An enzymatic inosine 5′-monophosphate assay of increased specificity

Abstract

The enzymatic inosine 5′-monophosphate assay described by Grassl [ in, Methods of Enzymatic Analysis (H. U. Bergman, ed.), pp. 2168–2171, 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 μg/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 Terjung [ Amer. J. Physiol. 237 C111–C118 (1979)] introduced a modification of Grassl's procedure, substituting 5′-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 5′-nucleotidase (instead of AP) and coformycin, in order to achieve a more specific assay, and one more suitable for use with whole tissue extracts.