Immunolocalization of 48K in rod photoreceptors. Light and ATP increase OS labeling.
An abundant, light-modified protein of Mr approximately equal to 51 kD, homologous to a previously described protein (termed 48K, arrestin, or S-antigen) of bovine retina, was identified and characterized in rod photoreceptors of the toad (Bufo marinus). Isolated, intact retinas were incubated in darkness, or irradiated under physiological conditions [dark-adapted (DA) and light-adapted (LA) retinas]. Using polyclonal antibodies raised in rabbit against purified toad 48K, and post-embedding immunoelectron microscopy (second antibody conjugated with 5 nm gold particles), we examined the localization of 48K in DA vs. LA rods. Physiological incubations and early fixation steps were carried out in the presence vs. absence of ATP (40 microM), a substance thought to support the activity of 48K in vivo. The distribution of 48K in rods was analyzed by quantitating the density of gold label within subcellular compartments. In DA rods, labeling was highest in the myoid region of the inner segment. Light adaptation increased 48K immunoreactivity within the outer segment, and decreased labeling within calycal processes. Labeling in outer segments of LA rods was maximal in the basal region. Treatment with ATP both amplified the light-dependent increase in basal OS labeling, and augmented the nonuniformity of basal vs. apical labeling in LA ROS. Morphometric analysis of labeling density over the length of DA vs. LA ROS indicated an approximately equal to 1.5-fold net increase in 48K label in LA ROS. By comparison, biochemical analysis of 48K level indicated an approximately equal to 1.8-fold increase in 48K in LA preparations. Together, the biochemical and immunocytochemical data indicate that the relative amount of 48K is increased in LA ROS, not just its immunoreactivity. The data are consistent with a selective movement of 48K, a cytoplasmic ROS protein, into ROS during LA.
D000222:Adaptation, Physiological; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002024:Bufo marinus; D003623:Dark Adaptation; D005136:Eye Proteins / Q000378:metabolism*; D007106:Immune Sera / Q000276:immunology; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D008027:Light; D008854:Microscopy, Electron; D008970:Molecular Weight; D010786:Photoreceptor Cells / Q000378:metabolism* / Q000648:ultrastructure; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D012995:Solubility
D005136:Eye Proteins; D007106:Immune Sera; D000255:Adenosine Triphosphate