Activation of cGMP phosphodiesterase in retinal rods: mechanism of interaction with the GTP-binding protein (transducin)

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Abstract
The mechanism of activation of the cGMP phosphodiesterase by the GTP-binding protein in the disc membrane of retinal rods has been investigated by measuring the light-induced phosphodiesterase activity in reconstituted systems where the concentration of either the GTP-binding protein or the phosphodiesterase is varied. The results are consistent with the existence of two activator sites per phosphodiesterase functional unit: binding of one G.alpha.GTP (.alpha. subunit of the G-protein with GTP bound) with high affinity (100 .+-. 50 nM) partially activates the enzyme (Vmax1 .apprx. 0.05Vmax to 0.10Vmax of trypsin-activated phosphodiesterase); binding of a second G.alpha.GTP with lower affinity (600 .+-. 100 nM) induces maximal activation (Vmax2 .apprx. Vmax of trypsin-activated phosphodiesterase). The two different states of activated phosphodiesterase have the same Km for cGMP and the same pH dependence; they differ in their sensitivity to GMP. Micromolar concentration of protamines increases the affinity of the two activator sites and slightly increases Vmax1. When G-protein is activated with GTP.gamma.S instead of GTP, the affinities of the two activator sites are not significantly modified, while Vmax1 appears to be increased.

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