Cytidylate cyclase activity in mouse tissues: the enzymatic conversion of cytidine 5′-triphosphate to cytidine 3′,5′-cyclic monophosphate (cyclic CMP)

Cytidylate cyclase activity, which enzymatically converts cytidine 5′-triphosphate (CTP) to cytidine 3′,5′-cyclic monophosphate (cyclic CMP), has been demonstrated in mouse tissue homogenates by use of a highly sensitive enzyme immunoassay (EIA) specific for cyclic CMP. Cyclic CMP formation is dependent on the amount of homogenate and on the incubation time. Although the enzyme activity was detected at wide ranges of pH from 6.8 to 11.5, the maximal activity was observed at around pH 9.4. The optimal temperature was 37°C. Cytidylate cyclase activity was almost completely lost if the homogenates were heated at 90°C for 3 min prior to use. The enzyme reaction exhibited typical Michaelis-Menten kinetics with an apparent K_m for CTP of approx. 0.31 mM. Cyclic CMP formation was greatly enhanced with 4 mM Mn²⁺, Mg²⁺, Co²⁺; Mn²⁺ was the most effective. Fe²⁺ and Ca²⁺ were without effect. Cu²⁺ and Zn²⁺ at a concentration of 0.1 to 0.5 mM were inhibitory to Mn²⁺-dependent activity. Moreover, the enzyme activity was inhibited by several nucleotides including ATP, ADP, 5′-AMP, and GTP. Cytidylate cyclase activity was found to be present in all homogenates from a variety of mouse tissues examined except heart, with the highest level found in brain, and the lowest in liver.