ACTIVATION BY PKC OF THE CA2-SENSITIVE GUANYLYL CYCLASE IN BOVINE RETINAL ROD OUTER SEGMENTS MEASURED WITH AN OPTICAL ASSAY()

cGMP and Ca2+ are intracellular messengers in vertebrate rod photorece ptors. cGMP is the excitatory messenger, while intracellular free Ca2 has been implied to be (one of) the messenger(s) in the process of li ght adaptation in vertebrate rod photoreceptors. The enzyme guanylyl c yclase (GC, EC 4.6.1.2.) catalyzes the reaction GTP --> cGMP + PPi. Bo vine retinal rod outer segments (ROS) contain a particulate GC which i s inhibited by an increase in free Ca2+ in the submicromolar range, al though the precise molecular mechanism underlying this inhibition is u nclear. We have developed an optical enzyme-coupled assay to study reg ulation of the particulate GC endogenous to bovine ROS. The particulat e GC exhibited a Ca2+-inhibited (IC50 83-144 nM) activity of 13-23 nmo l of PPi/(min .(mg of rhodopsin)). ATP increased the maximal velocity of GC by about 2-fold, and this increase was inhibited bp the specific PKC inhibitors chelerythrine and the pseudosubstrate-based peptide in hibitor PKC R10-31N. When the factor that mediated the ATP-dependent i ncrease in GC rate was removed by washing, the ATP-dependent increase in GC rate could be reestablished by addition of purified, constitutiv ely active PKC.