Static and kinetic studies on rabbit skeletal muscle troponin.
Fluorescence titration curves of 2-[4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled troponin (IAANS-labeled Tn) and troponin-1-anilinonaphthalene-8-sulfonic acid (Tn-ANS) complex indicated that the fluorescent moiety, IAANS or ANS, detects conformational change of troponin I (TnI) or Tn due to the Ca2+ binding or removal reaction with the low affinity Ca2+-binding sites of troponin C (TnC) component. A fluorescence stopped-flow study showed that the kinetic behavior of IAANS-labeled Tn reflects a change in state of the TnI component induced by the Ca2+ binding or removal reaction with the low affinity Ca2+-binding sites of TnC component. The state change of TnI induced by the Ca2+ binding was complete within the instrumental dead time. On the other hand, that induced by the Ca2+ removal had a rate constant of around 13 s-1. ANS, which is noncovalently bound to Tn, reflects the kinetic properties of both the TnI component and the low affinity Ca2+-binding region of TnC component. The fluorescence intensity change of ANS induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC was complete within the instrumental dead time, while that induced by the Ca2+ removal from the same sites was biphasic. The rate constants of the biphasic process were found to be 62 +/- 7 s-1 and 16 +/- 4 s-1. The former value corresponds to the rate constant of the Ca2+ removal reaction from the low affinity Ca2+-binding sites of TnC component, and the latter value to the rate constant observed in the case of IAANS-labeled Tn. Based on these experimental results and on the discussion in our previous paper (Iio, T. & Kondo, H. (1981) J. Biochem. 90, 163-175), we have refined the two-way information-transfer mechanism which we previously proposed in order to explain the biological function of Tn.
D000199:Actins / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D008958:Models, Molecular; D009132:Muscles / Q000378:metabolism*; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000378:metabolism*; D019210:Troponin I
D000199:Actins; D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D014336:Troponin; D019210:Troponin I; C011488:IAANS; C027132:1-anilino-8-naphthalenesulfonate; D002118:Calcium