Histidine and lysine residues and the activity of phospholipase A2 from the venom of Bitis gabonica.

Chemical modification of phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) from the venom of gaboon adder (Bitis gabonica) showed that histidine and lysine residues are essential for enzyme activity. Treatment with p-bromophenacyl bromide or pyridoxal 5'-phosphate resulted in the specific covalent modification of one histidine or a total of one lysine residue per molecule of enzyme, respectively, with a concomitant loss of enzyme activity. Competitive protection against modification and inactivation was afforded by the presence of Ca2+ and/or micellar concentrations of substrate analogue, lysophosphatidylcholine. Neither modification caused any significant conformational change, as judged from circular dichroic properties. Amino acid analyses and the alignment of peptides from cyanogen bromide and proteolytic cleavage of modified enzyme preparations delineated His-45 as the only residue modified by p-bromophenacyl bromide. However, pyridoxal 5'-phosphate was shown to have reacted not with a single lysine but with four different ones (residues 11, 33, 58 and 111) in such a manner that an overall stoichiometry of one modified lysine residue/molecule enzyme resulted. Apparently, the essential function of lysine could be fulfilled by any one out of these four residues.     

Role of phospholipid and protein-protein associations in activation and stabilization of soluble Ca2+-ATPase of sarcoplasmic reticulum

The effect of increasing concentrations of the nonionic detergent Triton X-100 on catalytic activity, stability, phospholipid content, and aggregational state of solubilized Ca2+ ion activated adenosinetriphosphatase (Ca2+-ATPase) of sarcoplasmic reticulum has been investigated. Increasing concentrations of Triton X-100 in the range 0.2-0.6% (w/v) inhibited ATP hydrolysis and p-nitrophenyl phosphate hydrolysis in parallel to the extent of 50% and 95%, respectively. Inactivation of p-nitrophenyl phosphate hydrolysis by preincubation in excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 25 degrees C was monophasic and first order at all concentrations of Triton X-100. The rate constant for inactivation increased sharply in the range 0.1-0.6% Triton X-100. At higher concentrations, the increase was less marked. Protein-protein associations of the solubilized ATPase were assessed by glutaraldehyde cross-linking and by ultracentrifugation in sucrose gradients. Both methods indicated a decrease in these associations in the 0.1-0.5% range. Cross-linking studies established that above 0.5% Triton X-100 the enzyme is greater than 90% monomeric. The amount of phospholipid associated with the ATPase, recovered from sucrose gradients, decreased from about 50 mol of phospholipid/mol of ATPase at 0.1% Triton X-100 to about 3 mol of phospholipid/mol of ATPase at 0.5% and higher concentrations. Monomeric ATPase and aggregated ATPase isolated from equilibrium mixtures of these components had similar phospholipid/protein ratios. The results indicated that with increasing Triton X-100 concentrations, inhibition of catalysis, destabilization, loss of protein-protein associations, and loss of phospholipid occur concurrently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatic microsomal epoxide hydrase. Involvement of a histidine at the active site suggests a nucleophilic mechanism

The effects of a wide variety of chemical modification reagents on the activity of purified rat liver microsomal epoxide hydrase have been investigated. Alkylating agents, such as the phenacyl bromides and benzyl bromide are potent inhibitors of epoxide hydrase. 2-Bromo-4'-nitroacetophenone (p-nitrophenacyl bromide) specifically and irreversibly inactivates epoxide hydrase. Pseudo-first order kinetics of inhibition is observed at higher inhibitor/enzyme ratios. The rate of inactivation is controlled by a group on the enzyme with an apparent pKa of 7.6. Inactivation of the enzyme with 14C-labeled 2-bromo-4'-nitroacetophenone leads to the incorporation of approximately 1 mol of radioactive inhibitor/mol of protein. Epoxide hydrase can be protected against this inactivation by the substrate phenanthrene-9,10-oxide. These results are consistent with the interpretation that 2-bromo-4'-nitroacetophenone acts as an active site-directed inhibitor. The site of alkylation by 2-bromo-4'-nitroacetophenone is a histidine residue of epoxide hydrase. The N-alkylated histidine derivative has been identified as 1-(p-nitrophenacyl)-4-histidine. A possible mechanism for the enzymatic hydration catalyzed by epoxide hydrase is discussed which involves a histidine residue of the enzyme serving as a general base catalyst for the nucleophilic addition of water.     

