Purification and properties of the ATPase solubilized from membranes of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius.
Purification and properties of the ATPase solubilized from membranes of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius.
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从嗜酸热古细菌（酸热硫化叶菌）膜中溶解的 ATP 酶的纯化和特性。
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发表时间：
1987
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通讯作者：
M. Yoshida
中科院分区：
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作者：
Jin Konishi;T. Wakagi;Tairo Oshima;M. Yoshida
一种新的ATP酶溶解从嗜酸热古细菌，硫化叶菌acidocaldarius，与低离子强度缓冲液含有EDTA的膜。通过疏水层析和凝胶过滤将酶纯化至均一。纯化的酶的分子量估计为360，000。在十二烷基硫酸钠存在下的纯化的酶的聚丙烯酰胺凝胶电泳显示，它由三种亚基，α，β和γ，其分子量分别约为69，000，54，000和28，000，和最可能的亚基化学计量是α 3 β 3 γ 1。纯化的ATP酶水解ATP、GTP、ITP和CTP，但不水解UTP、ADP、AMP和对硝基苯磷酸。该酶具有高度的热稳定性，最适温度为85 ℃。它的最佳pH值约为5，在中性pH值下活性很低，在pH 8.5处有另一个小活性峰。亚硫酸氢盐和碳酸氢盐离子显著刺激ATP酶活性，最适pH保持不变。Lineweaver-Burk图是线性的，ATP的Km和Vmax估计为1.6 mM和13 mumol Pi.mg.。1.min-1，在pH 5.2，60 ℃，在亚硫酸氢盐的存在下。化学修饰试剂7-氯-4-硝基苯并-2-氧杂-1，3-二唑使ATP酶活性失活，但酶不受N，N ′-二环己基碳二亚胺、N-乙基马来酰亚胺、叠氮化物或钒酸盐的抑制。这些结果表明，从S. acidocaldarius类似于其他古细菌ATP酶，尽管在后者中尚未发现γ亚基的对应物。S.讨论了酸热菌ATP酶与其它离子转运ATP酶如F0F1型或E1E2型ATP酶的相互作用。
A novel ATPase was solubilized from membranes of an acidothermophilic archaebacterium, 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 presence of sodium dodecyl sulfate revealed that it consisted of three kinds of subunits, alpha, beta, and gamma, whose molecular weights were approximately 69,000, 54,000, and 28,000, respectively, and the most probable subunit stoichiometry was alpha 3 beta 3 gamma 1. 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 degrees 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 mumol Pi.mg.-1.min-1, respectively, at pH 5.2 at 60 degrees 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 gamma 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.