Promotion of acid-induced membrane fusion by basic peptides. Amino acid and phospholipid specificities

The ability of oligo- and polymers of the basic amino acids L-lysine, L-arginine, L-histidine and L-ornithine to induce lipid intermixing and membrane fusion among vesicles containing various anionic phospholipids has been investigated. Among vesicle consisting of either phosphatidylinositol or mixtures of phosphatidic acid and phosphatidylethanolamine rapid and extensive lipid intermixing, but not complete fusion, was induced at neutral pH by poly-L-ornithine or L-lysine peptides of five or more residues. When phosphatidylcholine was included in the vesicles, the lipid intermixing was severely inhibited. Such lipid intermixing was also much less pronounced among phosphatidylserine vesicles. Poly-L-arginine provoked considerable leakage from the various anionic vesicles and caused significantly less lipid intermixing than L-lysine peptides at neutral pH. When the addition of basic amino acid polymer was followed by acidification to pH 5-6, vesicle fusion was induced. Fusion was more pronounced among vesicles containing phosphatidylserine or phosphatidic acid than among those containing phosphatidylinositol, and occurred also with vesicles whose composition resembles that of cellular membranes (i.e., phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine, 50:30:20, by mol). Liposomes with this composition are resistant to fusion by Ca2+ or by acidification after lectin-mediated contact. The tight interaction among vesicles at neutral pH, resulting in lipid intermixing, does not seem to be necessary for the fusion occurring after acidification, but the basic peptides nevertheless appear to play a more active role in the fusion process than simply bringing the vesicles in contact. However, protonation of the polymer side chains and transformation of the polymer into a polycation does not explain the need for acidification, since the pH-dependence was quite similar for poly(L-histidine)- and poly(L-lysine)-mediated fusion.     

Modulation of membrane fusion by calcium-binding proteins.

The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating fusion.     

Characterization of the fusogenic properties of glyceraldehyde-3-phosphate dehydrogenase: fusion of phospholipid vesicles

A fluorescence assay based on resonance energy transfer has been used to characterize the fusogenic properties of glyceraldehyde-3-phosphate dehydrogenase. The extent of phospholipid vesicles fusion induced by the protein increased with decreasing pH, being maximum at pH 4.5-5.0. Fusion reaction was temperature dependent with an activation energy of 10 Kcal/mol, and virtually completed within 1 min. at pH 5.0. Fusion is most efficient with vesicles bearing negative charge, however uncharged and even positively charged vesicles were fused. The negatively charged and uncharged vesicles showed the same pH dependence. These observations suggest the importance of hydrophobic interaction in the process of fusion, which was supported by a correlation between extent of fusion and exposure of hydrophobic region of the protein.     

Immobilized hybrids of glyceraldehyde-3-phosphate dehydrogenase.

Yeast glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) immobilized on CNBr-activated Sepharose 4-B has been subjected to dissociation to obtain matrix-bound dimeric species of the enzyme. Hybridization was then performed using soluble glyceraldehyde-3-phosphate dehydrogenase isolated from rat skeletal muscle. Immobilized hybrid tetramers thus obtained were demonstrated to exhibit two distinct pH-optima of activity characteristic of the yeast and muscle enzymes, respectively. The results indicate that under appropriate conditions the activity of each of the dimers composing the immobilized hybrid tetramer can be studied separately.     

Relationship between intact cell ATP levels and glucocorticoid receptor-binding capacity in the AtT-20 cell

A study was made on the correlation between the degree of membrane fusion and surface tension increase of phosphatidic acid membranes caused by divalent cations. Membrane fusion was followed by the Tb3+/dipicolinic acid assay, monitoring the fluorescent intensity for mixing of the internal aqueous contents of small unilamellar lipid vesicles. The surface tension and surface potential of monolayers made of the same lipids as used in the fusion experiments were measured as a function of divalent cation concentration. It was found that the 'threshold' concentration to induce massive vesicle membrane fusion was the same for Ca2+ and Mg2+, and that the surface tension increase in the monolayer, induced by changing divalent cation concentration from zero to a concentration which corresponds to its threshold value, inducing vesicle membrane fusion, was approximately the same: 6.3 dyn/cm for both Ca2+ and Mg2+. Both the divalent cation's threshold concentrations as well as the surface tension change corresponding to the threshold concentration for the phosphatidic acid membrane were smaller than those for the phosphatidylserine membrane. The different fusion capability of these divalent cations for phosphatidic acid and phosphatidylserine membranes is discussed in terms of the different ion binding capabilities of these ions to the membranes.     

