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

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

    Research output: Contribution to journalArticlepeer-review

    96 Scopus citations

    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 - Dec 1981

    ASJC Scopus subject areas

    • Biochemistry, Genetics and Molecular Biology(all)

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