[3H]ouabain autoradiography of frog retina

The kinetics and distribution of ouabain binding in retinas of Rana pipiens were examined quantitatively by scintillation counting and freeze-dry autoradiography. The time-course of binding at several concentrations was consistent with a bimolecular reaction. Estimated equilibrium binding levels gave a Michaelis-Menton relationship with a Km = 8.3 x 10(-8) M and a maximum binding level (Bmax) = 4.4 x 10(-8) mol/g protein. The distribution of binding sites measured autoradiographically varied considerably between layers. The photoreceptor, inner plexiform, and optic nerve fiber layers exhibited the heaviest binding. Within the photoreceptor layer, binding was nonuniform. Binding in the outer segment decreased distally, averaging approximately 4% of that in the proximal receptor layers (Bmax = 4.6 x 10(-6) M). The origin of the outer segment activity is uncertain at light microscope resolution, as it may be a result of inner segment calyceal processes. Binding within the proximal receptor layers was also nonuniform. Several peaks were observed, with those at the inner segment and synaptic layers being especially noticeable. Assuming an absence of glial cell binding in the proximal receptor layers, we calculated there to be 13 x 10(6) ouabain or Na+,K+ pump sites per rod receptor. Limited measurements suggest a Bmax of approximately 8 x 10(- 6) M for the inner plexiform layer.