Calcium dependence of the activation and inactivation kinetics of the light-activated phosphodiesterase of retinal rods.
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.
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
D000221:Adaptation, Ocular; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002024:Bufo marinus; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010786:Photoreceptor Cells / Q000187:drug effects / Q000201:enzymology*; D011494:Protein Kinases / Q000378:metabolism
D005136:Eye Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D010727:Phosphoric Diester Hydrolases; D019204:GTP-Binding Proteins; D002118:Calcium