Thorley-Lawson D A, Green N M
Biochem J. 1977 Dec 1;167(3):739-48. doi: 10.1042/bj1670739.
The ATPase (adenosine triphosphatase) from sarcoplasmic reticulum contains 20 thiol groups/115000 daltons, measured by using either N-ethyl[(14)C]maleimide or 5,5'-dithiobis-(2-nitrobenzoate) in sodium dodecyl sulphate. After reduction there were 26 thiol groups, in good agreement with 26.5 residues of cysteic acid found by amino acid analysis. The difference between this and the 20 residues measured before reduction implies the presence of three disulphide residues. The same number of disulphide residues was found by direct measurement. Three to six fewer thiol groups were found in preparations made in the absence of dithiothreitol. The missing residues were accounted for as cysteic acid. The distribution of disulphide bonds and of exposed and buried thiol groups among the tryptic fragments of the molecule was measured after labelling with N-ethyl[(14)C]-maleimide. The disulphides were confined to fragment B (mol.wt. 55000), whereas several thiol groups were present on each of the fragments (A, B, A(1) and A(2)). The kinetics of the reaction of the ATPase with 5,5'-dithiobis-(2-nitrobenzoate) showed that four or five of the thiol groups were unreactive in the absence of detergent and that 13 of the remainder reacted with a single first-order rate constant. In the presence of ATP and Ca(2+) the reaction rate of all but two groups of this class was uniformly decreased. In the presence or absence of ATP and Ca(2+) the rate constant for inactivation was close to the rate constant for this class, but was not identical with it. No selective protection of a specific active-site-thiol group was observed. Parallel experiments with sarcoplasmic reticulum gave similar results, except that the reaction rates were a little lower and there were two more buried groups. Solution of ATPase of sarcoplasmic reticulum in detergent greatly increased the reactivity of all thiol groups. The effects of low concentrations of deoxycholate were reversible. EGTA or low concentrations (0.02mm) of Ca(2+) of Mg(2+) had very little effect on the reactivity.
肌质网的ATP酶（腺苷三磷酸酶）含有20个巯基/115000道尔顿，这是通过在十二烷基硫酸钠中使用N-乙基[（14）C]马来酰亚胺或5,5'-二硫代双（2-硝基苯甲酸）测量得到的。还原后有26个巯基，与氨基酸分析发现的26.5个半胱氨酸残基高度一致。这与还原前测得的20个残基之间的差异意味着存在三个二硫键残基。通过直接测量也发现了相同数量的二硫键残基。在没有二硫苏糖醇的情况下制备的制剂中发现巯基少三到六个。缺失的残基被认为是半胱氨酸。用N-乙基[（14）C] - 马来酰亚胺标记后，测量了分子胰蛋白酶片段中二硫键以及暴露和埋藏的巯基的分布。二硫键局限于片段B（分子量55000），而每个片段（A、B、A1和A2）上都存在几个巯基。ATP酶与5,5'-二硫代双（2-硝基苯甲酸）反应的动力学表明，在没有去污剂的情况下，有四或五个巯基无反应性，其余的13个以单一的一级速率常数反应。在ATP和Ca（2+）存在下，这类中除两个基团外的所有基团的反应速率均均匀降低。在有或没有ATP和Ca（2+）的情况下，失活速率常数接近这类的速率常数，但与之并不相同。未观察到对特定活性位点巯基的选择性保护。对肌质网进行的平行实验给出了类似的结果，只是反应速率略低，并且有两个更多的埋藏基团。肌质网ATP酶在去污剂中的溶解大大增加了所有巯基的反应性。低浓度脱氧胆酸盐的作用是可逆的。EGTA或低浓度（0.02mM）的Ca（2+）或Mg（2+）对反应性影响很小。
