Phosphodiesterase efficiency: reconciling biochemical and electrophysiological measurements of the phototransduction cascade in the rod cells of the bullfrog Rana catesbeiana

The signal generated when a rod cell captures a photon is amplified via the phototransduction cascade. Specifically, two steps of the cascade are responsible for the observed amplification: the activation of numerous transducin G-proteins by one rhodopsin photoreceptor, and the hydrolysis of numerous cyclic GMP molecules by one molecule of phosphodiesterase (PDE). Direct biochemical measurements of the rate of the first step, combined with the literature efficiency value (k_cat/K_m) of the second step, fail to account for the amplification observed in electrophysiological recordings. In this work, the long-standing contradiction between electrophysiological and biochemical measurements of the amplification of rod phototransduction is resolved. Previous indirect estimates of the rate of PDE activation by the cascade are first confirmed by direct measurements to be ~120/s. The turnover number (k_cat) and the Michaelis constant (K_m) of rod PDE are then measured within disrupted rod outer segments while varying the level of PDE activation. While the turnover number remained rather constant throughout, the apparent Km of PDE declined with decreasing levels of PDE activation, finally stabilizing at ~10 µM when only 1-2% of PDE were activated. This last, stable value, 10 µM, was concluded to be the true Michaelis constant of rod PDE. At least 10-fold lower than published estimates, this true K_m value finally explains on a molecular level the efficiency of amplification seen in electrophysiological recordings