Caldesmon has two calmodulin-binding domains.
Chicken gizzard caldesmon was cleaved with chymotrypsin or CNBr, and the calmodulin-binding fragments were isolated using an affinity column. Limited chymotryptic digestion gives rise to a 38 kDa calmodulin-binding fragment (CT40) as described previously (Szpacenko, A. & Dabrowska, R., FEBS Lett. 202, 182-186, 1986; Fujii, T., Imai, M., Rosenfeld, G. C. & Bryan, J., J. Biol. Chem. 261, 16155-16160, 1987; Yazawa, M., Yagi, K. & Sobue, K., J. Biochem. 102, 1065-1073, 1987). In the case of CNBr cleavage a 37 kDa calmodulin-binding fragment (CB40) was obtained. Both CT40 and CB40 contain a reactive thiol group, but these thiols are apparently in different environments as judged by the responses of attached fluorescent labels to calmodulin-binding. A comparison of the N-terminal sequences of CB40 and CT40 with the complete sequence of caldesmon shows that the two calmodulin-binding fragments in fact originate from different parts of the parent molecule. Thus there exist two calmodulin-binding sites in caldesmon, one in the N-terminal half and the other in the C-terminal half of the molecule. This is consistent with the recent finding that up to two calmodulin molecules can be crosslinked to each caldesmon molecule (Wang, C.-L.A., Biochem. Biophys. Res. Commun., 156, 1033-1038, 1988).
Wang
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Department of Muscle Research, Boston Biomedical Research Institute, MA 02114.
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D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D005456:Fluorescent Dyes; D005895:Gizzard, Avian / Q000032:analysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010069:Oxadiazoles; D010446:Peptide Fragments / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D010446:Peptide Fragments; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; D002918:Chymotrypsin; D003488:Cyanogen Bromide