Kinetics of the hydrolysis of 8-bromo-cyclic GMP by the light-activated phosphodiesterase of toad rods.
The hypothesis that cyclic GMP is the internal transmitter of retinal rod phototransduction, when combined with the observations that 8-bromo-cyclic GMP opens the cyclic GMP-dependent outer segment conductance and that rods into which 8-bromo-cyclic GMP has been injected still respond to light, predicts that the light-activated phosphodiesterase (EC 3.1.4.17) must catalyze the hydrolysis of 8-bromo-cyclic GMP. This hypothesis was tested by measuring light-activated toad rod disk membrane phosphodiesterase with a pH assay technique. Phosphodiesterase-catalyzed hydrolysis of 8-bromo-cyclic GMP was confirmed: at pH 8.0, total proton production after flash activation was identical to total amount of 8-bromo-cyclic GMP added as substrate. Photoactivated phosphodiesterase was remarkably less efficient in catalyzing the hydrolysis of 8-bromo-cyclic GMP than of cyclic GMP: Vmax for 8-bromo-cyclic GMP was 0.063 M/M rhodopsin/s, whereas that for cyclic GMP was 11 M/M rhodopsin/s--170 times greater. The Km for 8-bromo-cyclic GMP was 160 microM, and for cyclic GMP, 590 microM. 8-bromo-cyclic GMP competitively inhibited phosphodiesterase-catalyzed hydrolysis of cyclic GMP with a Ki of 1.2 mM. Complete reaction progress curves were analyzed for obedience to Michaelis-Menten kinetics: cyclic GMP hydrolysis, 8-bromo-cyclic GMP hydrolysis, and cyclic GMP hydrolysis in the presence of 8-bromo-cyclic GMP as competitive inhibitor were found to follow the integrated form of the Michaelis-Menten equation over the time course of the reactions, assuming phosphodiesterase was activated as a step. The kinetic parameters extracted from reaction progress curves were consistent with those derived from analysis of the initial velocity.(ABSTRACT TRUNCATED AT 250 WORDS)
Barkdoll
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Department of Psychology, University of Pennsylvania, Philadelphia 19104.
Pugh
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EN
Sitaramayya
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A
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002025:Bufonidae; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D008027:Light; D010786:Photoreceptor Cells / Q000201:enzymology*
C016276:8-bromocyclic GMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP