Influence of glycophorin incorporation on Ca2+-induced fusion of phosphatidylserine vesicles

The effect of incorporation of glycophorin, the major integral sialoglycoprotein of the erythrocyte membrane, into bovine brain phosphatidylserine (PS) vesicles on the Ca2+-induced fusion of these vesicles has been investigated. Fusion was monitored by the terbium-dipicolinic acid fluorescence assay for the mixing of aqueous contents of the vesicles and by a resonance energy transfer assay that follows the intermixing of membrane lipids. The Ca2+-induced fusion of PS vesicles is completely prevented by incorporation of glycophorin (molar ratio of PS/glycophorin = 400-500:1) for Ca2+ concentrations up to 50 mM. The ability to fuse is partially restored after treating the glycophorin-containing vesicles with neuraminidase, which removes the negatively charged sialic acid residues of glycophorin. Fusion is further facilitated by trypsin treatment, removing the entire extravesicular glycosylated head group of glycophorin. However, Ca2+-induced fusion of enzyme-treated glycophorin-PS vesicles proceeds at a slower rate and to a smaller extent than fusion of protein-free PS vesicles. The influence of the aggregation state of the glycophorin molecules on fusion has been investigated in experiments using wheat germ agglutinin (WGA). Addition of WGA to the glycophorin-PS vesicles does not induce fusion. However, upon subsequent addition of Ca2+, distinct fusion occurs concomitantly with release of vesicle contents. The inhibition of Ca2+-induced fusion of PS vesicles by incorporation of glycophorin is explained by a combination of steric hindrance and electrostatic repulsion between the vesicles by the glycosylated head group of glycophorin and a direct bilayer stabilization by the intramembranous hydrophobic part of the glycophorin molecule.     

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side.

In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21 °C.

In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds.

Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds.

The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.     

Glycophorin-enriched vesicles obtained by a selective extraction of human erythrocyte membranes with a non-ionic detergent.

A method is described for isolating glycophorin-enriched vesicles from human erythrocytes by extracting membranes that were incubated for 30 min at 37 degrees C at pH 4.5 and washed at low and high ionic strength with the nonionic detergent Triton X-100. The extracts were 11.8 +/- 2.4 fold enriched in glycophorin and contained 325 +/- 69 microgram sialic acid/mg protein, which represented 61 +/- 16% of the total sialic acid. Upon removal of Triton X-100 one third of the total glycophorin forms glycophorin-enriched vesicles with coextracted, endogenous lipids as shown sedimintation, dextran-density gradient centrifugation, and electron microscopy. Addition of exogenous lipids increased the fraction of glycophorin-enriched vesicles up to 87%. The incorporation of glycophorin in the membrane was shown by hemagglutination inhibition assays using anti-M sera and by the accessibility of glycophorin to trypsin. Freeze-fractured vesicles did not reveal intramembranous particles. The selectivity of the extraction procedure is not simply due to chemical constraints introduced by disulfide cross-linkage of protein component 3, because only 20% of this protein undergo disulfide cross-linking. The selective extraction of glycophorin implies that glycophorin is segregated from protein component 3 and thus from intramembranous particles when erythrocyte membranes have been incubated at pH 4.5. This segregation may precede aggregation of intramembranous particles.     

Glycophorin facilitates the transbilayer movement of phosphatidylcholine in vesicles

The rate of transbilayer movement of dioleoylphosphatidylcholine in sonicated lipid vesicles is enhanced by at least two orders of magnitude upon incorporation of glycophorin in the bilayer.     

Preparation and spectroscopic characterization of molecular species of brain phosphatidylserines

