Kinetic studies suggest that light-activated cyclic GMP phosphodiesterase is a complex with G-protein subunits.
Cyclic GMP phosphodiesterase (PDE) in rod disk membranes has three subunits of molecular weight 88 000 (alpha), 84 000 (beta), and 13 000 (gamma). Physiological activation of the enzyme by light is mediated by a GTP binding protein (G protein). The enzyme can also be activated by controlled digestion with trypsin, which destroys the gamma subunit, leaving the activated enzyme as PDE alpha beta [Hurley, J. B., & Stryer, L. (1982) J. Biol. Chem. 257, 11094-11099]. Addition of purified gamma subunit to PDE alpha beta inhibited the enzyme fully. This suggested the possibility that G protein could also activate PDE by removing the gamma subunit and leaving the active enzyme in the form of PDE alpha beta. Should this be true, the properties of light- and trypsin-activated enzymes should be comparable. We found this not to be the case. The Km of light-activated enzyme for cyclic GMP was about 0.9-1.4 mM while that of trypsin-activated enzyme was about 140 microM. The cyclic AMP Km was also different for the two enzymes: 6.7 mM for light-activated enzyme and 2.0 mM for trypsin-activated enzyme. The inhibition of both enzymes by the addition of purified gamma subunit also differed significantly. Trypsin-activated enzyme was fully inhibited by the addition of about 200 nM gamma, but light-activated enzyme could not be fully inhibited even with 2600 nM inhibitor subunit. The Ki of the trypsin-activated enzyme for gamma was 15 nM and of the light-activated enzyme 440 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003624:Darkness; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D008970:Molecular Weight; D010786:Photoreceptor Cells / Q000201:enzymology*; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D046911:Macromolecular Substances; D012243:Rhodopsin; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D019204:GTP-Binding Proteins