Sodium-induced aggregation of phosphatidic acid and mixed phospholipid vesicles

Sodium-induced aggregations of sonicated vesicles prepared from synthetic phosphatidic acid and from its 1 : 1 mixtures with synthetic phosphatidylethanolamine and phosphatidylcholine were studied by turbidimetric measurements. The aggregation reactions were almost completely reversible on change in the Na⁺ concentration, pH or temperature. The threshold concentrations of Na⁺ for aggregation of pure dipalmitoylphosphatidic acid vesicles and mixed dipalmitoylphosphatidylenolamine- and dimyristoylphosphatidylcholine-dipalmitoylphosphatidic acid vesicles were found to be 200, 310 and 550 mM, respectively, at 25° and pH 7.2. The hydrocarbon chain lengths of phosphatidic acid and phosphatidylethanolamine had little effect on the threshold concentrations. The threshold concentrations for phospholipid vesicles composed of phosphatidic acid alone or its 1 : 1 mixture with phosphatidylethanolamine were changed by varying either the pH or temperature, while that for phosphatidylcholine-phosphatidic acid vesicles was almost independent of the pH and temperature, implying that aggregation of the latter vesicles is induced by a somewhat different mechanism.     

On the substrate specificity of rat liver phospholipase A1

The substrate specificity of purified phospholipase A1 was studied using mixed micelles of phospholipid and Triton X-100. The kinetic analysis employed determined Vmax, Ks (a dissociation constant for the phospholipase A1-mixed micelle complex), and Km (the Michaelis constant for the catalytic step which reflects the binding of the enzyme to the substrate in the interface). The order of Vmax values was phosphatidic acid greater than phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine. The order of Ks values was phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidic acid greater than phosphatidylserine; the order of Km values was phosphatidic acid greater than phosphatidylethanolamine = phosphatidylserine greater than phosphatidylcholine. When present together, phosphatidylcholine inhibited the hydrolysis of phosphatidylethanolamine but phosphatidylethanolamine did not affect the hydrolysis of phosphatidylcholine. Sphingomyelin, phosphatidylcholine plasmalogen, and phosphatidylethanolamine plasmalogen had no effect on the hydrolysis of phosphatidylethanolamine. The effects of the reaction products, lysolipids and/or fatty acids, were also considered for their influence on phosphatidylethanolamine hydrolysis catalyzed by phospholipase A1. Free fatty acid was found to inhibit, whereas lysophospholipids stimulated hydrolysis of phosphatidylethanolamine. In a mixture of 1,2- and 1,3-diacylglycerides in mixed micelles, only the acyl chain at the sn-1 position of the 1,2 compound was hydrolyzed. Surface charge did not modulate the hydrolysis of phosphatidylcholine vesicles or mixed micelles. In conclusion, it is hypothesized that steric hindrance at position 3 of the glycerol regulates substrate binding in the active site and that an acyl group in position 1 is favored over a vinyl ether linkage for binding.     

Hydrolysis of lipid mixtures by rat hepatic lipase

The hydrolysis of phospholipid mixtures by purified rat hepatic lipase, also known as hepatic triglyceride lipase, was studied in a Triton X-100/lipid mixed micellar system. Column chromatography of the mixed micelles showed elution of Triton X-100 and binary lipid mixtures of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine as a single peak. This indicated that the mixed micelles were homogenous and contained all components in the designated molar ratios. The molar ratio of Triton X-100 to lipid was kept constant at 4 to 1. Labeling one lipid with 3H and the other lipid with 14C enabled us to determine the hydrolysis of both components of these binary lipid mixed micelles. We found that the hydrolysis of phosphatidylcholine was activated by the inclusion of small amounts of phosphatidic acid (2.5-fold), phosphatidylethanolamine (1.5-fold) or phosphatidylserine (1.4-fold). The maximal activation of phosphatidylcholine hydrolysis was observed when 5 mol% of phosphatidylethanolamine, 7.5 mol% phosphatidic acid or 5 mol% phosphatidylserine was added to Triton X-100 mixed micelles. The hydrolysis of phosphatidic acid was activated 30%, and that of phosphatidylserine was inhibited 30% when the molar proportion of phosphatidylcholine was less than 50 mol%. The hydrolysis of phosphatidylethanolamine was slightly activated when the mol% of phosphatidylcholine was below 5. The hydrolysis of phosphatidylserine was inhibited by phosphatidylethanolamine when the mol% of the latter was 50 or less whereas phosphatidylethanolamine hydrolysis was not affected by phosphatidylserine. Under the conditions used sphingomyelin and cholesterol did not have a significant effect on the hydrolysis of the phospholipids studied. In agreement with our previous study (Kucera et al. (1988) J. Biol. Chem. 263, 1920-1928) these studies show that the phospholipid polar head group is an important factor which influences the action of hepatic lipase and that the interfacial properties of the substrate play a role in the expression of the activity of this enzyme. The molar ratios of phosphatidic acid, phosphatidylethanolamine and phosphatidylserine which activated phosphatidylcholine hydrolysis correspond closely to the molar ratios of these lipids found in the surface lipid film of lipoproteins e.g., high density lipoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Distribution of phospholipids around gramicidin and D-beta-hydroxybutyrate dehydrogenase as measured by resonance energy transfer