This study describes the first preparation and spectroscopic characterization of naturally occurring phospholipids separated according to degree of unsaturation. Phosphatidylserines (PS) have been prepared from bovine brain and shown to be pure by extensive thin layer chromatographic analysis as well as by infrared spectroscopy and fatty acid analysis. The PS has been separated according to degree of unsaturation and prepared using AgNO3-impregnated silica gel H thin-layer chromatography. Fatty acid analysis of the two principal PS subfractions indicates that they are enriched in the molecular species 1-octadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylserine and 1-octadecanoyl-2-octadecenoyl-sn-glycero-3-phosphorylserine. The identity of the two PS subfractions was further verified by rechromatographing on several thin layer systems and by infrared spectroscopy. With the use of a 100 MHz Fourier transform nuclear magnetic resonance (NMR) spectrometer, the spectra of bovine whole brain, white matter, gray matter, monoenoic, and hexaenoic PS were obtained. Distinct proton resonances were assigned to double bond protons, protons adjacent to a double bond, and protons between two double bonds, using fatty acid methyl ester standards. The various PS preparations gave different intensities of the various proton resonances which correlated with differences in fatty acid composition. The method provides a convenient, non-destructive spectroscopic method for distinguishing monoenoic and polyunsaturated species of intact phospholipids. Electron spin resonance studies of nitroxide-labelled cholestane in sonicated PS vesicles showed greater probe motion as the unsaturation of the acyl chains was increased. The hexaenoic PS vesicles were more fluid than monoenoic PS vesicles at all temperatures in the range 10-55 degrees C. These results suggest that neuronal membranes are more fluid than myelin membranes as neuronal membranes contain more hexaenoic phospholipids.     

Chemical modification of the CuA site affects the proton pumping activity of cytochrome c oxidase

Cytochrome c oxidase in which the CuA site has been perturbed by extensive modification of the enzyme with the thiol reagent p-(hydroxymercuri)benzoate has been reconstituted into phospholipid vesicles. The reconstituted vesicles lack respiratory control, and the orientation of the enzyme in the vesicles is similar to that of the native cytochrome c oxidase. In the proton translocation assay, the vesicles containing the modified enzyme behave as if they are unusually permeable to protons. When the modified and native proteins were coreconstituted, a substantial portion of the latter became uncoupled as revealed by low respiratory control and low overall proton pumping activity. These results suggest that the modified enzyme catalyzes a passive transport of protons across the membrane. When milder conditions were used for the chemical modification, a majority of the thiols reacted while the CuA site remained largely intact. Reconstitution of such a partially modified cytochrome c oxidase produced vesicles with respiratory control and proton translocating activity close to those of reconstituted native enzyme. It thus appears that the appearance of a proton leak is related to the perturbation of the CuA site. These observations suggest that the structure of CuA may be related to the role of this site in the proton pumping machinery of cytochrome c oxidase.     

Direct measurement of lactose/proton symport in Escherichia coli membrane vesicles: further evidence for the involvement of histidine residue(s)

Addition of lactose to Escherichia coli ML 308-225 membrane vesicles under nonenergized conditions induces transient alkalinization of the medium, and the initial rate of proton influx is stimulated by valinomycin and abolished by nigericin or carbonyl cyanide m-chlorophenylhydrazone. A functional lac y gene product is absolutely required as the effect is not observed in ML 308-225 vesicles treated with N-ethylmaleimide nor with vesicles from uninduced Escherichia coli ML 30. Furthermore, the magnitude of the phenomenon is enhanced about 3-fold in vesicles from Escherichia coli T206, which contain amplified levels of the lac carrier protein. Kinetic parameters for lactose-induced proton influx are the same as those determined for lactose-facilitated diffusion, and quantitative comparison of the initial rates of the two fluxes indicates that the stoichiometry between protons and lactose is 1:1. Treatment of ML 308-225 vesicles with diethyl pyrocarbonate causes inactivation of lactose-induced proton influx. Remarkably, however, treatment with the histidine reagent enhances the rate of lactose-facilitated diffusion in a manner suggesting that the altered lac carrier catalyzes lactose influx without the symport of protons. The results are consistent with the hypothesis that acylation of a histidyl residue(s) in the lac carrier protein dissociates lactose influx from proton influx and indicate that this residue(s) play(s) an important role in the pathway of proton translocation.     

