Synthesis and interaction of fluorescent thapsigargin derivatives with the sarcoplasmic reticulum ATPase membrane-bound region.

Fluorescent derivatives of thapsigargin (TG) were synthesized by replacing the C8-butanoyl chain with a dansyl (DTG) or eosin (ETG) moiety. DTG and ETG retain the inhibitory effect of TG on the sarcoplasmic reticulum (SR) ATPase, displaying a 2 and 10 microM Ki, respectively. Steady state and lifetime fluorescence measurements are consistent with energy transfer between tryptophanyl residues assigned to the ATPase membrane-bound region and DTG. This phenomenon exhibits saturation behavior, occurs in the presence of DTG concentrations producing ATPase inhibition, and is partially prevented by inhibitory concentrations of TG. Although long range conformational effects of TG binding affect the fluorescence properties of endogenous tryptophans as well as of a fluorescein 5'-isothiocyanate (FITC) label of the ATPase extramembranous region, no significant energy transfer was detected between DTG and the FITC label. It is concluded that the inhibitors partition within the membrane and the binding domain resides within or near the membrane-bound region of the ATPase.