Purification of membrane attachment and inhibitory subunits of the proton translocating adenosine triphosphatase from Escherichia coli.
Purification of membrane attachment and inhibitory subunits of the proton translocating adenosine triphosphatase from Escherichia coli.
复制标题
从大肠杆菌中纯化质子转位三磷酸腺苷的膜附着和抑制亚基。
作者：
J. Smith;P. Sternweis
大肠杆菌腺苷三磷酸酶(ATP酶)位于膜周围的部分(ECF1)由称为α、β、γ、δ和ε的五种独立的多肽组成。用吡啶处理纯化的ECF1沉淀了三种较大的多肽(α、β和γ)，但仅占ECF1的约10％的两种较小的多肽(δ和ε)保留在溶液中。除去吡啶后，δ和ε均具有活性，并且在单分子筛柱上进行色谱分离后均获得基本上纯的形式。 epsilon强烈抑制ECF1的ATP酶活性，表明epsilon对该酶具有调节作用。 ε抑制缺少δ的ECF1，表明δ对于ε的抑制来说不是必需的。然而，通过用蛋白酶处理ECF1制备的仅含有α和β亚基的酶具有完全的水解活性，但对ε的抑制完全不敏感。该结果表明γ多肽是ε抑制ATP酶所必需的。 δ恢复了缺失δ的ECF1与ECF1耗尽的膜囊泡重组的能力。通过添加的δ而附着至囊泡的ECF1在能量转导中发挥功能，如ATP水解与囊泡中的转氢酶反应的偶联所证明的。缺失δ的ECF1的重新结合与添加的δ的量成正比，直到膜中的所有ECF1受体被占据。 δ可以是将F1头件连接至膜的杆，因为ECF1至膜的附着表现出对δ的绝对依赖性。尽管在十二烷基硫酸钠存在下通过凝胶电泳已知δ具有约20,000的表观分子量，但从分子筛柱中洗脱出的活性δ具有约35,000的表观分子量，这表明活性形式的δ是二聚体或者更细长的形状。活性ε亚基从同一柱洗脱，表观分子量约为16,000。
The portion of Escherichia coli adenosine triphosphatase (ATPase) which is peripheral to the membrane (ECFl) is composed of five separate polypeptides referred to as alpha, beta, gamma, delta, and epsilon. Treating purified ECFl with pyridine precipitated the three larger polypeptides (alpha, beta, and gamma), but the two smaller ones (delta and epsilon), which represent only about 10% of ECFl, remained in solution. After removing the pyridine, both delta and epsilon were active and both were obtained in essentially pure form after chromatography on a single molecular-seive column. epsilon strongly inhibited the ATPase activity of ECFl, indicating that epsilon has a regulatory role in the enzyme. epsilon inhibited ECFl missing delta, indicating that delta is not required for inhibition by epsilon. However, enzyme containing just the alpha and beta subunits, which was prepared by treating ECFl with a protease, was fully active hydrolytically but not at all sensitive to inhibition by epsilon. This result suggests that the gamma polypeptide is required for the inhibition of the ATPase by epsilon. delta restored the capacity of ECFl missing delta to recombine with ECFl-depleted membrane vesicles. The ECFl, which became attached to the vesicles by the added delta, was functional in energy transduction, as evidenced by the coupling of ATP hydrolysis to the transhydrogenase reaction in the vesicles. The rebinding of ECFl missing delta was directly proportional to the amount of delta added until all the ECFl receptors in the membranes were occupied. delta may be a stalk which connects the Fl headpiece to the membrane, since the attachment of ECFl to the membrane exhibited an absolute dependence on delta. Although delta is known to have an apparent molecular weight of about 20,000 by gel electrophoresis in the presence of sodium dodecyl sulfate, the active delta eluted from a molecular-seive column with an apparent molecular weight of about 35,000, suggesting that in the active form delta is a dimer or rather elongated in shape. The active epsilon subunit eluted from the same column with an apparent molecular weight of about 16,000.