Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom

Valério, A.A.; Corradini, A.C.; Panunto, P.C.; Mello, S.M.; Hyslop, S.

Journal of Protein Chemistry 21(8): 495-503


ISSN/ISBN: 0277-8033
PMID: 12638651
DOI: 10.1023/a:1022414503995
Accession: 068774514

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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.