Annexin-mediated membrane fusion of human neutrophil plasma membranes and phospholipid vesicles.

Membrane fusion was studied using human neutrophil plasma membrane preparations and phospholipid vesicles approximately 0.15 microns in diameter and composed of phosphatidylserine and phosphatidylethanolamine in a ratio of 1 to 3. Liposomes were labeled with N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl (NBD) and lissamine rhodamine B derivatives of phospholipids. Apparent fusion was detected as an increase in fluorescence of the resonance energy transfer donor, NBD, after dilution of the probes into unlabeled membranes. 0.5 mM Ca2+ alone was sufficient to cause substantial fusion of liposomes with a plasma membrane preparation but not with other liposomes. Both annexin I and des(1-9)annexin I caused a substantial increase in the rate of fusion under these conditions while annexin V inhibited fusion. Fusion mediated by des(1-9)annexin I was observed at Ca2+ concentrations as low as approximately 5 microM, suggesting that the truncated form of this protein may be active at physiologically low Ca2+ concentrations. Trypsin treated plasma membranes were incapable of fusion with liposomes, suggesting that plasma membrane proteins may mediate fusion. Liposomes did not fuse with whole cells at any Ca2+ concentration, indicating that the cytoplasmic side of the membrane is involved. These results suggest that annexin I and unidentified plasma membrane proteins may play a role in Ca(2+)-dependent degranulation of human neutrophils.     

Changes in the phospholipid composition and phospholipid asymmetry of ram sperm plasma membranes after cryopreservation

The changes in the phospholipid composition of spermatozoa plasma membranes after freezing were determined by thin-layer chromatography. The results showed an augmentation of the diphosphatidylglycerol and a diminution of phosphatidylglycerol, phosphatidylserine, and phosphatidylethanolamine in sperm plasma membranes after freezing. In intact sperm cells we observed an elevation of the sphingomyelin and phosphatidylinositol levels and a diminution of the phosphatidylethanolamine and diphosphatidylglycerol levels. The effect of freezing on the phospholipid distribution between the inner and outer monolayers of the plasma membrane was also studied using exogenous phospholipases and trinitrobenzene sulfonate. The most important change we observed after freezing, was the translocation of diphosphatidylglycerol from the inner to the outer monolayer of the plasma membrane.     

Phospholipid composition and phospholipid asymmetry of ram spermatozoa plasma membranes

The phospholipid composition of ram spermatozoa plasma membranes has been investigated. An exclusively high participation of the choline- and ethanolamine-plasmalogens in the phosphatidylcholine and phosphatidylethanolamine fractions has been established. Phosphatidylcholine of ram spermatozoa plasma membranes contains a great amount of polyunsaturated fatty acids. The phospholipid distribution in spermatozoa plasma membrane was investigated. It was established that the choline containing phospholipids are situated mainly in the outer membrane lipid monolayer, whereas diphosphatidylglycerol and phosphatidylserine are localized predominantly in the inner monolayer. The rest of the phospholipids are evenly distributed among the two monolayers. Ram spermal plasma membranes exhibit high phospholipase A2 activity.     

Labeling of glucagon binding components in hepatocyte plasma membranes.

A photosensitive derivative of glucagon, ¹²⁵I-N^?-4-azido-2-nitrophenyl-glucagon, has been synthesized and used to specifically label glucagon binding proteins in hepatocyte plasma membranes. Photolysis of the derivative in the presence of a membrane suspension results in the incorporation of radioactivity primarily into membrane components with a molecular weight range of 23,000–25,000. The binding properties of the derivative are essentially identical to that observed for glucagon. The binding of ¹²⁵I-NAP-glucagon was completely inhibited in the presence of glucagon (3 μM) while greater than 90% of the covalent labeling was also inhibited in the presence of glucagon. These studies suggest that the labeled membrane protein may be a component of the glucagon receptor.     

