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

Cyclic GMP hydrolysis by the phosphodiesterase (PDE) of retinal rod ou ter segments (ROS) is a key amplification step in phototransduction. D efinitive estimates of the turnover number, k(cat), and of the K-m are crucial to quantifying the amplification contributed by the PDE. Publ ished estimates for these kinetic param eters vary widely; moreover, l ight-dependent changes in the K-m of PDE have been reported. The exper iments and analyses reported here account for most observed variations in apparent K-m, and they lead to definitive estimates of the intrins ic kinetic parameters in amphibian rods. We first obtained a new and h ighly accurate estimate of the ratio of holo-PDE to rhodopsin in the a mphibian ROS, 1:270. We then estimated the apparent kinetic parameters of light-activated PDE of suspensions of disrupted frog ROS whose str uctural integrity was systematically varied. Tn the most severely disr upted 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, th e apparent K-m increased to similar to 600 mu M, though k(cat) remaine d unchanged. In contrast, the K-m for cAMP was not shifted in the disc stack preparations. A theoretical analysis shows that the elevated ap parent K-m of suspensions of disc stacks can be explained as a consequ ence of diffusion with hydrolysis in the disc stack, which causes acti ve PDEs nearer the center of the stack to be exposed to a lower concen tration of cyclic GMP than PDEs at the disc stack rim. The analysis pr edicts our observation that the apparent K-m for cGMP is elevated with no accompanying decrease in k(cat). The analysis also predicts the la ck of a K-m shift for cAMP and the previously reported light dependenc e 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.