The kinetics of ouabain binding to Na-K pump and the distribution of pump sites were examined in the retina of Pseudemys scripta elegans. Binding to retinal slices followed bimolecular kinetics characterized by a KD of 1.5 × 10-6m and a maximum binding capacity of 11.2 × 10-8 mol g-1 of protein. Quantitative autoradiography of slices revealed a high concentration of bound ouabain in the inner segment, outer plexiform, inner plexiform and optic nerve layers, and correspondingly, a low level of binding in layers containing cell bodies. In the few instances that outer segments remained attached to cones, little or no binding to outer segments was observed. The membrane density of inner segment binding sites was measured by combining autoradiographic measurements of pump site concentration with stereological measurements of membrane concentration. The densities were 6.3 and 3.7 × 103 sites μm-2 of cone-ellipsoid and cone-fin cell membrane, respectively. The density of Müller cell microvilli was measured similarly but in enzymatically isolated cells and found to be 600 sites μm-2 of membrane. Measurements of cone-ellipsoid pump site density in enzymatically isolated cones were not different from measurements in slices. Calculation of Na-K pump site turnover number for the cone inner segment from pump site densities and published dark current measurements yielded a value of 30 Hz.
ASJC Scopus subject areas
- Cell Biology