Synthesis and breakdown of different sized retinal proteins in darkness and during photic stimulation

Abstract

Rabbit retinas were maintained in vitro in medium resembling cerebrospinal fluid and were exposed to (3)H-leucine or (14)C-leucine in double-labeling experiments designed to measure rates of protein turnover and to determine the effects of photic stimulation on protein synthesis and degradation. The retinas were solubilized, and the proteins separated according to size by polyacrylamide gel electrophoresis. The gels were cut into 95 slices and each slice differentially counted. Protein content of the slices was estimated from Coomassie Blue staining, and molecular weight (MW) from distribution along the gel of MW standards. The retinas appeared to function nearly normally in vitro for many hours. Protein renewal was rapid and reproducible at 0.55 +/- 0.01 (S.E.M.) %/h and remained quite constant for at least 7 h. Synthesis and degradation were approximately equal. Two retinas were maintained in vitro at 37 degrees C for 52 h, and showed good preservation of morphology, electrophysiological response to light, and protein synthesis. Total synthesis of new polypeptides was at the rate of 103 nmole per g of protein, per h; there was a sharp peak in the 33,000 to 43,000 MW range. Proteins in every size group were very heterogeneous with respect to breakdown coefficients (i.e. longevity) which were the prime determinants of the amount of each protein present. Fractional renewal showed a highly significance (p<0.0001) correlation with MW, due apparently to a reduction in maximal longevity as size increased. Neither synthesis nor degradation was significantly affected by intense continuous light or flashing light, though the latter increased uptake of 2-deoxyglucose by 38%.