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Inhibition by A23187 of conformational changes involved in the Ca(2+)-induced activation of sarcoplasmic reticulum
1Department of Biochemistry, Asahikawa Medical College, Hokkaido.
Insights
The ionophore A23187 inhibits sarcoplasmic reticulum Ca(2+)-ATPase by hindering its Ca(2+)-induced activation. Specifically, A23187 blocks the second slow phase of the enzyme
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Previous studies demonstrated that A23187 significantly inhibits sarcoplasmic reticulum Ca(2+)-ATPase at high ionophore/protein ratios.
- Understanding the mechanism of this inhibition is crucial for elucidating enzyme function and regulation.
Purpose of the Study:
- To investigate the effects of A23187 on the conformational changes associated with Ca(2+)-induced activation of sarcoplasmic reticulum Ca(2+)-ATPase.
- To determine which phase of the Ca(2+)-dependent conformational change is affected by A23187.
Main Methods:
- Purified rabbit skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase, both native and labeled with fluorescein 5-isothiocyanate (FITC), was used.
- Enzymes were preincubated with A23187 in the absence of Ca(2+).
- Ca(2+)-induced activation was assessed by measuring phosphoenzyme formation from ATP, and conformational changes were monitored by FITC fluorescence changes upon CaCl(2) addition.
Main Results:
- Preincubation with A23187 strongly inhibited the Ca(2+)-induced activation of the enzyme, indicating hindrance of activation.
- A23187 did not affect the first rapid phase of Ca(2+)-dependent fluorescence decrease in FITC-labeled enzyme.
- The second slow phase of the Ca(2+)-dependent fluorescence decrease was almost completely inhibited by A23187.
Conclusions:
- The Ca(2+)-dependent conformational change of sarcoplasmic reticulum Ca(2+)-ATPase is a biphasic process.
- A23187 inhibits the Ca(2+)-ATPase by specifically hindering the second slow phase of the Ca(2+)-dependent conformational change.
- This study provides mechanistic insight into A23187 inhibition of Ca(2+)-ATPase activity.
Abstract:
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.