Sensitive Fluorimetric Assay for Adenosine Deaminase with Formycin as Substrate; and Substrate and Inhibitor Properties of some Pyrazolopyrimidine and Related Analogues

Abstract

The nucleoside antibiotic formycin, 7-amino-3-(β-ᴅ-ribofuranosyl)pyrazolo(4,3-d)pyrimidine, a structural analogue of adenosine, is deaminated about 10-fold faster by adenosine deaminase than adenosine itself, and is therefore a superior substrate for both routine assays and kinetic studies with the purified enzyme. The luminescence properties of formycin have been profited from to develop a fluorimetric assay for adenosine deaminase which is considerably more sensitive than the spectrophotometric procedure widely employed with adenosine as substrate. Examples are presented of its application to routine assays of adenosine deaminase levels in cellular extracts, as well as to kinetic studies with the purified enzyme, including the properties of some pyrazolopyrimidine and purine substrates and inhibitors.