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. 1989 Jun 1;93(6):1091–1108. doi: 10.1085/jgp.93.6.1091

Calcium dependence of the activation and inactivation kinetics of the light-activated phosphodiesterase of retinal rods

PMCID: PMC2216249  PMID: 2549175

Abstract

The Ca2+ dependence of the kinetics and light sensitivity of light- activated phosphodiesterase was studied with a pH assay in toad and bovine rod disk membranes (RDM), and in a reconstituted system containing GTP-binding protein, phosphodiesterase and rhodopsin kinase. Three statistics, peak hydrolytic velocity, turnoff time, and time to peak velocity, were measured. ATP decreased phosphodiesterase light sensitivity nearly 10-fold and accelerated the dim-flash kinetics of cGMP hydrolysis when compared to those with GTP alone. CA2+ reversed all of the effects of ATP, Ca2+ increased peak velocity, turnoff time, and time to peak velocity, to the values obtained with GTP alone. The Ca2+ dependence of peak velocity and turnoff time can be characterized as hyperbolic saturation functions with a K0.5 for Ca2+ of 1.0-1.5 mM in toad RDM. In bovine RDM the Ca2+ dependence of peak velocity and turnoff time has a K0.5 of 0.1 mM Ca2+. The Ca2+ dependence in the reconstituted system is similar to that in bovine RDM for peak velocity (K0.5 = 0.1 mM Ca2+) but differs for turnoff time (K0.5 = 2.5 mM Ca2+). We tested the hypothesis that a soluble modulator, normally required to confer submicromolar Ca2+ sensitivity, was too dilute in our assay by comparing data obtained at one RDM concentration with those obtained at 10-fold higher RDM, and therefore a constituent protein, concentration. We observe no difference and present a formal analysis of these data that excludes the hypothesis that the soluble modulator binds its target protein with Kd less than 5 microM. The lack of submicromolar Ca2+ dependence of any of the steps in the cGMP cascade that underlie cGMP phosphodiesterase activation and inactivation in vitro argues against Ca2+ regulation of these steps having a significant role in the light adaptation of the intact rod.

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Selected References

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