ION-EXCHANGE COLUMN CHROMATOGRAPHIC METHOD FOR ASSAYING PURINE METABOLIC PATHWAY ENZYMES

High energy phosphate levels fall rapidly during cardiac ischemia and recover slowly (more than one week) during reperfusion. The slow recov ery of ATP may reflect a lack of purine metabolic precursors and/or in creased activity of purine catabolic enzymes such as 5'-nucleotidase ( 5'-NT, EC 3.1,3.5) and adenosine deaminase (ADA, EC 3.5.4.4). The acti vity of enzymes involved in both the catabolism of ATP precursors (5-N T and ADA) and the restoration of ATP from slow synthetic pathways [ad enosine kinase (AR, EC 2.7.1.20), adenine phosphoribusyl transferase ( APRT, EC 2.4.2.7) and hypoxanthine phosphoribosyl transferase (HPRT, E C 2.4.2.8)] may directly affect the rate of ATP recovery. Strategies t o enhance recovery will depend on the relative activity of these enzym es following ischemia. Their activity in different species and their r esponse to ischemia. are not well characterized. Hence, rapid assay me thods for-these enzymes would facilitate detailed time course studies of their activities in postischemic myocardium. WE modified a single i on-exchange column chromatographic method using DEAE-Sephadex to deter mine the products of incubation of 5'-NT, AK, APRT and HPRT with their respective substrates. The uniformity of the final product measuremen t procedure for all assays permits the activities of the foul enzymes to be rapidly determined io a single tissue sample and facilitates the study of a large number of samples. This technique should also be use ful fur enzymes of the pyrimidine metabolic pathway. (C) 1998 Elsevier Science B.V.