Measurements of half-life of rat cardiac myosin heavy chain with leucyl-tRNA used as precursor pool.

Abstract

The kinetics of labeling of myosin heavy chain, following a single intravenous injection of L-[4,5-3H]leucine, were analyzed with the help of a computer, in conjunction with the labeling kinetics of the specific radioactivities of the precursor amino acid pool. As precursor we used leucyl-tRNA which, as we show here, differs significantly from the intracellular free leucine pool. The half-life of myosin heavy chain was determined from the initial period (0 to 60 min) of incorporation of label into protein after a single injection of tritiated leucine, and also from the period (7 to 14 days) when there is exponential decay of the labeled protein. Myosin heavy chain was separated from other myofibrillar proteins by polyacrylamide gel electrophoresis before measurement of leucine specific radioactivity. The specific radioactivity was measured in both protein and precursor pools by a sensitive isotope dilution procedure (range, 100 to 1500 pmol). The values for the half-life of myosin heavy chain determined at both intervals were similar (5.4 and 5.9 days). Substitution of the specific radioactivity of the intracellular free leucine pool decreased the half-life to 2.7 dyas. Similar values were obtained when the half-life was calculated by simple graphical integration of the experimental curves.