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 apparent molecular masses of 57, 52, 35, 19, 15 and 4.8 kDa, as determined 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'-dicyclohexylcarbodiimide; the 4.8-kDa subunit specifically reacts with N,N'-dicyclohexyl[14C]carbodiimide, indicating that the enzyme is of the F1F0 type. The enzyme activity is dependent on MgATP (Km = 0.4), has a pH optimum of pH 7-9 and is stimulated by sulfite. ATP hydrolysis is strictly dependent on sodium ions with a Km for Na+ of 0.4 mM. The purified enzyme was reconstituted into liposomes. Upon addition of ATP, primary and electrogenic 22Na+ transport into the lumen of the proteoliposomes was determined. These experiments demonstrate that the ATPase of Acetobacterium woodii is a Na(+)-translocating F1F0-type ATPase.
Institut für Mikrobiologie, Göttingen, Germany.
J
J
Reidlinger
V
V
Müller
D000595:Amino Acid Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D016987:Gram-Positive Rods / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*
D008565:Membrane Proteins; D010446:Peptide Fragments; D011510:Proteolipids; C020485:proteoliposomes; D015636:Magnesium Chloride; D012964:Sodium; D006180:Proton-Translocating ATPases