Endosomes differ from plasma membranes in the phospholipid molecular species composition

125I-Labeled epidermal growth factor was incorporated into and highly concentrated in endosomes of Chinese hamster V79-UF cells during incubation at 37 degrees C for 8 min after binding to its receptors on the cell surface at 4 degrees C. From the labeled cells, endosomes were isolated by isopycnic centrifugation on a Percoll density gradient and then sucrose density gradients. The isolated endosomes were mostly free from contamination by Golgi, endoplasmic reticulum, lysosome, plasma membrane and mitochondria. Endosome membranes were found to differ from plasma membranes in the phospholipid composition. Sphingomyelin and phosphatidylserine were enriched in endosomes, compared with plasma membranes. Diacylphosphatidylcholine and diacylphosphatidylethanolamine were major phospholipids of the membranes in both organelles. The contents of molecular species of diacylphosphatidylcholine and diacylphosphatidylethanolamine with two monoenoic fatty acids were lower in endosomes than in plasma membranes. The differences in the polar head group and molecular species compositions of phospholipids between endosomes and plasma membranes did not change, regardless of whether or not the proportions of phospholipid molecular species in plasma membranes changed. The significance of the lipids in endosomes is discussed.     

Altered phospholipid composition of plasma membranes from thrombin-stimulated human platelets

The individual phospholipid concentrations, and their respective fatty acid distributions, in whole platelet lysates and plasma membranes derived from unstimulated and thrombin-stimulated intact human platelets were studied. This was of interest, since previous work had led to the suggestion that altered phospholipid concentrations in plasma membranes of intact stimulated cells may be of importance in mediating cellular responses. The concentrations (nmol/mg protein) of phosphatidylinositol in whole platelet lysates and plasma membranes derived from thrombin-activated platelets decreased by 37 and 45%, respectively, a compared to their corresponding controls. As well, concentrations of plasma membrane phosphatidylcholine and phosphatidylethanolamine in thrombin-stimulated platelets decreased by 20 and 9%, respectively, when compared with their control values. The amounts of phosphatidylserine and sphingomyelin in whole platelet lysates and plasma membranes were unchanged by exposure to thrombin. Fatty acid analyses revealed that thrombin stimulation of intact human platelets induced a decrease in the arachidonate content (from 37.7 to 33.1 wt.% of total fatty acid) of plasma membrane phosphatidylinositol. Similar shifts in the wt% of arachidonic acid in plasma membrane phosphatidylcholine were found. These results indicate that thrombin stimulation of intact human platelets produces a significant decrease in the mass of phosphatidylinositol in plasma membranes and raises the suggestion that the preferential depletion of the plasma membrane in arachidonoyl-containing phosphatidylinositol may be of importance in mediating cellular responses to external stimuli.     

Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.     

The interaction of phospholipid membranes and detergents with glutamate dehydrogenase.

1. The interaction of beef liver glutamate dehydrogenase with cardiolipin from both beef liver mitochondria and beef heart mitochondria, with phosphatidylcholine from both beef liver mitochondria and egg-yolk, and with beef brain phosphatidylserine was investigated by steady-state kinetic methods. 2. the phosphatidylcholine did not inhibit the enzyme under a wide range of conditions. The cardiolipins and phosphatidylserine inhibited the enzyme. The inhibition by these lipids was found to diminish with time if the lipids were prepared and the reaction was studied in either phosphate or Tris buffers, but in zwitterionic buffers these lipid brought about a rapid, reversible inhibition which remained stable with time for at least 150 min. 3. The kinetic type of the inhibition was difficult to determine because of variation between lipid sonicates. Complex mixed types of inhibition were found with cardiolipin, and with phosphatidylserine the inhibition approximated to a non-competitive interaction with Ki(app) values varying between (0.9-6.1) x 10(-6)M. 4. The extent of inhibition decreased with increasing pH and with increasing ionic strength. Basic proteins, such as cytochrome c, show a higher affinity for the anionic membranes and can dissociate the enzyme-lipid complexes. Cosonicates of the cardiolipin and phosphatidylcholine inhibited the enzyme, the extent of inhibition increasing in proportion to the amount of acidic lipid. 5.Sodium dodecylsulphate causes a time-dependent inhibition of the enzyme. The kinetics of this effect and its variation with detergent concentration were studied. 6. The relationship of these observations to the structure and function of the enzyme is discussed. It is suggested that their apparent regulation of the enzyme by oestrogens and other small molecules is due to their binding in vitro at sites on the enzyme designed for binding cardiolipin, when the enzyme is functioning in vivo. The association of the enzyme oligomer in vitro may, for similar reasons, be an artifact.     

