[3H]ouabain autoradiography of frog retina.

Abstract

The kinetics and distribution of ouabain binding in retinas of R. pipiens were examined quantitatively by scintillation counting and freeze-dry autoradiography. The time-course of binding at several concentrations was consistent with a biomolecular reaction. Estimated equilibration binding levels had a Km = 8.3 .times. 10-8 M and a maximum binding level (Bmax) = 4.4 .times. 10-8 mol/g protein. The distribution of binding sites measured autoradiographically varied considerably between layers. The photoreceptor, inner plexiform and optic fiber layers exhibited the heaviest binding. Within the photoreceptor layer, binding was nonuniform. Binding in the outer segment decreased distally, averaging .apprx. 4% of that in the proximal receptor layers (Bmax = 4.6 .times. 10-6 M). The origin of the outer segment activity is uncertain at light microscope resolution; it may result from inner segment calyceal processes. Binding within the proximal receptor layers was nonuniform. Several peaks were observed. At the inner segment and synaptic layers, peaks were especially noticeable. With an absence of glial cell binding in the proximal receptor layers, 13 .times. 106 ouabain or Na+ K+ pump sites were calculated to exist per rod receptor. Limited measurements suggest a Bmax of .apprx. 8 .times. 10-6 M for the inner plexiform layer.