THE TRYPTOPHAN FLUORESCENCE CHANGE UPON CONFORMATIONAL TRANSITION OF THE PHOSPHOENZYME INTERMEDIATE IN SARCOPLASMIC-RETICULUM CA2-ATPASE ISREVEALED IN THE ABSENCE OF K+ AND THE PRESENCE OF LASALOCID()

ATP-induced changes in the tryptophan fluorescence of the Ca2+-ATPase were determined with sarcoplasmic reticulum vesicles at pH 7.0 and 0 d egrees C by steady-state measurements in the presence of Ca2+ and the absence of K+ with and without added lasalocid (a carboxylic ionophore , 50 mu m), which was previously shown to cause a predominant accumula tion of the ADP-insensitive form of the phosphoenzyme intermediate (EP ) (Kawashima, T., Hare, H., and Kanazawa, T. (1990) J. Biol, Chem. 265 , 10993-10999), When ATP was added in the absence of lasalocid, the fl uorescence decreased by 1.7%. The addition of lasalocid quenched 71% o f the fluorescence but did not reduce the ATP-induced fluorescence dro p. 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 m uch 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 treat ment, the second phase disappeared, and the fluorescence drop entirely coincided with the formation of ADP sensitive EP. These findings demo nstrate 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-insensi tive form, The present results reveal the conditions that definitely d iscriminate these two phases.