Probing functional regions in cardiac isomyosins with monoclonal antibodies.
Seven Mabs prepared against subfragment 1 (S1) of either bovine cardiac beta-specific or rabbit fast skeletal muscle myosin were used to identify functional regions in cardiac isomyosin heavy chains. This approach was designed to improve the understanding of structure-function relationships within the myosin molecule and between alpha and beta myosin heavy chains (MHCs). We used bacterial expression of human beta myosin fragments and determined that the seven antibodies were localized within four different MHC subdomains: amino acid residues 33-37 (one beta-specific antibody), 67-84 (one alpha/beta-specific antibody), 85-106 (four alpha/beta-specific antibodies) and 215-248 (one alpha/beta-specific antibody). All epitopes were accessible on myosin and actomyosin with the same affinities. Therefore, none of these MHC epitopes were located on the interfaces between the myosin head and actin. Three antibodies reacting at three out of the four investigated epitopes enhanced acto-S1 ATPase activities but not myosin, S1, or actomyosin activities. One antibody, which was strictly beta-specific and bound to five amino acid residues near the most N-terminal MHC end, substantially inhibited all myosin or S1 ATPase activities measured with or without actin. The epitope of this antibody coincides with one difference cluster observed between both cardiac MHC isoforms [McNally et al (1989) J. Mol. Biol. 210, 665-671], suggesting that this small variable MHC area could be one of the structural bases to explain observed functional differences in cardiac alpha and beta myosin isoforms.
Eldin
P
P
Institut National de la Santé et de la Recherche Médicale, Unité 300, Faculté de Pharmacie, Montpellier, France.
Cathiard
A M
AM
Leger
J
J
Anoal
M
M
Pons
F
F
Mornet
D
D
Leger
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular; D006352:Heart Ventricles; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D015879:Myosin Subfragments / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D009218:Myosins / Q000032:analysis / Q000235:genetics* / Q000276:immunology; D009838:Oligodeoxyribonucleotides; D011817:Rabbits; D015183:Restriction Mapping
D000911:Antibodies, Monoclonal; D015879:Myosin Subfragments; D009838:Oligodeoxyribonucleotides; D009218:Myosins