The water-soluble venom of Bothrops asper Garman (San Juan Evangelista, Veracruz, México) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-l-arginine ethyl hydrolase, N-benzoyl-l-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded seven protein fractions, which were apparently less toxic than the whole venom (LD50=4.3μg/g mouse wt.). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH7.55) gave several fractions, two being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by gel disc electrophoresis yielding two more pure proteins designated phospholipase 2 and phospholipase 3. Analysis of phospholipids hydrolysed by these enzymes have shown that all three phospholipases belong to type A2. Amino acid analysis has shown that phospholipase A2 (type 1) has 97 residues with a calculated mol.wt. of 10978±11. Phospholipase A2 (type 2) has 96 residues with a mol.wt. of 10959±11. Phospholipase A2 (type 3) has 266 residues with 16 half-cystine residues and a calculated mol.wt of 29042±31. Automated Edman degradation showed the N-terminal sequence to be: Asx-Leu-Trp-Glx-Phe-Gly-Glx-Met-Met-Ser-Asx-Val- Met-Arg-Lys-Asx-Val-Val-Phe-Lys-Tyr-Leu- for phospholipase A2 (type 2).
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March 1980
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Research Article|
March 01 1980
Venom from the snake Bothrops asper Garman. Purification and characterization of three phospholipases A2
Alejandro C. Anagón;
Alejandro C. Anagón
*Departamento de Neurociencias, Centro de Investigaciones en Fisiología Celular and Departamento de Bioquímica, Facultad de Medicina, Apartado Postal 70-600, Universidad Nacional Autóma de México, 20 D.F., México
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Ricardo R. Molinar;
Ricardo R. Molinar
*Departamento de Neurociencias, Centro de Investigaciones en Fisiología Celular and Departamento de Bioquímica, Facultad de Medicina, Apartado Postal 70-600, Universidad Nacional Autóma de México, 20 D.F., México
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Lourival D. Possani;
Lourival D. Possani
*Departamento de Neurociencias, Centro de Investigaciones en Fisiología Celular and Departamento de Bioquímica, Facultad de Medicina, Apartado Postal 70-600, Universidad Nacional Autóma de México, 20 D.F., México
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Paul L. Fletcher, Jr.;
Paul L. Fletcher, Jr.
†Section of Cell Biology and Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06510, U.S.A.
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John E. Cronan, Jr.;
John E. Cronan, Jr.
†Section of Cell Biology and Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06510, U.S.A.
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Jordi Z. Julia
Jordi Z. Julia
‡Instituto Nacional de Higiene, S.S.A. 7 D.F., México
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1980 London: The Biochemical Society
1980
Biochem J (1980) 185 (3): 695–704.
Citation
Alejandro C. Anagón, Ricardo R. Molinar, Lourival D. Possani, Paul L. Fletcher, John E. Cronan, Jordi Z. Julia; Venom from the snake Bothrops asper Garman. Purification and characterization of three phospholipases A2. Biochem J 1 March 1980; 185 (3): 695–704. doi: https://doi.org/10.1042/bj1850695
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