Reconstitution of ATP-dependent calcium transport from streptococci

S. V. Ambudkar, A. R. Lynn, P. C. Maloney, B. P. Rosen

Research output: Contribution to journalArticle

Abstract

Membrane vesicles of three streptococcal strains (Streptococcus faecalis, Streptococcus lactis, and Streptococcus sanguis) were extracted with octyl-β-D-glucoside in the presence of Escherichia coli lipid and glycerol. For reconstitution, the detergent extract was mixed with bath-sonicated E. coli lipid, in the presence of octyl-β-D-glucoside, and proteoliposomes were formed by a 25-fold dilution. ATP-dependent calcium accumulation by proteoliposomes was comparable to that found in parent vesicles. Recovery of this calcium transport activity was dependent on the inclusion of an osmolyte protein stabilant (glycerol, etc.) during solubilization. The properties of ATP-driven calcium transport were studied in the reconstituted system. In proteoliposomes, ATP-linked calcium accumulation was not affected by the protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or by the ionophores, valinomycin and nigericin, in the presence of potassium, or by N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. On the other hand, calcium transport was completely blocked by micromolar levels of orthovanadate; half-maximal inhibitions were observed at 0.4, 4, and 4 μM vanadate, for S. faecalis, S. lactis, and S. sanguis, respectively. This marked sensitivity to orthovanadate suggests operation of an E1-E2-type ion-motive pump. These data demonstrate that, in a reconstituted system, calcium transport is not linked to an ATP-dependent proton circulation via the F0-F1-ATPase, but rather is driven by a calcium-translocating ATPase. Thus, calcium extrusion from the cytosol of enteric, lactic acid, or oral streptococci is mediated by an ATP-linked process analogous to the ion-motive ATPases of eukaryotic membranes.

Original languageEnglish (US)
Pages (from-to)15596-15600
Number of pages5
JournalJournal of Biological Chemistry
Volume261
Issue number33
StatePublished - Dec 1 1986

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Reconstitution of ATP-dependent calcium transport from streptococci'. Together they form a unique fingerprint.

  • Cite this

    Ambudkar, S. V., Lynn, A. R., Maloney, P. C., & Rosen, B. P. (1986). Reconstitution of ATP-dependent calcium transport from streptococci. Journal of Biological Chemistry, 261(33), 15596-15600.