The effect of general anesthetics on the proton and potassium permeabilities of liposomes

The pump-leak hypothesis of general anesthesia proposes that anesthetics act by increasing the functional proton permeability of membranes, particularly those of synaptic vesicles. Since transmembrane proton gradients are required for neurotransmitter accumulation, decay of such gradients by an uncompensated anesthetic-induced leak would result in loss of neurotransmitter from the vesicles, followed by synaptic block and anesthesia. We have tested this hypothesis by determining the effect of four different general anesthetics on the relative permeabilities of liposome membranes to protons and potassium ions. In all cases, physiologically relevant levels of anesthetics caused a 200 to 500 percent increment in ionic permeability. There was no marked preference for protons, suggesting that the anesthetics did not induce a leak specific for this ionic species. Instead the anesthetics appeared to produce a more general defect available to both protons and potassium ions which resulted in a functional increment in proton permeability. These observations were compared with available data on proton transport rates by synaptic vesicle ATPase enzymes. The magnitude of the anesthetic-induced leak could not be compensated by the ATPase, which is only capable of a 40 percent increase in rate when uncoupled. We consider these results to be consistent with the pump-leak hypothesis.     

The influence of lipid composition on glycophorin-induced bilayer permeability

Glycophorin was incorporated into large unilamellar vesicles and the bilayer permeability was measured as a function of the lipid composition. In agreement with previous data (Van der Steen, A.T.M., De Kruijff, B. and De Gier, J. (1982) Biochim. Biophys. Acta 691, 13-23) it was found that glycophorin greatly increased the bilayer permeability of DOPC vesicles. This effect was observed for a large variety of phosphatidylcholines, differing in their fatty acid composition and homogeneity. In sharp contrast, it was observed that variations in the polar headgroups by incorporation of DOPE, DOPS and, to a lesser extent, cholesterol, into the DOPC/glycophorin vesicles restored the barrier function. These results are compared to the size of the particles, revealed by freeze-fracture electron microscopy on the glycophorin-containing bilayer and are discussed in the light of various types of lipid-protein interactions and protein aggregation state.     

Isolation and assay of corn root membrane vesicles with reduced proton permeability

Conditions promoting the formation of sealed membrane vesicles from corn roots with reduced proton permeability were examined using the probe 9-aminoacridine as a rapid indicator of pH gradient formation and dissipation. Plasma membrane vesicles isolated by differential and density gradient centrifugation were leaky to protons and rapidly equilibrated when exposed to artificially imposed pH gradients. The leaky plasma membrane vesicles showed reduced proton permeability when incubated with calcium or with excess phospholipids. However, these vesicles were unable to form ATP-induced pH gradients. Sealed vesicles isolated by discontinuous Ficoll gradient centrifugation of a microsomal fraction displayed reduced proton permeability and were osmotically active. In contrast to purified plasma membrane vesicles, the microsomal-derived vesicles were more suitable for studies of active proton transport.     

Dynamics of proton diffusion within the hydration layer of phospholipid membrane

The diffusion of protons at the immediate vicinity of (less than 10 A from) a phospholipid membrane is studied by the application of the laser-induced proton pulse. A light-sensitive proton emitter (8-hydroxypyrene-1,3,6-trisulfonate) was trapped exclusively in the hydration layers of multilamellar vesicles made of egg phosphatidylcholine, and the protons were dissociated by a synchronizing laser pulse. The recombination of the proton with pyranin anion was monitored by time-resolved spectroscopy and analyzed by a diffusion-controlled formalism. The measured diffusion coefficient is only slightly smaller than the diffusion coefficient of proton in bulk water. Modulating the width of the hydration layer by external pressure had a direct effect on the diffusibility of the proton: the narrower the hydration layer, the slower is the diffusion of protons.     

Proton magnetic resonance studies of glutamate-alanine transaminase-catalyzed deuterium exchange. Evidence for proton conservation during prototropic transfer from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate.

