Phosphorylation of two sites on smooth muscle myosin. Effects on contraction of glycerinated vascular smooth muscle

J. R. Haeberle, T. A. Sutton, B. A. Trockman

Research output: Contribution to journalArticle

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Abstract

Contraction of glycerinated porcine carotid artery smooth muscle in response to calcium (20 μM), calmodulin (10 μM), and MgATP was associated with phosphorylation of the 20,000-dalton myosin light chain (LC20) to an average stoichiometry of 1.47 mol of PO4/mol of LC20. Tryptic and chymotryptic phosphopeptide maps of the mono- and diphosphorylated forms of LC20 purified from skinned muscles demonstrated the presence of a single phosphopeptide in all cases. Phosphoamino acid analysis indicated that the monophosphorylated form contained primarily phosphoserine, whereas the diphosphorylated form contained both phosphoserine and phosphothreonine. Thiophosphorylation of LC20 by adenosine 5'-O-(thiotriphosphate) resulted in the incorporation of 1 mol of thiophosphate into phosphoserine. Thiophosphorylated LC20 could be subsequently phosphorylated at a threonine residue to a stoichiometry of 1.7 mol of PO4/mol of LC20 by incubation in the presence of MgATP, calcium, and calmodulin. The extent of multiple site phosphorylation of LC20 was dependent upon both the ionic strength and the free Mg2+ concentration in the muscle bath; increasing either ionic strength (0.07-0.15 M) or [Mg2+] (1-20 mM) resulted in lower stoichiometries of LC20 phosphorylation. The effect of multiple site phosphorylation on contraction was examined in muscles which were sequentially phosphorylated at serine followed by threonine. Full activation (21°C) of both isometric force (1.4 newtons/cm2) and unloaded shortening velocity (0.016 L(o)/s) was achieved following thiophosphorylation to 1.1 mol of PO4/mol of LC20. No further activation of either isometric force (1.5 newtons/cm2) or unloaded shortening velocity (0.015 L(o)/s) occurred following phosphorylation to 1.7 mol of PO4/mol of LC20.

Original languageEnglish (US)
Pages (from-to)4424-4429
Number of pages6
JournalJournal of Biological Chemistry
Volume263
Issue number9
StatePublished - Jan 1 1988

Fingerprint

Smooth Muscle Myosins
Phosphorylation
Vascular Smooth Muscle
Muscle
Phosphoserine
Stoichiometry
Phosphopeptides
Threonine
Calmodulin
Ionic strength
Muscles
Osmolar Concentration
Adenosine Triphosphate
Chemical activation
Phosphothreonine
Phosphoamino Acids
Calcium
Myosin Light Chains
Baths
Carotid Arteries

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Phosphorylation of two sites on smooth muscle myosin. Effects on contraction of glycerinated vascular smooth muscle. / Haeberle, J. R.; Sutton, T. A.; Trockman, B. A.

In: Journal of Biological Chemistry, Vol. 263, No. 9, 01.01.1988, p. 4424-4429.

Research output: Contribution to journalArticle

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