ROD OUTER SEGMENT STRUCTURE INFLUENCES THE APPARENT KINETIC-PARAMETERS OF CYCLIC-GMP PHOSPHODIESTERASE

Cyclic GMP hydrolysis by the phosphodiesterase (PDE) of retinal rod outer segments (ROS) is a key amplification step in phototransduction. Definitive estimates of the turnover number, k(cat), and of the K-m are crucial to quantifying the amplification contributed by the PDE. Published estimates for these kinetic param eters vary widely; moreover, light-dependent changes in the K-m of PDE have been reported. The experiments and analyses reported here account for most observed variations in apparent K-m, and they lead to definitive estimates of the intrinsic kinetic parameters in amphibian rods. We first obtained a new and highly accurate estimate of the ratio of holo-PDE to rhodopsin in the amphibian ROS, 1:270. We then estimated the apparent kinetic parameters of light-activated PDE of suspensions of disrupted frog ROS whose structural integrity was systematically varied. Tn the most severely disrupted ROS preparation, we found K-m = 95 mu M and k(cat) = 4,400 cGMP.s(-1). In suspensions of disc-stack fragments of greater integrity, the apparent K-m increased to similar to 600 mu M, though k(cat) remained unchanged. In contrast, the K-m for cAMP was not shifted in the disc stack preparations. A theoretical analysis shows that the elevated apparent K-m of suspensions of disc stacks can be explained as a consequence of diffusion with hydrolysis in the disc stack, which causes active PDEs nearer the center of the stack to be exposed to a lower concentration of cyclic GMP than PDEs at the disc stack rim. The analysis predicts our observation that the apparent K-m for cGMP is elevated with no accompanying decrease in k(cat). The analysis also predicts the lack of a K-m shift for cAMP and the previously reported light dependence of the apparent K-m for cGMP. We conclude that the intrinsic kinetic parameters of the PDE do not vary with light or structural integrity, and are those of the most severely disrupted disc stacks.