Abstract
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 degrees C. Energy of activation (Ea) was calculated to be 164. The PDE-1 is a glycoprotein and exhibited basic pI. The V-max and K-m of PDE-1 calculated were 1.53 mu M/min/mg and 2.6x10(-3) M, respectively. The K-cat and K-sp values are 9.2(s-1) and 58.8 M-1 Min(-1) respectively. Cysteine caused a non-competitive inhibition, with a K-i 8.2x10(-3) M. The IC50 was 3.9 mM. The adenosine diphosphate (ADP) caused a competitive inhibition, having Ki 1.0x10(-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.