Membrane-associated and Solubilized ATPases of Streptococcus mutans and Streptococcus sanguis

Abstract

The proton-translocating, membrane ATPases of oral streptococci have been implicated in cytoplasmic pH regulation, acidurance, and cariogenicity. Membranes were isolated from Streptococcus mutans GS-5 and Streptococcus sanguis NCTC 10904 following salt-induced lysis of cells treated with lysozyme and mutanolysin. The ATPase activities of these membranes were 1.8 and 1.1 units per mg membrane protein, respectively. F₁ ATPases were washed free from the membranes and purified by fast protein liquid chromatography (FPLC). Hydrolytic activities of the F ₁ ATPases were maximal at pH values between 6.0 and 6.6, whereas the membrane-bound enzymes had pH maxima of 7.5 (S. sanguis) and 6.0 (S. mutans). The F₁ ATPases of the streptococci were similar to the well-characterized enzyme of Escherichia coli; they consisted of five different polypeptides and had apparent, aggregate molecular weights of from 335 to 350 Kd. The membrane-bound ATPases were characterized biochemically and found to be similar to those of proton-translocating ATPases of E. coli and Streptococcus faecalis. K_m values for the membranes with respect to ATP were found to be 0.9 and 1.0 mmol/L for S. mutans and S. sanguis, respectively. Both enzymes had specificities for purine triphosphates and were active with a variety of divalent cations, although optimal activity occurred with ATP and Mg. The membrane-associated enzymes were sensitive to the inhibitors dicyclohexylcarbodiimide (DCCD) and azide, but insensitive to ouabain and vanadate. Overall, it appears that the membrane-associated ATPases of S. mutans and S. sanguis are similar to the extensively studied proton-translocating ATPases of E. coli and S. faecalis, and that differences in pH sensitivities of the enzymes from the oral bacteria are greater for the membrane-bound, proton-translocating forms than for the soluble, F, forms.