Simple and specific radiochemical asays for adenylate kinase and AMP deaminase activities in crude tissue extracts are described. The radioactive substrate (AMP) is separated from the radioactive product (ADP or IMP) by chromatography on polyethyleneimine-cellulose thin layers. A rapid modification of the adenylate kinase assay is described in which samples of the reaction mixture are transferred directly to the polyethyleneimine-cellulose thin layer.
Radiochemical assays frequently possess advantages of sensitivity and specificity over other techniques of enzyme assay (1,2). Their application is, however, often limited by the relative complexity of the procedures which separate substrate from product. This paper describes relatively simple and rapid radiochemical assays for the enzymes adenylate kinase (EC 2.7.4.3) and AMP deaminase (EC 3.5.4.6) which are suitable for use on crude tissue extracts.
Adenylate kinase has been assayed in the direction of ATP formation by acontinuous spectrophotometric assay using hexokinase and glucose-6phosphate dehydrogenase (3). In the opposite direction, continuous assays employing pyruvate kinase and lactate dehydrogenase have been used (4). However, high activities of ATPases can interfere in such assays since this enzyme can remove the ATP formed or contribute to the ADP produced, The problem of ATPase interference has been reported with liver extracts (5). Sampling assays have been used in both directions and depend either on the enzymic measurement of the concentration of AMP produced (6,7) or on the spectrophotometric measurement of the concentration of adenine nucleotides after their separation (7-9). Two radiochemical assays have been described for adenylate kinase. Markland and Wadkins (10) separated the nucleotides by electrophoresis and eluted them before measuring their radioactivity.
Abdulla and McFarlane (11) employed thin-layer chromatography on cellulose for nucleotide separation. The radiochemical assay described below utilizes the conversion of