Changes in the fluoresence of N-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca2+-ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca2+ binding sites are located. Formation of the phosphoenzyme intermediates (EP) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes and EP formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps; conformational change in the calcium enzyme substrate complex, formation of ADP-sensitive EP, and transition of ADP-sensitive EP to ADP-insensitive EP. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca2+-binding sites in the transmembrane domain in these latter two steps.
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Review Article|
October 01 1995
Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase
Tohru Kanazawa;
Tohru Kanazawa
1Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan
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Hiroshi Suzuki;
Hiroshi Suzuki
1Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan
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Takashi Daiho;
Takashi Daiho
1Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan
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Kazuo Yamasaki
Kazuo Yamasaki
1Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan
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Publisher: Portland Press Ltd
Received:
September 14 1995
Online ISSN: 1573-4935
Print ISSN: 0144-8463
© 1995 Plenum Publishing Corporation
1995
Biosci Rep (1995) 15 (5): 317–326.
Article history
Received:
September 14 1995
Citation
Tohru Kanazawa, Hiroshi Suzuki, Takashi Daiho, Kazuo Yamasaki; Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase. Biosci Rep 1 October 1995; 15 (5): 317–326. doi: https://doi.org/10.1007/BF01788364
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