Exploring the action and specificity of cobra venom phospholipase A2 toward human erythrocytes, ghost membranes, and lipid mixtures

We have investigated the action and substrate specificity of phospholipase A2 (EC 3.1.1.4) purified from cobra venom (Naja naja naja) toward intact and Triton-solubilized human erythrocytes, toward ghost membranes, and toward extracted ghost lipids in mixed micelles with Triton X-100. We have found that: (i) phospholipids in the outer surface of intact erythrocytes are extremely poor substrates for the phospholipase, (ii) phospholipids in ghost erythrocyte membranes and in Triton-solubilized erythrocytes are suitable substrates for the enzyme, (iii) in these latter systems which contain a mixture of lipids, phosphatidylethanolamine is preferentially hydrolyzed, whereas in model studies on individual phospholipid species in mixed micelles with Triton, phosphatidylcholine is the preferred substrate of the enzyme, and (iv) the preferential hydrolysis of phosphatidylethanolamine is also observed for extracted ghost lipid mixtures in mixed micelles. These results demonstrate a dependence of phospholipase A2 activity on the ghosting procedure and a dependence of substrate specificity on the presence of other lipids. The relevance of these findings to the interpretation of membrane lipid asymmetry studies utilizing phospholipases is considered in detail.     

Specificity reversal in phospholipase A2 hydrolysis of lipid mixtures

The activity and specificity of phospholipase A₂ from cobra venom ($\text{Naja naja naja}$) toward binary mixtures of phosphatidylcholine and phosphatidylethanolamine in mixed micelles with the nonionic surfactant Triton X-100 were examined. In mixtures containing 5–50 mol % phosphatidylcholine, the rate for phosphatidylethanolamine hydrolysis was enhanced greatly over that for phosphatidylcholine. This is in marked contrast to previous studies with individual phospholipid species in mixed micelles where phosphatidylcholine was found to be the preferred substrate and phosphatidylethanolamine was found to be a very poor substrate. Possible explanations for this specificity reversal are considered.     

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)     

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.     

Comparative study on the properties of saturated phosphatidylethanolamine and phosphatidylcholine bilayers: Barrier characteristics and susceptibility to phospholipase A2 degradation

Comparative studies on bilayer systems of saturated phosphatidylcholines and phosphatidylethanolamines revealed a maximum in ionic permeability in phosphatidylcholine bilayers at the temperature of the gel to liquid-crystalline phase transition but such an increase in permeability was not detectable in bilayers of phosphatidylethanolamine. Furthermore, it was found that at the phase transition temperature the phosphatidylcholine bilayers are subject to rapid hydrolysis by pancreatic phospholipase A₂ whereas phosphatidylethanolamine bilayers are not. These differences are discussed in view of detailed information on the molecular organization in the gel and liquid crystalline phases of the two phospholipid classes.     

The transverse distribution of phospholipids in the membranes of Golgi subfractions of rat hepatocytes.

The transverse distribution of phospholipids in the membranes of subfractions of the Golgi complex was investigated by using phospholipase C and 2,4,6-trinitrobenzenesulphonic acid as probes. In trans-enriched Golgi membranes, 26% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate or for hydrolysis by phospholipase C, and 72% of the phosphatidylcholine is hydrolysed by phospholipase C. In cis-enriched Golgi membranes, 45% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate and for hydrolysis by phospholipase C, and 95% of the phosphatidylcholine is hydrolysed by phospholipase C. Under the conditions used with either probe the contents of the Golgi vesicles labelled with either [3H]palmitic acid or [14C]leucine were retained. Galactosyltransferase activity of the membrane vesicles was partially inhibited by the experimental procedures used to investigate the transverse distribution of phospholipids. However, the residual activity was latent, suggesting that the vesicles remained closed. Trinitrobenzenesulphonic acid caused no detectable morphological change in either Golgi fraction. Phospholipase C treatment caused morphological changes, including fusion of vesicles and the appearance of 'signet-ring' profiles in some vesicles; however, the vesicles remained closed and the bilayer was retained. It appears, therefore, that neither probe causes major disruption of the Golgi vesicles nor gains access to the inner surface of the membrane bilayer. These observations suggest that phospholipids have a transverse asymmetry in Golgi membranes, that this distribution differs in trans and cis membranes, and that the phospholipid structure of Golgi membranes is inconsistent with a simple flow of membrane bilayer from endoplasmic reticulum to Golgi membranes to plasma membrane.     

