Cobra venom phospholipase A2 inhibition by manoalide. A novel type of phospholipase inhibitor

Manoalide, an unusual nonsteroidal sesterterpenoid recently isolated from sponge, antagonizes phorbol-induced inflammation but not that induced by arachidonic acid, suggesting that manoalide acts prior to the cyclooxygenase step in prostaglandin synthesis, possibly by inhibiting phospholipase A2. We have now studied the inhibitory effect of manoalide on a homogeneous preparation of phospholipase A2 from cobra venom. For a given concentration of manoalide, the inhibition of phospholipase A2 activity toward dipalmitoylphosphatidylcholine/Triton X-100 mixed micelles is time-dependent and plateaus at about 85% inhibition of the initial velocity even after extensive preincubation. Metal ions (Ca2+, Ba2+, Mn2+) increase the inhibition, while lysophosphatidylcholine and substrate micelles protect. Increasing manoalide concentration shows increasing inhibition of the initial velocity until a plateau is reached, giving a typical saturation curve with a linear double-reciprocal plot. Under typical conditions (20-min preincubation, 40 degrees C, pH 7.1), 50% inhibition is achieved at a manoalide concentration of about 2 X 10(-6) M. The data indicate that manoalide is a potent inhibitor of the cobra venom phospholipase A2. Manoalide is now shown to react irreversibly with lysine residues in the enzyme. Surprisingly, the cobra venom phospholipase normally acts poorly on phosphatidylethanolamine as substrate, but after reaction with manoalide, the enzyme is somewhat more active toward this substrate rather than being inhibited. This suggests that a lysine residue may be important in understanding the substrate specificity of phospholipase A2.     

Manoalide, a natural sesterterpenoid that inhibits calcium channels

Manoalide is a marine natural product that has anti-inflammatory and anti-proliferative activities and is an irreversible inhibitor of phospholipase A2 and phospholipase C. It is now shown that the compound is a potent inhibitor of Ca2+ mobilization in several cell types. In A431 cells the increase in epidermal growth factor receptor-mediated Ca2+ entry and release from intracellular Ca2+ stores were blocked by manoalide in a time-dependent manner with an IC50 of 0.4 microM. The effect of manoalide on phosphoinositide metabolism, namely the production of inositol monophosphate, did not coincide with its effect on the epidermal growth factor response. In GH# cells, manoalide blocked the thyrotropin-releasing hormone-dependent release of Ca2+ from intracellular stores without inhibition of the formation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate. Manoalide also blocked the K+ depolarization-activated Ca2+ channel in these cells as well as the activation of the channel by Bay K8644 with an IC50 of 1 microM. In addition, manoalide also inhibited the Ca2+ influx induced by concanavalin A in mouse spleen cells in a time- and temperature-sensitive manner with an IC50 of 0.07 microM. However, neither forskolin-activated adenylate cyclase in A431 cells nor the distribution of the potential sensitive dye, 3,3'-dipropylthiodicarbocyanide iodide in GH3 cells was affected by manoalide. Thus, manoalide acts as a Ca2+ channel inhibitor in all cells examined. This action may account for its effects on inflammation and proliferation and may be independent of its effect on phospholipases.     

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.     

Definition of crucial structural factors of acetogenins, potent inhibitors of mitochondrial complex I

Some natural acetogenins are the most potent inhibitors of bovine heart mitochondrial complex I. These compounds are characterized by two functional units (i.e. hydroxylated tetrahydrofuran (THF) and alpha,beta-unsaturated gamma-lactone ring moieties) separated by a long alkyl spacer. To elucidate which structural factors of acetogenins including their active conformation are crucial for the potent inhibitory effect, we synthesized a series of novel acetogenin analogues possessing bis-THF rings. The present study clearly demonstrated that the natural gamma-lactone ring is not crucial for the potent inhibition, although this moiety is the most common structural unit among a large number of natural acetogenins and has been suggested to be the only reactive species that directly interacts with the enzyme (Shimada et al., Biochemistry 37 (1998) 854-866). The presence of free hydroxy group(s) in the adjacent bis-THF rings was favorable, but not essential, for the potent activity. This was probably because high polarity (or hydrophilicity), rather than hydrogen bond-donating ability, around the bis-THF rings is required to retain the inhibitor in the active conformation. Interestingly, length of the alkyl spacer proved to be a very important structural factor for the potent activity, the optimal length being approximately 13 carbon atoms. The present study provided further strong evidence for the previous proposal (Kuwabara et al., Eur. J. Biochem. 267 (2000) 2538-2546) that the gamma-lactone and THF ring moieties act in a cooperative manner on complex I with the support of some specific conformation of the spacer.     

