Isoform-specific Interaction of the Myosin-binding Proteins (MyBPs) with Skeletal and Cardiac Myosin Is a Property of the C-terminal Immunoglobulin Domain*
- From the ‡Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021 and the‖Department Bioquimica, Instituto de Quimica, Universidade de São Paulo, CP 20.780, CEP 01498, São Paulo, Brazil
Abstract
Full-length cDNAs encoding chicken and human skeletal MyBP-H and MyBP-C have been isolated and sequenced (1–5). All are members of a protein family with repetitive immunoglobulin C2 and fibronectin type III motifs. The myosin binding domain was mapped to a single immunoglobulin motif in cardiac MyBP-C and skeletal MyBP-H. Limited α-chymotryptic digestion of cardiac MyBP-C generated three peptides, similar in relative mobility to those of skeletal MyBP-C: ∼100, 40, and 15 kDa. Tryptic digestion of MyBP-H yielded two peptides: ∼50 and 14 kDa. Partial amino acid sequences proved that the 15- and 14-kDa fragments are located at the C termini of cardiac MyBP-C and skeletal MyBP-H, respectively. Only the 14- and 15-kDa peptides bound to myosin. Thus, the myosin binding site in all three proteins resides within an homologous, C-terminal immunoglobulin domain. Binding reactions (2) between the skeletal and cardiac MyBPs and corresponding myosin isoforms demonstrated saturable binding of theMyBP proteins and their C-terminal peptides to myosin, but there are higher limiting stoichiometries with the homologous isoform partners. Evidence is presented indicating that MyBP-H and -C compete for binding to a discrete number of sites in myosin filaments.
Footnotes
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↵* This work was supported by National Institutes of Health Grant AR32147 and by funds from the American Heart Association, the Muscular Dystrophy Association, and David Cofrin.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Present address: Thrombosis Research Laboratory, Cornell University Medical College, Dept. of Veterans Affairs Medical Center, 423 East 23rd St., New York, NY 10010.
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↵¶ Investigator of the American Heart Association.
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↵** International Fellow of the Rockefeller Foundation.
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↵‡ To whom correspondence should be addressed: Dept. of Cell Biology and Anatomy, Cornell University Medical College, 1300 York Ave., New York, NY 10021.
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↵1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; Fn, fibronectin; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; FPLC, fast protein liquid chromatography.
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- Received March 12, 1997.
- Revision received May 15, 1997.
