Lateral diffusion in inhomogeneous membranes. Model membranes containing cholesterol.

The problem of lateral diffusion in inhomogeneous membranes is illustrated by a theoretical calculation of the lateral diffusion of a fluorescent lipid probe in binary mixtures of phosphatidylcholine and cholesterol under conditions of temperature and composition such that this lipid mixture consists of alternating parallel domains of fluid and solid lipid, having separations that are small compared with the distance scale employed in photobleaching experiments. The theoretical calculations clearly illustrate how inhomogeneities in membrane composition affecting the lateral motion of membrane components on a small (10-100 nm) distance scale can give complex diffusive responses in experiments such as fluorescence photobleaching that employ comparatively macroscopic distances (10-100 micrometers) for the measurement of diffusive recovery. The theoretical calculations exhibit the unusual dependence of the apparent lateral diffusion coefficient of a fluorescent lipid probe on lipid composition in binary mixtures of cholesterol and phosphatidylcholines as reported by Rubenstein et al. (1979, Proc. Natl. Acad. Sci. U.S.A., 76:15-18).     

Affinity partitioning of membranes. Cholinergic receptor-containing membranes from Torpedo californica.

Affinity partitioning has been employed in the purification of membranes rich in cholinergic receptor from Torpedo californica electric organs. The procedure involves a modification of poly(ethylene oxide)-dextran aqueous phase partitioning systems where a ligand selective for the receptor is conjugated to the poly(ethylene oxide). Specific partitioning of the receptor-containing membranes into the poly(ethylene oxide)-rich phase occurs when bis-alpha,omega-trimethylamino poly(ethylene oxide) or bis-rho-tri-methylammonium phenylamino poly(ethylene oxide) was added to the phase system in low mole ratio. bis-alpha,omega-Methylamino poly(ethylene oxide), which should impart equivalent interfacial electromotive potential to the system but bind poorly to the receptor sites, was much less effective in producing phase distribution changes. The ligand-polymer-dependent phase distribution shifts were blocked by bisquaternary methonium ligands at concentrations consistent with their relative affinities for the cholinergic receptor. Titration or receptor sites with cobra alpha-toxin decreased the phase distribution changes in a linear fashion up to the point of stoichiometry. These observations are consistent with the phase distribution changes being consequent to ligand-polymer association with the pharmacologically important site on the receptor. The affinity partitioning procedure, when employed following an initial purification of the membranes by differential and density gradient centrifugation, yields membrane preparations with a high degree of morphological uniformity and a specific activity between 2.9 and 4.6 nmol of bound cobra alpha-toxin/mg of protein.     

Bilayer-gel membranes. Formation and some properties.

We developed a new procedure which induces multifunctional reagents to crosslink at one interface between a black bilayer and the adjacent water phase. This procedure yields "bilayer-gel" membranes, i.e. membranes consisting of a bilayer and a polymer layer. The bilayer-gel membrane may tentatively be considered to be a new membrane system, because the formation of the polymer layer changes some bilayer properties. We studied bilayer-gel membranes composed of a bilayer of oxidized cholesterol and of a polymer layer of poly-L-lysine crosslinked by glutardialdehyde. Compared to unmodified bilayers, this membrane system has an electrical conductance of the same magnitude, the same electrical capacity and similar shapes of current-voltage dependences. However, this system is asymmetrical and differs in ion selectivity and increased stability from an unmodified bilayer.     

Colicins and bacterial membranes: structures and functions. II. Studies on reconstituted homologous and hybrid membranes prepared from cytoplasmic membranes of untreated and colicin K-treated bacteria.

Cytoplasmic membranes of Escherichia coli K12 C600 treated and not treated with colicin K were dissociated into unsolubilized and solubilized fractions. Neither fraction catalyzed ATP-linked transhydrogenase activity. Mixtures of unsolubilized fractions of the untreated bacteria with solubilized fractions of either the treated or untreated bacteria yielded reconstituted membranes with restored ATP-linked transhydrogenase activity. The level of the activity was similar to that of the undissociated membranes of untreated bacteria. The membranes which were reconstituted from unsolubilized fractions of the treated bacteria and the solubilized fraction of the treated or the untreated bacteria showed impairment of activity. The impairment is not due to an inability to bind ATPase of the soluble fraction or to an incorrect binding of the ATPase. The impaired, reconstituted membranes showed striking decreases in the relative amounts of three proteins with apparent molecular weights of 122,000, 73,000, and 62,000. The affected proteins were found to be components of the unsolubilized membrane fraction. It is, thus, concluded that the impaired activity is due to the defective nature of the unsolubilized membrane fraction of colicin-treated cells.     

Lymphocyte plasma membranes. III. Composition of lymphocyte plasma membranes from normal and immunized rats

Isolated plasma membranes of thymic and splenic lymphocytes from unimmunized and immunized rats of the inbred ACI and F344 strains were analyzed for chemical and enzymatic composition, for membrane protein patterns by polyacrylamide gel electrophoresis and for membrane-associated immunoglobulins. After immunization, the thymic and splenic lymphocyte membranes from F344 rat contained less carbohydrate and higher phospholipid contents than control animals. In both ACI and F344 inbred rat strains the membrane phospholipid to cholesterol weight ratio increased significantly after immunization. The electrophoretic patterns of solubilized membrane proteins and of iodinated external membrane proteins were similar in unimmunized and immunized animals.When thymic and splenic lymphocytes of normal or immunized animals were surface radioiodinated, solubilized in Triton X-100, NP-40 or 10 M urea in 1.5 M acetic acid and analyzed by immunoprecipitation, labeled IgM immunoglobulin was recovered from thymic lymphocytes but both labeled IgG and IgM were recovered from splenic lymphocytes. However, when unlabeled isolated plasma membranes were solubilized in 1% Triton X-100 and analyzed by immunodiffusion in agarose gels, both IgG and IgM were identified in thymic and splenic cells.     

Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes.

Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.     

Fusion of synaptic vesicle membranes with planar bilayer membranes.

The interaction of synaptic vesicles with horizontal bilayer lipid membranes (BLMs) was investigated as a model system for neurotransmitter release. High concentrations (200 mM) of the fluorescent dye, calcein, were trapped within synaptic vesicles by freezing and thawing. In the presence of divalent ions (usually 15 mM CaCl2), these frozen and thawed synaptic vesicles (FTSVs) adhere to squalene-based phosphatidylserine-phosphatidylethanolamine BLMs whereupon they spontaneously release their contents which is visible by fluorescence microscopy as bright flashes. The highest rate of release was obtained in KCl solutions. Release was virtually eliminated in isotonic glucose, but could be elicited by perfusion with KCl or by addition of urea. The fusion and lysis of adhering FTSVs appears to be the consequence of stress resulting from entry of permeable external solute (KCl, urea) and accompanying water. An analysis of flash diameters in experiments where Co+2, which quenches calcein fluorescence, was present on one or both sides of the BLM, indicates that more than half of the flashes represent fusion events, i.e., release of vesicle contents on the trans side of the BLM. A population of small, barely visible FTSVs bind to BLMs at calcium ion concentrations of 100 microM. Although fusion of these small FTSVs to BLMs could not be demonstrated, fusion with giant lipid vesicles was obvious and dramatic, albeit infrequent. Addition of FTSVs or synaptic vesicles to BLMs in the presence of 100 microM-15 mM Ca2+ produced large increases in BLM conductance. The results presented demonstrate that synaptic vesicles are capable of fusing with model lipid membranes in the presence of Ca+2 ion which, at the lower limit, may begin to approach physiological concentrations.