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PubMed1986

Inhibition of phospholipase A2 by protein I.

The 36 kDa substrate of several tyrosine protein kinases has been shown to exist in monomeric and oligomeric (362102) forms. Partial sequence data has suggested that the oligomer, referred to as protein I, is homologous to a group of phospholipase A2 inhibitory proteins, collectively called lipocortins. In the present communication we demonstrate that protein I inhibits bovine pancreas phospholipase A2 with similar potency to that of lipocortin. Approximately 44 pmol protein I was required to produce 50% inhibition of 7.2 pmol of phospholipase A2. Inhibition of phospholipase A2 activity by calmodulin, S-100, calregulin, parvalbumin, troponin-C, or CAB-48 was not observed. These results indicate that protein I is a potent and specific inhibitor of phospholipase A2 activity, and thus shares functional homology with the lipocortin proteins. We therefore propose that this protein be named lipocortin-85.

Amino Acid Sequence↗
PubMed1982

Cardiolipin-protein interactions in the phosphate binding activity of a proteolipid from yeast mitochondria. Action of phospholipases A2 and C and of cations.

A proteolipid able to bind phosphate has been isolated from yeast mitochondria. During the purification the active protein was always associated with cardiolipin. The cardiolipin requirement for the phosphate binding activity of this proteolipid has been studied using controlled lipid depletion with two phospholipases A2 and with phospholipase C. Only phospholipase A2 from pig pancreas, that deacylated cardiolipin, promoted inhibition of the proteolipid activity (but never more than 70 per cent). Phospholipase A2 from snake venom did not inhibit the binding activity of the proteolipid. Using thin layer chromatography with two sequential solvents it was possible to separate two cardiolipin subspecies; one of them was preferentially hydrolysed by phospholipase C of Bacillus cereus, leading to inhibition of the proteolipid activity. Ca2+ complexed to cardiolipin stoichiometrically (1-1) inhibited the proteolipid activity. This effect could be due to a conformational change in the cardiolipin-protein association.

Animals↗
PubMed2000

Isolation and characterization of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a new Lys49 phospholipase A2 homologue.

Myotoxic phospholipases A2 of class II are commonly found in the venoms of crotalid snakes. As an approach to understanding their structure-activity relationship, diverse natural variants have been characterized biochemically and pharmacologically. This study describes a new myotoxic phospholipase A2 homologue, isolated from the venom of Atropoides (Bothrops) nummifer from Costa Rica. A. nummifer myotoxin 1 is a basic protein, with an apparent subunit molecular mass of 16 kDa, which migrates as a dimer in sodium dodecylsulfate-polyacrylamide gel electrophoresis under nonreducing conditions. It is strongly recognized by antibodies generated against Bothrops asper myotoxin II, by enzyme-immunoassay. The toxin induces rapid myonecrosis upon intramuscular injection in mice (evidenced by an early increase in plasma creatine kinase activity), and significant edema in the footpad assay. It also displays cytolytic activity upon cultured murine endothelial cells. The toxin (up to 50 microg) has no detectable phospholipase A2 activity on egg yolk phospholipids, and does not show an anticoagulant effect on sheep platelet-poor plasma in vitro. N-terminal sequence determination (53 amino acid residues) demonstrated that A. nummifer myotoxin II is a new Lys49 variant of the family of myotoxic, class II phospholipases A2. Sequence comparison with other phospholipases A2 revealed Asn53 as a novel substitution. In addition, this myotoxin is the first Lys49 variant presenting Asn in its N-terminus. Consequently, these findings suggest that neither Ser1 or Lys53, usually found in this family of proteins, are essential amino acid residues for their myotoxic, cytolytic, or edema-inducing effects.

Amino Acid Sequence↗
PubMed1998

Phorbol ester-induced production of prostaglandin E2 from phosphatidylcholine through the activation of phospholipase D in UMR-106 cells.

To determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on phospholipase D (PLD) activity in osteoblast-like UMR-106 cells, we used cells prelabeled with [3H] myristic acid or [3H] arachidonic acid, which were preferentially incorporated to phosphatidylcholine. The treatment of [3H] myristate-labeled cells with TPA in the presence of 1% ethanol caused a dose-dependent formation of [3H] phosphatidylethanol (PEt), a product specific to PLD, suggesting an activation of this enzyme. Pretreatment of the cells with protein kinase C (PKC) inhibitors (GF109203X, staurosporine or H-7) abolished the TPA-dependent formation of PEt. The PEt formation in response to TPA treatment was not observed after the pretreatment of the cells with TPA to downregulate PKC. These results suggest the involvement of PKC in the TPA-induced activation of PLD. With [3H] arachidonate-labeled cells, TPA treatment in the absence of ethanol resulted in the liberation of [3H] arachidonic acid, which was gradually converted to prostaglandin E2 (PGE2), but the accumulations of [3H] phosphatidic acid (PA) and [3H] diacylglycerol (DAG) were very small and temporary. In contrast, PA was linearly accumulated following TPA treatment, when the cells were pretreated with an inhibitor of phosphatidate phosphohydrolase (PAP), propranolol, with no accumulation of either DAG or arachidonic acid. The TPA treatment of the cells pretreated with a DAG lipase inhibitor, RHC-80267, caused the generation of DAG after a lag period of approximately 5 min, with a very small and temporary accumulation of PA. The TPA treatment of cells pretreated with a cyclooxygenase (COX) inhibitor, indomethacin, blocked the PGE2 production. The TPA-induced PGE2 production was not affected by the pretreatment of cells with a phospholipase A2 inhibitor, p-bromophenacylbromide, or with a phospholipase C inhibitor, D-609. TPA also stimulated PGE2 production in osteoblastic cells that were enzymatically isolated from adult rat calvaria, and the experiments with lipid metabolizing enzyme inhibitors gave the same profile of inhibition of TPA-induced PGE2 production as was observed in UMR-106 cells. These results suggest that PA formed as a consequence of the activation of PLD by TPA is rapidly converted to arachidonic acid via a PAP/DAG lipase pathway, followed by a gradual conversion of arachidonic acid to PGE2 by COX in both UMR-106 cells and isolated adult osteoblastic cells, and that neither phospholipase A2 nor phospholipase C is involved in the TPA-induced PGE2 production. To the best of our knowledge, this is the first report that shows that the activation of PKC in osteoblastic cells leads to the production of PGE2 via a PLD/PAP/DAG lipase/COX pathway.

Animals↗
PubMed1992

Calcium ion independent membrane leakage induced by phospholipase-like myotoxins.

The two snake venom myotoxins ammodytin L and myotoxin II, purified respectively from Vipera ammodytes ammodytes and Bothrops asper, have phospholipase-like structures but lack an Asp-49 in the active site and are without normal phospholipase activity. The interaction of these proteins with different types of liposomes indicated that the myotoxins were able to provoke rapid and extensive release of the aqueous content of liposomes. Leakage was measured by two different methods: fluorescence dequenching of liposome-entrapped carboxyfluorescein and ESR measurement of intravesicular TEM-POcholine reduction by external ascorbate. The process was independent of Ca2+ and took place without any detectable phospholipid hydrolysis. Nonmyotoxic phospholipases tested under the same conditions were unable to induce liposome leakage, which could be detected only when Ca2+ was added to the medium and with the concomitant hydrolysis of phospholipids. The kinetics of Ca(2+)-dependent and Ca(2+)-independent leakage were completely different, indicating two different mechanisms of interaction with the lipid bilayer. Studies using diphenylhexatriene as a probe of lipid membrane organization indicated that the myotoxins gave rise to a profound perturbation of the arrangement of the lipid chains in the membrane interior, whereas interaction of Naja naja phospholipase A2 with the membrane surface did not affect lipid organization. On the basis of these results we suggest that a new type of cytolytic reaction mechanism is responsible for the effects of phospholipase-like myotoxins in vivo.

Calcium↗
PubMed1985

Aggregation studies on fluorescein-coupled cobra venom phospholipase A2.

Phospholipase A2 from Naja naja naja venom (Indian cobra) undergoes a concentration-dependent aggregation, and at an assay concentration of 1 microgram mL-1, it exists as a monomer. However, there is some evidence that the enzyme is actually active as a dimer or higher order aggregate. Previous attempts to determine the aggregation state of the enzyme under actual assay conditions were thwarted by experimental difficulties due in part to the low enzyme concentrations required. This aggregation has now been studied by using fluorescence polarization. The extrinsic probe fluorescein isothiocyanate was coupled to the enzyme to serve as the fluorescence marker. Steady-state polarization measurements were made to determine changes in the aggregation state of the fluorescently tagged enzyme. The phospholipases A2 from Crotalus adamanteus (rattlesnake) and porcine pancreas, whose states of aggregation are known, were also labeled with fluorescein isothiocyanate and used as controls. It was found that the divalent metal ions Ca2+, a phospholipase cofactor, and Ba2+, an inhibitor, caused an increase in the cobra venom enzyme polarization, while Mn2+, Mg2+, and Co2+ did not. The water-soluble substrate diheptanoylphosphatidylcholine and the lipid analogue dodecylphosphocholine, when present below their respective critical micelle concentrations, also increased the polarization of the phospholipase-fluorescein conjugate. Thus, both cofactor and substrate caused an increase in the polarization, which implies an increase in the aggregation state. It is concluded that under assay conditions the phospholipase A2 exists in an aggregated form.