Protein-induced fusion of phospholipid vesicles of heterogeneous sizes.

We have investigated the fusion of phospholipid vesicles induced by lysozyme and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vesicles were composed of dimyristoylphosphatidylcholine/dioleoylphosphatidylethanolamine/ cholesterol (DMPC:DOPE:Chol, 2:1:1). Small unilamellar vesicles (SUV, diameter ca. 30 nm) obtained by extensive sonication or large unilamellar vesicles (LUV, diameters ranged from 100 to 400 nm) obtained by extrusion methods were used. Fusion of LUV induced by lysozyme and GAPDH was drastically decreased when the diameter of the vesicles increased over a value of 100 nm. Lysozyme effect was stopped at the aggregation step while GAPDH effect was stopped at the fusion (lipid mixing) step. Fusion of heterogeneous vesicle populations (SUV with LUV) was observed only with GAPDH and this happened only when the lipids were in the liquid-crystalline state.     

Lysine peptides induce lipid intermixing but not fusion between phosphatidic acid-containing vesicles

Penta(L-lysine) and poly(L-lysine) (but not di- or tri(-L-lysine)) were found to induce rapid and extensive phospholipid intermixing between unilamellar vesicles consisting largely of phosphatidic acid and phosphatidylethanolamine, without causing significant intermixing of, nor leakage from, the encapsulated aqueous spaces. Neither did the size of such vesicles increase after treatment with lysine peptide, as determined by gel chromatography. The results suggest that lysine oligo-(or poly-) peptides may induce reversible semifusion between vesicles, but not complete membrane fusion.

Pentalysine Polylysine Phosphatidic acid Lipid intermixing Membrane fusion     

Phosphatidylethanol counteracts calcium-induced membrane fusion but promotes proton-induced fusion

The susceptibility of phosphatidylethanol-containing lipid vesicles towards Ca2+- and proton-induced fusion has been investigated, using a system of interacting vesicles. The results show that phosphatidylethanol-rich vesicles are quite resistant to Ca2+-induced fusion while being highly sensitive to proton-induced fusion. Inclusion of phosphatidylethanol was also found to promote and inhibit, respectively, the proton-induced and Ca2+-induced fusion of bilayer vesicles containing also phosphatidylethanolamine and either phosphatidylserine or phosphatidic acid. Thus, phosphatidylethanol affected Ca2+- and proton-induced fusion in opposite directions, in contrast to the naturally occurring anionic phospholipids phosphatidic acid, phosphatidylserine and phosphatidylinositol, which affect the sensitivity to Ca2+- and H+-induced fusion in the same direction. However, the fusion competence of phosphatidylethanol vesicles in response to both Ca2+ and H+ was inversely related to the apparent thickness of the polar headgroup layer, determined by using lectin-glycolipid interaction as a steric probe, as previously found for vesicles containing naturally occurring anionic phospholipids.     

Interaction between glyceraldehyde-3-phosphate-dehydrogenase and lactate dehydrogenase

Physical interaction between rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase was detected by means of matrix immobilization technique. Glyceraldehyde-3-phosphate dehydrogenase covalently bound to CNBr-activated Sepharose 4B was capable of forming a complex with soluble lactate dehydrogenase with a stoichiometry of 0.8 mole of lactate dehydrogenase per mole of glyceraldehyde-3-phosphate dehydrogenase and KD of 0.385 microM at pH 6.5. The bienzyme association weakened when pH changed to 7.0 (the KD increased to 1.25 microM).     

Structural studies on glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle

The NH2-terminal amino acid sequence of rat skeletal muscle glyceraldehydephosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NAD+ oxidoreductase(physphorylating), EC 1.2.1.12) was determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Ser-Ser-(-)-(-)--Val-Asx-Ile-Val-Ala-Ile. The presence of Asn instead of Asp in position 6 differentiates this enzyme from other glyceraldehyde-3-phosphate dehydrogenases so far sequenced with the exception of the enzymes isolated from liver. The location of Asn in position 6 has been considered as a specific property of liver glyceraldehyde-3-phosphate dehydrogenase (Kulbe, K.D., Jackson, K.W. and Tang, J. (1975) Biochem. Biophys. Res. Commun. 67, 35--42); this suggestion is not sustained by the results of the present investigation. The amino acid composition of the rat skeletal muscle dehydrogenase demonstrates the unusually low histidine content of this enzyme as compared to other mammalian muscle glyceraldehyde-phosphate dehydrogenases.     

