Purification and reconstitution of the F1F0-ATP synthase from alkaliphilic Bacillus firmus OF4. Evidence that the enzyme translocates H+ but not Na+.

摘要: The F1F0-ATP synthase from the alkaliphilic Bacillus firmus OF4 was purified in a reconstitutively active form, good yield and with high specific ATPase activity when appropriately activated. purification procedure involved octyl glucoside extraction of washed membrane vesicles presence 20% glycerol asolectin followed by ammonium sulfate fractionation sucrose density gradient centrifugation. preparation resolved into seven bands sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to five F1 subunits, alpha, beta, gamma, delta, epsilon, b c subunits F0. Two-dimensional sulfate-poly-acrylamide analysis revealed candidate for alpha subunit MgATPase B. F1F0 barely detectable but could be stimulated, optimally more than 100-fold, sulfite, methanol, thioglucoside. enzyme inhibited N,N'-dicyclohexylcarbodiimide azide, not aurovertin, an inhibitor Escherichia coli. reconstituted proteoliposomes catalyzed activity, ATP-Pi exchange, ATP-dependent delta pH psi formation. ATP hydrolysis stimulated protonophores while other activities were abolished protonophores. These neither dependent on added ions nor significantly affected them. made crude extracts that also contained Na+/H+ antiporter shown catalyze Na+ uptake completely sensitive carbonyl cyanide m-chlorophenyl-hydrazone; absent containing lacking antiporter. data show translocates protons does substitute H+ energy coupling.