CONDITIONS FOR FORMATION, PARTIAL PURIFICATION AND ASSAY OF AN INHIBITOR OF ADENOSINE 3',5'-MONOPHOSPHATE

Particle preparations from dog and rat heart and dog liver were used to catalyze the formation of 3′,5′-AMP from ATP in the presence of Mg2+. These incubation mixtures also formed a maternal which noncompetitively inhibited the 3′, 5′-AMP-stimulated activation of phosphorylase in extracts of liver and heart. Inhibitory activity was quantified using an assay based on this effect. Conditions which increased the accumulation of the inhibitor were very similar to those that enhanced the accumulation of 3′,5′-AMP. These included the presence of a low-speed particle fraction, ATP, Mg2+, l-epinephrine, F- and caffeine. The β-adrenergic-blocking agent [I-(3,4-dichlorophenyl)-2-isopropylaminoethanol·HCl] prevented the stimulating effect of epinephrine on accumulation of the inhibitor and 3′,5′-AMP. With heart preparations, carbachol decreased the accumulation of both compounds. With liver preparations, glucagon increased the formation of both. Added 3′,5′-AMP also increased the formation of the inhibitor, whereas N6-monobutyryl 3′,5′-AMP prevented its formation. Both the inhibitor and 3′,5′-AMP were inactivated by treatment with 3′,5′-nucleotide phosphodiesterase, but were unaffected by several phosphataes. The inhibitor was not separated from 3′,5′-AMP by a number of chromatographic procedures. Both materials were, however, separated from other compounds that interfered with the effect of 3′,5′-AMP in the assay. The inhibitor was found in acidic extracts from brain, heart, liver, lung, adipose tissue and skeletal muscle, and the levels paralleled the 3′,5′-AMP concentration. © 1969.