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 [C-14]dicyclohexylcarbodiimide, subunit c of the enzyme complex became specifically labeled. The proteoliposomes catalyzed the Mg2+ -dependent incorporation of [P-32]phosphate into ATP by ATP/[P-32]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.