The lipid requirement of the (Ca2+ + Mg2+)-ATPase in the human erythrocyte membrane, as studied by various highly purified phospholipases.

1. When complete hydrolysis of glycerophosphlipids and sphingomyelin in the outer membrane leaflet is brought about by treatment of intact red blood cells with phospholipase A2 and sphingomyelinase C, the (Ca2+ + Mg2+)-ATPase activity is not affected. 2. Complete hydrolysis of sphingomyelin, by treatment of leaky ghosts with spingomyelinase C, does not lead to an inactivation of the (Ca2+ + Mg2+)-ATPase. 3. Treatment of ghosts with phospholipase A2 (from either procine pancreas of Naja naja venom), under conditions causing an essentially complete hydrolysis of the total glycerophospholipid fraction of the membrane, results in inactivation of the (Ca2+ + Mg2+)-ATPase by some 80--85%. The residual activity is lost when the produced lyso-compounds (and fatty acids) are removed by subsequent treatment of the ghosts with bovine serum albumin. 4. The degree of inactivation of the (Ca2+ + Mg2+)-ATPase, caused by treatment of ghosts with phospholipase C, is directly proportional to the percentage by which the glycerophospholipid fraction in the inner membrane layer is degraded. 5. After essentially complete inactivation of the (Ca2+ + Mg2+)-ATPase by treatment of ghosts with phospholipase C from Bacillus cereus, the enzyme is reactivated by the addition of any of the glycerophospholipids, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine or lysophosphatidylcholine, but not by addition of sphingomyeline, free fatty acids or the detergent Triton X-100. 6. It is concluded that only the glycerophospholipids in the human erythrocyte membrane are involved in the maintenance of the (Ca2+ + Mg2+)-ATPase activity, and in particular that fraction of these phospholipids located in the inner half of the membrane.     

Interaction of Trimeresurus flavoviridis phospholipase A2 and its fragment with calcium ion

Dimeric T. flavoviridis phospholipase A2 has been studied in terms of the interaction with essential Ca2+ by equilibrium gel filtration, ultraviolet difference spectroscopy, fluorescence measurements, and chemical modifications with p-bromophenacyl bromide. The subunit bound to Ca2+ with a 1:1 molar ratio and no cooperative binding was observed. The hypochromic effect produced upon the binding of Ca2+ is due to perturbation of (a) specific tryptophan residue(s) located in the vicinity of the active site and appears to be characteristic of this enzyme. On the basis of the pH dependence of the dissociation constants, it has been found that the alpha-amino group (pKa 8.7) controls the binding of Ca2+. Deprotonation of the alpha-amino group is possibly accompanied by conformational transition to the active form which is able to bind Ca2+. This is in contrast to the case of bovine pancreatic phospholipase A2 in which Asp-49 (pKa 5.2) is responsible for the metal ion binding (Fleer et al. (1981) Eur. J. Biochem. 113, 283-288). Des-octapeptide(1-8)-phospholipase A2 (L-fragment) was found to be capable of binding Ca2+ under the control of a group with a pKa of 7.6. This pKa value was similar to an apparent pKa of 7.5 determined for the histidine residue in the active site of the native enzyme by way of p-bromophenacyl bromide modification. It appears that the N-terminal (octapeptide) sequence affects the binding mode of Ca2+, possibly because of conformational transition arising from its removal. The reinvestigation showed that the N-terminal octapeptide sequence is Gly-Leu-Trp-Gln-Phe-Glu-Asn-Met.     