Design syntheses and mitochondrial complex I inhibitory activity of novel acetogenin mimics.

Some natural acetogenins are the most potent inhibitors of mitochondrial complex I. These compounds are characterized by two functional units [i.e. hydroxylated tetrahydrofuran (THF) and alpha, beta-unsaturated gamma-lactone ring moieties] separated by a long alkyl spacer. To elucidate which structural factors of acetogenins, including their active conformation, are crucial for the potent inhibitory activity we synthesized a novel bis-acetogenin and its analogues possessing two gamma-lactone rings connected to bis-THF rings by flexible alkyl spacers. The inhibitory potency of the bis-acetogenin with bovine heart mitochondrial complex I was identical to that of bullatacin, one of the most potent natural acetogenins. This result indicated that one molecule of the bis-acetogenin does not work as two reactive inhibitors, suggesting that a gamma-lactone and the THF ring moieties act in a cooperative manner on the enzyme. In support of this, either of the two ring moieties synthesized individually showed no or very weak inhibitory effects. Moreover, combined use of the two ring moieties at various molar ratios exhibited no synergistic enhancement of the inhibitory potency. These observations indicate that both functional units work efficiently only when they are directly linked by a flexible alkyl spacer. Therefore, some specific conformation of the spacer must be important for optimal positioning of the two units in the enzyme. Furthermore, the alpha,beta-unsaturated gamma-lactone, the 4-OH group in the spacer region, the long alkyl tail attached to the THF unit and the stereochemistry surrounding the hydroxylated bis-THF rings were not crucial for the activity, although these are the most common structural features of natural acetogenins. The present study provided useful guiding principles not only for simplification of complicated acetogenin structure, but also for further wide structural modifications of these molecules.     

A phospholipase inhibitor, manoalide, and a G-protein activator, Mas-7, both affect the turnover of phototransductive membranes by crab retinas in darkness With a model for the regulation of rhabdomeral membrane turnover

(1) In vitro retinas of a crab, Leptograpsus, were treated with a phospholipase inhibitor, manoalide, or a G-protein activator, Mas-7. Both drugs address early stages of the phototransduction cascade. (2) Manoalide inhibited the light-dependent reduction of rhabdoms during the `day' phase of the light cycle, but did not induce rhabdom overgrowth. Following a period of darkness manoalide failed to affect the diminution of illuminated rhabdoms. (3) The diminution of rhabdoms that follows photoreceptor depolarisation induced by 100 mmol · l<sup>−1</sup> K<sup>+</sup> in darkness was not affected by 2␣μmol · l<sup>−1</sup> manoalide. (4) When retinas in the `night' phase were treated with Mas-7 in darkness, rhabdom diameters were augmented, concurrently with endocytosis of photoreceptor plasma membranes. (5) The results of combining manoalide and Mas-7 with actinomycin D, U-57908 or okadaic acid, drugs used in previous studies to manipulate steps notionally lower in the transduction cascade, lead to a hypothetical model for the regulation of phototransductive membrane turnover by arthropods. Accepted: 3 October 1996     

Purification of a Class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja Naja) venom

A platelet aggregation inhibitor phospholipase A(2) (NND-IV-PLA(2)) was isolated from Naja naja (Eastern India) venom by a combination of cation and anion exchange chromatography. NND-IV-PLA(2) is the most catalytically active enzyme isolated from the Indian cobra venom. The acidic PLA(2) profile of Eastern regional Indian cobra venom is distinctly different from that of the western regional venom. However the acidic PLA(2)s from both the regions follow the pattern of increasing catalytic activity with increase in acidic nature of the PLA(2) isoform. NND-IV-PLA(2) is a Class B1 platelet aggregation inhibitor and inhibits platelet aggregation induced by ADP, collagen and epinephrine. Modification of active site histidine abolishes both catalytic activity and platelet aggregation inhibition activities while aristolochic acid, a phospholipase A(2) inhibitor has only partial effect on the two activities.     

