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Myosin light chain kinase structure function analysis using bacterial expression.

Publication ,  Journal Article
Bagchi, IC; Kemp, BE; Means, AR
Published in: J Biol Chem
September 25, 1989

A 40-kDa fragment of chicken smooth muscle myosin light chain kinase was produced and partially purified from a bacterial expression system. This fragment exhibits calmodulin binding and substrate phosphorylation properties similar to those of the isolated chicken gizzard enzyme. A series of 3'-deletion mutants was prepared and used to produce proteins with the same NH2 terminus but with COOH termini varying over 180 amino acids. Results show that truncation of the enzyme at Ser-512 (based on the amino acid numbering system described for the partial cDNA clone by Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381) does not alter calmodulin binding, calmodulin regulation, or enzymatic properties. Removal of an additional 5 residues from the COOH terminus completely inhibits calmodulin binding and results in an inactive kinase that can be fully activated by limited proteolysis. Site specific mutations within these 5 residues demonstrate that Gly-508 and Arg-509 are independently involved in calmodulin-dependent binding and activation of myosin light chain kinase. Truncation of the enzyme at residues within the protein kinase catalytic domain results in inactive protein that cannot be activated by proteolysis.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

September 25, 1989

Volume

264

Issue

27

Start / End Page

15843 / 15849

Location

United States

Related Subject Headings

  • Plasmids
  • Myosin-Light-Chain Kinase
  • Mutation
  • Muscle, Smooth
  • Molecular Weight
  • Gizzard, Avian
  • Genetic Vectors
  • Genes
  • Escherichia coli
  • DNA
 

Citation

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MLA
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Bagchi, I. C., Kemp, B. E., & Means, A. R. (1989). Myosin light chain kinase structure function analysis using bacterial expression. J Biol Chem, 264(27), 15843–15849.
Bagchi, I. C., B. E. Kemp, and A. R. Means. “Myosin light chain kinase structure function analysis using bacterial expression.J Biol Chem 264, no. 27 (September 25, 1989): 15843–49.
Bagchi IC, Kemp BE, Means AR. Myosin light chain kinase structure function analysis using bacterial expression. J Biol Chem. 1989 Sep 25;264(27):15843–9.
Bagchi, I. C., et al. “Myosin light chain kinase structure function analysis using bacterial expression.J Biol Chem, vol. 264, no. 27, Sept. 1989, pp. 15843–49.
Bagchi IC, Kemp BE, Means AR. Myosin light chain kinase structure function analysis using bacterial expression. J Biol Chem. 1989 Sep 25;264(27):15843–15849.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

September 25, 1989

Volume

264

Issue

27

Start / End Page

15843 / 15849

Location

United States

Related Subject Headings

  • Plasmids
  • Myosin-Light-Chain Kinase
  • Mutation
  • Muscle, Smooth
  • Molecular Weight
  • Gizzard, Avian
  • Genetic Vectors
  • Genes
  • Escherichia coli
  • DNA