Pulsed Fourier transform proton magnetic resonance spectroscopy was used to study the glutamate-alanine transaminase-catalyzed incorporation of deuterium from solvent deuterium oxide into the alpha and beta positions of L-alanine. It was found that the beta proton resonance signal initially disappears slightly faster than the signal due to the alpha proton, but whereas the alpha proton signal decays exponentially, that due to the beta proton signal does not. Eventually, the rate of decrease of the alpha proton signal becomes greater than that for the beta proton. This change in the relative rates is ascribed to a deuterium isotope effect upon substitution of an alpha proton by a deuteron. Furthermore, as deuterium begins to replace hydrogen, two classes of alanine become distinguishable, i.e. alanine which contains deuterium in the alpha position and hydrogen in the beta position, and alanine which contains hydrogen in the alpha position and deuterium in the beta position. Thus, removal of all 3 beta protons is not contingent upon loss of an alpha proton from the same molecule. The two classes of deuterated alanine may conceivably arise by a scrambling mechanism in which protons are transferred from the alpha to the beta position and vice versa. Present evidence excludes this scramblong mechanism and leads to the conclusion that deuterium incorporation into L-alanine involves, (a) the reversible enzymatic conversion of the classical ketimine enzymes intermediate to an enaminetype structure, and (b) considerable conservation of label during the prototropic shift from the alpha carbon of L-alanine to the C4-position of pyridoxal 5'-phosphate. It is also postulated that alanine binds at the active site in such a way as to bring the beta protons into close contact with a basic group on the enzyme surface. This group is distinct from that used in abstraction of an alpha proton. The beta protons of glutamate are not enzymatically removed; presumably glutamate binds in such a way that the beta protons cannot effectively interact with an enzyme base. Similar studies were carried out on soluble glutamate-aspartate transaminase; no evidence was found for significant enzyme-catalyzed deuterium incorporation into the beta position of L-glutamate, L-aspartate, and L-alanine.     

Kinetic analysis of proton transport by the vanadate-sensitive ATPase from maize root microsomes

Proton transport by the nitrate-insensitive, vanadate-sensitive ATPase in Kl-washed microsomes and reconstituted vesicles from maize (Zea mays L.) roots was followed by changes in acridine orange absorbance in the presence of either KNO₃ or KCl. Data from such studies obeyed a kinetic model in which net proton transport by the pump is the difference between the rate of proton transport by the action of the ATPase and the leak of protons from the vesicles in the direction opposite from the pump. After establishing the steady state proton gradient, the rate of return of transported protons was found to obey first-order kinetics when the activity of the ATPase was completely and rapidly stopped. The rate of return of these protons varied with the quencher used. When the substrate Mg:ATP was depleted by the addition of either EDTA or hexokinase, the rate at which the proton gradient collapsed was faster than when vanadate was used as the quencher. These trends were independent of the anion accompanying the K and the transport assay used.     

Exocytosed protons feedback to suppress the Ca2+ current in mammalian cone photoreceptors.

A proton pump acidifies synaptic vesicles and provides the electrochemical gradient for transmitter uptake. Although external protons can modulate membrane voltage- and ligand-gated conductances, the fate of the protons released when vesicles fuse with the plasma membrane is unclear. In the dark, the glutamate-laden vesicles of cone photoreceptors fuse continuously with the plasma membrane. I now show that vesicular protons feed back to block the nearby calcium channels that mediate release. This local proton-mediated feedback is a novel mechanism through which neurons may regulate the release of transmitter.     

Glycophorin A interacts with interleukin-2 and inhibits interleukin-2-dependent T-lymphocyte proliferation.

Sialoglycolipids shed by tumor cells have been implicated in tumor-induced inhibition of T-lymphocyte responses to interleukin-2 (IL-2). In the present study, we have used glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, to investigate whether shedding of glycoproteins might also contribute to immunosuppression. Glycophorin A inhibited IL-2-stimulated proliferation of the IL-2-dependent cell lines HT-2 and CTLL-2 in a dose-dependent manner. Time course studies on synchronized cell populations indicated that the glycoprotein acted early in the activation process. On the other hand, glycophorin A had essentially no effect on IL-1-mediated stimulation of the IL-1-sensitive thymocyte cell line EL-4 NOB-1. Gel filtration FPLC demonstrated that IL-2 was able to bind to glycophorin aggregates under physiological conditions. Reconstituted vesicles containing glycophorin were also shown to bind IL-2. In addition, both soluble glycophorin aggregates and lipid vesicles containing glycophorin blocked binding of IL-2 to high-affinity cellular IL-2 receptors. Taken together, these results suggest that shedding of tumor sialoglycoproteins with oligosaccharide chains similar to glycophorin A might contribute to negative modulation of IL-2-mediated immune responses.     

