An enzymatic inosine 5′-monophosphate assay of increased specificity

✍ Scribed by Harold T. Kyriazi; R.E. Basford

• Publisher: Elsevier Science
• Year: 1985
• Tongue: English
• Weight: 422 KB
• Volume: 144
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(85)90144-7

✦ Synopsis

The enzymatic inosine S-monophosphate assay described by Grass1 [in, Methods of Enzymatic Analysis (H. U. Bergman, ed.), pp. 2 168-2 17 1, Academic Press, New York (1974)] is highly nonspecific, as ITP, ATP, ADP, AMP, and adenosine react stoichiometrically. The reactivity with the adenine derivatives is due to the tri-and diphosphatase activity of alkaline phosphatase (AP), coupled with adenosine deaminase (and possibly AMP deaminase) contamination of commercially available preparations of AP, purine-nucleoside phosphorylase, and/or xanthine oxidase. The inclusion of coformycin (0.05 pg/ml), a potent inhibitor of these deaminases, completely eliminated the cross-reactivity. ITP, however, still reacted stoichiometrically due to the tri-and diphosphatase activity of AP. Meyer and Tejung [Amer. J. Physiol. 237 Cl 1 I-CI 18 (1979)] introduced a modification of Grassi's procedure, substituting S-nucleotidase for AP. It has been found that this disallows reactivity with ATP, ADP, and ITP but that AMP and adenosine still react completely. Coformycin prevents this cross-reactivity. It is therefore recommended that the assay be carried out with S-nucleotidase (instead of AP) and coformycin, in order to achieve a more specific assay, and one more suitable for use with whole tissue CXt~CtS.