Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme

Vincent Guerriero, David R. Rowley, Anthony R. Means

Research output: Contribution to journalArticle

95 Citations (Scopus)

Abstract

A specific precipitating antibody against chicken gizzard myosin light chain kinase (MLCK) has been produced in rabbits. The antibody inhibited enzyme activity in a dose-dependent manner with 11 moles of antibody required to inhibit 80% of the activity of one mole MLCK. Crude homogenates from various chicken tissues were analyzed by SDS-polyacrylamide gel electrophoresis, and the proteins were transferred onto nitrocellulose sheets and reacted with antibody. In each case, the antibody bound to only one protein with a molecular weight of approximately 130,000. These data suggest the MLCK present in all types of muscle as well as non-muscle tissues is of the same molecular weight. Indirect immunofluorescent microscopy of non-muscle tissue culture cells revealed MLCK to be localized in the spindle apparatus and midbody of mitotic cells and on the stress fibers and in the nucleolus of interphase cells. The nucleolar localization was confirmed by electron microscopy and shown to be restricted to the fibrillar region. In myofibrils isolated from skeletal and cardiac muscle, anti-MLCK decorated the actin-containing I bands of the sarcomere. These results are consistent with the suggestion that MLCK and calmodulin are intermediates in the regulation of cell motility by calcium.

Original languageEnglish (US)
Pages (from-to)449-458
Number of pages10
JournalCell
Volume27
Issue number3 PART 2
DOIs
StatePublished - 1981
Externally publishedYes

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Myosin-Light-Chain Kinase
Antibody Formation
Antibodies
Enzymes
Muscle
Chickens
Molecular Weight
Molecular weight
Cell Nucleolus
Cardiac Myosins
Tissue
Avian Gizzard
Tissue culture
Stress Fibers
Sarcomeres
Spindle Apparatus
Collodion
Myofibrils
Interphase
Enzyme activity

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme. / Guerriero, Vincent; Rowley, David R.; Means, Anthony R.

In: Cell, Vol. 27, No. 3 PART 2, 1981, p. 449-458.

Research output: Contribution to journalArticle

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abstract = "A specific precipitating antibody against chicken gizzard myosin light chain kinase (MLCK) has been produced in rabbits. The antibody inhibited enzyme activity in a dose-dependent manner with 11 moles of antibody required to inhibit 80{\%} of the activity of one mole MLCK. Crude homogenates from various chicken tissues were analyzed by SDS-polyacrylamide gel electrophoresis, and the proteins were transferred onto nitrocellulose sheets and reacted with antibody. In each case, the antibody bound to only one protein with a molecular weight of approximately 130,000. These data suggest the MLCK present in all types of muscle as well as non-muscle tissues is of the same molecular weight. Indirect immunofluorescent microscopy of non-muscle tissue culture cells revealed MLCK to be localized in the spindle apparatus and midbody of mitotic cells and on the stress fibers and in the nucleolus of interphase cells. The nucleolar localization was confirmed by electron microscopy and shown to be restricted to the fibrillar region. In myofibrils isolated from skeletal and cardiac muscle, anti-MLCK decorated the actin-containing I bands of the sarcomere. These results are consistent with the suggestion that MLCK and calmodulin are intermediates in the regulation of cell motility by calcium.",
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AB - A specific precipitating antibody against chicken gizzard myosin light chain kinase (MLCK) has been produced in rabbits. The antibody inhibited enzyme activity in a dose-dependent manner with 11 moles of antibody required to inhibit 80% of the activity of one mole MLCK. Crude homogenates from various chicken tissues were analyzed by SDS-polyacrylamide gel electrophoresis, and the proteins were transferred onto nitrocellulose sheets and reacted with antibody. In each case, the antibody bound to only one protein with a molecular weight of approximately 130,000. These data suggest the MLCK present in all types of muscle as well as non-muscle tissues is of the same molecular weight. Indirect immunofluorescent microscopy of non-muscle tissue culture cells revealed MLCK to be localized in the spindle apparatus and midbody of mitotic cells and on the stress fibers and in the nucleolus of interphase cells. The nucleolar localization was confirmed by electron microscopy and shown to be restricted to the fibrillar region. In myofibrils isolated from skeletal and cardiac muscle, anti-MLCK decorated the actin-containing I bands of the sarcomere. These results are consistent with the suggestion that MLCK and calmodulin are intermediates in the regulation of cell motility by calcium.

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