Protein turnover in retina

Abstract

Rabbit retinas were exposed in vitro to 0.5-h pulses of [3H]leucine or [14C]leucine. Some retinas were harvested promptly after labeling to measure synthesis. These were combined, in double-labeling experiments, with retinas that had been returned to unlabeled medium for a subsequent 1 h or 3.75 h to measure degradation. All of the proteins were solubilized, and separated according to size by gel electrophoresis. The gels were cut into 95 slices, and each slice was differentially counted. The amount of protein in the slice was estimated from the Coomassie blue staining, and its molecular weight from the distribution of molecular weight (MW) standards. Turnover rates of the various sizes of proteins were calculated from these data using certain well-defined assumptions. Retinal protein contained about 32 X 10(3) nmol of polypeptide per g, with a median MW of 27,000. Total synthesis was at the rate of 103 nmol/g of protein/h, with the most rapid synthesis in the 33,000--43,000 MW range, at 2 nmol/g/h for every 1000 increment in MW. Protein renewal averaged 0.52%/h, but varied directly (p < 0.0001) with MW, so that proteins of 10,000 MW were being renewed at about 0.1%/h and proteins of 140,000 MW at about 1.4%/h. Taken together, the measurements of fractional renewal and the measurements of degradation of the newly synthesized proteins demonstrated that each slice contained proteins with markedly different breakdown coefficients, and provided enough information to characterize the proteins in the slice in terms of a fast and slow subgroup. This analysis indicated that: breakdown coefficients varied much more than rates of synthesis and were therefore the prime determinant of the amount of each protein that was present; as MW increased, breakdown coefficients of the long-lived proteins increased (p < 0.0001), accounting in major part for the correlation between size and turnover; most staining bands were due to proteins with peculiarly long lifespans; the proteins with the slowest turnover of all appeared to be histones; there was an unusually rapid synthesis of a 138,000 MW polypeptide with a moderately short half-life (about 3 h).