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

Am J Physiol. 1998 Dec;275(6):E1092-9. doi: 10.1152/ajpendo.1998.275.6.E1092.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / biosynthesis
  • Actins / isolation & purification*
  • Adult
  • Animals
  • Electrophoresis, Polyacrylamide Gel / methods
  • Female
  • Gas Chromatography-Mass Spectrometry / methods
  • Humans
  • Keto Acids / metabolism
  • Leucine / metabolism
  • Male
  • Muscle Proteins / biosynthesis
  • Muscle, Skeletal / chemistry*
  • Muscle, Skeletal / metabolism
  • Myosin Heavy Chains / biosynthesis
  • Myosin Heavy Chains / isolation & purification*
  • Rats

Substances

  • Actins
  • Keto Acids
  • Muscle Proteins
  • alpha-ketoisocaproic acid
  • Myosin Heavy Chains
  • Leucine