PROTEIN SYNTHESIS IN CENTRAL NERVOUS TISSUE: STUDIES ON RETINA IN VITRO1

Abstract

Rabbit retinas were maintained in vitro in medium that resembled CSF but with leucine varied from 2 to 1000 microM. Both leucine and threonine were isotopically labelled. When leucine in the medium was 100-1000 microM, leucine was incorporated into protein at 2.03 +/- 0.04 (S.E.M.) mumol/g dry wt./h, a turnover per h of 0.55% of the leucine in retinal protein. Incorporation was constant for at least 7 h. It was reduced 34% when the other amino acids were omitted from the medium and 24% when they were increased 15 fold above physiological levels. When medium leucine was reduced to 2 microM with other amino acids constant, 14C-leucine incorporation fell 70% without significant change in 3H-threonine incorporation, indicating a fall in intracellular specific activity of leucine. The intracellular/extracellular concentration ratio of labelled leucine was 4:1 with medium leucine 23 microM. It fell markedly when medium leucine was reduced to 2 microM or increased to 1000 microM. The concentration ratio of labelled threonine was 15:1 with medium leucine at physiological levels but fell to 6:1 when medium leucine was increased to 1000 microM. Decarboxylation removed 1.5% of free intracellular leucine per min and, at physiological concentrations, was 7.7% the rate of protein incorporation. The ratio of protein synthesis/breakdown, estimated from changes in leucine and 7 other essential amino acids in the medium, was nearly unity. The potential of this preparation for study of CNS protein metabolism is discussed.