Purification and characterization of myosin light-chain kinase from porcine myometrium and its phosphorylation and modulation by cyclic amp-dependent protein kinase

Kenji Higashi, Kohji Fukunaga, Kazuo Matsui, Masao Maeyama, Eishichi Miyamoto

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16 Citations (Scopus)

Abstract

Myosin light-chain kinase was purified from porcine myometrium to apparent homogeneity at about 262-fold with an Mr of 130 000 as determined by SDS-polyacrylamide gel electrophoresis and a sedimentation coefficient of 4.5 S. The approximate content of the soluble myosin light-chain kinasee was estimated to be about 0.85 μ M. The purified enzyme exhibited strict substrate specificity only for 20-kDa myosin light chain and a values of 0.6 nM and 0.3 μM for calmodulin and Ca2+, respectively. The enzyme was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a decrease in the affinity for calmodulin of 4-7-fold without effect on the Vmax. The maximal amount of phosphate incorporated into the enzyme was 0.5-0.8 and 1.0-1.4 mol per mol of the enzme in the presence and absence of Ca2+ and calmodulin, respectively. In the presence of a subsaturating concentration of calmodulin, the enzyme showed a lower sensitivity for Ca2+ by phosphorylation.

Original languageEnglish
Pages (from-to)232-240
Number of pages9
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume747
Issue number3
DOIs
Publication statusPublished - 1983 Sep 28
Externally publishedYes

Keywords

  • (Procine myometrium)
  • Calmodulin sensitivity
  • Myosin light-chain kinase
  • Phosphorylation

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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