Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans
Braz. j. med. biol. res
; 41(12): 1047-1053, Dec. 2008. ilus, tab
Artículo
en Inglés
| LILACS
| ID: lil-502156
Biblioteca responsable:
BR1.1
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 percent and did not allow differentiation between F- and P-type ATPase activities by use of the standard inhibitors vanadate (3 µM) and oligomycin (4 µg/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.
Texto completo:
Disponible
Colección:
Bases de datos internacionales
Base de datos:
LILACS
Asunto principal:
Solventes
/
Streptococcus mutans
/
Tolueno
/
Permeabilidad de la Membrana Celular
/
ATPasas de Translocación de Protón Bacterianas
Idioma:
Inglés
Revista:
Braz. j. med. biol. res
Asunto de la revista:
Biologia
/
Medicina
Año:
2008
Tipo del documento:
Artículo
/
Documento de proyecto
País de afiliación:
Brasil
Institución/País de afiliación:
Universidade de São Paulo/BR
/
Universidade de Uberaba/BR