Isolation of human skeletal muscle myosin heavy chain and actin for measurement of fractional synthesis rates

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we have developed a simple method to isolate myosin heavy chain (MHC) and actin from small (60-80 mg) human skeletal muscle samples for the determina tion of their fractional synthesis rates. The amounts of MHC and actin isol ated are adequate for the quantification of [C-13]leucine abundance by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Frac tional synthesis rates of mixed muscle protein (MMP), MHC, and actin were d etermined in six healthy young subjects (27 +/- 1 yr) after they received a 14-h intravenous infusion (prime = 7.58 mu mol/kg body wt, constant infusi on = 7.58 mu mol.kg.body wt(-1).h(-1)) of [1-C-13]leucine. The fractional s ynthesis rates of MMP, MHC, and actin were found to be 0.0468 +/- 0.0048, 0 .0376 +/- 0.0033, and 0.0754 +/- 0.0078%/h, respectively. Overall, the synt hesis rate of MHC was 20% lower (P = 0.012), and the synthesis rate of acti n was 61% higher (P = 0.060, not significant) than the MMP synthesis rate. The isolation of these proteins for isotope abundance analysis by GC-C-IRMS provides important information about the synthesis rates of these specific contractile proteins, as opposed to the more general information provided by the determination of MMP synthesis rates.