The kinetics of calcium binding to calmodulin: Quin 2 and ANS stopped-flow fluorescence studies

• Published in: Biochemical and biophysical research communications Vol. 120; no. 1; pp. 185 - 191
• Main Authors: Bayley, Peter, Ahlstrőm, Peter, Martin, Stephen R., Forsen, Sture
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.1984
• Subjects: Aminoquinolines; Anilino Naphthalenesulfonates; Animals; calcium; Calcium - metabolism; calmodulin; Calmodulin - metabolism; Cattle; fluorescence; Kinetics; Male; Models, Chemical; Protein Binding; Spectrometry, Fluorescence; testes; Testis - metabolism; EDTA; ANS; CaM; BAPTA; EGTA
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/0006-291X(84)91431-1

The rate of calcium dissociation from bovine testis calmodulin was measured by fluorescence stopped-flow using the calcium indicator Quin 2 or the fluorescence probe 8-anilinonaphthalene sulphonate. Two processes are resolved with Quin 2 corresponding to dissociation from the high affinity sites (k diss 2 to 9 s −1 for T = 11 to 28°C) and from the low affinity sites (k diss 293 to 550 s −1 for T = 11 to 19°C). These rates and the activation parameters as determined for the slow process ΔH‡ = 59 ± 10 kJ.mol −1 and ΔS‡ = 30 ± 30 JK −1.mol −1 are in good agreement with values determined from the 43 Ca NMR exchange rates. These experiments provide confirmation that the calcium induced conformational change cannot be resolved kinetically from the calcium binding or dissociation, and by inference this conformational change is not a rate-limiting process in the function of calmodulin.