Increased cyclic nucleotide phosphodiesterase activity in a mutant S49 lymphoma cell. Characterization and comparison with wild type enzyme activity.

We have characterized the markedly elevated cyclic AMP and cyclic GMP phosphodiesterase activities of a recently isolated S49 mouse lymphoma mutant, termed K30a, and compared both activities to enzyme activities in the parental wild type S49 cell. cAMP phosphodiesterase activity in K30a appears to be slightly larger than the major wild type CAMP-hydrolyzing enzyme, in sucrose gradient sedimentation and gel filtration. Both cAMP phosphodiesterase activities elute from DEAEcellulose columns at a 0.4 M salt concentration. cAMP phosphodiesterase in K30a, as compared to wild type cells, exhibits a component of activity with higher affinity for substrate (K,,, = 0.15 f 0.02 versus 0.53 2 0.05 p ~ ) , much greater sensitivity to competitive inhibition by cGMP (Ki = 0.04 p~ versus greater than 100 p~ in wild type) and cyclic IMP, and greatly reduced sensitivity to inhibition by the synthetic inhibitor, RO 20-1724. By the same criteria, cGMP-hydrolyzing activities in the two cell types appeared similar or identical, although K30a cells contain much more high affinity cGMP-hydrolyzing activity than do wild type cells. Both cGMP-hydrolyzing enzymes exhibited a high affinity component with a K, of 0.04 p~ for cGMP as substrate. The CAMPand cGMP-hydrolyzing activities of K30a probably are catalyzed by the same enzyme, because the two activities co-migrate exactly on DEAEcellulose, sucrose gradients, and gel filtration, and because hydrolysis with either substrate is inhibited by low concentrations of the other substrate. From these results, we propose that the K30a mutation caused increased expression of a cyclic nucleotide phosphodiesterase different from the predominant CAMP-hydrolyzing activity of wild type S49 cells. Wild type S49 cells express, in addition to the predominant CAMP-hydrolyzing activity, a phosphodiesterase similar to or identical with the enzyme whose expression is increased in K30a.