Cyclic nucleotide phosphodiesterases associated with bovine retinal outer-segment fragments

Abstract

ATP-dependent cyclic GMP phosphodiesterase activity (EC 3.1.4.16) associated with bovine retinal outer-segment fragment preparations was stimulated an order of magnitude by light, confirming the results of Miki et al. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3820-3824 at Yale for the frog system. In contrast to the results of the Yale group, however, light stimulation was not observed for cyclic AMP as substrate. A direct relationship of bovine rhodopsin bleaching to phosphodiesterase activation differs from a previous report by the Yale group that full activation of the frog enzyme was achieved by bleaching of a maximum of 2% rhodopsin. Phosphodiesterase activity could be qualitatively removed from the fresh outer-segment preparations with isotonic sucrose which apparently did not disrupt the plasmalemma or discs. Activity recovered from the washing was not light sensitive. Two Km values were determined for cyclic AMP, 5 and 0.05 mM; for cyclic GMP a Km of 0.22 mM was found. All Km values were determined in the presence of 1 mM ATP in the dark. Sonication of fresh outer segments or storing at -20 degrees C abolished the light response. However, storage at -76 degrees C fully preserved it.