A resonance energy transfer method was developed to study the distribution of phospholipids around integral membrane proteins. The method involved measuring the extent of energy transfer from tryptophan residues of the proteins to different phospholipids labeled with a dansyl moiety in the fatty acid chain. No specific interactions were observed between gramicidin and dansyl-labeled phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid. The results were consistent with a random distribution of each phospholipid in the bilayer in the presence of gramicidin. However, a redistribution of both gramicidin and dansyl-labeled phospholipids was easily observed when a phase separation was induced by adding Ca2+ to vesicles made up of phosphatidylcholine and phosphatidic acid. Polarization measurements showed that in the presence of Ca2+ a rigid phosphatidic acid rich region and a more fluid phosphatidylcholine-rich region were formed. Energy-transfer measurements from gramicidin to either dansylphosphatidylcholine or dansylphosphatidic acid showed gramicidin preferentially partitioned into the phosphatidylcholine-rich regions. Energy-transfer measurements were also carried out with D-beta-hydroxybutyrate dehydrogenase reconstituted in a vesicle composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid. Although the enzyme has a specific requirement for phosphatidylcholine for activity, the extent of energy transfer decreased in the order dansylphosphatidic acid, dansylphosphatidylcholine, dansylphosphatidylethanolamine. Thus, the enzyme reorganized the phospholipids in the vesicle into a nonrandom distribution.     

Hydrolysis of thioester analogs by rat liver phospholipase A1

The hydrolysis of thioester containing phospholipids by rat liver plasmalemma phospholipase A1 was measured in a continuous spectrophotometric assay. In this assay thioester substrates were employed which, upon hydrolysis, liberated a free thiol which was reacted with 4,4'-dithiopyridine to yield the product 4-thiopyridone that absorbs at 324 nm. Thioester substrates, prepared by chemical synthesis, were used in phospholipid and Triton X-100 micelles for kinetic analysis carried out according to the method of Hendrickson and Dennis (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739). Vmax, Ks, and Km values obtained for various isomers and racemic mixtures of the synthetic thioester analogs are compared with corresponding oxyester substrates. Unnatural sn-1 isomers competitively inhibited the hydrolysis of natural sn-3 isomers of phosphatidylethanolamine and phosphatidic acid. Furthermore, the sn-1 isomer of phosphatidic acid was hydrolyzed by phospholipase A1, but with lower catalytic efficiency than the sn-3 isomer. The presence of a thioester at the sn-1 position did not change the Vmax significantly, as compared to the oxyester phospholipids. When two thioesters were present on the phospholipid molecule, the Vmax was decreased significantly. A convenient synthesis of 1-monothioester analogs of phospholipids is reported. The results presented show the usefulness of the spectrophotometric assay for measuring phospholipase A1 activity as well as the influence of racemic mixtures and thioesters on the hydrolytic rate.     

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.     

Regulators of G-protein signaling (RGS) 4, insertion into model membranes and inhibition of activity by phosphatidic acid

