A phosphodiesterase-1 (PDE-1, EC 3.1.4.1) have been purified from King cobra; Ophiophagus hannah (O. hannah) venom by preparative native PAGE. A single protein band was observed in analytical native PAGE and SDS-PAGE. The molecular mass was found to be 148 kDa. The enzyme was free from 5’-nucleotidase and alkaline phosphatase activities. The enzyme showed an optimum pH 10.0 in Tris-HCl buffer. The optimum temperature was found to be 50 ºC. Energy of activation (Ea) was calculated to be 164. The PDE-1 is a glycoprotein and exhibited basic pI. The Vmax and Km of PDE-1 calculated were 1.53 μM/min/mg and 2.6×10-3 M, respectively. The Kcat and Ksp values are 9.2s-1 and 58.8 M-1 Min-1 respectively. Cysteine caused a non-competitive inhibition, with a Ki 8.2×10−3M. The IC50 was 3.9 mM. The adenosine diphosphate (ADP) caused a competitive inhibition, having Ki 1.0×10−3 M. The IC50 was 12.0 mM. Glutathione, o-phenanthroline, Zinc and EDTA inhibited the PDE-1 activity, whereas magnesium slightly potentiated the activity. The enzyme hydrolyzed thymidine 5’-mono phosphate p- nitro-phenyl ester most readily (10 fold) while cyclic 3’-5’-AMP was least readily hydrolyzed substrate. The PDE-1 up to 4.0 mg/Kg i.p was not lethal in mice. The PDE-1 exhibited an anticoagulant effect whereas the crude venom showed strong coagulant effect. The above mentioned studies show that the O. hannah PDE-1 is very similar to that isolated from other snake venoms.
SAMI ULLAH KHAN ,MOHAMMAD ASHRAF ,SAAD SM ALSALEH ,
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