Differential compartmentation of leucine for oxidation and for protein synthesis in cultured skeletal muscle.

Leucine oxidation and incorporation into protein were studied in cultured skeletal muscle cells to develop methods for measurement of absolute rates of these processes and to define the functional compartmentation of leucine within the muscle cell. The composition of the oxidation precursor pool and absolute rates of oxidation were determined from measurement of production of "CO2 from externally supplied [1-'4C]leucine and from [1-1'4C]leucine previously incorporated into protein. Absolute rates of leucine incorporation into protein were based on measurements of radiolabeled leucine bound to tRNA. In cells cultured in buffered saline at 0.05 m~ leucine, 70% of the oxidized leucine originated extracellularly, while 60% of the leucine for protein synthesis was derived from degraded protein. The preferential use of extracellular leucine for oxidation was enhanced at higher external leucine concentrations, but tRNA leucine showed little change in source when medium leucine was raised as high as 5 mm. The intracellular, free leucine pool was significantly different in derivation from both the oxidative and synthetic precursors. Skeletal muscle cultures oxidized appreciable quantities of leucine compared to leucine utilization for protein synthesis. The rate of oxidation was decreased by the presence of other oxidizable substrates but increased when the external leucine was raised above physiological levels. The rate of protein synthesis did not vary over a 100-fold range of medium leucine concentrations. These studies underscore the need to obtain accurate information on the isotopic content of the immediate precursor when measuring absolute or even relative rates of leucine oxidation or protein synthesis.