Activation by PKC of the Ca(2+)-sensitive guanylyl cyclase in bovine retinal rod outer segments measured with an optical assay.
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.
Wolbring
G
G
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Schnetkamp
P P
PP
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010770:Phosphotransferases; D011493:Protein Kinase C / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D013053:Spectrophotometry / Q000379:methods
D000255:Adenosine Triphosphate; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase; D011493:Protein Kinase C; D006162:Guanylate Cyclase; D002118:Calcium