Murphy A J
Biochemistry. 1976 Oct 5;15(20):4492-6. doi: 10.1021/bi00665a025.
Modification of calcium-translocating sarcoplasmic reticulum membranes (SR) with 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) reveals four classes (kinetic sets) of sulfhydryl groups. Of the 25 mol/1.5 X 10(5) G OF SR protein (i.e., containing 1 mol of ATPase protein) estimated in the presence of sodium dodecyl sulfate, 8 mol are unreactive, while 7, 8, and 2 mol display pseudo-first-order rate constants (k1) of 0.16, 0.68, and 8.3 min(-1), respectively (25 decrees C, pH 7.8, 4 MM Nbs2). Under these conditions, the Ca-ATPase activity is lost with k1 = 0.73 min(-1), whereas the Ca-independent ATPase activity is essentially unchanged. These results are little changed by the presence of Mg2+ or Ba2+ in the modification mixture, while Ca2+ or Sr2+ causes all 16-17 reactable sulfhydryls to be modified with k1 = 0.50 and 0.53 min(-1), respectively. The corresponding values for the loss of Ca-ATPase activity are 0.53 and 0.67 min(-1); this suggests that blocking of only one of the 16-17 SH groups inactivates the enzyme, i.e., that there is a single "essential" SH group. The midpoint of the transition between the Ca2+-free and Ca2+-modification patterns occurs at a free Ca2+ concentration of about 0.9 muM, implying that it is Ca2+ binding at the active sites (KD = 0.1 muM), rather than at the low-affinity nonspecific sites, that effects a conformation change in the ATPase protein (which contains greater than 90% of the cysteines). A calcium-induced conformation change is also suggested by increased ultraviolet absorbance spectrum of the purified ATPase protein upon calcium binding. If protein-lipid interaction is disrupted with deoxycholate or Triton X-100 (which does not destroy the Ca-ATPase activity and hence presumably leaves the tertiary structure of the ATPase protein largely intact), 95% of the sulfhydryls react with Nbs2 considerably faster; thus, at 2 mg/ml o- deoxycholate, 14 groups react with k1 greater than 20, 5 with k1 = 2.3, and 5 with k1 = 0.4 min(-1). These results suggest that the inaccessibility of SH groups in the absence of detergents is due to extensive interaction of the bilayer phospholipids with the ATPase protein.
用5,5'-二硫代双（2-硝基苯甲酸）（Nbs2）修饰钙转运肌浆网（SR）膜，可揭示四类（动力学组）巯基。在十二烷基硫酸钠存在下估计每1.5×10⁵克SR蛋白（即含1摩尔ATP酶蛋白）有25摩尔，其中8摩尔无反应，而7、8和2摩尔的假一级速率常数（k1）分别为0.16、0.68和8.3分钟⁻¹（25℃，pH 7.8，4毫摩尔Nbs2）。在此条件下，钙ATP酶活性以k1 = 0.73分钟⁻¹丧失，而钙非依赖性ATP酶活性基本不变。修饰混合物中存在Mg²⁺或Ba²⁺时，这些结果变化不大，而Ca²⁺或Sr²⁺使所有16 - 17个可反应巯基分别以k1 = 0.50和0.53分钟⁻¹被修饰。钙ATP酶活性丧失的相应值为0.53和0.67分钟⁻¹；这表明仅阻断16 - 17个SH基团中的一个就会使酶失活，即存在一个单一的“必需”SH基团。无钙和钙修饰模式之间转变的中点出现在游离钙浓度约为0.9微摩尔处，这意味着是活性位点（KD = 0.1微摩尔）而非低亲和力非特异性位点的钙结合导致ATP酶蛋白（含超过90%的半胱氨酸）发生构象变化。钙结合后纯化的ATP酶蛋白紫外吸收光谱增加也表明存在钙诱导的构象变化。如果用脱氧胆酸盐或 Triton X - 100破坏蛋白质 - 脂质相互作用（这不会破坏钙ATP酶活性，因此推测ATP酶蛋白的三级结构基本保持完整），95%的巯基与Nbs2反应快得多；因此，在2毫克/毫升脱氧胆酸盐存在下，14个基团以k1大于20反应，5个以k1 = 2.3反应，5个以k1 = 0.4分钟⁻¹反应。这些结果表明，在没有去污剂时SH基团难以接近是由于双层磷脂与ATP酶蛋白的广泛相互作用。
