Date of Award

Spring 2002

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Richard H Cote

Abstract

Rod photoreceptor phosphodiesterase (PDE) is the central enzyme of visual transduction. PDE is a heterodimer (Palphabeta) with two associated inhibitory gamma subunits, and is bound to the disk membrane by isoprenyl groups. Each catalytic subunit contains a catalytic site and a high-affinity cGMP binding site. In this research, two independent mechanisms of PDE regulation are addressed. The first part examines transducin activation of PDE, focusing on the interactions between the gamma subunits and Palphabeta. The second part examines the 17 kDa delta protein believed to regulate bovine PDE. We found that transducin activation of PDE induces heterogeneity at the active sites as well as the cGMP binding sites, and that there is a strong correlation between cGMP binding and gamma binding to PDE. The PDE activation induced by transducin is approximately half of the maximum rate of Palphabeta lacking bound gamma inhibitory subunits. This research supports a model in which transducin interacts with gamma bound to only one of the catalytic subunits. The relationship between cGMP and gamma binding to PDE may play a role in light adaptation of rod photoreceptor cells.

The delta protein is hypothesized to regulate adaptation of rod photoreceptor cells by delta binding to the prenyl groups on PDE and releasing PDE from the disk membrane and thereby preventing activation by transducin. The frog homologue of the bovine 17 kDa delta protein was identified in frog retina, but the amount of the delta in rod cells was low (0.03 +/- 0.01 mol delta per mol PDE). This is consistent with frog PDE being >95% membrane-associated. Recombinant frog delta bound to PDE and was able to release PDE from the membrane. The majority of the delta in frog ROS is soluble, and the remainder does not co-purify with PDE when extracted from the membranes. Immunocytochemical evidence indicates that delta localizes to the cone cells. We hypothesize that delta may serve to direct the intracellular membrane trafficking for various prenylated proteins.

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