The Tryptophan Fluorescence Change upon Conformational Transition of the Phosphoenzyme Intermediate in Sarcoplasmic Reticulum Ca2+-ATPase Is Revealed in the Absence of K+ and the Presence of Lasalocid

ATP-induced changes in the tryptophan fluorescence of the Ca(2+)-ATPase were determined with sarcoplasmic reticulum vesicles at pH 7.0 and 0 degrees C by steady-state measurements in the presence of Ca2+ and the absence of K+ with and without added lasalocid (a carboxylic ionophore, 50 microM), which was previously shown to cause a predominant accumulation of the ADP-insensitive form of the phosphoenzyme intermediate (EP) (Kawashima, T., Hara, H., and Kanazawa, T. (1990) J. Biol. Chem. 265, 10993-10999). When ATP was added in the absence of lasalocid, the fluorescence decreased by 1.7%. The addition of lasalocid quenched 71% of the fluorescence but did not reduce the ATP-induced fluorescence drop. The fluorescence drop and the EP formation were also determined in the presence of lasalocid by stopped-flow spectrometry and continuous-flow rapid quenching. The observed fluorescence drop was biphasic. The first phase coincided with the formation of EP, which was largely ADP-sensitive in this early stage of the reaction. The second phase was much slower than the first phase and coincided with the accumulation of ADP-insensitive EP. When the transition of EP from the ADP-sensitive form to the ADP-insensitive form was blocked by N-ethylmaleimide treatment, the second phase disappeared, and the fluorescence drop entirely coincided with the formation of ADP-sensitive EP. These findings demonstrate that the first phase of the fluorescence drop is attributed to the formation of ADP-sensitive EP, the second phase being attributed to the transition of EP from the ADP-sensitive form to the ADP-insensitive form. The present results reveal the conditions that definitely discriminate these two phases.