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

doi:10.1016/0092-8674(81)90386-X

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 journal › Article › peer-review

100 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 language	English (US)
Pages (from-to)	449-458
Number of pages	10
Journal	Cell
Volume	27
Issue number	3 PART 2
DOIs	https://doi.org/10.1016/0092-8674(81)90386-X
State	Published - Dec 1981

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology

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10.1016/0092-8674(81)90386-X

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@article{6732017cd7f0429fa828e85056a24f4e,

title = "Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme",

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.",

author = "Vince Guerriero and Rowley, {David R.} and Means, {Anthony R.}",

note = "Funding Information: We wish to thank D. J. Tindall from this department for his enthusiastic support and advice during the course of this work. We thank Elizabeth MacDougall for performing the indirect immunofluorescent microscopy and Mick Scheib for help in preparing the manuscript. Purified CAMP-dependent protein kinase was kindly supplied by J. S. Tash from this department. We thank Don Blumenthal and Jim Stull (Moss Heart Center and Department of Pharmacology, University of Texas Health Science Center, Dallas) for supplying purified rabbit skeletal muscle MLCK. This research was supported by grants from the National institutes of Health. V. G., Jr. was a recipient of a Muscular Dystrophy postdoctoral fellowship. The costs of publication of this article was defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.",

year = "1981",

month = dec,

doi = "10.1016/0092-8674(81)90386-X",

language = "English (US)",

volume = "27",

pages = "449--458",

journal = "Cell",

issn = "0092-8674",

publisher = "Cell Press",

number = "3 PART 2",

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TY - JOUR

T1 - Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme

AU - Guerriero, Vince

AU - Rowley, David R.

AU - Means, Anthony R.

N1 - Funding Information: We wish to thank D. J. Tindall from this department for his enthusiastic support and advice during the course of this work. We thank Elizabeth MacDougall for performing the indirect immunofluorescent microscopy and Mick Scheib for help in preparing the manuscript. Purified CAMP-dependent protein kinase was kindly supplied by J. S. Tash from this department. We thank Don Blumenthal and Jim Stull (Moss Heart Center and Department of Pharmacology, University of Texas Health Science Center, Dallas) for supplying purified rabbit skeletal muscle MLCK. This research was supported by grants from the National institutes of Health. V. G., Jr. was a recipient of a Muscular Dystrophy postdoctoral fellowship. The costs of publication of this article was defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

PY - 1981/12

Y1 - 1981/12

N2 - 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.

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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U2 - 10.1016/0092-8674(81)90386-X

DO - 10.1016/0092-8674(81)90386-X

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AN - SCOPUS:0019781401

SN - 0092-8674

VL - 27

SP - 449

EP - 458

JO - Cell

JF - Cell

IS - 3 PART 2

ER -

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

Abstract

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