Rod Outer Segment cGMP Phosphodiesterase Research Articles

Carriers of the mouse rd gene have reduced levels of the beta subunit of the retinal cyclic GMP phosphodiesterase

Polyclonal antipeptide antisera have been utilized to quantitate the amount of retinal rod outer segment cGMP phosphodiesterase α and β catalytic subunits present in retinas from C57BL/6J mice which are normal or carriers for the rd gene defect. Results suggest that the quantity of PDE-β subunit is reduced in carrier mice while PDE-α and PDE-γ are not affected. In 21-day-old mice, the PDE-β was reduced by about one-half while adult carrier mice had even more reduced levels of PDE-β. Since PDEα was not reduced, this suggests that synthesis of PDEα and PDEβ may not be coordinately controlled.

Transient response of rod outer segment cGMP phosphodiesterase to actinic light pulses: II. Detailed quantitative kinetic model

Abstract In the accompanying article (Schmidt, J.A., and Yguerabide, J. (1989) J. Biol. Chem. 264, 19790-19803), we presented a minimal quantitative kinetic model with one rate-limiting step for the transient response of rod outer segment (ROS) phosphodiesterase (PDE) to stimulating light pulses of low fractional bleach (linear response range) and showed that the model was in excellent quantitative agreement with experimental results. The model characterizes the PDE response in terms of the specific rate constant of the rate-limiting step, kL, the lifetime of photoactivated rhodopsin, tau R, and the lifetime of activated PDE, tau P, but makes no predictions on how these kinetic parameters should depend on the concentrations of the various reactive species involved in the PDE response to light and does not reveal the nature of the rate-limiting step. However, we established by curve fitting experimental data to theoretical expressions from the model that kL increases hyperbolically with [GTP], tau R decreases with [GTP], and tau P is independent of GTP. In this report we present three detailed kinetic models which make specific quantitative predictions on how the kinetic parameters of the minimal model should depend on nucleotide and G protein concentrations and test the models against experimental data. Each model consists of one rate-limiting step. The first detailed model postulates that the rate-limiting step is the dissociation of R*GT into R* and GT (T stands for GTP). The second model postulates that the rate-limiting step is the binding of GTP to R*G, and the third model postulates that the rate-limiting step is the encounter rate of R* and G on the ROS disc membrane. We find that only the first detailed model is consistent with the experimental results as characterized by the minimal model. Using this detailed model we (a) define kL and tau R in terms of more fundamental equilibrium and rate parameters, (b) develop a theory for the systematic evaluation of amplification or gain of the PDE light response from light-stimulated GTP-binding data as well as v(t) versus t graphs, and (c) clarify methods which have been used in the past to evaluate gain experimentally.

A Monoclonal Antibody against the Rod Outer Segment Guanyl Nucleotide-binding Protein, Transducin, Blocks the Stimulatory and Inhibitory G Proteins of Adenylate Cyclase

GTP-binding proteins have been implicated as transducers of a variety of biological signaling processes. These proteins share considerable structural as well as functional homology. Due to these similarities, it was thought that a monoclonal antibody that inhibits the light activation of the rod outer segment GTP-binding protein, tranducin (Gt), might exert some functional effect upon the G proteins that regulate the adenylate cyclase system. Antibody 4A, raised against the alpha subunit of Gt, cross-reacted (by hybridization on nitrocellulose) with purified alpha subunits of other G proteins (Gi and Gs, regulatory guanyl nucleotide-binding proteins that mediate inhibition and stimulation of adenylate cyclase, respectively) as long as they were not denatured. This antibody, which interferes with rod outer segment cGMP phosphodiesterase activation by blocking interaction between rhodopsin and Gt, also interfered with actions of both the stimulatory and inhibitory G proteins of adenylate cyclase from rat cerebral cortex membranes. Effects of monoclonal antibody (mAb) 4A were dose-dependent and not reversed by washing. mAb 4A also blocked the Gi-mediated inhibition of adenylate cyclase in the cyc- variant of S49 lymphoma and in doing so raised the level of adenylate cyclase activity in both the cyc- variant and the S49 wild type. There was no effect of mAb 4A on adenylate cyclase activity of the resolved catalytic subunit. These results suggest that the well known sequence homologies among the G proteins involved in cellular signal transduction may extend to the sites that interact with other members of signal-transducing cascades (receptors and effector molecules). Therefore, antibody 4A may serve as a useful tool to probe the similarities and differences among the various systems.

Effect of fluoride on retinal rod outer segment cGMP phosphodiesterase and G-protein

The effects of fluoride on ROS phosphodiesterase and G-protein have been studied using membrane-free extracts. When G-protein was present NaF, at millimolar concentrations, stimulated PDE activity however, in a G-protein free extract, cGMP hydrolysis was inhibited by high fluoride concentrations. Fluoride was also found to profoundly inhibit the ability of G-protein to bind a GTP analogue, GTPγS, both in the presence and absence of rhodopsin. Aluminium greatly modified these effects of fluoride on PDE and G-protein. The possibility that fluoride activates PDE through its effect on G-protein is discussed.

Methylation of proteins in photoreceptor rod outer segments.

A set of proteins in bovine rod outer segments is specifically methylated by S-adenosyl-L-methionine. The reaction can be demonstrated in the intact retina as well as in fragmented preparations of isolated rod outer segments. The apparent molecular weights of these proteins are 88,000, 61,000, and a subset between 21,000 and 26,000. The Mr = 88,000 protein is shown to be the alpha subunit of the rod outer segment cGMP phosphodiesterase by peptide mapping, two-dimensional gel electrophoresis, the ionic strength dependence of its interaction with the membrane, and immunoprecipitation by antiserum raised against purified phosphodiesterase. For each of these proteins, the incorporated methyl groups are hydrolyzed in alkali to yield methanol, indicating that the proteins are carboxymethylated.

Isolation and recombination of bovine rod outer segment cGMP phosphodiesterase and its regulators

cGMP phosphodiesterase extracted from rod outer segments can be activated by GTP in the presence of phospholipid vesicles containing bleached rhodopsin. I have separated the phosphodiesterase from a phosphodiesterase inhibitory protein and a GTPase also present in the crude extracts from rods. The GTPase can be activated by bleached rhodopsin. However, in the absence of the GTPase and inhibitor, the phosphodiesterase was not activated by GTP in the presence of bleached rhodopsin. Recombination with these proteins partially restored the activation by GTP and bleached rhodopsin.