Synthesis and inhibitory activity of ubiquinone-acetogenin hybrid inhibitor with bovine mitochondrial complex I

To elucidate the inhibitory action of acetogenins, the most potent inhibitors of mitochondrial complex I, we synthesized an acetogenin analogue which possesses a ubiquinone ring (i.e., the physiological substrate of complex I) in place of the alpha,beta-unsaturated gamma-lactone ring of natural acetogenins, and named it Q-acetogenin. Our results indicate that the gamma-lactone ring of acetogenins is completely substitutable with the ubiquinone ring. This fact is discussed in light of the inhibitory action of acetogenins.     

Manoalide,a Phospholipase A2 Inhibitor,Inhibits Arachidonate Incorporation and Turnover in Brain Phospholipids of the Awake Rat

The Fatty Acid method was used to determine whether incorporation of plasma radiolabeled arachidonic acid into brain phospholipids is controlled by phospholipase A₂. Awake rats received an i.v. injection of a phospholipase A₂ inhibitor, manoalide (10 mg/kg), and then were infused i.v. with [1-¹⁴C]arachidonate or [³H]arachidonate. Animals were killed after infusion by microwave irradiation, and tracer distribution was analyzed in brain phospholipid, neutral lipid and acyl-CoA pools. Calcium-independent phospholipase A₂ activity in brain homogenate was reduced by manoalide, whereas phospholipase C activity was unaffected. At 60 min but not at 20 or 40 min after its injection, manoalide had significantly decreased by 50% incorporation of unesterified arachidonate into and turnover within brain phospholipids, taking into account dilution of the brain arachidonoyl-CoA pool by recycled arachidonate. Manoalide also increased by 100% the net rate of unesterified arachidonate incorporation into brain triacylglycerol. This study indicates that manoalide can be used to inhibit brain phospholipase A₂ in vivo, and that phospholipase A₂ plays a critical role in arachidonate turnover in brain phospholipids and neutral lipids.     

Synthesis and inhibition mechanism of Delta lac-acetogenins, a novel type of inhibitor of bovine heart mitochondrial complex I

We have synthesized Deltalac-acetogenins that are new acetogenin mimics possessing two n-alkyl tails without an alpha,beta-unsaturated gamma-lactone ring and suggested that their inhibition mechanism may be different from that of common acetogenins [Hamada et al. (2004) Biochemistry 43, 3651-3658]. To elucidate the inhibition mechanism of Deltalac-acetogenins in more detail, we carried out wide structural modifications of original Deltalac-acetogenins and characterized the inhibitory action with bovine heart mitochondrial complex I. In contrast to common acetogenins, both the presence of adjacent bis-THF rings and the stereochemistry around the hydroxylated bis-THF rings are important structural factors required for potent inhibition. The inhibitory potency of a derivative possessing an n-butylphenyl ether structure (compound 7) appeared to be superior to that of the original Deltalac-acetogenins and equivalent to that of bullatacin, one of the most potent natural acetogenins. Double-inhibitor titration of steady-state complex I activity showed that the extent of inhibition of compound 7 and bullatacin is not additive, suggesting that the binding sites of the two inhibitors are not identical. Competition tests using a fluorescent ligand indicated that the binding site of compound 7 does not overlap with that of other complex I inhibitors. The effects of compound 7 on superoxide production from complex I are also different from those of other complex I inhibitors. Our results clearly demonstrate that Deltalac-acetogenins are a novel type of inhibitor acting at the terminal electron-transfer step of bovine complex I.     

