Energy-transducing H+-ATPase of Escherichia coli. Purification, reconstitution, and subunit composition

Foster, D.L.; Fillingame, R.H.

Journal of Biological Chemistry 254(17): 8230-8236

1979


ISSN/ISBN: 0021-9258
PMID: 38249
Accession: 068518681

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Abstract
The energy-transducing ATPase complex (H+-ATPase) in membranes of E. coli was purified to an extent where 8 distinct subunits were observed following electrophoresis on acrylamide gels containing dodecyl sulfate. The complex was solubilized from the membrane with deoxycholate and 1 M KCl. The solubilized extract was fractionated with (NH4)2SO4 and centrifuged on sucrose gradients containing deoxycholate and dispersed phospholipid. The ATPase activity of the purified complex was inhibited by more than 80% with dicyclohexylcarbodiimide [DCCD]. After reconstitution with additional phospholipid and removal of detergent by dialysis, the purified complex demonstrated enhanced [32P]Pi-ATP exchange activity and ATP-driven quenching of quinacrine fluorescence. Both of these energy-transducing activities were blocked by dicyclohexylcarbodiimide. Eight distinct subunits were observed on dodecyl sulfate electrophoresis of the purified complex when it was prepared from cells grown on glucose. Five of these bands corresponded to subunits of the extrinsic (F1) ATPase component of the complex. The other 3 bands electrophoresed with apparent MW of 24,000, 19,000 and 8400 and are likely to be subunits of the Fo sector of the complex. The 8400-dalton subunit was identified as the DCCD-reactive proteolipid. When the complex was purified from cells grown on succinate/acetate/malate rather than glucose as a C source, several additional bands were observed on dodecyl sulfate electrophoresis. All but 1 of these bands were concluded to be contaminants because their amounts varied, relative to the 8 subunits listed above, when different fractions of the sucrose gradient were analyzed. The 14,000-dalton band in this preparation co-purified with invariant stoichiometry and thus cannot be excluded as a possible subunit of the complex in cells grown on succinate/acetate/malate.