ACTA, A FLUORESCENT ANALOG OF THAPSIGARGIN, IS A POTENT INHIBITOR ANDA CONFORMATIONAL PROBE OF SKELETAL-MUSCLE CA2-ATPASE()

Thapsigargin is a highly potent and selective inhibitor of sarco-endop lasmic reticulum (SERCA) family of Ca2+ ATPases and a useful tool in r esearch concerning the function of intracellular Ca2+ stores. We descr ibe here a novel fluorescent derivative 8-O-(4-aminocinnamoyl)-8-O-deb utanoylthapsigargin, termed ACTA) of this compound, acting as a Ca2+-A TPase inhibitor with a potency approaching that of thapsigargin. Bindi ng of ACTA to the skeletal muscle sarcoplasmic reticulum vesicles resu lts in a strong fluorescence enhancement, approximately 66% of which d epends on ACTA association with Ca2+-ATPase. This specific component o f ACTA fluorescence is sensitive to the E-1-E-2 conformational equilib rium of the pump. The combined properties of high potency and binding- dependent fluorescence suggest ACTA to be a useful probe for a range o f studies involving the SERCA class of ATPases. (C) 1998 Federation of European Biochemical Societies.