Phospholipase C from Clostridium novyi type A. II. Factors influencing the enzyme activity

The apparent activity of phospholipase C[EC 3.1.4.3] of Clostridium novyi type A toward phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine increased in the presence of sodium deoxycholate (SDC). The effects of divalent cations on phospholipase C activity were examined in detail at various concentrations of these cations. These effects varied with substrate. Hydrolysis of phosphatidylcholine by this enzyme significantly increased in the presence of Mg2+ or Ca2+. Hydrolysis of sphingomyelin was inhibited by Ca2+, but increased in the presence of Mg2+. Phosphatidylethanolamine-hydrolyzing activity increased only slightly in the presence of Mg2+ and Ca2+. Zn2+ rather inhibited hydrolysis of these substrates. The effects of divalent cations and detergent appear to be directly related to the physical state of the phospholipid micelles used as substrates. When phosphatidylcholine, sphingomyelin, or phosphatidylethanolamine was used as a substrate, phospholipase C activity was completely inhibited by 2.5 mM EDTA or o-phenanthroline (concentration in the final incubation mixture: 0.5 mM), and was fully restored by Zn2+ alone. Both Ca2+ and Mg2+ were ineffective for reactivation. The isoelectric point of the enzyme was 7.1 +/- 0.1.     

Study of phospholipase A2 specificity in substrate-containing structures having different supramolecular organization

The specificity of snake venom phospholipase A2(PLA2) towards a number of phospholipid (PL) substrates, e. g., phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) organized in Triton X-100 mixed micelles, liposomes and proteoliposomes was studied. PC was shown to be more rapidly hydrolyzed in micelles. For other PLs, the rate of hydrolysis decreased in the following sequence: PC greater than PI greater than PE greater than PG. The incorporation into micelles of a non-hydrolyzable by PLA2 sphinogomyelin which, similar to PC, has a choline group, resulted in an increase of PLA2 specificity towards PL that are known to be devoid of this group: PE greater than PI greater than PG greater than PC. Quite a different picture was observed in bilayer liposomal structures: PI congruent to PE greater than PC greater than PG. The incorporation of cytochrome P-450 into liposomes caused the acceleration of PE and PG hydrolysis. The course of the PLA2-catalyzed hydrolysis in model membrane structures seems to be governed primarily by the supramolecular organization and localization of the substrate in the bilayer, but not by its chemical structure.     

Phospholipid topology and flip-flop in intestinal brush-border membrane

The topological distribution of the two major phospholipids of brush-border membrane, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), has been investigated using brush-border membrane vesicles from rabbit small intestine. Bee venom phospholipase A2 and phosphatidylcholine exchange protein from bovine liver were used as membrane probes. It is shown that the brush-border membrane retains its integrity under conditions of phospholipase hydrolysis and intermembrane phospholipid exchange. Kinetic analysis of the data of phospholipase hydrolysis and phospholipid exchange at temperatures under 10 degrees C shows that both PC and PE occur in two pools: a minor (about 25%) more readily accessible pool and a major one (about 75%) less readily available. The rate of PC exchange between these two pools is relatively fast. The half-time derived under conditions of phospholipase hydrolysis is of the order of 20 min. Under conditions of phospholipid exchange the exchange rates may be even faster. The difference in exchange kinetics observed with the two methods of probing is probably due to changes in membrane properties such as the bilayer fluidity induced by the probing process itself. It is proposed that the two pools represent the transverse distribution of the phospholipids. The two major phospholipids of brush-border membranes, PC and PE, would be distributed mainly on the inner (cytoplasmic) side of the brush-border membrane. The phospholipid exchange between the brush-border vesicles and unilamellar phosphatidylcholine vesicles in the presence of phosphatidylcholine exchange protein reveals that significant quantities of phospholipid are taken up by brush-border membrane independently, i.e., in a separate process independent of the exchange protein-catalyzed phosphatidylcholine exchange.     

Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidylcholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A₂ of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphingomyelin which is not hydrolysed by the former enzyme.     