Regulators of G-protein signaling (RGS) proteins are critical for attenuating G protein-coupled signaling pathways. The membrane association of RGS4 has been reported to be crucial for its regulatory activity in reconstituted vesicles and physiological roles in vivo. In this study, we report that RGS4 initially binds onto the surface of anionic phospholipid vesicles and subsequently inserts into, but not through, the membrane bilayer. Phosphatidic acid, one of anionic phospholipids, could dramatically inhibit the ability of RGS4 to accelerate GTPase activity in vitro. Phosphatidic acid is an effective and potent inhibitor of RGS4 in a G alpha(i1)-[gamma-(32)P]GTP single turnover assay with an IC(50) approximately 4 microm and maximum inhibition of over 90%. Furthermore, phosphatidic acid was the only phospholipid tested that inhibited RGS4 activity in a receptor-mediated, steady-state GTP hydrolysis assay. When phosphatidic acid (10 mol %) was incorporated into m1 acetylcholine receptor-G alpha(q) vesicles, RGS4 GAP activity was markedly inhibited by more than 70% and the EC(50) of RGS4 was increased from 1.5 to 7 nm. Phosphatidic acid also induced a conformational change in the RGS domain of RGS4 measured by acrylamide-quenching experiments. Truncation of the N terminus of RGS4 (residues 1-57) resulted in the loss of both phosphatidic acid binding and lipid-mediated functional inhibition. A single point mutation in RGS4 (Lys(20) to Glu) permitted its binding to phosphatidic acid-containing vesicles but prevented lipid-induced conformational changes in the RGS domain and abolished the inhibition of its GAP activity. We speculate that the activation of phospholipase D or diacylglycerol kinase via G protein-mediated signaling cascades will increase the local concentration of phosphatidic acid, which in turn block RGS4 GAP activity in vivo. Thus, RGS4 may represent a novel effector of phosphatidic acid, and this phospholipid may function as a feedback regulator in G protein-mediated signaling pathways.     

Phosphatidic acid distribution on the external surface of mixed vesicles.

A new method has been developed to detect the distribution of phosphatidic acid on the external surface of mixed phospholipid vesicles. Some positive dyes undergo large absorbance changes when the spatial separation between two or more dye molecules is smaller than a critical distance. When these dyes interact with mixed phospholipid vesicles, the absorbance changes may be utilized to calculate the amount of phosphatidic acid molecules which, on the external surface, occupy nearby positions not exceeding the critical dye distance, i.e., the amount of paired phosphatidic acid molecules. This amount was found to be higher than that calculated by statistical methods, indicating that phosphatidic acid molecules tend to be associated, in spite of the electrostatic repulsion between negative groups. The dependence of the amount of paired phosphatidic acid molecules on the pH, phosphatidylcholine:phosphatidic acid ratio, and temperature has been also analyzed.     

Vinculin phosphorylation by the src kinase. Interaction of vinculin with phospholipid vesicles

Vinculin phosphorylation by pp60src is stimulated by anionic phospholipids (Ito, S., Richert, N., and Pastan, I. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4628-4631). We have examined whether vinculin interacts with phospholipids, the specificity of the interactions, and a possible mechanism for the enhancement of vinculin phosphorylation by these phospholipids. 3H-labeled vinculin binds to phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. No binding to phosphatidylcholine or phosphatidylethanolamine was observed. The phospholipid binding specificity correlated with the ability of these phospholipids to enhance vinculin phosphorylation by the src kinase. Chlorpromazine (0.1 and 0.3 mM) inhibited both vinculin binding to phosphatidylinositol and the enhanced phosphorylation of vinculin by pp60src in the presence of phosphatidylinositol. Tryptic peptide maps of vinculin phosphorylated in the absence of phospholipid revealed three phosphorylated peptides. The same three peptides were phosphorylated in the presence of phospholipid. However, phosphorylation at one site was markedly increased. In the presence of phospholipid proteolysis of vinculin with both chymotrypsin and V8 protease was markedly enhanced and different peptide maps of vinculin were generated. Microheterogeneity of vinculin was observed with isoelectric focusing. All the isoforms (pI 5.45-5.8) were found to bind phospholipids and undergo phosphorylation by the src kinase. These results suggest that one way anionic phospholipids can enhance vinculin phosphorylation is by binding to vinculin and inducing a conformational change in the vinculin molecule.     

Action of Phospholipase A2 and Phospholipase C on Bacillus subtilis Protoplasts

Protoplasts prepared from Bacillus subtilis by lysozyme digestion lysed in the presence of pure pancreatic phospholipase A(2). The phospholipids cardiolipin, phosphatidylethanolamine, phosphatidylglycerol and lysylphosphatidylglycerol, which are present in the membrane, are degraded by phospholipase A(2) only after removal of the cell wall, giving free fatty acids and lyso derivatives. The four phospholipids are hydrolyzed equally well at a given enzyme concentration. Differences in the phospholipid composition of the protoplasts were obtained by variations in the growth medium, time of harvesting, and preincubation time with lysozyme. The extent of hydrolysis appeared to depend on the initial phospholipid composition. A relative increase in acidic phospholipids in the membrane facilitated the action of phospholipase A(2), whereas the rate of hydrolysis was diminished when protoplasts were tested which contained a relatively high amount of positively charged phospholipid. Pure phospholipase C from B. cereus preferentially hydrolyzed phosphatidyl-ethanolamine in the B. subtilis membrane. More than 80% of this phospholipid was converted into diglyceride, whereas only 30% of the cardiolipin was hydrolyzed. Such a loss of phospholipids, however, was not followed by lysis of the protoplasts. Liposomes were prepared from the lipid extracts of B. subtilis and incubated with both phospholipases. The hydrolysis pattern of the phospholipids in these model membrane systems was identical to the hydrolysis pattern of the phospholipids in the protoplast membrane. Phospholipase A(2) hydrolyzed all the phospholipids in the liposomes equally well, whereas phospholipase C preferentially degraded phosphatidylethanolamine.     

