Abstract
[2- 3H]Inosinic acid ([2- 3H]IMP) has been biosynthesized in good yield from [2- 3H]hypoxanthine and PRPP via the action of a partially purified preparation of hypoxanthine/guanine phosphoribosyl transferase from mouse brain. The product was purified in one step by ascending paper chromatography, and used to assess the activity of IMP dehydrogenase. To conduct the assay, tritiated substrate is admixed with enzyme in a final volume of 10 μl; NAD is present to serve as cofactor for the reaction, and allopurinol to inhibit the oxidation of any hypoxanthine generated as a consequence of side reactions. After an appropriate period of incubation, the 3H 2O arising from the oxidation of tritiated IMP via [ 3H]NAD is isolated by quantitative microdistillation. Performed as described, the assay is facile, sensitive, and accurate, with the capability of detecting the dehydrogenation of as little as 1 pmol of [ 3H]IMP. Using it, measurements have been made of IMP dehydrogenase in a comprehensive array of mouse organs. Of these, pancreas contained the enzyme at the highest specific activity.
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