Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom.
A phosphodiesterase was purified from the venom of the snake Bothrops alternatus by a combination of gel filtration and ion exchange chromatographies. In SDS-PAGE, the enzyme gave a single band with a molecular mass of 105 kDa, which was unaltered in the presence of beta-mercaptoethanol, indicating that the protein contained no subunits. A single protein band was also observed in native PAGE. There were no contaminating 5'-nucleotidase, alkaline phosphatase and protease activities. The enzyme was recognized by commercial bothropic antiserum and gave a single band in immunoblotting. The enzyme had a pH optimum in the range of 7.5-9.5 and the optimum temperature was 60 degrees C, with activity being rapidly lost within 1 min at > or = 70 degrees C. The Km of the enzyme was 2.69 mM. PDE activity was potentiated by cobalt and, to a lesser extent, by calcium, whereas copper, manganese, zinc, EDTA, and beta-mercaptoethanol were inhibitory. These properties show that this enzyme is very similar to that isolated from other snake venoms.
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Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazil.
Corradini
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AC
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Mello
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Hyslop
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D000818:Animals; D017837:Bothrops; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification*; D012685:Sepharose / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D015032:Zinc / Q000494:pharmacology
D008345:Manganese; D008623:Mercaptoethanol; D003300:Copper; D012685:Sepharose; D004492:Edetic Acid; D010727:Phosphoric Diester Hydrolases; D015032:Zinc