Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom.
Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.
Higuchi
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University of Mogi das Cruzes, Av Dr Candido Xavier de Almeida Souza 200, Centro Cívico, CEP 08780-911 Mogi das Cruzes, São Paulo, Brazil.
Barbosa
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CM
Bincoletto
C
C
Chagas
J R
JR
Magalhaes
A
A
Richardson
M
M
Sanchez
E F
EF
Pesquero
J B
JB
Araujo
R C
RC
Pesquero
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects; D017837:Bothrops / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002286:Carcinoma, Ehrlich Tumor / Q000139:chemically induced / Q000201:enzymology / Q000473:pathology; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004797:Enzyme-Linked Immunosorbent Assay; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020014:K562 Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010974:Platelet Aggregation / Q000187:drug effects; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012910:Snake Venoms / Q000201:enzymology* / Q000494:pharmacology
D000925:Anticoagulants; D006454:Hemoglobins; D007527:Isoenzymes; D012910:Snake Venoms; D010741:Phospholipases A; D002118:Calcium