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Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of
G Thedei1, D P S Leitão, M Bolean
1Universidade de Uberaba, Uberaba, MG, Brasil.
Abstract:
Streptococcus mutans membrane-bound P- and F-type ATPases are responsible for H+ extrusion from the cytoplasm thus keeping intracellular pH appropriate for cell metabolism. Toluene-permeabilized bacterial cells have long been used to study total membrane-bound ATPase activity, and to compare the properties of ATPase in situ with those in membrane-rich fractions. The aim of the present research was to determine if toluene permeabilization can significantly modify the activity of membrane-bound ATPase of both F-type and P-type. ATPase activity was assayed discontinuously by measuring phosphate release from ATP as substrate. Treatment of S. mutans membrane fractions with toluene reduced total ATPase activity by approximately 80% and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 microM) and oligomycin (4 microg/mL). Transmission electron microscopy shows that, after S. mutans cells permeabilization with toluene, bacterial cell wall and plasma membrane are severely injured, causing cytoplasmic leakage. As a consequence, loss of cell viability and disruption of H+ extrusion were observed. These data suggest that treatment of S. mutans with toluene is an efficient method for cell disruption, but care should be taken in the interpretation of ATPase activity when toluene-permeabilized cells are used, because results may not reflect the real P- and F-type ATPase activities present in intact cell membranes. The mild conditions used for the preparation of membrane fractions may be more suitable to study specific ATPase activity in the presence of biological agents, since this method preserves ATPase selectivity for standard inhibitors.
Insights
Toluene permeabilization significantly damages Streptococcus mutans cells, reducing membrane-bound ATPase activity and hindering the differentiation of P- and F-type ATPases. This method is unsuitable for accurate ATPase studies.
Area of Science:
- Microbiology
- Biochemistry
Background:
- Streptococcus mutans utilizes membrane-bound P- and F-type ATPases for H+ extrusion, maintaining intracellular pH crucial for cell metabolism.
- Toluene-permeabilized bacterial cells are traditionally used to study ATPase activity in situ and in membrane fractions.
Purpose of the Study:
- To investigate the impact of toluene permeabilization on the activity of membrane-bound P- and F-type ATPases in Streptococcus mutans.
- To assess whether toluene treatment affects the ability to differentiate between F- and P-type ATPase activities using specific inhibitors.
Main Methods:
- ATPase activity was measured by quantifying phosphate release from ATP.
- Streptococcus mutans cells were permeabilized using toluene.
- Transmission electron microscopy was employed to examine cellular damage.
- Standard inhibitors, vanadate and oligomycin, were used to differentiate ATPase types.
Main Results:
- Toluene treatment reduced total ATPase activity by approximately 80%.
- Toluene permeabilization prevented the differentiation of F- and P-type ATPase activities using standard inhibitors.
- Transmission electron microscopy revealed severe injury to the cell wall and plasma membrane, leading to cytoplasmic leakage, loss of viability, and disrupted H+ extrusion.
Conclusions:
- Toluene permeabilization causes significant cellular damage in Streptococcus mutans, compromising ATPase activity and inhibitor selectivity.
- While effective for cell disruption, toluene permeabilization is not suitable for accurately studying P- and F-type ATPase activities in situ.
- Mild preparation methods for membrane fractions are recommended for preserving ATPase selectivity and studying specific activities in the presence of biological agents.
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