggesting a mosaic pattern for the organization of the lipids and proteins in membranes.

A model, based on the available chemical, electron microscopic, and x-ray diffraction data, has been developed for the structure of the retinal rod outer segment membranes. The model consists of a double layer of globular protein molecules, with lipid bilayer filling the spaces between the proteins. The electron density across the membrane has been calculated and shown to be in agreement with that found by x-ray diffraction. The protein molecules "float" half submerged in a lipid field; this serves to orient them in the same way at all times relative to incident radiation in the intact retina.

Bimolecular membranes are formed from two lipid monolayers at an air-water interface by the apposition of their hydrocarbon chains when an aperture in a Teflon partition separating two aqueous phases is lowered through the interface. Formation of the membrane is monitored by an increase of the electrical capacity, as measured with a voltage clamp. Electrical resistance of the unmodified membrane is analogous to that of conventional planar bilayers (black lipid membranes) prepared in the presence of a hydrocarbon solvent, i.e., 10(6)-10(8) ohm cm(2); the resistance can be lowered to values of 10(3) ohm cm(2) by gramicidin, an antibiotic that modifies the conductance only when the membranes are of biomolecular thickness. In contrast to the resistance, there is a significant difference between the capacity of bilayers made from mono-layers and that of hydrocarbon-containing bilayers made by phase transition; the average values are 0.9 and 0.45 muF cm(-2), respectively. The value of 0.9 muF cm(-2) approximates that of biological membranes. Assuming a dielectric constant of 2.1 for the hydrocarbon region, the dielectric thickness, as calculated from a capacity of 0.9 muF cm(-2), is 22 A. This value is 6-10 A smaller than the actual thickness of the hydrocarbon region of bilayers and cell membranes, as determined by x-ray diffraction. The difference may be due to a limited penetration of water into the hydrocarbon region near the ester groups that would lower the electrical resistance of this region and reduce the dielectric thickness. Asymmetric membranes have been formed by adjoining two lipid monolayers of different chemical composition.