Cyclic GMP-specific, high affinity, noncatalytic binding sites on light-activated phosphodiesterase.
Two classes of high affinity, cGMP-specific binding sites have been found in association with a peripheral membrane protein in rod outer segments. [3H]cGMP and a photoaffinity label, 8-N3-[32P]cIMP, have been used to study these cGMP binding sites. The cGMP binding sites co-migrated with rod outer segment phosphodiesterase (EC 3.1.4.17) upon Bio-Gel A-0.5m column chromatography, sucrose density gradient centrifugation, and isoelectric focusing (pI 5.35). Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 8-N3-[32P]cIMP-labeled protein also migrated in a position identical with that of purified phosphodiesterase. Scatchard analysis, using purified phosphodiesterase, revealed the presence of two classes of cGMP binding sites with apparent KD values of 0.16 and 0.83 microM. A number of observations indicated that these high affinity, cGMP-specific binding sites are distinct from the phosphodiesterase catalytic site. cAMP, which is a substrate for phosphodiesterase, did not bind to the high affinity cGMP specific sites. Limited tryptic proteolysis of phosphodiesterase resulted in a striking activation of the catalytic activity and a 96% loss of cGMP binding. 1-Methyl-3-isobutylxanthine inhibited phosphodiesterase activity and enhanced the specific binding of cGMP. Mg2+ was necessary for phosphodiesterase activity, but not for high affinity cGMP binding. Finally, phosphodiesterase activity and the cGMP-specific high affinity sites showed different stabilities on storage in phosphate buffer. These specific high affinity cGMP binding sites may be involved in the regulation of phosphodiesterase activity.
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D006152:Cyclic GMP; D007289:Cyclic IMP / Q000031:analogs & derivatives; D007292:Inosine Nucleotides; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D010446:Peptide Fragments; D010786:Photoreceptor Cells / Q000201:enzymology*; D011485:Protein Binding; D011892:Rana catesbeiana; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D007292:Inosine Nucleotides; D008565:Membrane Proteins; D010446:Peptide Fragments; D007289:Cyclic IMP; C024279:8-N(3)cIMP; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D014357:Trypsin; D006152:Cyclic GMP