Uniform preparations of large unilamellar vesicles containing anionic lipids

A general procedure for the preparation of large unilamellar vesicles of selected sizes has been developed. The procedure consists of dissolving the lipid in organic solvent, washing with mild acid, removing the solvent, adding salt (0.15 M KCl) solution, and adjusting the pH (raising it to about pH 10 and lowering it immediately to pH 7.55). The procedure takes less than 30 min. The resulting unilamellar vesicles are of a single size with a rather low standard deviation. The sizes of these preparations range between 150 and 1000 nm in diameter. Sizes and polydispersities were measured to within 1-2% by photon correlation spectroscopy. Vesicle size varies with the phospholipid structure, the composition of the phospholipid mixture, the ionic strength of the medium, the alkyl chain composition, the cholesterol content of the phospholipid mixture, and the timing of the pH adjustment procedure. Uniform preparations of vesicles have been obtained from the dioleoyl esters of phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine, from diphytanyl ethers of glycolipid sulfate, phosphatidylglycerol, phosphatidylglycerol phosphate, and phosphatidylglycerol sulfate, from bovine liver phosphatidylinositol, from Escherichia coli phosphatidylethanolamine, from membrane lipid extracts from E. coli and Holabacterium cutirubrum, and from dodecanesulfonate-alkanol mixtures and free oleic acid. The preparation of unilamellar vesicles from oleic acid is novel, and the size range is 600-3000 nm; the preparations are relatively uniform. Vesicles of phospholipids in which sucrose and trehalose replace salt as the impermeant do not differ significantly from those prepared in pentaerythritol.     

Effect of surface composition on triolein hydrolysis in phospholipid vesicles and microemulsions by a purified acid lipase

Sonicated dispersions of egg yolk phosphatidylcholine and triolein as vesicles and microemulsions have been used as substrates for the assay of a purified acid lipase. Previous studies have also shown that triolein localized in the surface phase of emulsions is the preferred substrate. In this study, we examined enzyme activity following several surface modifications using both vesicles and microemulsions. When the acidic phospholipids phosphatidylserine and phosphatidic acid were incorporated into both vesicles and microemulsions at up to 10 mol % of the total phospholipid, a dose-dependent reduction in the apparent Km was observed. Using the vesicles as substrate, a dose-dependent decrease in Vmax was also observed. Agarose gel electrophoresis was used to verify suspected changes in net particle charge. Analogous inclusion of phosphatidylethanolamine, sphingomyelin, or cholesterol did not affect kinetic parameters. Addition of oleic acid to sonication mixtures produced vesicles with a decreased apparent Km and Vmax, but triolein hydrolysis in microemulsions was not significantly altered. Triolein-containing vesicles prepared by using dimyristoyl- or dipalmitoylphosphatidylcholine were hydrolyzed maximally at the gel liquid-crystalline transition temperatures of the appropriate phospholipid. Differential scanning calorimetry was used to verify the temperatures of transition in these vesicles. The results indicate that acid lipase activity is influenced by the charge or physical state of the surface phase of model substrates and suggest that degradation of core components of naturally occurring substrates such as lipoprotein may be influenced by chemical changes on the surface of these particles.     

Interfacial catalysis by phospholipase A2: substrate specificity in vesicles.

