ISOLATION OF HUMAN SKELETAL MUSCLE MYOSIN HEAVY CHAIN &ACTIN

Washington University, Saint Louis MO

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Abstract

We have developed a simple method to isolate mysoin heavy chain (MHC) and actin from small (60-80 mg) human skeletal muscle samples for determinatin of their fractional synthesis rates, utilizing polyacrylamide gel electrophoresis (PAGE). The amounts of MHC and actin isolated are adequate for the quantification of [13C]leucine abundance by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS). Fractional synthesis rates of mixed muscle protein (MMP), MHC, and actin were determined in six healthy young subjects (27 ( 3 yr) after they received a 14-hr intravenous infusion (prime = 7.58 (mol.kg-1, constant infusion = 7.58 (mol.kg-1.hr-1) of [1-13C]leucine. Overall, the synthesis rate of MHC was 20% lower (p=0.012) and the synthesis rate of actin was 61% higher (p=0.060, N.S.) than the MMP synthesis rate. The isolation of these proteins for isotope abundance analysis by GC/C/IRMS provides valuable information about the synthesis rates of these s pecific c ontractile proteins as opposed to the more general information provided by previous studies of MMP synthesis rates.