Separation of the glycoprotein and ganglioside components of thyrotropin receptor activity in plasma membranes.

The two components of thyroid plasma membranes known to interact with thyrotropin, i.e., a glycoprotein with specific thyrotropin binding activity and the gangliosides of the thyroid membranes, are shown to segregate differently when membranes are solubilized with lithium diiodosalicylate. Individually examined, the interaction of each component with thyrotropin exhibits a different sensitivity to salts. The data suggest that the thyrotropin receptor on the thyroid membrane is a complex which is composed of both glycoprotein and ganglioside components and that its properties are derived from each component.     

Identification of the calmodulin-binding components in bovine lens plasma membranes

Lens membranes, purified from calf lenses, have been labeled by covalent cross-linking to membrane-bound 125I-calmodulin with dithiobis(succinimidyl propionate). Electrophoretic analysis in sodium dodecyl sulfate demonstrated two major 125I-containing products of Mr = 49 000 and 36 000. That the formation of these two components was specifically inhibited by unlabeled calmodulin, or calmodulin antagonists, would indicate that the formation of these components was calmodulin-specific. The size of these two 125I-labeled components was unchanged over a range of 125I-calmodulin or dithiobis(succinimidyl propionate) concentrations indicating that they represent 1:1 complexes between 125I-calmodulin (Mr = 17 000) and Mr-32 000 and Mr-19 000 lens membrane components respectively. Although formation of both cross-linked components exhibited an absolute dependence on Mg2+, the autoradiographic intensity of these components was enhanced when Ca2+ was included with Mg2+ during the cross-linking reaction. Labeling was maximal in 10 mM MgCl2 and approximately 1 microM Ca2+. Treatment of lens membranes with chymotrypsin resulted in the cleavage of MP26 (the major lens membrane protein), with the appearance of a major proteolytic fragment of Mr = 22 000. This proteolysis was not associated with any significant change in either the size or amount of the 125I-calmodulin-labeled membrane components. These results suggest that calmodulin interacts with two membrane proteins, but not significantly with MP26, in the intact lens cell membrane. Our results indicate the need to maintain caution in interpreting direct calcium plus calmodulin effects on MP26 and lens cell junctions.     

Fluorimetric detection of phospholipid vesicles bound to planar phospholipid membranes.

The first step in the fusion of two phospholipid membranes culminates in the aggregation of the two lipid bilayers. We have used a custom-built fluorimeter to detect multilamellar vesicles (liposomes) containing the fluorescent dye, 6-carboxyfluorescein (6-CF), bound to a planar lipid bilayer (BLM). Liposomes were added to one side of the BLM, and unbound vesicles were perfused out. This left a residual fluorescence from the BLM, but only when the membranes contained anionic lipids, and then only when millimolar levels of calcium were present. This residual fluorescence was consistently detected only when calcium was included in the buffer during the perfusion. This residual fluorescence originated from liposomes bound to the BLM. Breaking the BLM or lysing the adsorbed vesicles with distilled water abolished it. free 6-CF and/or calcium in the absence of liposomes resulted in no residual fluorescence. No residual fluorescence was detected when both the liposomes and the BLM were composed entirely of zwitterionic lipids. This was found to result from the insensitivity of the fluorimeter to a small number of liposomes adsorbed to the BLM. For this system, we conclude that calcium is necessary for both the initiation and maintenance of the state in which the vesicle membrane is bound to the planar bilayer when the membranes contain negatively charged lipids. This attachment is stronger than the interaction between zwitterionic membranes.     

The antimicrobial peptide trichogin and its interaction with phospholipid membranes.

