Inhibition by A23187 of conformational changes involved in the Ca(2+)-induced activation of sarcoplasmic reticulum Ca(2+)-ATPase.
We previously showed that A23187 in high ionophore/protein ratios almost completely inhibits the sarcoplasmic reticulum Ca(2+)-ATPase [Hara, H. & Kanazawa, T. (1986) J. Biol. Chem. 261, 16584-16590]. In an attempt to obtain information on the mechanism of this inhibition, the effects of A23187 on conformational changes involved in the Ca(2+)-induced activation of the enzyme were investigated. The purified enzyme from sarcoplasmic reticulum of rabbit skeletal muscle as well as the purified enzyme labeled with fluorescein 5-isothiocyanate (FITC) were preincubated with A23187 in the absence of Ca2+ at pH 7.0 and 0 degrees C for 45 min. The activation of the enzyme following addition of CaCl2 was assessed by determining the capacity for rapid formation of phosphoenzyme from ATP. This activation was strongly inhibited by the preincubation with A23187. This indicates that the previously observed inhibition of the Ca(2+)-ATPase is mostly due to hindrance of the Ca(2+)-induced activation of the enzyme. In the control, in which the FITC-labeled enzyme was preincubated without A23187, the fluorescence intensity of the bound FITC decreased in a biphasic manner upon addition of CaCl2. The first rapid phase of this fluorescence drop was unaffected by A23187, whereas its second slow phase was almost completely inhibited by this drug. These results show that the Ca(2+)-dependent conformational change is biphasic and that the second slow phase (but not the first rapid phase) of this conformational change is inhibited by A23187. This suggests that the observed inhibition of Ca2+ activation is attributed to hindrance of the second slow phase of the Ca(2+)-dependent conformational change.
Ohmiya
H
H
Department of Biochemistry, Asahikawa Medical College, Hokkaido.
Kanazawa
T
T
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate; D066298:In Vitro Techniques; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D000001:Calcimycin; D004533:Egtazic Acid; D014364:Tryptophan; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium