ADENYL CYCLASE ASSAY IN FAT CELL GHOSTS

An assay of adenyl cyclase in the fat cell ghost preparation is described that permits measurement of enzyme in the presence of 10,000-fold excess of ATPase activity. The assay system contains ATP-α-32P as the substrate, phosphoenol pyruvate and pyruvate kinase to regenerate the ATP hydrolyzed, and cold adenosine-3′,5′-cyclic monophosphate (cAMP) to trap the radioactive cAMP formed. cAMP is separated by either two-dimensional cellulose thin-layer chromatography or by anion-exchange thin-layer chromatography (using polyethyleneimine-impregnated cellulose) and measured by liquid scintillation counting. The cyclase had an apparent Km of 0.1-0.2 × 10-3 M in the presence and absence of 10 mM NaF; Vm was increased 6- to 10-fold by NaF. General aspects of radioactive cyclase assays relating to radioactive impurities of the substrate ATP and the formation of radioactive side products are discussed. © 1969.