The binding equilibrium of phospholipase A2 (PLA2) to the substrate interface influences many aspects of the overall kinetics of interfacial catalysis by this enzyme. For example, the interpretation of kinetic data on substrate specificity was difficult when there was a significant kinetic contribution from the interfacial binding step to the steady-state catalytic turnover. This problem was commonly encountered with vesicles of zwitterionic phospholipids, where the binding of PLA2 to the interface was relatively poor. The action of PLA2 on phosphatidylcholine (PC) vesicles containing a small amount of anionic phospholipid, such as phosphatidic acid (PA), was studied. It was shown that the hydrolysis of these mixed lipid vesicles occurs in the scooting mode in which the enzyme remains tightly bound to the interface and only the substrate molecules present on the outer monolayer of the target vesicle became hydrolyzed Thus the phenomenon of scooting mode hydrolysis was not restricted to the action of PLA2 on vesicles of pure anionic phospholipids, but it was also observed with vesicles of zwitterionic lipids as long as a critical amount of anionic compound was present. Under such conditions, the initial rate of hydrolysis of PC in the mixed PC/PA vesicles was enhanced more than 50-fold. Binding studies of PLA2 to vesicles and kinetic studies in the scooting mode demonstrated that the enhancement of PC hydrolysis in the PC/PA covesicles was due to the much higher affinity of the enzyme toward covesicles compared to vesicles of pure PC phospholipids. A novel and technically simple protocol for accurate determination of the substrate specificity of PLA2 at the interface was also developed by using a double-radiolabel approach. Here, the action of PLA2 in the scooting mode was studied on vesicles of the anionic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol that contained small amounts of 3H- and 14C-labeled phospholipids. From an analysis of the 3H and 14C radioactivity in the released fatty acid products, the ratio of substrate specificity constants (kcat/KMS) was obtained for any pair of radiolabeled substrates. These studies showed that the PLA2s from pig pancreas and Naja naja naja venom did not discriminate between phosphatidylcholine and phosphatidylethanolamine phospholipids or between phospholipids with saturated versus unsaturated acyl chains and that the pig enzyme had a slight preference for anionic phospholipids (2-3-fold). The described protocol provided an accurate measure of the substrate specificity of PLA2 without complications arising from the differences in binding affinities of the enzyme to vesicles composed of pure phospholipids.     

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.     

Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium

Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The five methods gave concordant results, showing that vesicles containing only 10% phosphatidic acid can be induced to fuse by millimolar concentrations of Ca2+. When the fusing capability of several soluble proteins was assayed, it was found that concanavalin A, bovine serum albumin, ribonuclease, and protease were inactive. On the other hand, lysozyme, L-lactic dehydrogenase, and muscle and yeast glyceraldehyde-3-phosphate dehydrogenase were capable of inducing vesicle fusion. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, the most extensively studied protein, proved to be very effective: 0.1 microM was enough to induce complete intermixing of bilayer phospholipid vesicles. Under conditions used in this work, fusion was accompanied by leakage of internal contents. The fusing capability of glyceraldehyde-3-phosphate dehydrogenase was not affected by 5 mM ethylenediaminetetraacetic acid. The Ca2+ concentration in the medium, as determined by atomic absorption spectroscopy, was 5 ppm. Heat-denatured enzyme was incapable of inducing fusion. We conclude that glyceraldehyde-3-phosphate dehydrogenase is a soluble protein inherently endowed with the capability of fusing phospholipid vesicles.     

Studies on the activation of rat liver pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase by adrenaline and glucagon. Role of increases in intramitochondrial Ca2+ concentration.

The association of different phospholipids with a lipid-depleted oligomycin-sensitive ATPase from bovine cardiac mitochondria [Serrano, Kanner & Racker (1976) J. Biol. Chem. 251, 2453-2461] has been examined using three approaches. First, reconstitution of the ATPase with different synthetic diacyl phospholipids resulted in a 2-10-fold stimulation of ATPase specific activity depending upon the particular phospholipid employed. The phospholipid headgroup region displayed the following order of ATPase reactivation potential: dioleoylphosphatidylglycerol greater than dioleoylphosphatidic acid greater than dioleoylphosphatidylcholine. Furthermore, the ATPase showed higher levels of specific activity when reconstituted with dioleoyl phospholipid derivatives compared with dimyristoyl derivatives. Second, examination of the phospholipid remaining associated with the lipid-depleted ATPase upon purification showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were present. No relative enrichment of any of these phospholipids (compared with their distribution in submitochondrial particles) was noted. Therefore, no preferential association between the ATPase and any one phospholipid could be found in the mitochondrial ATPase. Third, the sodium cholate-mediated phospholipid exchange procedure was employed for studying the phospholipid requirements of the ATPase. Replacement of about 50% of the mitochondrial phospholipid remaining with the lipid-depleted ATPase could be achieved utilizing either synthetic phosphatidic acid or phosphatidylcholine. Examination of the displaced mitochondrial phospholipid showed that phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol were replaced with equal facility.     

