title: Isolation of human skeletal muscle myosin heavy chain and actin for measurement of fractional synthesis rates.
abstract: 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 determination of their fractional synthesis rates. 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 +/- 1 yr) after they received a 14-h intravenous infusion (prime = 7.58 micromol/kg body wt, constant infusion = 7.58 micromol. kg body wt-1. h-1) of [1-13C]leucine. The fractional synthesis 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 synthesis rate of MHC was 20% lower (P = 0.012), and the synthesis rate of actin 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.
authors.affiliation: Division of Endocrinology, Diabetes and Metabolism, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Hasten
authors.forename: G S
authors.initials: GS
authors.lastname: Morris
authors.forename: S
authors.initials: S
authors.lastname: Ramanadham
authors.forename: K E
authors.initials: KE
authors.lastname: Yarasheski
mesh_terms: D000199:Actins / Q000096:biosynthesis / Q000302:isolation & purification*; D000328:Adult; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D007651:Keto Acids / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008297:Male; D009124:Muscle Proteins / Q000096:biosynthesis; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000096:biosynthesis / Q000302:isolation & purification*; D051381:Rats
chemical_list: D000199:Actins; D007651:Keto Acids; D009124:Muscle Proteins; C013082:alpha-ketoisocaproic acid; D018995:Myosin Heavy Chains; D007930:Leucine