Effect of phospholipases A2 from bee and cobra venom on the phospholipid composition and Na+-,K+-ATPase activity of synaptosomes

The bee and cobra venom phospholipases A2 as well as partially acetylated cobra venom phospholipase A2 are studied for their effect on phospholipid composition of synaptosomes and their Mg2+- and Na+,K+-ATPase activity. It is established that these phospholipases induce the splitting of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine, inhibition of the Na+,K+-ATPase activity and activation of Mg2+-ATPase. Bee venom phospholipase A2 is more effective than cobra venom phospholipase A2, the both phospholipases splitting phosphatidylethanolamine most intensively. The ATPase activity may be partially or completely restored by exogenic phosphatidylcholine and phosphatidylserine; exogenic phosphatidylethanolamine is not efficient in this respect.     

Inhibitory effects on mitochondrial complex I of semisynthetic mono-tetrahydrofuran acetogenin derivatives

Modifications in the terminal alpha,beta-unsaturated gamma-methyl-gamma-lactone moiety or in the alkyl chain that links this terminal gamma-lactone with the alpha,alpha'-dihydroxylated THF system of the natural mono-tetrahydrofuranic acetogenins, annonacin and annonacinone, led to the preparation of eight semisynthetic derivatives. Their inhibitory effects on mitochondrial complex I is discussed and compared with that of the classical complex I inhibitor, rotenone.     

Kinetic characterization of phospholipase A2 modified by manoalogue

Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivate the phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, B. P., Hite, E. D., Mihelich, E. D., & Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172) by reacting with enzyme lysine residues. In the present study, the inactivation of the phospholipases A2 from pig pancreas, bee venom, and cobra (Naja naja naja) venom by manoalogue was studied in detail. Manoalogue-treated enzymes were examined in the scooting mode on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol. Here the native enzymes bound irreversibly to the vesicles and hydrolyzed all of the phospholipids in the outer monolayer without leaving the surface of the interface. All three manoalogue-treated enzymes showed reduced catalytic turnover for substrate hydrolysis in the scooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease in the fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospholipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine epsilon-amino groups have been amidinated was not inactivated by manoalogue, indicating that the modification of lysine residues and not the amino-terminus is required for the inactivation. Several studies indicated that the manoalogue-modified enzymes contain a functional active site. For example, studies that monitored the protection by ligands of the active site from attack by a alkylating agent showed that manoalogue-modified pig phospholipase A2 was capable of binding calcium, a substrate analogue, lipolysis products, and a competitive inhibitor. Furthermore, relative to native enzymes, manoalogue-modified enzymes retained significantly higher catalytic activities when acting on water-soluble substrates than when acting on vesicles in the scooting mode. Intact manoalogue had no affinity for the catalytic site on the enzyme as it did not inhibit the enzyme in the scooting mode and it did not protect the active site from alkylation. Pig pancreatic phospholipase A2 bound to micelles of 2-hexadecyl-sn-glycero-3-phosphocholine was resistant to inactivation by manoalogue, suggesting that the modification of lysine residues on the interfacial recognition surface of the enzyme was required for inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of a glycoprotein inhibitor of toxic phospholipase from Withania somnifera

A phospholipase inhibitor (WSG) has been purified from Withania somnifera using gel-filtration and ion-exchange chromatographies. The WSG is an acidic glycoprotein. Its molecular mass as determined by SDS-PAGE was 27kDa. It neutralized the enzyme activity and pharmacological properties such as cytotoxicity, edema, and myotoxicity of a multi-toxic Indian cobra venom phospholipase (NNXIa-PLA) but failed to neutralize the neurotoxicity. The glycan part of the molecule does not appear to be involved in any of the pharmacological properties studied. The results suggest that the neutralization of the pharmacological effects of the toxic phospholipase is brought about by inhibition of the enzyme activity by formation of a complex between the WSG and the toxic phospholipase. We report the purification and characterization of a glycoprotein phospholipase A inhibitor from Withania somnifera, medicinal plant.     