1-14C]oleate-labeled autoclaved yeast: a membranous substrate for measuring phospholipase A2 activity in vitro

Radiolabeled, autoclaved yeast were tested as a substrate for mammalian phospholipase A2 activity because the only other membranous substrate used for this purpose, autoclaved Escherichia coli, totally lacks a major mammalian phospholipid, phosphatidylcholine. Candida albicans were grown in the presence of [1-14C]oleate and then autoclaved. Sixty three percent of the incorporated label was in yeast phospholipid, and more than 95% of that was in the 2-acyl position. The distribution of label in the yeast phospholipids (phosphatidylcholine and -ethanolamine, -serine + -inositol, and phosphatidic acid corresponded closely to the chemical distribution of phosphorus in those phospholipids. Snake venom (Naja naja) and human synovial fluid phospholipase A2 hydrolyzed yeast phospholipid exclusively to release 14C-labeled fatty acid. When 50-60% of the yeast phospholipid was hydrolyzed, the radioactive fatty acids as determined by gas-liquid chromatographic analysis were predominantly oleate (45%) and linoleate (greater than 54%). Hydrolysis of yeast phospholipid by both enzymes was near-linear with protein and time under conditions of optimal pH (neutral-alkaline) and Ca2- (1-5 mM) previously reported for optimal hydrolysis of autoclaved E. coli phospholipid. N. naja phospholipase A2 showed less preference for phosphatidylethanolamine than -choline as liposomes or yeast phospholipid as compared to human synovial fluid phospholipase A2 which clearly preferred phosphatidylethanolamine to -choline as a liposome or yeast phospholipid. These results illustrate that radiolabeled phospholipids of autoclaved yeast, enriched in phosphatidylcholine, are readily hydrolyzed by snake venom and human nonpancreatic phospholipases A2 and may, therefore, be useful in the measurement of in vitro enzymatic activity.     

Phospholipid and detergent effects on (Ca2+ + Mg2+)ATPase purified from human erythrocytes

(Ca2+ + Mg2+)ATPase (EC 3.6.1.3) was solubilized from human erythrocyte membranes by detergent extraction with Triton N-101 (0.5 mg/mg membrane protein) and purified by calmodulin affinity chromatography. ATPase activity was assayed in mixtures of Triton N-101 and phospholipid, without reconstitution into bilayer vesicles. At low levels of phospholipid (5 micrograms/ml), the ATPase activity was highly sensitive to the detergent concentration, with maximal activity occurring at or near the critical micelle concentration of the detergent. With increased amounts of phospholipid (50 micrograms/ml), detergent concentrations greater than the critical micelle concentration were required for maximal activity. Detergent alone did not support ATPase activity. Sonicated phospholipid in the form of vesicles was equally ineffective. Activity seemed to be dependent on the presence of detergent/phospholipid mixed micelles. The acidic phospholipids, phosphatidylserine and phosphatidylinositol, as well as the commercial phospholipid preparation, Asolectin, gave activities five to eight times greater than the same amount of phosphatidylcholine. Mixtures of phosphatidylserine and phosphatidylcholine produced intermediate ATPase activities, with the maximal value dependent on the phosphatidylserine concentration. Addition of phosphatidylcholine to fixed concentrations of phosphatidylserine caused a rise in activity that was independent of the ratio of the two phospholipids or the total phospholipid concentration. Phosphatidylcholine may therefore be irreplaceable for some aspect of ATPase function. The number of phospholipid molecules present in mixed micelles at maximal ATPase activity was calculated to be near 50. This value implied that the hydrophobic surface of the ATPase molecule must be completely coated by a single layer of phospholipid molecules for maximum activity to occur.     

Interaction with phospholipids of a membrane thiol peptidase that is essential for the signal transduction of mating pheromone in Rhodosporidium toruloides