Synergistic effects of micromolar concentrations of Zn2+ and Ca2+ on membrane fusion

Resonance Energy Transfer between N-(7-nitro-2,1,3 benzoxadiazol -4 yl) phosphatidyl ethanolamine and N-Lissamine-Rhodamine B sulfonyl) phosphatidyl ethanolamine embedded in two different populations of small unilamellar vesicles made of phosphatidyl serine has been used to study the fusion process induced by Zn2+ and Ca2+. Lipid intermixing demonstrating fusion of liposome membranes can already be observed at 125 and 250 mumol/l of Zn2+. After short time pre-incubations with micromolar concentrations of Zn2+ as low as 150 mumol/l, Ca2+ induces an instantaneous increase of vesicle fusion. The lipid intermixing induced by micromolar concentrations of Ca2+ (250-500 mumol/l) could be increased up to 4 times when pre-incubated with 150 or 200 mumol/l of Zn2+. The effect of 1 mM of Ca2+ alone on lipid intermixing can be mimicked by 150 mumol/l of Zn2+ followed by 500 mumol/l of Ca2+. Our data demonstrate that Zn2+ and Ca2+ act synergistically to affect cation-induced membrane fusion. We suggest that Zn2+ specifically alters the physical state of phospholipid membranes making them more prone to calcium-triggered fusion.     

Immobilization of glyceraldehyde-3-phosphate dehydrogenase by non-covalent binding to specific antibodies and Fab-fragments coupled to Sepharose]

Rabbit antibodies to rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase, as well as monovalent Fab fragments of these antibodies were coupled to CNBr-activated Sepharose 4B. Rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase was then immobilized on a matrix by non-covalent binding to specific antibodies. Immobilized enzyme retains approximately 90% catalytic activity of the soluble dehydrogenase; pH optimum of activity and the Km value observed are changed as compared to the enzyme in solution. Glyceraldehyde-3-phosphate dehydrogenase immobilized on specific antibodies is shown to undergo adenine nucleotide-induced dissociation into dimers. The immobilized dimeric form of the enzyme thus obtained is catalytically active and capable of reassociating with the dimers of apoglyceraldehyde-3-phosphate dehydrogenase added in solution to the suspension of Sepharose.     

Chemical and functional properties of the native and reconstituted forms of the membrane-bound, aerobic glycerol-3-phosphate dehydrogenase of Escherichia coli.

A simple purification for the membrane-associated, flavin-linked, glycerol-3-phosphate dehydrogenase of Escherichia coli has been developed which yields homogeneous enzyme in a detergent-solubilized state. 1. The dissociated form of the enzyme has a molecular weight of 58,000 and contains 0.5 mol of FAD/mol of protein monomer. 2. The solubilized enzyme-catalyzed reaction has a pH profile and temperature dependence similar to that observed for the membrane-bound enzyme. 3. The most efficient electron acceptor is potassium ferricyanide but phenazine methosulfate, methylene blue, menadione, and dichlorophenolindophenol can also be utilized. 4. The reaction is competitively inhibited by dihydroxyacetone phosphate, phosphoenolpyruvate, phosphoglycolic acid, glyceraldehyde-3-phosphate, and D-2- and D-3-phosphoglyceric acid. 5. The activity of the enzyme is regulated in a complex manner by ATP and GTP. 6. Detergent-depleted enzyme can be functionally reconstituted with Escherichia coli membrane vesicles to support glycerol-3-phosphate-dependent active transport of L-proline. 7. Detergent-depleted enzyme requires exogenous phospholipid or nondenaturing detergent for electron transfer activity.     

Membrane-bound and soluble extracellular alpha-amylase from Bacillus subtilis.

Extracellular alpha-amylase was purified to homogeneity from a Marburg strain of Bacillus subtilis. The enzyme is a single polypeptide chain of molecular weight approximately 67,000. Its NH2-terminal amino acid sequence is Leu-Thr-Ala-Pro-Ser-Ile-Lys. A membrane-derived alpha-amylase was solubilizing from membrane vesicles by treatment with Triton X-100 and was highly purified by chromatography on an anti-alpha-amylase-protein A-Sepharose column. Membrane-derived alpha-amylase was indistinguishable from the soluble extracellular enzyme by sodium dodecyl sulfate-gel electrophoresis and radioimmunoassay. The membrane-derived enzyme contains phospholipid. Approximately 30 to 80% of the phospholipid was extracted from the purified enzyme by chloroform:methanol. The extracted phospholipid was predominately phosphatidylethanolamine. Treatment with phospholipase D released phosphatidic acid. Membrane-bound alpha-amylase was latent in membrane vesicles. Release of membrane-bound alpha-amylase from vesicles by an endogenous enzyme was maximal at pH 8.5, was inhibited by metal chelators and diisopropyl fluorophosphate and was stimulated by Ca2+ and Mg2+. The amount of membrane-bound alpha-amylase was related to the level of secretion.     