Studies on phospholipase A inhibitor in blood plasma. II. Interaction of phospholipase A inhibitor with phospholipase A and its specificity

Non-competitive inhibition of snake venom phospholipase A2 which has been exhibited by bovine plasma phospholipase A inhibitor, a kind of lipoprotein, was not observed unless the inhibitor was preincubated with the enzyme. The inhibition seemed to be due to the formation of the enzyme-inhibitor complex, which was identified by immunoelectrophoresis. The enzyme-inhibitor interaction was observed maximally on incubation at physiological pH, but not below pH 5. The inhibitor was inactivated by trypsin digestion and heat treatment. It suppressed the phospholipase A2 activities of rat blood plasma as well as of the snake venom and porcine pancreas, but not the enzyme activities such as those of phospholipase C of Bacillus cereus, lipase of porcine pancreas, trypsin, and papain. The inhibitor also showed the ability to decrease membrane-bound phospholipase A1 and A2 activities in intracellular organelles such as plasma membranes, mitochondria, lysosomes, and microsomes. In view of these facts, it was concluded that the plasma inhibitor is specific for phospholipase A.     

The interaction of phospholipase A2 with phospholipid analogues and inhibitors

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)     

The anticoagulant activity of Malayan cobra (Naja naja sputatrix) venom and venom phospholipase A2 enzymes

Malayan cobra (Naja naja sputatrix) venom was found to exhibit an in vitro anticoagulant activity that was much stronger than most common cobra (genus Naja) venoms. The most potent anticoagulants of the venom are two lethal phospholipase A2 enzymes with pI's of 6.15 and 6.20, respectively. The anticoagulant activity of the venom is due to the synergistic effect of the venom phospholipase A2 enzymes and polypeptide anticoagulants. Bromophenacylation of the two phospholipase A2 enzymes reduced their enzymatic activity with a concomitant drop in both the lethal and anticoagulant activities.     

Inhibition studies on the membrane-associated phospholipase A2 in vitro and prostaglandin E2 production in vivo of the macrophage-like P388D1 cell. Effects of manoalide, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacyl bromide

In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostaglandin production in the intact cell from which it is derived.     

1-Stearyl,2-stearoylaminodeoxy phosphatidylcholine, a potent reversible inhibitor of phospholipase A2

1-stearyl, 2-stearoylaminodeoxy phosphatidylcholine, a structurally modified phospholipid substrate analog exhibits potent and reversible inhibition of phospholipase A2 from cobra venom (N. naja naja). The apparent KI values determined in two different assay systems employing phosphatidylcholine-surfactant mixed micelles are in reasonable agreement (40 microM and 16 microM) and indicate that the inhibitor binds to the enzyme as much as two orders of magnitude more tightly than does dipalmitoyl phosphatidylcholine. With phosphatidylethanolamine as substrate, the kinetics are more complicated as the analog also exhibits activation, presumably at a second binding site on the enzyme.     

Activation, aggregation, and product inhibition of cobra venom phospholipase A2 and comparison with other phospholipases

The kinetics of phospholipid hydrolysis by cobra venom phospholipase A2 were examined and compared to those of phospholipase A2 from porcine pancreas, Crotalus adamanteus (rattlesnake) venom, and bee venom. Only the enzyme from Naja naja naja (cobra) venom was found to be activated significantly by phosphorylcholine-containing compounds when hydrolyzing phosphatidylethanolamine. The cobra venom enzyme was also the only one in which these activators induced protein aggregation. The parallel specificity for activators and aggregators suggests that these two phenomena are linked. Product effects were also shown to vary between these four phospholipases. These effects manifest themselves in nonlinear time courses, in changes in steady state velocity, and in the differential effects of serum albumin on reaction rates. Different effects were even seen for the same enzyme when acting on different substrates. A model is presented to account for these observations; its main features are enzyme activation by an activator molecule, whose specificity depends on the enzyme, and an activator-induced aggregation of the enzyme.