Different phosphorylated forms of myosin in contracting tracheal smooth muscle

A. Persechini, K. E. Kamm, J. T. Stull

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Abstract

Calmodulin-dependent myosin light chain kinase phosphorylates two light chain subunits on each myosin molecule. We have developed a method for measuring nonphosphorylated, monophosphorylated, and diphosphorylated forms of myosin in smooth muscle. Four protein bands were separated in tissue extracts by nondenaturing polyacrylamide gel electrophoresis in the presence of pyrophosphate. Immunoblots demonstrated that three forms (designated M, MP, and MP2) reacted with rabbit antisera prepared against the purified phosphorylated light chain (P-light chain) from bovine tracheal smooth muscle. Evidence was obtained that M, MP, and MP2 represented nonphosphorylated, monophosphorylated, and diphosphorylated myosin, respectively, and that the other protein band was probably filamin. The formation of different phosphorylated forms of myosin was measured in bovine trachealis strips neurally stimulated from 1.0 to 3.5 s and quick-frozen. There was no detectable MP or MP2 in unstimulated muscles; the extent of P-light chain phosphorylation measured directly was 0.02 ± 0.01 mol of phosphate/mol of P-light chain. After 2.5-s stimulation, maximal values of 0.63 ± 0.06 mol of phosphate/mol of P-light chain and 0.40 ± 0.06 MP2/myosin(total) were obtained. During continuous neural stimulation from 1.0 to 3.5 s, the relationship between the extent of P-light chain phoshorylation (measured directly or calculated) and the relative amount of MP2 is consistent with a random phosphorylation process.

Original languageEnglish (US)
Pages (from-to)6293-6299
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number14
StatePublished - 1986

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Myosins
Smooth Muscle
Muscle
Light
Phosphorylation
Phosphates
Filamins
Smooth Muscle Myosins
Myosin-Light-Chain Kinase
Tissue Extracts
Calmodulin
Electrophoresis
Immune Sera
Polyacrylamide Gel Electrophoresis
Proteins
Rabbits
Muscles
Molecules

ASJC Scopus subject areas

  • Biochemistry

Cite this

Different phosphorylated forms of myosin in contracting tracheal smooth muscle. / Persechini, A.; Kamm, K. E.; Stull, J. T.

In: Journal of Biological Chemistry, Vol. 261, No. 14, 1986, p. 6293-6299.

Research output: Contribution to journalArticle

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