A structural study of pig liver glyceraldehyde-3-phosphate dehydrogenase.

A substantial portion of the primary structure of pig liver glyceraldehyde-3-phosphate dehydrogenase has been investigated and the results compared with those previously reported for the pig muscle enzyme. Liver and muscle glyceraldehyde-3-phosphate dehydrogenases show the same amino acid content, and the first N-terminal residues occur in the same sequence. No differences in N-terminal residues and amino acid composition have been evidenced by analysis of several tryptic peptides, which account for about 50% of the total amino acid sequence. From the electrophoretic mobilities of peptides T8 T9 and T25 it is concluded that residues Asp 60, Asp 67 and Glu 220 in the reported sequence of the pig muscle enzyme must be present as amides in the liver enzyme. The NAD+ content was found to be 2 mol per tetramer, while higher values have been reported for the muscle enzyme from various mammalian sources. The reactivity of lysyl side chains towards pyridoxal 5'-phosphate has been examined: the results indicate that Lys 212 is the main site reacted in fully inactivated pig liver holoenzyme. A similar result has been found for rabbit muscle apoenzyme, whereas rabbit muscle holoenzyme reacts at Lys 212 and 191.     

Identification of the mammalian DNA-binding protein P8 as glyceraldehyde-3-phosphate dehydrogenase.

The DNA-binding protein P8 from transformed hamster fibroblasts (line NIL-1-hamster sarcoma virus) has been purified to homogeneity by DNA-cellulose and phosphocellulose chromatography. The molecular weight of dissociated P8 is 36000, the same as that reported for the subunits of glyceraldehyde-3-phosphate dehydrogenase, and the mobility of these proteins in polyacrylamide gels is identical. The amino acid composition of P8 is very similar to that of glyceraldehyde-3-phosphate dehydrogenase. When assayed for glyceraldehyde-3-phosphate dehydrogenase activity the P8 preparation had a specific activity of 54.6 units/mg, a value comparable to that of the crystalline enzyme from several sources. Furthermore, serum prepared against P8 crossreacts with glyceraldehyde-3-phosphate dehydrogenase from hamster muscle. These results show that P8 is glyceraldehyde-3-phosphate dehydrogenase. The interaction of P8 from transformed fibroblasts and glyceraldehyde-3-phosphate dehydrogenase from hamster and rabbit muscle with DNA has been studied using a Millipore filtration technique. These proteins have affinity for single-stranded DNA but not for double-stranded DNA.     

Alkylacetylglycerophosphocholine stimulates Na+-Ca2+ exchange, protein phosphorylation and polyphosphoinositide turnover in rat ileal plasmalemmal vesicles

The novel ether phospholipid, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), isometrically contracted helically cut rat ileal smooth muscle strips in a dose- and time-dependent manner. Utilizing an enriched plasma membrane vesicular preparation from rat ileal longitudinal smooth muscle, AGEPC specifically stimulated Na+-Ca2+ exchange in a dose- and time-dependent manner. Concomitant with the AGEPC stimulation of Na+-dependent Ca2+ influx in plasma membrane vesicles is an enhanced turnover of the polyphosphoinositides, an elevated concentration of phosphatidic acid and also an enhanced phosphorylation of an Mr 40,000 plasmalemmal protein. The mechanisms by which AGEPC may regulate ileal plasmalemmal Ca2+ flux and contractility are considered.     

Relationship between fluorescence and conformation of epsilonNAD+ bound to dehydrogenases.

