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Staphylococcus aureus adenosine triphosphatase: inhibitor sensitivity and release from membrane
Abstract:
Homogeneous preparations of cytoplasmic membrane isolated from Staphylococcus aureus 6538P exhibited membrane-associated adenosine triphosphatase (ATPase) activity. Membrane ATPase activity was activated by divalent cations (4.0 mM: Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+), and ATP was hydrolyzed more readily than other nucleoside triphosphates and phosphorylated substrates. The pH optimum for the membrane ATPase was 6.5. The ATPase could not be released from the membrane by differential osmotic treatments, but detergent treatment effectively solubilized active enzyme. The nonionic detergent Triton X-100 (1%) released a protein with ATPase activity, after substrate-dependent staining in polyacrylamide gels, that differed slightly in electrophoretic migration when compared to the active enzyme solubilized with sodium dodecyl sulfate (0.1%). Membrane-associated ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (0.001 to 1 mM) and NaF (50% inhibition at 5 mM NaF). Azide and trypsin inhibited activity, whereas ouabain had a slight inhibitory effect. Diethylstilbestrol showed appreciable activation of the membrane ATPase over the range employed (0.001 to 1 mM).
Insights
Staphylococcus aureus cytoplasmic membranes possess an adenosine triphosphatase (ATPase) enzyme. This membrane-bound ATPase is activated by divalent cations and influenced by pH, detergents, and specific inhibitors.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Cytoplasmic membranes of Staphylococcus aureus harbor enzymatic activities crucial for cellular functions.
- Understanding membrane-associated enzymes like adenosine triphosphatase (ATPase) is vital for comprehending bacterial physiology.
Purpose of the Study:
- To characterize the membrane-associated adenosine triphosphatase (ATPase) activity in Staphylococcus aureus.
- To investigate the properties and modulators of this bacterial membrane ATPase.
Main Methods:
- Isolation of homogeneous cytoplasmic membrane preparations from Staphylococcus aureus.
- Assay of ATPase activity under varying conditions (divalent cations, pH, substrates).
- Solubilization of the enzyme using detergents (Triton X-100, SDS) and characterization via polyacrylamide gel electrophoresis.
- Assessment of inhibition and activation by various chemical agents (N,N'-dicyclohexylcarbodiimide, NaF, azide, trypsin, ouabain, diethylstilbestrol).
Main Results:
- Membrane-associated ATPase activity was observed, with optimal activity at pH 6.5.
- Activity was activated by divalent cations, with Mg2+ showing the highest effect, and preferred ATP hydrolysis.
- The enzyme was detergent-solubilized, and its electrophoretic behavior differed slightly depending on the detergent used.
- Specific inhibitors (N,N'-dicyclohexylcarbodiimide, NaF, azide, trypsin) and an activator (diethylstilbestrol) were identified.
Conclusions:
- Staphylococcus aureus possesses a distinct membrane-associated ATPase enzyme.
- The enzyme's characteristics, including cation dependency, pH optimum, and susceptibility to inhibitors/activators, provide insights into its function.
- Detergent-based solubilization and electrophoretic analysis offer methods for further purification and characterization of this important bacterial enzyme.