Purification and Characterization of Cyclic GMP-stimulated Cyclic Nucleotide Phosphodiesterase from Calf Liver

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase purified >13,000-fold to apparent homogeneity from calf liver exhibited a single protein band (M. 102,000) on polyacrylamide gel electrophoresis under denaturing conditions. Enzyme activity comigrated with the single protein peak on anaIyticaI polyacrylamide gel electrophoresis, sucrose density gradient centrifugation, and gel filtration. From the sedimentation coefficient of 6.9 S and Stokes radius of 67 A, an M, of 201,000 and frictional ratio (f/fo) of 1.7 were calculated, suggesting that the native enzyme is a nonspherical dimer of similar, if not identical, peptides. The effectiveness of Mg2+, Mn2+, and Coz+ in supporting catalytic activity depended on the concentration of cGMP and cAMP present as substrate or effector. Over a wide range of substrate concentrations, optimal concentrations for M 8 + , MnZ+, and Co2+ were about 10, 1, and 0.2 mM, respectively. At concentrations higher than optimal, Mg2+ inhibited activity somewhat; inhibition by Co2+ (and in some instances by Mn2+) was virtually complete. At low substrate concentrations, activity with optimal Mn2+ was equal to or greater than that with Co2+ and always greater than that with M8+. With 20.5 PM cGMP or 20 to 300 PM cAMP and for CAMP-stimulated cGMP or cGMP-stimulated cAMP hydrolysis, activity with M8+ > Mn2+ > Co2+. In the presence of Mg2+, the purified enzyme hydrolyzed cGMP and cAMP with kinetics suggestive of positive cooperativity. Apparent K , values were 15 and 33 PM, and maximal velocities were 200 and 170 pmol/min/mg of protein, respectively. Substitution of Mn2+ for Mg2+ increased apparent K,,, and reduced Vma, for cGMP with little effect on K , or V,,, for CAMP. Co2+ increased K,,, and reduced V,,, for both. cGMP stimulated cAMP hydrolysis -32-fold in the presence of Mg2+, much less with Mn2’ or Co”. In the presence of Mg2+, Mn2+ and Co2+ at concentrations that increased activity when present singly inhibited cGMPstimulated cAMP hydrolysis. It appears that divalent cations as well as cyclic nucleotides affect cooperative interactions of this enzyme. Whereas Co2* effects were observed in the presence of either cyclic nucleotide, Mn2+ effects were especially prominent when cGMP was present (either as substrate or effector).