Quantitation of myosin light chain phosphorylation in small tissue samples.

P. J. Silver, J. T. Stull

Research output: Contribution to journalArticle

54 Citations (Scopus)

Abstract

A method to quantitate the extent of phosphorylation of the 20,000-dalton phosphorylatable myosin light chain (P-light chain) in cardiac, smooth, or skeletal muscle samples which have limited amounts of tissue and myosin is presented. Native myosin is isolated from other cellular proteins in crude homogenates prepared from a few milligrams of muscle by pyrophosphate-polyacrylamide gel electrophoresis. The extent of P-light chain phosphorylation is maintained throughout this procedure by the inclusion of myosin light chain kinase and phosphatase inhibitors. Myosin obtained by pyrophosphate-gel electrophoresis is subjected to isoelectric focusing on polyacrylamide gels to separate the phosphorylated from the nonphosphorylated forms of the P-light chain. Following staining with ammonial-silver phosphorylation is quantitated on a mole of phosphate incorporated per mol of P-light chain basis by densitometric scanning of the isoelectric focusing gels. Direct comparison of this methodology with another method used for quantitating phosphorylation revealed no difference between the techniques in measuring the extent of P-light chain phosphorylation in muscle biopsy samples. This methodology provides the means for examination of the possible regulatory roles of P-light chain phosphorylation in contraction of cardiac, smooth, skeletal muscle.

Original languageEnglish (US)
Pages (from-to)6137-6144
Number of pages8
JournalJournal of Biological Chemistry
Volume257
Issue number11
StatePublished - Jun 10 1982

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Myosin Light Chains
Phosphorylation
Tissue
Muscle
Myosins
Isoelectric Focusing
Electrophoresis
Smooth Muscle
Skeletal Muscle
Gels
Myosin-Light-Chain Phosphatase
Myosin-Light-Chain Kinase
Muscles
Biopsy
Silver
Polyacrylamide Gel Electrophoresis
Myocardium
Phosphates
Staining and Labeling
Scanning

ASJC Scopus subject areas

  • Biochemistry

Cite this

Quantitation of myosin light chain phosphorylation in small tissue samples. / Silver, P. J.; Stull, J. T.

In: Journal of Biological Chemistry, Vol. 257, No. 11, 10.06.1982, p. 6137-6144.

Research output: Contribution to journalArticle

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