Assay of ATPase and Na,K-ATPase activity using high-performance liquid chromatographic determination of ADP derived from ATP

An HPLC assay for determination of ATPase activity was developed and valida ted. After stopping the enzyme reaction of the enzyme source (rat renal cor tical basolateral membranes) with ATP, products derived from ATP were analy zed by two methods; HPLC determination of ADP derived from ATP, and colorim etry of inorganic phosphorus (P-i) released from ATP. This HPLC procedure w as precise and linear over the range of protein amount of the enzyme source studied, and the intra- and inter-assay variations were lower than 10%. Th e values that were obtained by the two methods revealed a significant corre lation. Also, even when the samples contained P-i or were contaminated with P-i, this HPLC method allowed determination of ATPase activity. In additio n, when ouabain was used as an inhibitor, the HPLC method was found to be a pplicable for Na,K-ATPase determination; This indicated that this HPLC assa y would enable determination of ATPases other than Na,K-ATPase, when other inhibitors are employed instead of ouabain. (C) 2000 Elsevier Science B.V. All rights reserved.