Conditions for assay of ATPase from biological materials using a bioluminescence technique

Abstract

Conventional methods for determining the ATPase activity of different tissues are generally based on the measurement of inorganic phosphate derived from enzymatitally hydrolyzed ATP used as the substrate in the reaction mixture. The high phosphate background of most tissues disturbs this determination. The assay of phosphate, normally as the phosphomolybdate complex, is interfered with by e.g. formation of colourless complexes of molybdate [ 121. Furthermore, enzymatic hydrolysis of ATP, catalyzed by other phosphate ester hydrolases than ATPase in the tissue during the incubation, must also be taken into account. Using bioluminescence techniques it is possible to determine the ATP level of different biological materials. Naturally, this type of assay may be used also in the determination of ATP-forming or ATP-consuming enzyme activities directly from tissue homogenates. The experimental conditions for the assay of ATPase activities from biological materials have, however, not been determined so far. Therefore, the optimal conditions for the bioluminescence assay of membrane ATPase and extracts from mineralized and unmineralized connective tissue were analyzed.