Molecular and Cellular Biochemistry

, Volume 204, Issue 1, pp 127–134

Purification and characterization of intestinal adenosine deaminase from mice

  • Lisam Singh
  • Ramesh Sharma
Article

DOI: 10.1023/A:1007087905192

Cite this article as:
Singh, L. & Sharma, R. Mol Cell Biochem (2000) 204: 127. doi:10.1023/A:1007087905192

Abstract

Adenosine deaminase (ADA) was isolated from small intestine of mice and purified to utmost homogeneity. SDS-PAGE of purified ADA gave a molecular weight of 41 kDa. Western blot analyses gave a single reactive band at 41 kDa and the other band was an associated ADA binding protein. The purified enzyme was more stable in the alkaline pH. The optimum pH and the pI values were about 7.0 and 4.96, respectively. Km values of the small intestinal ADA for adenosine and 2′-deoxyadenosine were 23 and 16μM, respectively. Purine riboside was a competitive inhibitor with Ki of 5 μM, whereas 2′-3′-o-isopropylidene adenosine acted as an uncompetitive inhibitor (Ki 66 μM). Activity of ADA was inhibited by the presence of theophylline (-40%), caffeine (-30%), and L-cysteine (-50%). Significantly, Hg2+ (100 μM) inhibited 98% of the initial ADA activity. In addition, various purine analogs such as inosine, purine, α-adenosine and adenine showed variable inhibitions on the activity of ADA. Relative ADA activity towards 3′-deoxyadenosine and 6-chloropurine riboside was lower by 30% and 40%, respectively. However, the activity towards 2′-o-methyl adenosine was higher (30%) compared to the activity obtained using adenosine.

intestinal adenosine deaminase mice purification physicochemical and kinetic characterization 

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Lisam Singh
    • 1
  • Ramesh Sharma
    • 1
  1. 1.Department of BiochemistryNorth Eastern Hill UniversityShillongIndia

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