PURIFICATION OF ATP SYNTHASE FROM ACETOBACTERIUM-WOODII AND IDENTIFICATION AS A NA-TRANSLOCATING F1F0-TYPE ENZYME()

The ATPase of Acetobacterium woodii was purified after solubilization of membranes with Triton X-100 by poly(ethylene glycol) precipitation and gel filtration. The enzyme consists of at least six subunits of ap parent molecular masses of 57, 52, 35, 19, 15 and 4.8 kDa, as determin ed by SDS/PAGE. The 52-kDa band is immunologically related to the F1F0 -ATPase beta subunit of Escherichia coli. The enzyme is not inhibited by vanadate but is inhibited by nitrate, azide and N,N'-dicyclohexylca robodiimide; the 4.8-kDa subunit specifically reacts with N,N'-dicyclo hexyl[C-14]carbodiimide, indicating that the enzyme is of the F1F0 typ e. The enzyme activity is dependent on MgATP (K-m = 0.4), has a pH opt imum of pH 7-9 and is stimulated by sulfite. ATP hydrolysis is strictl y dependent on sodium ions with a K-m for Na+ of 0.4 mM. The purified enzyme was reconstituted into liposomes. Upon addition of ATP, primary and electrogenic Na-22(+) transport into the lumen of the proteolipos omes was determined. These experiments demonstrate that the ATPase of Acetobacterium woodii is a Na+-translocating F1F0-type ATPase.