Role of catalytic function in the antiplatelet activity of phospholipase A(2) cobra (Naja naja naja) venom

Three acidic phospholipases A(2) from Indian cobra (Naja naja naja) venom inhibited platelet aggregation in platelet rich plasma induced separately by ADP, collagen and epinephrine with different potencies. The order of inhibition was epinephrine > collagen > ADP. They did not inhibit platelet aggregation induced by arachidonic acid (10 muM). The inhibition was dependent on concentration of the protein and the time of incubation of the phospholipases A(2) with platelet rich plasma. Parabromophenacyl bromide modified PLA(2) enzymes lost their enzymatic activity as well as platelet aggregation inhibition activity suggesting the involvement of catalytic function in platelet aggregation inhibitory activity

Comparison of proteomic profiles of the venoms of two of the \u27Big Four\u27 snakes of India, the Indian Cobra (Naja naja) andthe common krait (Bungarus caeruleus), and analyses of their toxins

Snake venoms are mixtures of biologically-active proteins and peptides, and several studies have described the characteristics of some of these toxins. However, complete proteomic profiling of the venoms of many snake species has not yet been done. The Indian cobra (Naja naja) and common krait (Bungarus caeruleus) are elapid snake species that are among the ‘Big Four’ responsible for the majority of human snake envenomation cases in India. As understanding the composition and complexity of venoms is necessary for successful treatment of envenomation in humans, we utilized three different proteomic profiling approaches to characterize these venoms: i) one-dimensional SDS-PAGE coupled with in-gel tryptic digestion and electrospray tandem mass spectrometry (ESI-LC-MS/MS) of individual protein bands; ii) in-solution tryptic digestion of crude venoms coupled with ESI-LC-MS/MS; and iii) separation by gel-filtration chromatography coupled with tryptic digestion and ESI-LC-MS/MS of separated fractions. From the generated data, 81 and 46 different proteins were identified from N. naja and B. caeruleus venoms, respectively, belonging to fifteen different protein families. Venoms from both species were found to contain a variety of phospholipases A2 and three-finger toxins, whereas relatively higher numbers of snake venom metalloproteinases were found in N. naja compared to B. caeruleus venom. The analyses also identified less represented venom proteins including L-amino acid oxidases, cysteine-rich secretory proteins, 5’-nucleotidases and venom nerve growth factors. Further, Kunitz-type serine protease inhibitors, cobra venom factors, phosphodiesterases, vespryns and aminopeptidases were identified in the N. naja venom, while acetylcholinesterases and hyaluronidases were found in the B. caeruleus venom. We further analyzed protein coverage (Lys/Arg rich and poor regions as well as potential glycosylation sites) using in-house software. These studies expand our understanding of the proteomes of the venoms of these two medically-important species

Isolation and characterization of an endogenous inhibitor of phospholipase A(2) from Indian cobra (Naja naja naja) venom

A PLA(2)-inhibitor has been purified from Indian cobra (Naja naja naja) venom by the combination of ion-exchange and gel-filtration chromatography. The inhibitor, NN-I-3 was a peptide with mol.wt 6500 and has a fluorescence emission maxima cn 340 nm. NN-I-3 specifically inhibited the enzyme activity of the three acidic PLA? from the same venom. The inhibition of NN-I-2d-PLA(2) and NN-I-2e-PLA(2) by NN-I-3 was of mixed type and NN-I-2c-PLA(2) was of uncompetitive type, Neither the inhibitor nor the individual mixtures of acidic PLA(2) with the inhibitor (1:1 w/w or 1:2 mol:mol) were lethal to mice when injected intraperitoneally in doses up to 10 mgkg(-l) body weight. (C) 1998 Elsevier Science Ltd. All rights reserved

Purification and characterization of three acidic, cytotoxic phospholipases A(2) from Indian cobra (Naja naja naja) venom

Three acidic phospholipases A(2) (NN-I-2c-PLA(2), NN-I-2d-PLA(2) and NN-I-2e-PLA(2)) were purified by successive chromatography of Indian cobra (Naja naja naja) venom on CM-Sephadex C-25, Sephadex G-50 and QAE-Sephadex A-25 columns. They have molecular weights of 13 000-14 500 by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. They showed tryptophan specific fluorescence emission spectra (similar to 345 nm). All the three phospholipases A(2) were enzymatically highly active with specific activities 9-17 mu mol min(-1) mg(-1) They were non-lethal to mice when injected intraperitoneally in doses up to 10 mg kg(-1) body weight. They induced edema in mouse foot pads and were cytotoxic to Ehrlich ascites tumour cells. They did not exhibit direct haemolytic and anticoagulant activities. (C) 1998 Elsevier Science Ltd. All rights reserved

Antibodies to a Phospholipase-A2 from Vipera-Russelli selectively Neutralize Venom Neurotoxicity

Polyclonal antibodies to a purified neurotoxic phospholipase A2 (PLA2), VRV PL-VIIIa, from Vipera russelli venom were raised in rabbits. Anti-PL-VIIIa-Ig (gamma-globulin fraction of rabbit antiserum injected with VRV PL-VIIIa) selectively neutralized the neurotoxicity of VRV PL-VIIIa, VRV PL-V, VRV PL-VI (neurotoxic PLA2 of V. russelli venom) and whole V. russelli venom without affecting their PLA2 activity, which clearly demonstrates that the catalytic site and the neurotoxic site (the site through which the PLA2 binds to the nervous system) are distinct on a PLA2 molecule. Anti-PL-VIIIa Ig did not have any effect on the oedema-inducing activity and indirect haemolytic activity of VRV PL-VIIIa, VRV PL-V and VRV PL-VI, which are attributed to the PLA2 activity of the peptide, but inhibited their anti-coagulant potency