Abstract
A novel ATPase was solubilized from membranes of an acidothermophilic archae-bacterium, Sulfolobus acidocaldarius, with low ionic strength buffer containing EDTA. The enzyme was purified to homogeneity by hydrophobic chromatography and gel filtration. The molecular weight of the purified enzyme was estimated to be 360, 000. Polyacrylamide gel electrophoresis of the purified enzyme in the pres-ence of sodium dodecyl sulfate revealed that it consisted of three kinds of subunits, α, β, and γ, whose molecular weights were approximately 69, 000, 54, 000, and 28, 000, respectively, and the most probable subunit stoichiometry was α3β3γ1. The purified ATPase hydrolyzed ATP, GTP, ITP, and CTP but not UTP, ADP, AMP, or p-nitrophenylphosphate. The enzyme was highly heat stable and showed an optimal temperature of 85°C. It showed an optimal pH of around 5, very little activity at neutral pH, and another small activity peak at pH 8.5. The ATPase activity was significantly stimulated by bisulfite and bicarbonate ions, the optimal pH remaining unchanged. The Lineweaver-Burk plot was linear, and the Km for ATP and the Vmax were estimated to be 1.6 mM and 13 μmol P1•mg•-1•min-1, respectively, at pH 5.2 at 60°C in the presence of bisulfite. The chemical modification reagent, 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole, caused inactivation of the ATPase activity although the enzyme was not inhibited by N, N'-dicyclohexylcarbodiimide, N-ethyl-maleimide, azide or vanadate. These results suggest that the ATPase purified from membranes of S. acidocaldarius resembles other archaebacterial ATPases, although a counterpart of the γ subunit has not been found in the latter. The relationship of the S. acidocaldarius ATPase to other ion-transporting ATPases, such as F0F1, type or E1E2 type ATPases, was discussed.