Animals↗
PubMed1985

Mechanism of cationic amphiphilic drug inhibition of purified lysosomal phospholipase A1.

Cationic amphiphilic drugs like chlorpromazine, propranolol, and chloroquine inhibit lysosomal phospholipase A in vitro. Some workers have proposed that cationic amphiphilic drugs inhibit the activity of phospholipase A1 by forming substrate-drug complexes which cannot be degraded while others have reported competitive inhibition implying drug effects on the enzyme. To analyze the mechanism of inhibition, we examined the binding ability of these drugs to unilamellar vesicles of dioleoylphosphatidylcholine and correlated these results with a detailed kinetic analysis of phospholipase A. Chlorpromazine and propranolol bound to small unilamellar liposomes of dioleoylphosphatidylcholine substrate in a positive cooperative way consistent with two binding sites: a high-affinity site with low capacity and a low-affinity site with high capacity. The affinity of chlorpromazine for the high-affinity site was 2 times greater than that of propranolol (KA = 13 807 +/- 1722 vs. 8481 +/- 1078 M-1), and the saturation number for chlorpromazine was 3 times greater than for propranolol (N = 0.20 +/- 0.004 vs. 0.07 +/- 0.02 mol of drug/mol of phosphatidylcholine). Chloroquine did not bind to unilamellar liposomes of dioleoylphosphatidylcholine. We carried out detailed kinetic studies using purified lysosomal phospholipase A1 from rat liver. In the case of chloroquine inhibition, the Lineweaver-Burk double-reciprocal plots showed straight lines, but the slope replots were curved, indicating the formation of complexes having 2 mol of chloroquine/mol of enzyme (EI2 complexes). Thus, chloroquine is a competitive inhibitor which forms EI2 complexes with phospholipase A1. However, in the case of chlorpromazine and propranolol, the observed kinetic data do not fit to the same equilibrium used for the case of chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Structural and functional properties of a phospholipase A2 purified from an inflammatory exudate.

The cell-free supernatant of sterile inflammatory peritoneal exudates contains a phospholipase A2 that participates in the digestion of Escherichia coli killed by polymorphonuclear leukocytes or by the purified bactericidal/permeability increasing protein (BPI) of these cells. This phospholipase A2 has been purified, and the sequence of the NH2-terminal 39 amino acids has been determined and compared with sequences of both BPI-responsive and BPI-nonresponsive phospholipases A2 from snake venoms and mammalian pancreas. The high concentration and location of basic residues in the NH2-terminal region is a common feature of BPI-responsive phospholipases A2 and may characterize those phospholipases A2 participating in inflammatory events.

Amino Acid Sequence↗
PubMed1987

Synthesis of prodan-phosphatidylcholine, a new fluorescent probe, and its interactions with pancreatic and snake venom phospholipases A2.

A new fluorescent probe, prodan-PC, was synthesized by incubating thio-PC, a thiol ester analogue of phosphatidylcholine [1,2-bis(decanoylthio)-1,2-dideoxy-sn-glycero-3-phosphocholine], with acrylodan, a fluorescent thiol-reactive reagent [6-acryloyl-2-(dimethylamino)naphthalene], in the presence of phospholipase A2, which served to generate lysothio-PC in situ. Prodan-PC (PPC) showed maximum absorption in ethanol at 370 nm. The fluorescence emission spectrum showed maximum emission at 530 nm in water and at 498 nm in ethanol. In the presence of a saturating amount of phospholipase A2, the emission maximum shifted to about 470 nm. PPC showed a critical micellar concentration around 5 microM, with evidence of premicellar aggregation above 1 microM. Binding of PPC to Crotalus adamanteus phospholipase A2 was evidenced by an increase in emission at 480 nm and an increase in fluorescence anisotropy. An apparent dissociation constant of 0.323 microM was calculated for this enzyme complex. Binding was dependent on the presence of calcium ion and was abolished by blocking the active site with p-bromophenacyl bromide. Binding was also followed by energy transfer from tryptophan in the enzyme to PPC. Apparent dissociation constants for PPC complexes with phospholipases A2 from Naja naja naja and porcine pancreas and the prophospholipase A2 from porcine pancreas were 0.509, 0.107, and 0.114 microM, respectively. PPC was shown to inhibit the activity of pancreatic phospholipase A2 in thio-PC-sodium cholate mixed micelles. Inhibition studies were complicated because PPC can also serve as an activator of the snake venom enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Naphthylamine↗
PubMed1987

