Characterization of the carboxyl-terminal 10-kDa cyanogen bromide fragment of caldesmon as an actin-calmodulin-binding region.
A pair of 10-kDa peptides, designated CB-a and CB-b, was isolated by calmodulin-Sepharose chromatography from a total CNBr digest of turkey gizzard caldesmon. CB-a encompasses the COOH-terminal segment of residues 659-756, according to the sequence of adult chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R.G., and Lin, W.G. (1989) J. Biol. Chem. 264, 13873-13879), whereas CB-b comprises the same structure but was a few amino acids shorter at its COOH terminus. Both peptides cosedimented with F-actin, and their binding was increased by smooth muscle tropomyosin. The Kd values were 1.3 and 0.5 microM, in the absence and presence of tropomyosin, respectively, with a maximum binding capacity of 6.9 actins/mol of peptides. The CB-a/CB-b fragments inhibited, in a tropomyosin-sensitive and Ca2(+)-calmodulin-dependent manner, the skeletal actomyosin subfragment 1 ATPase activity to a level close but not identical to that observed for the parent caldesmon. Ca2(+)-calmodulin was selectively cross-linked to either caldesmon or the CNBr peptides with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide producing 1:1 covalent complexes that were retained neither by phenyl-Sepharose nor by immobilized calmodulin. Moreover, the cross-linked caldesmon bound weakly to F-actin and did not inhibit the actomyosin subfragment 1 ATPase in the absence of Ca2+. The results suggest that the CB-a/CB-b peptide region contains major regulatory determinants of caldesmon.
Institut, National de la Santé et de la Recherche Médicale U. 249, Université de Montpellier I, France.
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Bartegi
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Fattoum
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Derancourt
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Kassab
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005895:Gizzard, Avian / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011817:Rabbits; D014335:Tropomyosin / Q000302:isolation & purification / Q000378:metabolism; D014422:Turkeys
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D014335:Tropomyosin; D003488:Cyanogen Bromide