Retinas of 9-10-day-old rd and control mice were incubated for 2 hr with [14C]leucine along with either tritiated palmitic acid or galactose to investigate the acylation or glycosylation, respectively, of rhodopsin. Although other laboratories have reported that phosphorylation of rhodopsin is not detectable in rd retinas, the two post-translational modifications of rhodopsin investigated in the present work are detectable. The rod outer segments (ROS) were separated from the retinal debris containing the rough endoplasmic reticulum (RER) of photoreceptor cells by vortexing and then by linear sucrose gradients. The rhodopsin from the RER was purified by affinity chromatography and gel electrophoresis. In the acylation studies, the mean ratio of palmitate to leucine in the rd mouse was nearly twice that of controls (11·73 ± 2·84 v. 6·81 ± 1·04). Possible explanations for the disparity between the two groups could include: (1) a diminished internal pool size of the fatty acid; or (2) acylation of amino acids such as serine or threonine which normally are not acylated in rhodopsin. Treatment of purified rhodopsin with 1 m hydroxylamine released similar amounts of palmitate from the rd mice and controls. Hence, the higher ratio of palmitate to leucine in rd mice is apparently due to a diminished internal pool size. In the glycosylation studies, the ratio of galactose to leucine was very similar between rd mice and controls, 1·7 ± 0·43 v. 2·47 ± 0·74. Protein content and specific activity were determined for the crude ROS preparations and for the remaining retinal debris. Although the amount of ROS protein differed significantly between the two groups, the specific activities did not. Determinations of the amount of protein and specific activity in retinal debris pellets were similar for the two groups. The results suggest that building of the core structure of the N-linked oligosaccharide of rhodopsin proceeds normally in the rd mouse and that acylation of rhodopsin is not defective in the rd mouse.
- rd mouse
- retinal degeneration
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience