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Sensitive Radiochemical Assay for Inosine 5'-Monophosphate Dehydrogenase and Determination of Activity in Murine Tumor and Tissue Extracts¹

Department of Medical Oncology, City of Hope National Medical Center, Duarte, California 91010

Crude tissue or tumor extracts either do not contain sufficient inosine 5'-monophosphate dehydrogenase (IMPD) activity to be measured spectrophotometrically, or interfering enzyme activities prevent the use of a more sensitive radiochemical assay. A modified assay system which incorporates ,ß-methylene adenosine 5'-diphosphate, an inhibitor of 5'-nucleotidase; allopurinol, an inhibitor of xanthine oxidase; and ethylenediaminetetraacetate, an inhibitor of alkaline phosphatase, has been developed. [¹⁴C]Xanthine monophosphate produced during the assay was separated from [¹⁴C]hypoxanthine monophosphate by thin-layer chromatography on flexible diethylaminoethyl-cellulose sheets. Xanthine monophosphate formation was linear for at least 40 min and was inhibited by greater than 95% in the presence of mycophenolic acid, a specific IMPD inhibitor. Partial purified IMPD from murine EMT6 tumors was used to compare assay rates obtained with the radiochemical and spectrophotometric assays under identical conditions. The reaction rate of the radiochemical assay was 0.92 ± 0.07 (S.E.) of the rate of xanthine monophosphate formation as determined spectrophotometrically at 290 nm, indicating that both assays are measuring product formation with an equal degree of accuracy. The improved radiochemical assay was used to determine IMPD specific activity in supernatants from EMT6 tumors and several normal mouse tissues. The observed activities (nmol/min/mg protein) were: EMT6 tumor, 0.303; spleen, 0.029; brain, 0.022; kidney, 0.015; lung, 0.009; liver, 0.008; and heart and skeletal muscle, <0.004.

¹ This work was supported in part by the Biomedical Research Grant Program at the City of Hope National Medical Center.

² Present address: Vestar Research, Inc., 939 East Walnut Street, Pasadena, Calif. 91106. To whom requests for reprints should be addressed.

³ Present address: Department of Physiology, University of California at Davis, Davis, Calif. 95616.

Received 2/25/82. Accepted 1/ 6/83.

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