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Inhibition of sarcoplasmic reticulum Ca2+-ATPase by miconazole
Antonio Lax1, Fernando Soler, Francisco Fernandez-Belda
1Departamento de Bioquímica y Biología Molecular A, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Espinardo, Murcia, Spain.
Abstract:
The inhibition of sarcoplasmic reticulum Ca2+-ATPase activity by miconazole was dependent on the concentration of ATP and membrane protein. Half-maximal inhibition was observed at 12 microM miconazole when the ATP concentration was 50 microM and the membrane protein was 0.05 mg/ml. When ATP was 1 mM, a low micromolar concentration of miconazole activated the enzyme, whereas higher concentrations inhibited it. A qualitatively similar response was observed when Ca2+ transport was measured. Likewise, the half-maximal inhibition value was higher when the membrane concentration was raised. Phosphorylation studies carried out after sample preequilibration in different experimental settings shed light on key partial reactions such as Ca2+ binding and ATP phosphorylation. The miconazole effect on Ca2+-ATPase activity can be attributed to stabilization of the Ca2+-free enzyme conformation giving rise to a decrease in the rate of the Ca2+ binding transition. The phosphoryl transfer reaction was not affected by miconazole.
Insights
Miconazole affects sarcoplasmic reticulum Ca2+-ATPase activity and calcium transport. Its impact depends on ATP and protein concentrations, potentially stabilizing enzyme conformations.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzyme Kinetics
Background:
- Sarcoplasmic reticulum Ca2+-ATPase (SERCA) is crucial for muscle contraction.
- Miconazole is an antifungal agent with known interactions with cellular processes.
Purpose of the Study:
- To investigate the effect of miconazole on sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity.
- To elucidate the mechanism underlying miconazole's interaction with SERCA.
Main Methods:
- Enzyme kinetics assays measuring Ca2+-ATPase activity.
- Calcium transport measurements.
- Phosphorylation studies to analyze reaction intermediates.
Main Results:
- Miconazole's inhibition of SERCA activity is concentration-dependent on ATP and membrane protein.
- Miconazole exhibited biphasic effects: activation at low concentrations and inhibition at higher concentrations with 1 mM ATP.
- Ca2+ binding was identified as the primary target of miconazole's inhibitory action, stabilizing a Ca2+-free enzyme conformation.
Conclusions:
- Miconazole modulates SERCA activity by altering Ca2+ binding kinetics.
- The drug's effect is influenced by substrate and protein concentrations.
- Miconazole does not affect the phosphoryl transfer step in the SERCA catalytic cycle.