Utilization of troponin C as a model calcium-binding protein for mapping of the calmodulin-binding sites of caldesmon.
Troponin C, a structural analogue of calmodulin, was used for mapping the calmodulin-binding sites of caldesmon. The apparent Kd values for the formation of the caldesmon-calcium-binding-protein complex as determined by native gel electrophoresis were 0.5, 1.2 and 3.9 microM for calmodulin, rabbit skeletal muscle troponin C and bovine cardiac troponin C respectively. Troponin C induced a 4-6 nm blue shift of the Trp fluorescence of caldesmon without affecting the amplitude of fluorescence. In the presence of Ca2+, troponin C induced partial displacement of caldesmon from actin tropomyosin complexes. Addition of 5,5'-dithiobis(nitrobenzoic) acid to an equimolar complex of caldesmon and troponin C induced disulphide cross-linking between Cys-98 of rabbit skeletal muscle troponin C and the single Cys residue of duck gizzard caldesmon, located in a position analogous to Cys-580 of the chicken gizzard protein. The cross-linked caldesmon-troponin C complex was ineffective in inhibiting actomyosin ATPase activity. It is concluded that Cys-580 of caldesmon can be located close to both the central helix of calcium-binding proteins and the C-terminal domain of actin. This may be important for the regulation of actomyosin ATPase activity by caldesmon.
Polyakov
A A
AA
Department of Biochemistry, School of Biology, Moscow State University, Russian Federation.
Gusev
N B
NB
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D003432:Cross-Linking Reagents / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004372:Ducks; D004591:Electrophoresis, Polyacrylamide Gel; D009132:Muscles / Q000378:metabolism; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000378:metabolism* / Q000494:pharmacology
D000199:Actins; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D019209:Troponin C; D000251:Adenosine Triphosphatases