Investigations on the mechanism of action of crotoxin

Crotoxin, the major toxic protein of Crotalus durissus terrificus, is composed of a basic phospholipase, component-B, and of an acidic subunit, component-A. The crotoxin complex is insensitive to an active site directed reagent, p-bromophenacyl bromide, while its isolated enzymatic component-B is rapidly and irreversibly inactivated. We observed that crotoxin possesses an intrinsic phospholipase A2 activity on monodispersed substrates, indicating that the active site of component-B is not masked by component-A in the complex. The inactivation of component-B by p-bromophenacyl bromide follows pseudo-first order kinetics, with a rate constant proportional to the concentration of phospholipase, as expected for a reaction of second order with respect to the protein. On the basis of a detailed kinetic analysis of this reaction, and of physico-chemical studies of component-B in various experimental conditions, we propose that (1) an equilibrium exists between reactive dimers of component-B and preponderant but non-reactive monomer; (2) component-A protects component-B against inactivation by p-bromophenacyl bromide by preventing the formation of reactive dimers. When comparing the reactivity of component-B with that of other phospholipases, we observed that the enzymes which have not been shown to produce dimers all react with p-bromophenacyl bromide with similar low rates of reaction, while phospholipases which have been reported to form dimers react much more rapidly.     

Phospholipid binding and the activation of group IA secreted phospholipase A2

Equilibrium dialysis was used to study the binding of two nonhydrolyzable, short chain phospholipid analogues to the secreted group IA phospholipase A(2) (PLA(2)), which has been shown to contain several phospholipid binding sites that dramatically affect activity. This study provides new insight into how these activations occur. One analogue contained a phosphorylethanolamine (DiC(6)SNPE) headgroup, while the other contained a phosphorylcholine (DiC(6)SNPC) headgroup. Using phospholipase D, we incorporated tritium into each analogue. No binding of DiC(6)SNPE to PLA(2) was observed under submicellar conditions. Addition of submicellar amounts of Triton X-100 resulted in a linear nonsaturating response to lipid concentration, suggestive of premicellar aggregation of the DiC(6)SNPE with Triton X-100 and PLA(2). Binding of DiC(6)SNPE when presented as Triton X-100 mixed micelles saturated at 0.93 binding sites per PLA(2) with a K(D) of 38 microM. Addition of sphingomyelin, a potent activator of PLA(2) hydrolysis of phosphorylethanolamine containing compounds, resulted in a 13-fold decrease in the K(D), to 2.8 microM. This suggests that changes in the catalytic site binding affinity contribute to "phosphatidylcholine activation". Binding of DiC(6)SNPC with 2.0 mM Triton X-100 showed positive cooperativity (Hill coefficient of 1.7), which saturated at 2.0 binding sites per PLA(2). No binding of either analogue was observed when the catalytic site was alkylated with p-bromophenacyl bromide. Since p-bromophenacyl bromide does not physically block the phosphatidylcholine activator site, this indicates that the two phosphatidylcholine binding sites interact. The binding studies show that DiC(6)SNPC binds cooperatively to two sites on group IA PLA(2), while DiC(6)SNPE binds to only one site.     

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.     

Lipid-induced aggregation of phospholipase A2: sucrose density gradient ultracentrifugation and crosslinking studies

The aggregation behavior of cobra venom (Naja naja naja) phospholipase A2 in the presence of lipids and Ca2+ was examined using ultracentrifugation and crosslinking techniques. Velocity sedimentation experiments were performed in sucrose gradients. The sedimentation coefficients of the cobra phospholipase A2 and various controls, including bovine serum albumin (BSA), malate dehydrogenase, carbonic anhydrase and pancreatic phospholipase A2, were calculated both in the presence and absence of ligands. The monomeric phospholipid, diheptanoylphosphatidylcholine, and the phospholipid analogue, dodecylphosphocholine (DPC), increased the sedimentation coefficient of the cobra phospholipase A2 from 2.2 S to 2.9 S, a value that is consistent with the formation of an enzyme dimer. The control proteins were unaffected by the presence of phospholipid, except for BSA, which apparently binds large amounts of DPC. Crosslinking experiments with glutaraldehyde showed that in the presence of diheptanoylphosphatidylcholine or DPC, the amount of crosslinked enzyme increased. Ca2+ had no effect on the aggregation state of the enzyme as measured by either technique. Both the ultracentrifugation data and crosslinking data are consistent with the hypothesis that the cobra venom phospholipase A2 exists as a dimer or higher-order aggregate in the presence of lipid substrate, although it is yet to be determined whether the functional subunit is a monomer, dimer or higher-order oligomer.     