The interaction of the antimicrobial peptide trichogin GA IV with phospholipid bilayers has been studied. A series of analogs of trichogin was synthesized in which the nitroxide spin label, 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC), replaced one of the three alpha-aminoisobutyric acid (Aib) residues in the sequence. These modified peptides were used to assess the location of different residues of the peptide in a phospholipid bilayer composed of egg phosphatidylcholine containing 0.4 mol% of a fluorescently labelled phospholipid. We demonstrate that the substitution of Aib residues with TOAC does not alter the manner in which the peptide affects membrane curvature or induces vesicle leakage. The proximity of the nitroxide group on the peptide to the 4,4-difluoro-4-bora-3a,4a-diaza-S-indacene (BODIPY) fluorophore attached to the phospholipid was estimated from the extent of quenching of the fluorescence. By this criterion it was concluded that the peptide penetrates into the bilayer and that Aib4 is the most deeply inserted of the Aib residues. The results suggest that the helix axis of the peptide is oriented along the plane of the membrane. All of the peptides were shown to raise the bilayer to the hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine, indicating that they promote positive membrane curvature. This is a property observed with peptides that do not penetrate deeply into the bilayer or are oriented along the bilayer normal. We also demonstrate trichogin-promoted leakage of the aqueous contents of liposomes. These results indicate that the peptides cause bilayer destabilization. The extent of leakage induced by trichogin is very sensitive to the peptide to lipid ratio over a narrow range.     

Membrane fusion. Transfer of phospholipid molecules between phospholipid bilayer membranes

Fusion of phosphatidylcholine (PC) vesicles and of PC-phosphatidylserine (PS) vesicles has been studied using spin-labeled PC and PS. Analysis of ESR spectra indicated transfer of phospholipid molecules between phospholipid vesicles at the instant of membrane contact by vesicular collision. The transfer rate of PC was not greatly affected by the presence of the anionic lipid in the membranes. The rate of PC transfer between PS-PC vesicles was nearly the same as that of PS transfer. Calcium ion greatly enhanced the transfer of phospholipid molecules between PS-PC vesicles. The enhancement of PS transfer occurred instantaneously. The phospholipid transfer is related to the fusion of vesicles.     

Interaction of the P-type cardiotoxin with phospholipid membranes.

The cardiotoxin (cytotoxin II, or CTII) isolated from cobra snake (Naja oxiana) venom is a 60-residue basic membrane-active protein featuring three-finger beta sheet fold. To assess possible modes of CTII/membrane interaction 31P- and 1H-NMR spectroscopy was used to study binding of the toxin and its effect onto multilamellar vesicles (MLV) composed of either zwitterionic or anionic phospholipid, dipalmitoylglycerophosphocholine (Pam2Gro-PCho) or dipalmitoylglycerophosphoglycerol (Pam2Gro-PGro), respectively. The analysis of 1H-NMR linewidths of the toxin and 31P-NMR spectral lineshapes of the phospholipid as a function of temperature, lipid-to-protein ratios, and pH values showed that at least three distinct modes of CTII interaction with membranes exist: (a) nonpenetrating mode; in the gel state of the negatively charged MLV the toxin is bound to the surface electrostatically; the binding to Pam2Gro-PCho membranes was not observed; (b) penetrating mode; hydrophobic interactions develop due to penetration of the toxin into Pam2Gro-PGro membranes in the liquid-crystalline state; it is presumed that in this mode CTII is located at the membrane/water interface deepening the side-chains of hydrophobic residues at the tips of the loops 1-3 down to the boundary between the glycerol and acyl regions of the bilayer; (c) the penetrating mode gives way to isotropic phase, stoichiometrically well-defined CTII/phospholipid complexes at CTII/lipid ratio exceeding a threshold value which was found to depend at physiological pH values upon ionization of the imidazole ring of His31. Biological implications of the observed modes of the toxin-membrane interactions are discussed.     

Characterization of two insulin-binding components of rat-liver plasma membranes

We have identified and isolated two forms of insulin receptor from rat-liver plasma membranes. The smaller (M _r= 90k) is a single polypeptide. The same poly-peptide appears to be the insulin-binding site of the largerM _r=280k). Only the larger, multisubunit, receptor shows high-affinity binding of insulin and negative cooperativity in its dissociation kinetics.