Triggering of the activity of phospholipase A2 by an electric field.

In this paper we show that the action of phospholipase A2 can be triggered by applying an electric field across a 1,2-didodecanoyl-sn-3-phosphoglycerol monolayer residing between an alkylated silicon surface and water. When the silicon wafer served as a cathode, rapid activation of porcine pancreatic phospholipase was observed and did depend on the magnitude of the applied potential. The degree of activation was different for the pancreatic phospholipase A2 and snake and bee venom enzymes. Maximally, a 7-fold activation of pancreatic phospholipase A2 was observed when the applied potential was 75 V. The effective field over the lipid film could be estimated to be approximately 25-175 mV, i.e., in the range of membrane potentials found in cells. On the basis of these results, we suggest that changes in membrane potential might be an important factor in the regulation of the action of intracellular phospholipases A2 in vivo.

Animals↗
PubMed1981

Photoaffinity labeling of Crotalus atrox phospholipase A2 by a substrate analogue.

A photolabile analogue of phosphatidylethanolamine (photolabile PE analogue), 1,2-di-O-hexylglycero-3-(ethyl diazomalonamidoethyl phosphate), was synthesized in nonisotopic and 14C-radiolabeled form and in both the L configuration (that of the naturally occurring phospholipids) and the racemic form. When the unlabeled racemic compound was photolyzed in the presence of phospholipase A2 of Crotalus atrox, extensive enzyme inactivation was observed. The rate of inactivation was stimulated by Ca2+ and by formation of micelles of the photolabile compound. The dihexyl ether analogue of phosphatidylethanolamine protected the enzyme from inactivation. Phospholipase A2 gave rise to a covalently labeled polypeptide when irradiated in the presence of either L or racemic 14C-labeled photolabile PE analogue. The racemic compound labeled both the N-terminal region (residues 1--15) and the central region (residues 43--97) of the polypeptide while the L compound labeled only the N-terminal region. The lone methionine at position 10 of the C. atrox phospholipase A2 permitted degradation by cyanogen bromide, which showed that labeling by the L compound was restricted to the first ten amino acid residues at the N-terminal end. Phospholipase A2 has an absolute specificity for L-phospholipids, and D-phospholipids are competitive inhibitors. The results of these studies underscore the importance of the head-group region of the phospholipid in phospholipase--substrate interactions and suggest that the two optical isomers of the substrate may be rather differently oriented on the enzyme surface.

Affinity Labels↗
PubMed1998

Agonist-specific alterations in receptor-phospholipase coupling following inactivation of Gi2alpha gene.

Different forms of phospholipase A2, together with pertussis toxin-sensitive G-proteins, [Ca2+]i (intracellular Ca2+ concentration), protein kinase C, calmodulin, protein tyrosine kinases, mitogen-activated protein kinases and Ca2+/calmodulin-dependent protein kinase appear to play a role in agonist-mediated release of arachidonic acid. Here we report that fibroblasts from 14-day-old mouse embryos with inactivated Gi2alpha (alpha-subunit of the heterotrimeric G-protein Gi2) gene display a marked decrease in the ability of lysophosphatidic acid, thrombin and Ca2+ ionophores to release arachidonic acid compared with their normal counterparts. The requirement for Gi2alpha in the release of arachidonic acid following increased [Ca2+]i may be explained by the incomplete translocation of cytosolic phospholipase A2 observed in Gi2alpha-deficient cells. Paradoxically, inactivation of the Gi2alpha gene resulted in up-regulation of bradykinin receptors and their coupling to increased arachidonic acid release, phospholipase C activity and [Ca2+]i. A concomitant increase in basal phospholipase C activity was also observed in the Gi2alpha-deficient cells. These observations establish a pleiotropic and essential role for Gi2alpha in receptor-phospholipase coupling that contrasts with its less obligatory participation in agonist-mediated inhibition of adenylate cyclase.