A basic phospholipase A from the venom of the Australian king brown snake (Pseudechis australis) showing diverse activities against membranes

1. A basic phospholipase A (MSPA) was isolated from the venom of the Australian king brown snake, Pseudechis australis. 2. MSPA had an approximate Mr of 13,000 and consisted of a single polypeptide chain of 119 amino acid residues cross-linked by seven disulphide bridges. 3. MSPA exhibited direct haemolytic, anticoagulant and myotoxic activities. 4. Treatment of MSPA with p-bromophenacyl bromide modified a single histidine residue, resulting in complete loss of enzyme activity.     

Characterization of an active site peptide modified by the substrate analogue 3-bromo-2-ketoglutarate on a single chain of dimeric NADP+-dependent isocitrate dehydrogenase

The substrate analogue 3-bromo-2-ketoglutarate reacts with pig heart NADP+-dependent isocitrate dehydrogenase to yield partially inactive enzyme. Following 65% inactivation, no further inactivation was observed. Concomitant with this inactivation, incorporation of 1 mol of reagent/mol of enzyme dimer was measured. The dependence of the inactivation rate on bromoketoglutarate concentration is consistent with reversible binding of reagent (KI = 360 microM) prior to irreversible reaction. Manganous isocitrate reduces the rate of inactivation by 80% but does not provide complete protection even at saturating concentrations. Complete protection is obtained with NADP+ or the NADP+-alpha-ketoglutarate adduct. By modification with [14C]bromoketoglutarate or by NaB3H4 reduction of modified enzyme, a single major radiolabeled tryptic peptide was obtained by high performance liquid chromatography with the sequence: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Evidence in the following paper (Bailey, J.M., Colman, R.F. (1987) J. Biol. Chem. 262, 12620-12626) indicates that X is glutamic acid. Enzyme modified at the coenzyme site by 2-(bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the presence of manganous isocitrate is not further inactivated by bromoketoglutarate. Bromoketoglutarate-modified enzyme exhibits a stoichiometry of binding isocitrate and NADPH equal to 1 mol/mol of enzyme dimer, half that of native enzyme. These results indicate that bromoketoglutarate modifies a residue in the nicotinamide region of the coenzyme site proximal to the substrate site and that reaction at one catalytic site of the enzyme dimer decreases the activity of the other site.     

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.     

Detergent-resistant phospholipase A of Escherichia coli K-12. Purification and properties.

Detergent-resistant phospholipase A, which is tightly bound to the outer membranes of Escherichia coli K-12 cells, was purified approximately 2000-fold to near homogeneity by solubilization with sodium dodecylsulfate and butan-1-ol, acid precipitation, acetone fractionation and column chromatographies on Sephadex G-100 in the presence of sodium dodecylsulfate and on DEAE-cellulose in the presence of Triton X-100. The final preparation showed a single band in the sodium dodecylsulfate gel system. The enzyme hydrolyzes both the 1-acyl and 2-acyl chains of phosphatidylethanolamine or phosphatidylcholine. It also attacks 1-acyl and 2-acylglycerylphosphorylethanolamine. Thus, this enzyme shows not only phospholipase A1 and lysophospholipase L1 activities but also phospholipase A2 and lysophospholipase L2 activities. The enzyme lost its activity completely on incubation at 80 degrees C for 5 min at either pH 6.4 or pH 8.0. It was stable in 0.5% sodium dodecylsulfate at below 40 degrees C. The enzyme was inactivated on incubation for 5 min at 90 degrees C in 1% sodium dodecylsulfate/1% 2-mercaptoethanol/4 M urea. The native and inactivated enzymes showed different protein bands with RF values corresponding to Mr 21 000 and Mr 28 000 respectively, in a sodium dodecylsulfate gel system. Triton X-100 seemed to protect the enzyme from inactivation. The purified enzyme was fully active on phosphatidylethanolamine in the presence of 0.0002% or 0.05% Triton X-100. The enzyme requires Ca2+. From its properties this enzyme seems to be identical with the enzyme purified from crude extracts of Escherichia coli B by Scandella and Kornberg. However, it differs from the latter in its positional specificity and susceptibility to sodium dodecylsulfate. Possible explanation of the difference of positional specificity of the two preparations is also described.     

