Purification and Properties of Reconstitutively Active and Inactive Adenosinetriphosphatase from Escherichia coli

Abstract

The Mg2+-and Ca"-stimulated ATPase (EC 3.6.1.3; ATP phosphohydrolase) (bacterial coupling factor) was purified from two strains of E. coli by two different procedures: (a) method of Nelson, Kanner, and Gutnick [Proc. Nat. Acad. Sci. USA (1974) 71, 2720-27241 and (b) a modified procedure described in this paper. The ATPase purified from E. coli K12 (X) by the first procedure had 4 subunits (a, ,, -y, and e). It did not bind to a deficient membrane, nor did it reconstitute ATP-driven transhydrogenase activity. Our modified procedure (b) yielded 5 subunits (a, ,, -y, 6, and e). This ATPase could bind to a deficient membrane and reconstitute ATP-driven transhydrogenase. This finding suggests that the 5 subunit is required for the reaction with the membrane. The molecular weight of the 4-subunit ATPase was significantly lower than that of the 5-subunit ATPase, as judged by equilibrium centrifugation. The specific ATPase activities of both preparations were about the same. These two procedures were also applied to E. coli ML308-225.

Subunit equivalence in Escherichia coli and bovine heart mitochondrial F1F0 ATPases

“…The extra subunit in the rat liver enzyme has a Mr of 26 500 and has properties in common with the bovine oscp. Complexes diminished in 8 have been prepared from E.coli [15] and chloroplasts [30]. So it appears that the partitioning of the 8-subunit in E.coli and chloroplasts or of the oscp in beef heart (and other) mitochondria during extraction of Fl is dependent upon the experimental conditions.…”

“…be almost absent [15]; in others it is present in a 1:1 stoichiometry with the ~,-and c-subunits [16]. Bacterial e-subunit is also important for binding Fl to F0 [17,18].…”

Transmembrane topology of Escherichia coli H+‐ATPase (ATP synthase) subunit a

“…These cells were cultured in a rich medium (L-broth) containing 50 btg/ml ampicillin, harvested by centrifugation, and suspended in 50 mM Tris-C1 buffer (pH 8.0) containing 10 mM MgC12, 0.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.2 mM phenylmethylsulfonylfluoride, 5 ~tg/ml leupeptin and 5 I.tg/ml pepstatin A [17]. Insideout membrane vesicles were prepared by passing cells through a French press [18]. Spheroplasts (right side-out vesicles) were prepared by lysozyme treatment [19], and suspended in phosphate-buffered saline comprising 148 mM NaC1, 2.7 mM KCI, 1.5 mM KH2PO4, 8.1 mM NaHPO4 (pH 7.2) (PBS) containing 20% (w/v) sucrose and 0.1 mg/ml chloramphenicol.…”

“…Published procedures were used for polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate [21], immunoblotting [17], purification of FoF1 [17], ATP-dependent H + transport [17], hydrolysis of ATP [17], and depletion of F1 from inside-out membrane vesicles [18].…”

Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site

“…To prepare F o F " -containing membrane vesicles, strains were grown at 37 mC until mid-exponential phase in minimal medium containing 0.4 % glucose ; membranes were isolated as described previously [40]. Protein concentrations were determined by the method of Lowry et al [41].…”