Adenylate Cyclase Toxin↗
PubMed1980

A phospholipase A2 inhibitory protein in rabbit neutrophils induced by glucocorticoids.

When rabbit peritoneal neutrophils were treated with glucocorticoids, their chemotactic response to stimulation by the chemoattractant fMet-Leu-Phe was markedly reduced. Preincubation of cells with glucocorticoids also decreased phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) activity in situ as measured by the release of [1-14C]arachidonic acid previously incorporated into phospholipids. The inhibitory potencies of glucocorticoids on phospholipase A2 activity correlated well with their anti-inflammatory activities and their abilities to bind to glucocorticoid receptors. Inhibitors of RNA and protein synthesis suppressed the inhibitory effect of glucocorticoids on phospholipase A2 activity. Digestion of the glucocorticoid-treated cells by Pronase overcame the inhibitory activity. Phospholipase A2 activity induced by Ca2+ ionophore A23187 was not affected by Pronase treatment. Gel filtration of proteins from neutrophil membranes labeled with [3H]lysine showed an induction of protein(s) (about 40,000 daltons) after glucocorticoid treatment. This protein inhibited a partially purified pancreatic phospholipase A2 and reduced the peptide-initiated chemotactic response of neutrophils.

Animals↗
PubMed1986

Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine.

Human platelets stimulated by epinephrine undergo enhanced turnover of phosphatidylinositol 4,5-bisphosphate, accumulate inositol trisphosphate, diacylglycerol, and phosphatidic acid, and phosphorylate a 47-kDa protein. All of these phenomena indicate stimulation of phospholipase C. These responses are blocked completely by inhibitors of alpha 2-adrenergic receptors (yohimbine), cyclooxygenase (aspirin or indomethacin), phospholipase A [2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082)], Na+/H+ exchange [ethylisopropylamiloride (EIPA)], fibrinogen binding to glycoprotein IIb/IIIa (antibody A2A9), Ca2+/Mg+ binding (EDTA), or removal of fibrinogen. Epinephrine evokes (i) an increased turnover of ester-linked arachidonic acid in aspirin treated platelets that is inhibited by ONO-RS-082, EDTA, yohimbine, or the absence of fibrinogen and (ii) a rapid cytoplasmic alkalinization that is inhibited partially by blockage of cyclooxygenase activity and completely by A2A9 or EIPA. In contrast, when incubated with subaggregatory concentrations of the prostaglandin H2/thromboxane A2 analogue [(15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic acid (U46619) and epinephrine, aspirin-treated platelets show a potentiation of phospholipase C activation that is unaffected by the above inhibitors. We propose that epinephrine, in promoting exposure of glycoprotein IIb/IIIa sites for fibrinogen binding, leads to a cytoplasmic alkalinization, which, in conjunction with local shifts in Ca2+, promotes low-level activation of phospholipase A. The resulting free arachidonic acid is converted to cyclooxygenase products, which, potentiated by epinephrine, activate phospholipase C. This further amplifies the initial stimulatory response.

Arachidonic Acid↗
PubMed2001

The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gqalpha -dependent ERK-independent pathway.

The Ca(2+)-sensing receptor (CaR) stimulates a number of phospholipase activities, but the specific phospholipases and the mechanisms by which the CaR activates them are not defined. We investigated regulation of phospholipase A(2) (PLA(2)) by the Ca(2+)-sensing receptor (CaR) in human embryonic kidney 293 cells that express either the wild-type receptor or a nonfunctional mutant (R796W) CaR. The PLA(2) activity was attributable to cytosolic PLA(2) (cPLA(2)) based on its inhibition by arachidonyl trifluoromethyl ketone, lack of inhibition by bromoenol lactone, and enhancement of the CaR-stimulated phospholipase activity by coexpression of a cDNA encoding the 85-kDa human cPLA(2). No CaR-stimulated cPLA(2) activity was found in the cells that expressed the mutant CaR. Pertussis toxin treatment had a minimal effect on CaR-stimulated arachidonic acid release and the CaR-stimulated rise in intracellular Ca(2+) (Ca(2+)(i)), whereas inhibition of phospholipase C (PLC) with completely inhibited CaR-stimulated PLC and cPLA(2) activities. CaR-stimulated PLC activity was inhibited by expression of RGS4, an RGS (Regulator of G protein Signaling) protein that inhibits Galpha(q) activity. CaR-stimulated cPLA(2) activity was inhibited 80% by chelation of extracellular Ca(2+) and depletion of intracellular Ca(2+) with EGTA and inhibited 90% by treatment with W7, a calmodulin inhibitor, or with KN-93, an inhibitor of Ca(2+), calmodulin-dependent protein kinases. Chemical inhibitors of the ERK activator, MEK, and a dominant negative MEK, MEK(K97R), had no effect on CaR-stimulated cPLA(2) activity but inhibited CaR-stimulated ERK activity. These results demonstrate that the CaR activates cPLA(2) via a Galpha(q), PLC, Ca(2+)-CaM, and calmodulin-dependent protein kinase-dependent pathway that is independent the ERK pathway.