Suicide-inhibitory bifunctionally linked substrates (SIBLINKS) as phospholipase A2 inhibitors. Mechanistic implications

Novel phospholipids that function as mechanism-based inhibitors for phospholipase A2 (PLA2) are described. PLA2-catalyzed hydrolysis of the sn-2 ester of these suicide-inhibitory bifunctionally linked substrates (SIBLINKS) followed by a cyclization reaction generates a cyclic anhydride at the active site of the enzyme which leads to inhibition. Structure/activity relationships for the SIBLINKS substituents in the sn-1 and sn-2 position are delineated. Time courses and efficiency of SIBLINKS inhibition are reported and compared for extracellular PLA2s obtained from Naja naja naja, porcine pancreas, bee venom, Crotalus atrox and Crotalus adamanteus. SIBLINKS-inhibited PLA2s cannot process either monomeric or micellar substrates consistent with inhibition at the catalytic site. Some SIBLINKS efficiently inactivate 1 mol of N. naja naja and C. adamanteus PLA2/6-10 mol of SIBLINKS hydrolyzed. Inhibition of N. naja naja PLA2 can be reversed by hydroxylamine, suggesting that a tyrosine residue is acylated.     

1H-NMR and protection studies of interactions between ligands and bovine pancreatic phospholipase A2

A number of long-chain amines and naphthylamine sulfonates have been studied for their ability to inhibit bovine pancreatic phospholipase A2 (PLA2) and to protect PLA2 against alkylation of the active site histidine by p-bromophenacyl bromide. Their areas of interaction on the enzyme were further delineated using observations of chemical shift changes of assigned aromatic signals in the 1H-NMR spectrum of PLA2, while the bound conformations of two amine inhibitors were revealed using transferred nuclear Overhauser effects. The alkyl amines bind rather non-specifically on the surface of the enzyme, over the active site cleft and the interface recognition site.     

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.     

Inhibition of venom phospholipases A2 by manoalide and manoalogue. Stoichiometry of incorporation

We have previously described the irreversible inhibition of cobra venom phospholipase A2 (PLA2) by the marine natural product manoalide (MLD) (Lombardo, D., and Dennis, E. A. (1985) J. Biol. Chem. 260, 7234-7240) and by its synthetic analog, manoalogue (MLG) (Reynolds L. J., Morgan, B. P., Hite, G. A., Mihelich, E. D., and Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172-5177). We have now made a direct comparison of the action of these two inhibitors on PLA2 from cobra, bee, and rattlesnake venoms and have found that MLG behaves kinetically similarly to MLD in all cases with only minor differences. The time courses of inactivation differ significantly between the three enzymes, however, with the inactivation of bee and rattlesnake PLAs2, occurring much faster than does the inactivation of the cobra venom enzyme. The enzymes also differ in their sensitivity to the presence of Ca2+ during the inactivation. Of the three enzymes, the most Ca(2+)-sensitive is the rattlesnake enzyme, which shows a much faster rate of inactivation in the presence of Ca2+ than in the presence of EGTA. However, the same rate of inactivation was also observed when the inhibitor Ba2+ was substituted for Ca2+, indicating that catalytic activity is not required for inactivation of the enzyme. To probe the mechanism of inactivation and to determine the stoichiometry of incorporation, we have synthesized 3H-labeled MLG and have found that inactivation of cobra PLA2 is accompanied by an incorporation of 3.8 mol of [3H]MLG/mol of enzyme. The same amount of 3H incorporation was observed when p-bromophenacyl bromide-inactivated PLA2 was incubated with [3H]MLG, again indicating that catalytic activity is not required for the reaction of PLA2 with MLG. All together, these results suggest that MLD and MLG are not suicide inhibitors of PLA2. A portion of the incorporated radioactivity was acid-labile, and dialysis of the radiolabeled PLA2 under acidic conditions resulted in a loss of about one-third of the enzyme-associated radioactivity, leaving 2.4 mol of [3H]MLG/mol of PLA2. In previous studies, amino acid analysis, which also included acid treatment, indicated that MLG-modified cobra phospholipase A2 contained 2.8 mol of Lys less than the native enzyme. Thus, 1 mol of [3H]MLG is incorporated per mol of Lys lost. The implications of this 1:1 stoichiometry of MLG to Lys on the mechanism of reaction of these inhibitors is discussed.     