Proton pumping by vesicles reconstituted from two fractions of solubilized rose-cell plasma membrane ATPase

Two forms of K⁺ -stimulated ATPase, which can be solubilized from purified plasma membrane preparations of suspension-cultured rose cells and separated by molecular sieve chromatography, both catalyze the ATP-dependent accumulation of protons into artificial phospholipid/cholesterol vesicles. The higher-molecular weight form of ATPase is highly sensitive to ultraviolet light, and the proton pumping ability of this form is similarly sensitive.     

Proton movements across the membranes of sarcoplasmic reticulum during the uptake of calcium ions

A burst of proton ejection was observed during the initial steps of Ca²⁺ uptake by sarcoplasmic reticulum vesicles. The initial rate of this proton ejection is considerably higher than the initial rate of Ca²⁺ uptake, and is independent of the amount of accumulated Ca²⁺. The ejection of protons is a transmembrane event, since it is dissipated by the ionophore X-537A, and does not occur when the ionophore is added before the initiation of the transport of Ca²⁺. The low proton permeability of the membranes is largely increased by X-537A. The studies of facilitated diffusion of protons in the presence of the ionophore permitted the estimation of the pH within the vesicles. A fast alkalinization occurs within the vesicles during the initial steps of Ca²⁺ uptake, as revealed by sequestered bromothymol blue. The change in absorbance of this dye corresponds to a change of 0.15 pH unit within the vesicles, and a maximal transmembrane ΔpH of about 0.5 may build up. Since such a gradient may not account energetically for the transmembrane gradients of Ca²⁺, I suggest that a transmembrane electrical potential may develop as a consequence of proton ejection.     

Determination of the size of the packing defects in dimyristoylphosphatidylcholine bilayers,present at the phase transition temperature

Multilamellar liposomes of dimyristoylphosphatidylcholine, containing 4 mol% egg phosphatidic acid show at the phase transition temperature an increased permeability for non-electrolytes of M_r values up to 900. This indicates that the packing defects occurring at the liquid crystalline/gel state phase boundary have a similar pore diameter (15–18 A) as the packing defects present in glycophorin—dioleoylphos-phatidylcholine vesicles. This suggests that packing defects at the protein—lipid interphase are the major permeation pathway of the glycophorin—dioleoylphosphatidylcholine vesicles.     

Plasmenylcholine and phosphatidylcholine membrane bilayers possess distinct conformational motifs

The conformation of plasmenylcholine near the hydrophobic-hydrophilic interface in membrane bilayers was deduced by determination of critical internuclear distances utilizing truncated driven nuclear Overhauser enhancement. These experiments demonstrated that the beta-vinyl ether proton in plasmenylcholine was in close spatial proximity and nearly equidistant (approximately 3 A) to both the alpha- and beta-methylene protons of the sn-2 aliphatic chain. In contrast, the distances between the alpha-vinyl ether proton and the alpha- and beta-methylene protons of the sn-2 aliphatic chain were greater than or equal to 5 A. Furthermore, the distance between the N-CH3 protons in the polar head group and the methylene protons of the glycerol backbone in plasmenylcholine vesicles is larger than that present in phosphatidylcholine vesicles. Although the proximal portion of the sn-2 acyl chain in phosphatidylcholine is bent, conformational analysis utilizing these distance constraints demonstrated that the carbon atoms which comprise the proximal portion of the sn-2 aliphatic chain in plasmenylcholine are nearly coplanar, in register, and parallel to the sn-1 aliphatic chain. Taken together, these observations indicate that modest covalent alterations in the proximal portion of the sn-1 aliphatic chain in choline glycerophospholipids result in substantial changes in the molecular conformation and packing of hydrated phospholipid bilayers.     

Light-dependent proton and rubidium translocation in membrane vesicles from Halobacterium halobium.

A procedure for the isolation of membrane vesicles after sonication of $\text{Halobacterium halobium}$ is described. Upon illumination these vesicles took up rubidium. This process was stimulated 3 to 7 fold by valinomycin, and inhibited by uncouplers of oxidative phosphorylation or by nigericin. In the light, these vesicles extruded protons. However, on addition of low concentrations of uncoupler the direction of proton movement was reversed. All proton movements were abolished by high concentrations of uncoupler or by nigericin. These observations suggest that part of the vesicle population was inverted and less sensitive to uncouplers.