Arachidonic Acid↗
PubMed1987

Purification and characterization of phospholipase A2 from Trimeresurus gramineus venom.

Phospholipase A2 was purified to homogeneity from the venom of Trimeresurus gramineus (the Green Habu snake) via three steps consisting of Sephadex G-75, DEAE-cellulose, and DEAE-Toyopearl 650M column chromatographies. Molecular weight determinations showed that the enzyme consists of a single polypeptide chain with a molecular weight of approximately 14,000. The isoelectric point was 4.5. The enzyme is characterized by high contents of acidic amino acids, glycine, and half-cystine. Calcium ion was essential for eliciting activity. The enzyme was inactivated by alkylation of a single histidine residue with p-bromophenacyl bromide following pseudo first-order kinetics, and the rate of the inactivation was depressed in the presence of Ca2+. The N-terminal sequence of this enzyme determined to the 40th residue was found to be highly homologous to that of Trimeresurus okinavensis phospholipase A2 but not to that of Trimeresurus flavoviridis phospholipase A2. The phenylalanine residue at the 27th position of T. gramineus phospholipase A2 is noteworthy because all other phospholipases A2, with only two exceptions, contain a tyrosine residue at this position.

Acetophenones↗
PubMed1989

Isolation and fundamental properties of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis.

Phospholipase A2 inhibitor was purified from the blood plasma of Habu, Trimeresurus flavoviridis, by Sephadex G-200 gel filtration, DEAE-cellulose chromatography, and Blue-Sepharose CL-6B column chromatography. The purified inhibitor was shown to be a glycoprotein with a molecular weight of about 100K. It was found to consist of four subunits whose molecular weights were around 20-24K. In order to examine the inhibition mechanism of the inhibitor, the interaction of the inhibitor with a phospholipase A2 from T. flavoviridis venom was examined by Sephadex G-100 gel filtration. One inhibitor molecule was found to bind directly to one phospholipase A2 molecule in both the presence and absence of Ca2+. The inhibitor inhibited the phospholipase A2 from T. flavoviridis venom with an apparent dissociation constant, Ki, of 1.7 X 10(-10) M, but not the porcine pancreas enzyme or the Agkistrodon halys blomhoffii enzyme belonging to the same family, Crotalidae, as T. flavoviridis, or the phospholipase C from Bacillus cereus.

Animals↗
PubMed1989

Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2.

We have studied the role of Tyr-69 of porcine pancreatic phospholipase A2 in catalysis and substrate binding, using site-directed mutagenesis. A mutant was constructed containing Phe at position 69. Kinetic characterization revealed that the Phe-69 mutant has retained enzymatic activity on monomeric and micellar substrates, and that the mutation has only minor effects on kcat and Km. This shows that Tyr-69 plays no role in the true catalytic events during substrate hydrolysis. In contrast, the mutation has a profound influence on the stereospecificity of the enzyme. Whereas the wild-type phospholipase A2 is only able to catalyse the degradation of sn-3 phospholipids, the Phe-69 mutant hydrolyses both the sn-3 isomers and, at a low (1-2%) rate, the sn-1 isomers. Despite the fact that the stereospecificity of the mutant phospholipase has been altered, Phe-69 phospholipase still requires Ca2+ ions as a cofactor and also retains its specificity for the sn-2 ester bond. Our data suggest that in porcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69 serves to fix and orient the phosphate group of phospholipid monomers by hydrogen bonding. Because no such interaction can occur between the Phe-69 side-chain and the phosphate moiety of the substrate monomer, the mutant enzyme loses part of its stereospecificity but not its positional specificity.

Animals↗