R Blough's research while affiliated with University of Chicago and other places

The principles of chemical kinetics were applied to turnover studies soon after tracers had been introduced into biological experiments, and several reviews on this subject are available. However, rigorous precursor-product analysis of protein turnover was seldom attempted in the past, mostly because it was assumed that the radioactive precursors of proteins are rapidly eliminated from the organisms. The estimate of protein half-life therefore was based on measurements of decay of the protein radioactivity only. Although such experiments clearly confirmed the dynamic state of protein molecules, it was later recognized that this approach leads to a gross overestimate of protein half-lives because the tracer amino acids are not eliminated as fast as had been thought. The omission of precursor specific radioactivity in calculations of turnover rates thus is not justified. Consequently, any meaningful measure of protein turnover requires determination of the specific radioactivity of the amino acid, both in the protein molecule and in the precursor pool.

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.

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 days. Similar values were obtained when the half-life was calculated by simple graphical integration of the experimental curves.