title: Kinetic and pharmacologic characterization of phospholipases A2 from Bothrops neuwiedii venom.
abstract: Two phospholipases A2 (PLA2) (EC 3.1.1.4) were purified from Bothrops neuwiedii venom (isoenzymes P-1 and P-2). The molecular weights of P-1 and P-2 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 15,000 and 16,200 and the isoelectric points were 4.8 and 4.6, respectively. The N-terminal 14-amino-acid sequences determined were Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Met-Ile-Ala-Gly and Ser-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Met-Ile-Ala-Gly for P-1 and P-2, respectively. Since both show sequence almost identical to that of a PLA2 from Crotalus atrox it was tentatively classified as being of group II. The enzymatic activity of P-1 and P-2 toward lipid monolayers was studied. The hydrolysis of dilauroylphosphatidylcholine (dlPC) shows a broad optimum between 7 and 18 mN m-1 and a cut-off pressure of 22 mN m-1. The activity toward dlPC displays a maximum at pH 8 and is dependent on the presence of Ca2+ with an apparent Kd of 0.1 mM, for both enzymes. P-1 and P-2 are heat-stable enzymes, unable to hydrolyze dilauroylphosphatidic acid monolayers. The enzymes are not lethal to mice at doses up to 5 micrograms/g body weight by intraperitoneal injection and they do not show myotoxic (up to 40 micrograms) or hemolytic activity (up to 8.5 micrograms/ml). Both lack anti-coagulant activity, determined by absence of changes in the recalcification time of platelet poor plasma (up to 100 micrograms/ml), and are not able to induce platelet aggregation (up to 50 micrograms/ml). However, both isoenzymes exhibit an important edema-inducing activity that is not altered at short times by the irreversible chemical inactivation of the hydrolytic activity with phenacyl bromide. P-1 and P-2 are able to release arachidonic acid from membrane phospholipids of neutrophils, property that is lost by the inactivation of the enzyme. This suggests that the edema-inducing activity of the active but not the inactive forms may be partly due to arachidonic acid-derived mediators. The edema-inducing activity of the active or inactive forms of the enzymes is inhibited by antagonists of histamine, suggesting that histamine plays an important role in both the active and the inactive B. neuwiedii PLA2s-induced edema. The results suggest that the inflammatory and the catalytic activity of these enzymes constitute separate properties.
authors.affiliation: Departamento de Química Biológica-CIQUIBIC, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina.
authors.forename: J J
authors.initials: JJ
authors.lastname: Daniele
authors.forename: I D
authors.initials: ID
authors.lastname: Bianco
authors.forename: G D
authors.initials: GD
authors.lastname: Fidelio
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017837:Bothrops / Q000235:genetics / Q000378:metabolism*; D003435:Crotalid Venoms / Q000235:genetics / Q000378:metabolism*; D004487:Edema / Q000209:etiology; D006801:Humans; D066298:In Vitro Techniques; D007526:Isoelectric Point; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D054467:Phospholipases A2; D017382:Reactive Oxygen Species / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D003435:Crotalid Venoms; D007527:Isoenzymes; D017382:Reactive Oxygen Species; D010741:Phospholipases A; D054467:Phospholipases A2