Purification and characterization of Human Intestinal alkaline phosphatase and its role in the colonization of Helicobacter pylori in the duodenum

Order of Publishing in Issue: 
6
Volume :3
Issue :4
October, 2009 - December, 2009
Page No: 
389-395
Authors: 
P. V. G. K. Sarma [1]*, O. Hari Prasad, U. V. Prasad, M. Manohar Reddy, M. Kumar Swamy Reddy [2] and G. Subramanyam [3].
Address: 
[1] Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences; Tirupati, India
Address: 
[2] Department of Pathology, Sri Venkateswara Institute of Medical Sciences; Tirupati, India
Address: 
[3] Department of Cardiology, Director, Sri Venkateswara Institute of Medical Sciences, Tirupati, India
Email-ID: 
pvgksarma@gmail.com

Abstract : Intestinal alkaline phosphatase (IAP) from  normal duodenum was concentrated by 0-35% ammonium sulphate and was fractionated in DEAE cellulose column and thus, partially purified IAP was purified by passing through Sephadex G-75 column in all the steps the purification was monitored through enzyme activity. The purity of the IAP was established on C-18 RPHPLC which gave single peak at a retention time of 15 minutes and SDS-PAGE analysis showed single band with molecular weight of 66 KD. Further, PAS staining confirmed the glycoprotein nature of IAP. It is very well known that IAP dephosphorylates lipid- A moiety of lipopolysaccharide layer present in the gram-negative bacteria and protects from gram negative sepsis. However, in the present study it was observed that colonisation of H. pylori in the duodenum showed decrease in the intestinal alkaline phosphatase activity and increased Km (IAP from normal tissue 0.98 ?M of PNP/ml/min and Km =0.4?M. IAP from H. pylori infected tissue and Km = 0.48 ?M of PNP/ ml/min and Km 1 ?M). The reverse-transcript PCR results indicated that, the expression of IAP was normal in both H. pylori infected tissue and normal intestinal tissue. Therefore, it can be concluded that colonisation of H. pylori in the intestine resulted in the lowering of IAP activity

Keywords: 
H. pylori, Intestinal alkaline phosphatase, Sepsis, Colonisation.
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