Effects of divalent cations bound to the catalytic site on ATP-induced conformational changes in the sarcoplasmic reticulum Ca(2+)-ATPase: stopped-flow analysis of the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to cysteine-674.
Cys-674 of the sarcoplasmic reticulum Ca(2+)-ATPase was labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The steady-state fluorescence of the label decreased with increasing concentration of Mg.ATP, Mn.ATP, or Ca.ATP in the presence of Ca2+ and 0.1 M KCl at 0 degree C, pH 7.0. The maximum extent of the decrease was 23%. The Mg.ATP-, Mn.ATP-, or Ca.ATP-induced fluorescence drop was also determined by the stopped-flow spectrofluorometry. The Ca.ATP-induced fluorescence drop manifested a biphasic time course. The first phase reflects the conformational change which was previously shown [Suzuki, H., Obara, M., Kuwayama, H., & Kanazawa, T. (1987) J. Biol. Chem. 262, 15448-15456] to occur upon formation of the calcium-enzyme-substrate complex. The amplitude of the second phase was 2.5% of the fluorescence before addition of Ca.ATP. This phase of the fluorescence drop coincided with the phosphoenzyme formation, which was determined by the continuous flow-rapid quenching method. The phosphoenzyme formed was largely sensitive to ADP. When adenosine 5'-(beta,gamma-methylenetriphosphate) (a nonhydrolyzable ATP analog incapable of phosphorylating the enzyme) was added in the presence of 5 mM CaCl2 without added MgCl2 the steady-state fluorescence decreased rapidly by 20%. However, this drop lacked the second phase. When phosphoenzyme isomerization from the ADP-sensitive form to the ADP-insensitive form was prevented by the N-ethylmaleimide treatment, the second phase of the Ca.ATP-induced fluorescence drop again coincided with the phosphoenzyme formation.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry, Asahikawa Medical College, Japan.
H
H
Suzuki
S
S
Nakamura
T
T
Kanazawa
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent; D003545:Cysteine / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D005456:Fluorescent Dyes; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009282:Naphthalenesulfonates; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D002413:Cations, Divalent; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D003545:Cysteine; D005033:Ethylmaleimide