Stimulation of prostaglandin E2 synthesis by exogenous phospholipase A2 and C in rabbit kidney medulla slices.

We have investigated the effects of phospholipase A2 and C on the synthesis of prostaglandin E2 in rabbit kidney medulla and the release of fatty acids from the medulla slices. Exogenous phospholipase A2 [from Naja naja (Indian cobra) venom] and phospholipase C (from Clostridium welchii) stimulated prostaglandin E2 production in a dose-dependent manner. At the maximal effective concentrations (0.5 unit of phospholipase A2/ml, 2 units of phospholipase C/ml), phospholipase C increased prostaglandin E2 formation to the level observed with phospholipase A2. Phospholipase A2 enhanced the release only of unsaturated fatty acids, whereas phospholipase C stimulated the release of individual free fatty acids (C 16:0, C 18:0, C 18:1, C 18:2 and C 20:4). Moreover, p-bromophenacyl bromide inhibited phospholipase A2-stimulated prostaglandin E2 production and the release of fatty acids, but it had no influence on prostaglandin E2 formation and the release of fatty acids increased by phospholipase C, indicating that the stimulatory effect of phospholipase C is not mediated through the activation of endogenous phospholipase A2. These results suggest the presence of diacylglycerol lipase and monoacylglycerol lipase in the kidney and the importance of this pathway in prostaglandin synthesis by the kidney.     

Surface-active phospholipase A2 in mouse spermatozoa

The partial characterization of a calcium-dependent phospholipase A2 associated with membranes of mouse sperm is described. Intact and sonicated sperm had comparable phospholipase A2 activity which was maximal at pH 8.0 using [1-14C]oleate-labeled autoclaved Escherichia coli or 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine as substrates. More than 90% of the activity was sedimented when the sperm sonicate was centrifuged at 100 000 X g, indicating that the enzyme is almost totally membrane-associated. The activity is stimulated 200% during the ionophore-induced acrosome reaction and is almost equally distributed between plasma/outer acrosomal and inner acrosomal membrane fractions. The membrane-associated phospholipase A2 had an absolute requirement for low concentrations of Ca2+; Sr2+, Mg2+ and other divalent and monovalent cations would not substitute for Ca2+. In the presence of optimal Ca2+, zinc and gold ions inhibited the activity while Cu2+ and Cd2+ were without effect. Incubation of sperm sonicates with 1-[1-14C]stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine in the presence and absence of sodium deoxycholate demonstrated the presence of phospholipase A2 and lysophospholipase activities. No phospholipase A1 activity was detectable. Indomethacin, sodium meclofenamate and mepacrine, but not dexamethasone or aspirin, inhibited the sperm phospholipase A2 activity. Preincubation with p-bromophenacyl bromide inhibited phospholipase A2, suggesting the presence of histidine at the active site. The enzyme may play an important role in the membrane fusion events in fertilization.     

Studies on phospholipase A2 in human seminal plasma.

1. Human seminal plasma and posterior lobe of prostate was found to have phospholipase A2 (PLA2) activity hydrolysing phosphatidylethanolamine with 14C-labelled linoleic and arachidonic acid. 2. A negative relationship was between sperm count and PLA2 activity in human seminal plasma. 3. The purified PLA2 from human seminal plasma showed high affinity to heparin, sensitivity toward p-bromophenacyl bromide, Pb2+, dithioerythritol and EDTA and it was activated by Ca2+ and Mn2+. 4. The purified PLA2 had alkaline pH optimum (7.5-10.0) and pI-value of 5.3. In SDS-PAGE enzyme preparation resulted in two bands with mol. wt of 14,000 and 16,000.