The ATP-induced change of tryptophan fluorescence reflects a conformational change upon formation of ADP-sensitive phosphoenzyme in the sarcoplasmic reticulum Ca(2+)-ATPase. Stopped-flow spectrofluorometry and continuous flow-rapid quenching method.
The ATP-induced change in the tryptophan fluorescence of the Ca(2+)-ATPase was determined with sarcoplasmic reticulum vesicles at pH 7.0 in the presence of Ca2+ under various conditions by steady-state measurements and stopped-flow spectrofluorometry. Formation of the phosphoenzyme intermediate was also determined by the continuous flow-rapid quenching method. The steady-state fluorescence at 0 degrees C decreased by 1.1% on addition of ATP, whereas no fluorescence change was induced by adenosine 5'-(beta,gamma-methylene)triphosphate (a nonhydrolyzable ATP analog incapable of phosphorylating the enzyme). The time course of the ATP-induced fluorescence drop agreed well with that of the phosphoenzyme formation under all of the conditions tested, and the phosphoenzyme formed was largely sensitive to ADP. When phosphoenzyme isomerization from the ADP-sensitive form to the ADP-insensitive form was almost completely prevented by N-ethylmaleimide treatment, the time course of the ATP-induced fluorescence drop again agreed with that of the phosphoenzyme formation. These results show that the ATP-induced fluorescence drop occurs upon formation of the ADP-sensitive phosphoenzyme. The results further indicate that the tryptophan fluorescence of this enzyme is insensitive to the conformational change which was previously shown (Suzuki, H., Obara, M., Kuwayama, H., and Kanazawa, T. (1987) J. Biol. Chem. 262, 15448-15456) to occur upon formation of the calcium-enzyme-substrate complex. Thus, we conclude that the ATP-induced drop in the tryptophan fluorescence reflects a conformational change occurring upon formation of the ADP-sensitive phosphoenzyme.
Nakamura
S
S
Department of Biochemistry, Asahikawa Medical College, Japan.
Suzuki
H
H
Kanazawa
T
T
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D005453:Fluorescence; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry
C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000001:Calcimycin; D004533:Egtazic Acid; D014364:Tryptophan; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D000252:Calcium-Transporting ATPases; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D002118:Calcium