Mapping of the functional domains in the amino-terminal region of calponin.
Three chymotryptic fragments accounting for almost the entire amino acid sequence of gizzard calponin (Takahashi, K., and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288) were isolated and characterized. They encompass the segments of residues 7-144 (NH2-terminal 13-kDa peptide), 7-182 (NH2-terminal 22-kDa peptide), and 183-292 (COOH-terminal 13-kDa peptide). They arise from the sequential hydrolysis of the peptide bonds at Tyr182-Gly183 and Tyr144-Ala145 which were protected by the binding of F-actin to calponin. Only the NH2-terminal 13- and 22-kDa fragments were retained by immobilized Ca(2+)-calmodulin, but only the larger 22 kDa entity cosedimented with F-actin and inhibited, in the absence of Ca(2+)-calmodulin, the skeletal actomyosin subfragment-1 ATPase activity as the intact calponin. Since the latter peptide differs from the NH2-terminal 13-kDa fragment by a COOH-terminal 38-residue extension, this difference segment appears to contain the actin-binding domain of calponin. Zero-length cross-linked complexes of F-actin and either calponin or its 22-kDa peptide were produced. The total CNBr digest of the F-actin-calponin conjugate was fractionated over immobilized calmodulin. The EGTA-eluted pair of cross-linked actin-calponin peptides was composed of the COOH-terminal actin segment of residues 326-355 joined to the NH2-terminal calponin region of residues 52-168 which seems to contain the major determinants for F-actin and Ca(2+)-calmodulin binding.
Mezgueldi
M
M
Centre de Recherches de Biochimie Macromoléculaire du Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale U 249, Université de Montpellier, France.
Fattoum
A
A
Derancourt
J
J
Kassab
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D011817:Rabbits; D014336:Troponin / Q000378:metabolism; D014422:Turkeys; D000096985:Calponins
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002918:Chymotrypsin; D003432:Cross-Linking Reagents; D008840:Microfilament Proteins; D009124:Muscle Proteins; D010446:Peptide Fragments; D014336:Troponin; D000096985:Calponins