Please use this identifier to cite or link to this item: http://nopr.niscair.res.in/handle/123456789/4583
Title: Biochemical characterization and some biological properties of the phosphodiesterase I purified from <i>Agistrodon bilineatus</i> venom
Authors: Al-Saleh, Saad S M
Khan, Sami Ullah
Ashraf, Mohammad
Keywords: <i style="">Agistrodon bilineatus</i>
Snake
Venom
Phosphodiesterase I<b></b>
Issue Date: Jun-2009
Publisher: CSIR
Abstract: The venom phosphodiesterase I (PDE-I, EC 3.1.4.1) is useful in the elucidation of the structure and nucleotide sequence of nucleic acids. In the present study, PDE-I was purified from <i style="">Agistrodon bilineatus</i> venom by preparative native-PAGE. A single protein band was observed in analytical native-PAGE. The enzyme also gave a single band in SDS-PAGE with a molecular mass of 140 kDa. The position of the band was not altered in the presence of β-mercaptoethanol, suggesting the protein did not contain subunits. The enzyme was free from 5’-nucleotidase and alkaline phosphatase activities. It showed a broad optimum pH range (9.0-11.0), whereas the optimum temperature was found to be 60<sup>0</sup>C, with activity decreasing at >65<sup>0</sup>C. Energy of activation (E<sub>a</sub>) was calculated to be 0.31. The PDE-I was a glycoprotein having 14% of carbohydrate content. The <i style="">V</i><sub>max, </sub><i style="">K</i><sub>m,</sub> <i style="">K</i><sub>cat</sub> and <i style="">K<sub>s</sub></i><sub>p </sub>values<sup> </sup>of the enzyme were 3.85 μM/min/mg, 8.3 × 10<sup>-3</sup> M, 23<sup>s-1</sup> and 46.4 M<sup>-1</sup> Min<sup>-1</sup> respectively. Cysteine caused a non-competitive inhibition with a <i style="">K</i><sub>i </sub>6.3 × 10<sup>−3</sup> M (IC<sub>50 </sub>of 1.6 mM), whereas ADP caused a competitive inhibition having <i style="">K</i><sub>i </sub>0.8 × 10<sup>−3</sup> M (IC<sub>50</sub> 5.4 mM). Glutathione, <i style="">o</i>-phenanthroline, zinc and EDTA inhibited the enzyme activity, whereas Mg<sup>2+</sup> slightly potentiated the activity. The enzyme hydrolyzed thymidine 5’-monophosphate <i style="">p</i>-nitro-phenyl ester most readily (10-fold), while 3’-5’-cAMP was least readily hydrolyzed substrate. The enzyme up to 4.0 mg/Kg i.p was not lethal in mice. It exhibited an anticoagulant effect, and increased the normal clotting time of normal citrated human plasma, whereas the crude venom showed strong coagulant effect. The above results showed that the <i style="">A. bilineatus</i> PDE-I was very similar to that isolated from other snake venoms. The purification procedure described here is simple, rapid and reproducible and may prove useful to isolate pure protein for investigation into the contribution of this enzyme to the biological activities of <i style="">A. bilineatus </i>venom and PDE-I insight, in general.
Description: 221-229
URI: http://hdl.handle.net/123456789/4583
ISSN: 0975-0959 (Online); 0301-1208 (Print)
Appears in Collections:IJBB Vol.46(3) [June 2009]

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