Serrahima-Zieger M, Monteil H
Biochim Biophys Acta. 1978 Jun 8;502(3):445-57. doi: 10.1016/0005-2728(78)90077-4.
The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step. The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s20,w of 13,4 and an amino acid composition very similar to bacterial ATPases already studied. After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (alpha) and 57 000 (beta) with different charges. Kinetic studies showed that Ca2+ or Zn2+ are required for ATPase activity, although Mg2+ was uneffective. At optimal Ca2+ concentration, the Mg2+ has an inhibitory effect. The Km for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na+ + K+)-ATPase are studied.
枯草芽孢杆菌的膜ATP酶（EC 3.6.1.3）可通过冲击洗涤过程溶解。本文报道了两种纯化溶解酶的方法。在溶解和纯化步骤中引入了一种蛋白酶抑制剂——苯甲基磺酰氟。通过密度梯度离心法纯化得到的ATP酶分子量为315000，沉降系数s20,w为13.4，其氨基酸组成与已研究的细菌ATP酶非常相似。在十二烷基硫酸钠（SDS）、8M尿素或SDS - 尿素存在的情况下进行聚丙烯酰胺凝胶电泳后，纯化的ATP酶可解离为分子量分别为59000（α）和57000（β）且电荷不同的两个不同亚基。动力学研究表明，ATP酶活性需要Ca2+或Zn2+，而Mg2+无效。在最佳Ca2+浓度下，Mg2+具有抑制作用。ATP的米氏常数为1.3 mM。本文还研究了氧化磷酸化、线粒体ATP酶和（Na+ + K+）-ATP酶的抑制剂。
