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Interaction of a fluorescent N-dansylazi ... absence and presence of calcium
Interaction of a fluorescent N-dansylaziridine derivative of troponin I with calmodulin in the absence and presence of calcium.
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Rabbit skeletal muscle troponin I was covalently labeled with N-dansylaziridine, resulting in a fluorescent labeled protein. This derivative (DANZTnI) and native troponin I (TnI) inhibited calmodulin (CaM) stimulation of bovine heart Ca2+-sensitive cyclic nucleodite phosphodiesterase with identical inhibition constants. Association of DANZTnI with calmodulin was monitored directly by changes in flourescence intensity in the presence of Ca2+ and by changes in fluorescence anisotropy in the absence of Ca2+. Quantitation of the affinity of calmodulin for calmodulin-binding proteins in both the presence and absence of Ca2+ is necessary for prediction of the extent of interaction of both Ca2+ and calmodulin-binding proteins with calmodulin in vivo. The dissociation constants for the DANZTnI-calmodulin-l4Ca2+ and DANZTnI-calmodulin complexes were 20 nM and 70 micrometers, respectively. These dissociation constants define a free energy coupling of-4.84 kcal/mol of troponin I for binding of Ca2+ and troponin I to calmodulin. The Ca2+ dependence for troponin I-calmodulin complex formation predicted from these experimentally determined parameters was closely approximated by the Ca2+ dependence for complex formation between troponin I and fluorescent 5-[[[(iodoacetyl)amino]ethyl]-amino]-1-napthalenesulfonic acid derivatized calmodulin as determined by fluorescence anisotropy. Complex formation occurred over a relatively narrow range of Ca2+ concentration, indicative of positive heterotropic cooperativity for Ca2+ and troponin I binding to calmodulin.
Olwin BB, Keller CH, Storm DR
Biochemistry
1982-10-26 00:00
21
22
5669-75
3',5'-Cyclic-Nucleotide Phosphodiesterase,Animals,Aziridines,Azirines,Calcium,Calcium-Binding Proteins,Calmodulin,Calmodulin-Binding Proteins,Carrier Proteins,Dansyl Compounds,Fluorescence Polarization,Muscle Proteins,Muscles,Rabbits,Thermodynamics,Troponin,Troponin I,Aziridines,Azirines,Calcium-Binding Proteins,Calmodulin,Calmodulin-Binding Proteins,Carrier Proteins,Dansyl Compounds,Muscle Proteins,Troponin,Troponin I,dansylaziridine,Calcium,3',5'-Cyclic-Nucleotide Phosphodiesterase
Biochemistry
NIAID AI 00310, NIGMS GM-07270, NHLBI HL 05933
0006-2960
878
True
6293554