This work reports on the interaction of the fluorescent nicotinamide 1,N6-ethenoadenine dinucleotide (epsilonNAD+) with horse liver alcohol dehydrogenase, octopine dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase from different sources (yeast, lobster muscle, and rabbit muscle). The coenzyme fluorescence is enhanced by a factor of 10-13 in all systems investigated. It is shown that this enhancement cannot be due to changes in the polarity of the environment upon binding, and that it must be rather ascribed to structural properties of the bound coenzyme. Although dynamic factors could also be important for inducing changes in the quantum yield of epsilonNAD+ fluorescence, the close similarity of the fluorescence enhancement factor in all cases investigated indicates that the conformation of bound coenzyme is rather invariant in the different enzyme systems and overwhelmingly shifted toward an open form. Dissociation constants for epsilonNAD+-dehydrogenases complexes can be determined by monitoring the coenzyme fluorescence enhancement or the protein fluorescence quenching. In the case of yeast glyceraldehyde-3-phosphate dehydrogenase at pH 7.0 and t = 20 degrees the binding plots obtained by the two methods are coincident, and show no cooperativity. The affinity of epsilonNAD+ is generally lower than that of NAD+, although epsilonNAD+ maintains most of the binding characteristics of NAD+. For example, it forms a tight complex with horse liver alcohol dehydrogenase and pyrazole, and with octopine dehydrogenase saturated by L-arginine and pyruvate. One major difference in the binding behavior of NAD+ and epsilonNAD+ seems to be present in the muscle glyceraldehyde-3-phosphate dehydrogenase. In fact, no difference was found for epsilon NAD+ between the affinities of the third and fourth binding sites. The results and implications of this work are compared with those obtained recently by other authors.     

Agglutination and fusion of globoside GL-4 containing phospholipid vesicles mediated by lectins and calcium ions

We have investigated the interaction of five N-acetylgalactosamine (GalNAc) specific lectins with the glycosphingolipid globoside GL-4, inserted into phospholipid vesicles composed of phosphatidyl-ethanolamine and phosphatidic acid, with respect to their ability to induce vesicle agglutination, fusion, and destabilization. The following lectins were used: soybean agglutinin (SBA); Sophora japonica agglutinin (SJA); Helix pomatia agglutinin (HPA); Ricinus communis agglutinin II (RCAII); and Codium fragile agglutinin (CFA). SBA and SJA caused rapid vesicle agglutination while HPA, CFA, and RCAII were ineffective. However, in the presence of RCAII, but not HPA and CFA, the addition of Ca2+ caused vesicle agglutination which was specifically inhibited by the haptenic sugar GalNAc, while ethylenediaminetetraacetic acid (EDTA) dissociated the vesicle complex. RCAII/Ca2+-induced vesicle agglutination was accomplished by binding of Ca2+ to RCAII after the lectin/receptor interaction. The rate of SBA-induced vesicle agglutination was increased in the presence of Ca2+, independent of the order of Ca2+ addition, and was not reversed by EDTA, indicating that the mechanism by which Ca2+ stimulated agglutination in this case was different from that observed in the presence of RCAII. In contrast to RCAII/Ca2+, SBA/Ca2+ induced of the vesicles, which occurred only when Ca2+ was added after lectin addition. Close approach of adjacent bilayers was accomplished by nonspecific interactions of SBA with the bilayer after lectin binding to the receptor as revealed by a limited extent of SBA-induced fusion and an enhanced membrane permeability upon lectin binding. The phenomena observed can be explained in terms of a Ca2+-modulated reorientation of the carbohydrate head group, causing it to adopt a more perpendicular orientation with respect to the plane of the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acceleration of glycolysis in the presence of the non-phosphorylating and the oxidized phosphorylating glyceraldehyde-3-phosphate dehydrogenases

Mild oxidation of glyceraldehyde-3-phosphate dehydrogenase in the presence of hydrogen peroxide leads to oxidation of some of the active site cysteine residues to sulfenic acid derivatives, resulting in the induction of acylphosphatase activity. The reduced active sites of the enzyme retain the ability to oxidize glyceraldehyde-3-phosphate yielding 1,3-diphosphoglycerate, while the oxidized active sites catalyze irreversible cleavage of 1,3-diphosphoglycerate. It was assumed that the oxidation of glyceraldehyde-3-phosphate dehydrogenase by different physiological oxidants must accelerate glycolysis due to uncoupling of the reactions of oxidation and phosphorylation. It was shown that the addition of hydrogen peroxide to the mixture of glycolytic enzymes or to the muscle extract increased production of lactate, decreasing the yield of ATP. A similar effect was observed in the presence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase catalyzing irreversible oxidation of glyceraldehyde-3-phosphate into 3-phosphoglycerate. A role of glyceraldehyde-3-phosphate dehydrogenase in regulation of glycolysis is discussed.