The ATPase of Bacillus alcalophilus. Reconstitution of energy-transducing functions.
The purified ATPase of Bacillus alcalophilus (F1F0) was reconstituted into proteoliposomes by gradual removal of the detergent Triton X-100 with Amberlite XAD-2. The reconstitution was apparently highly asymmetric with nearly 100% of the F1 portion of the ATPase becoming oriented to the outside. Similar to results obtained with the soluble enzyme, the membrane-bound ATPase required Mg2+ and methanol for maximum activity. With Ca2+ or Mg2+ without methanol, 25% and 1%, respectively, of the maximum activity were observed. The ATPase was unable to pump Na+ ions but catalyzed the translocation of protons into the reconstituted proteoliposomes. Optimum proton translocation required the presence of Mg2+, not Ca2+, as divalent metal ion. The proton pump was inhibited by dicyclohexylcarbodiimide, venturicidin and NaN3. On incubation of the reconstituted ATPase with [14C]dicyclohexylcarbodiimide, subunit c of the enzyme complex became specifically labeled. The proteoliposomes catalyzed the Mg2(+)-dependent incorporation of [32P]phosphate into ATP by ATP/[32P]phosphate exchange. This exchange was little affected by monensin, but was completely abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Protons and not Na+ are thus the coupling ions of the ATPase of B. alcalophilus.
Hoffmann
A
A
Mikrobiologisches Institut, Eidgenössischen Technischen Hochschule, Zürich, Switzerland.
Dimroth
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D001693:Biological Transport, Active; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D008081:Liposomes; D008274:Magnesium / Q000378:metabolism; D008567:Membranes, Artificial; D000432:Methanol; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism
D005456:Fluorescent Dyes; D008081:Liposomes; D008567:Membranes, Artificial; D000255:Adenosine Triphosphate; D012964:Sodium; D006180:Proton-Translocating ATPases; D008274:Magnesium; D000432:Methanol