Interaction with phospholipids of a membrane thiol peptidase [referred to as trigger peptidase (TPase), T. Miyakawa et al. (1987) J. Bacteriol. 169, 1626-1631] that plays a key role in the signalling of a lipopeptidyl mating pheromone at the cell surface of pheromone-target cell (mating type a) of Rhodosporidium toruloides was studied. The activity of highly purified TPase which requires phospholipids was restored by reconstitution of the enzyme into liposomes prepared with phospholipids extracted from the yeast cell. The presence of Ca2+ was essential for both the reconstitution process and the catalytic reaction of TPase. Triton X-100 mixed micelles containing phospholipids also activated the enzyme. The specificity and stoichiometry of activation by phospholipids was investigated by determination of TPase in the presence of mixed micelles that contained defined classes and numbers of phospholipid molecules in the Triton X-100 micelles. It was demonstrated that TPase is activated by mixed micelles containing 2-6 molecules of phosphatidylserine or phosphatidylethanolamine. Other phospholipids of the membranes of this organism, such as phosphatidylcholine and phosphatidylglycerol, had little effect on activation, indicating that the amino group of the phospholipids may be required for the function of TPase. Direct evidence for the interaction of TPase and Triton X-100/phosphatidylserine mixed micelles was obtained by molecular sieve chromatography on Sephacryl S-200. These data established that a phospholipid bilayer is not a requirement for TPase activation, and that the purified enzyme can be activated by a relatively small number of phospholipid molecules of specific classes.     

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.     

Correlation between changes in the membrane organization and susceptibility to phospholipase C attack induced by ATP depletion of rat erythrocytes

About 20 and 43% of the total membrane phospholipids are hydrolized in fresh rat erythrocytes by treatment with phospholipase C (Bacillus cereus), or both sphingomyelinase and phospholipase C, respectively, without causing cell lysis. Treatment of ATP-depleted cells with phospholipase C alone results in 50% hydrolysis and extensive lysis. Depletion of ATP causes a marked increase in the aggregation of intramembranous particles accompanied by a similar increase in the smooth area between the particle clusters as revealed by the freeze-etch technique. Such changes are not induced by extensive phospholipid hydrolysis in absence of cell lysis in fresh cells.Based on these and additional data, it is suggested that the membrane phospholipid organization can be divided into 3 types: phospholipids exposed to phospholipase C; phospholipids protected against phospholipase C by presence of sphingomyelin; phospholipids which can be exposed following alteration of the proteinlipid interactions. Such alterations which might be induced by a variety of means, including ATP depletion, might result in clustering of intramembranous particles and increase of the free lipid bilayer phase of the membrane.     

Preferential interaction of alpha-tocopherol with phosphatidylcholines in mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine.

The effect of alpha-tocopherol on the structure and phase behaviour of mixed aqueous dispersions of phosphatidylcholine and phosphatidylethanolamine has been examined by synchrotron X-ray diffraction and freeze-fracture electron microscopy. Equimolar mixtures of fully saturated derivatives of phospholipids that show gel phase immiscibility were examined including dimyristoylglycerophosphocholine/dipalmitoylglycerophosphoethanolamin e and distearoylglycerophosphocholine/dilauroylglycerophosphoethanolamine++ +. Analysis of the X-ray scattering intensities recorded at wide angles during heating scans of mixed aqueous dispersions containing 2.5 or 5 mol% alpha-tocopherol showed that alpha-tocopherol disordered the acyl chain packing of the phosphatidylcholine to a greater extent than the phosphatidylethanolamine component of the mixture. This suggested that alpha-tocopherol preferentially interacts with phosphatidylcholine rather than phosphatidylethanolamine, irrespective of whether this was the high or low melting point component of the mixture. The presence of 20 mol% alpha-tocopherol in either phospholipid mixture prevented gel phase separation during the prior cooling scan and no conclusions could be drawn as to the distribution of alpha-tocopherol in these mixtures.     

Cobra venom phospholipase A2 immobilized to porocus glass beads.

Phospholipase A₂ (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A₂ toward phosphatidylcholine is about 160 μmol min^?1 ml^?1 of glass beads, and the specific activity is about 13 μmol min^?1 mg^?1 of protein in this assay system. The pH rate profile and apparent pK_a in 10 mm Ca²⁺ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A₂ from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered.     

The alkaline phospholipase A1 of rat liver cytosol.