Phospholipid interactions with cytochrome P-450 in reconstituted vesicles Preference for negatively-charged phosphatidic acid

Cytochrome $\text{P-450}$ LM2 was reconstituted by the cholate-dialysis method into vesicles containing a mixture of either phosphatidylcholine or phosphatidylethanolamine with up to 50 mol% of phosphatidic acid. Phase transition curves in the presence or absence of cytochrome $\text{P-450}$ were obtained from electron paramagnetic resonance experiments by measuring the partitioning of 2,2,6,6-tetramethylpiperidine-1-oxyl. Protein-free phospholipid vesicles exhibit a phase separation into domains of gel phase enriched in phosphatidic acid in a surrounding fluid matrix containing mainly phosphatidylcholine. The phase transition of the phosphatidic acid domains disappeared following incorporation of cytochrome $\text{P-450}$ into the bilayers. In contrast, in vesicles containing mixtures of egg-phosphatidic acid and dimyristoyl phosphatidylcholine, the phase transition of the domains enriched in dimyristoyl phosphatidylcholine was less sharp than in the corresponding vesicles containing cytochrome $\text{P-450}$. The results of both of these experiments could be explained by a redistribution of the mol fraction of the two phospholipids in the gel phase due to preferential binding of the egg-phosphatidic acid to the cytochrome $\text{P-450}$. For comparison, incorporation of cytochrome $\text{P-450}$ into uncharged vesicles of dimyristoyl phosphatidylcholine and egg-phosphatidylethanolamine did not alter the     

Catabolism of N-Acylethanolamine Phospholipids by Dog Brain Preparations

Abstract: N -Acylphosphatidylethanolamine, incubated with dog brain homogenate or microsomes, was hydroyzed to phosphatidic acid and N -acylethanolamine by a phosphodiesterase of the phospholipase D type. In the absence of F⁻, phosphatidic acid was further hydrolyzed to diacylglycerol and P_i while N -acylethanolamine was hydrolyzed by an amidase to fatty acid and ethanolamine. The phosphodiesterase showed an alkaline pH optimum and was also active towards N -acetylphosphatidyletha-nolamine, N -acyl-lysophosphatidylethanolamine, and glycerophospho( N -acyl)ethanolamine but showed little activity toward phosphatidylethanolamine and phosphati-dylcholine. Ca²⁺ stimulated slightly at low concentrations but inhibited at higher concentrations. Triton X-100 stim ulated the hydrolysis of N -acylphosphatidylethanol-amine, inhibited that of N -acyl-lysophosphatidyletha-nolamine and glycerophospho( N -acyl)ethanolamine, and had no effect on phosphatidylethanolamine or phospha-tidylcholine hydrolysis. The N -acylethanolamine hydrolase (amidase) was also present in the microsomal fraction and exhibited a pH optimum of 10.0. In addition to hydrolysis by the phosphodiesterase, N -acylphosphati-dylethanolamine was also catabolized by microsomal phospholipases A₁ and/or A₂ to N -acyl-lysophosphati-dylethanolamine, some of which was further hydrolyzed to glycerophospho( N -acyl)ethanolamine.     

Phosphatidylinositol synthase from Saccharomyces cerevisiae. Reconstitution, characterization, and regulation of activity

Purified membrane-associated phosphatidylinositol synthase (CDP diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) from Saccharomyces cerevisiae was reconstituted into unilamellar phospholipid vesicles. Reconstitution of the enzyme was performed by removing detergent from an octylglucoside/phospholipid/Triton X-100/enzyme mixed micelle mixture by Sephadex G-50 superfine column chromatography. The average diameter of the vesicles was 40 nm and chymotrypsin treatment of intact vesicles indicated that over 90% of the reconstituted enzyme had its active site facing outward. The enzymological properties and reaction mechanism of reconstituted phosphatidylinositol synthase were determined in the absence of detergent. The reconstituted enzyme was used as a model system to study the regulation of activity. Phosphatidylinositol synthase was constitutive in wild type cells grown in the presence of water-soluble phospholipid precursors as determined by enzyme activity and immunoblotting. Reconstituted enzyme was not effected by water-soluble phospholipid precursors or nucleotides. Maximum activity was found when the enzyme was reconstituted into phosphatidylcholine: phosphatidylethanolamine: phosphatidylinositol: phosphatidylserine vesicles. Phosphatidylserine stimulated reconstituted activity, suggesting that the local phospholipid environment may regulate phosphatidylinositol synthase activity.