Activation by PKC of the Ca(2+)-sensitive guanylyl cyclase in bovine retinal rod outer segments measured with an optical assay.

Abstract

cGMP and Ca2+ are intracellular messengers in vertebrate rod photoreceptors. cGMP is the excitatory messenger, while intracellular free Ca2+ has been implied to be (one of) the messenger(s) in the process of light adaptation in vertebrate rod photoreceptors. The enzyme guanylyl cyclase (GC, EC 4.6.1.2.) catalyzes the reaction GTP-->cGMP + PPi. Bovine retinal rod outer segments (ROS) contain a particulate GC which is inhibited by an increase in free Ca2+ in the submicromolar range, although the precise molecular mechanism underlying this inhibition is unclear. We have developed an optical enzyme-coupled assay to study regulation of the particulate GC endogenous to bovine ROS. The particulate GC exhibited a Ca(2+)-inhibited (IC50 83-144 nM) activity of 13-23 nmol of PPi/(min-(mg of rhodopsin)). ATP increased the maximal velocity of GC by about 2-fold, and this increase was inhibited by the specific PKC inhibitors chelerythrine and the pseudosubstrate-based peptide inhibitor PKC R10-31N. When the factor that mediated the ATP-dependent increase in GC rate was removed by washing, the ATP-dependent increase in GC rate could be reestablished by addition of purified, constitutively active PKC.