1. Rat liver cytosol contains a heat-sensitive phospholipase A1 active against phosphatidylethanolamine, 1-acylglycerophosphoethanolamine and, to a very much lesser extent, phosphatidylcholine and phosphatidylinositol. 2. Activity towards a pure phosphatidylethanolamine substrate is invoked by the presence of water-soluble cations that do not precipitate at the pH optimum of the enzyme (9.5). In this activation bivalent cations, e.g. Mg2+, Ca2+, Mn2+, Sr2+ and Ba2+, are effective at much lower concentrations (2.5-5 mM) than univalent cations K+, Na+ and NH4+ (100 mM). 3. In the absence of such cations the enzyme can be activated by cationic amphiphiles containing quaternary nitrogen or by basic proteins. 4. It is concluded that these agents activate the enzyme by reducing the negative zeta potential on the substrate at the high pH optimum (9.5) and allow interaction with the enzyme whose isoelectric point is at 7.15. 5. The activated enzyme is markedly inhibited by mixing the phosphatidylethanolamine substrate with many other phospholipids that exist in cell membranes, e.g. phosphatidylcholine, phosphatidylinositol. On the other hand, both phosphatidylcholine and phosphatidylinositol can be hydrolysed much more readily if they are mixed with an excess of phosphatidylethanolamine. 6. Such results on the inhibition and substrate specificity of the enzyme, coupled with birefringence measurements, allow the tentative conclusion that phospholipid substrates are only attacked when they exist in a hexagonal or non-bilayer structure and not in the bilayer (lamellar) form.     

Pancreatic porcine phospholipase A2 catalyzed hydrolysis of phosphatidylcholine in lecithin-bile salt mixed micelles: kinetic studies in a lecithin-sodium cholate system

Pancreatic porcine phospholipase A2 catalyzed hydrolysis of phosphatidylcholine in bile salt lecithin mixed micelles has been studied, utilizing a series of assay mixtures for which the micellar size, weight, and composition had been experimentally determined. Under these conditions the enzymatic hydrolysis is dependent on the phosphatidylcholine-to-sodium cholate molar ratio within the mixed micelle rather than the bulk concentration of the phospholipid in the mixture: at 5 mM phosphatidylcholine, variation of the NPC/NNaCh ratio from 0.2 to 2.0 increases the enzymatic activity from 82 to 933 mumol/min/mg protein. The initial rates are linear throughout the entire series of assay mixtures, the activity vs micellar concentration curves exhibit saturation behavior, and treatment of the data according to the "surface-as-cofactor" theory provides linear double-reciprocal plots which intersect in one point. The assay system should be applicable for detailed kinetic studies of lipolytic enzymes, including mammalian phospholipases which exhibit rather low activities toward lecithin-Triton X-100 mixed micelles. The system should also provide a convenient basis for mechanistic studies involving the use of inhibitory phospholipid substrate analogs.     

Kinetic study of the action of snake venom phospholipase A2 on human serum high density lipoprotein 3.

The hydrolysis of the phospholipids of intact human serum high density lipoprotein 3 (HDL3) by pure alpha-phospholipase A2 from Crotalus adamanteus was studied by pH-stat titration. The enzyme quantitatively hydrolyzed phosphatidylcholine and phosphatidylethanolamine and left sphinogomyelin intact, yielding a stable and water-soluble modified HDL. Lysophospholipids and free fatty acids, the products of hydrolysis, remained in the lipoprotein. When 1 mol of defatted bovine serum albumin/mol of substrate phospholipids was added to the reaction mixture, up to 60% of the fatty acids and 85% of the lysophospholipids were removed from the modified lipoprotein. The immunological reactivity of the hydrolyzed HDL remained unaltered in both the presence and absence of albumin. The changes in the physical properties of the lipoprotein during hydrolysis were rather small, the most notable being an increase in the hydrated density and in the electrophoretic mobility in alkaline buffers. The hydrolysis followed an apparent first order time course with product inhibition (KI) and yielded values of kcat/Km = 7 X 10(5 M(-1)s(-1) and KI congruent to 1 X 10(-4) M. Addition of albumin to the reaction mixture relieved the product inhibition without any alteration of the kinetic parameters. High concentrations of albumin protected some of the substrate phospholipids from hydrolysis, presumably through complexation to the lipoprotein. The Arrhenius plot for the experimental first order rate constant in the absence of albumin (kexp = kcat (KI/Km)) was linear between 15 degrees and 47 degrees, indicating the absence of any phospholipid phase transitions and yielding an activation energy of 15.2 kcal/mol. From the accessibility of the HDL phospholipids to phospholipase A2 one concludes that the phosphatidylcholine and phosphatidylethanolamine are located at, or are in rapid equilibrium with, the surface of this lipoprotein. It also appears that these phospholipids are not essential for maintaining the supramolecular properties of the lipoprotein in vitro. Thsu the study of the modified Hdl should provide valuable information concenring the structure and function of this lipoprotein particularly with regard to the role played by shiingomyelin.