Reithmeier R A, MacLennan D H
J Biol Chem. 1981 Jun 25;256(12):5957-60.
The (Ca2+ + Mg2+)-adenosine triphosphatase (ATPase) of sarcoplasmic reticulum contains a cysteine residue at position 12 of its sequence. This sulfhydryl group was 1 out of a total of 10-11 that were labeled by treatment of sarcoplasmic reticulum vesicles with N-[3H]ethylmaleimide under saturating conditions. This was shown by isolating a 31-residue NH2-terminal peptide from a tryptic digest of the succinylated ATPase, prepared from N-[3H]ethylmaleimide-labeled vesicles. Reaction of the vesicles with glutathione maleimide, parachloromercuribenzoic acid, or parachloromercuriphenyl sulfonic acid, membrane-impermeant reagents, prevented further reaction of sulfhydryl groups with N-ethylmaleimide. This result indicates that all sulfhydryl groups that are reactive with N-ethylmaleimide are on the outside of the vesicles. Since Cys12 is located in a hydrophilic NH2-terminal portion of the ATPase, the labeling results suggest that the NH2 terminus of the ATPase is on the cytoplasmic side of the membrane. These results are consistent with earlier observations (Reithmeier, R. A. F., de Leon, S., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 11839-11846) that the (Ca2+ + Mg2+)-ATPase is synthesized without an NH2-terminal signal sequence.
肌浆网的（Ca2+ + Mg2+）-腺苷三磷酸酶（ATP酶）在其序列的第12位含有一个半胱氨酸残基。在饱和条件下用N-[3H]乙基马来酰亚胺处理肌浆网小泡时，该巯基是总共10 - 11个被标记的巯基之一。这是通过从由N-[3H]乙基马来酰亚胺标记的小泡制备的琥珀酰化ATP酶的胰蛋白酶消化物中分离出一个31个残基的NH2末端肽段来证明的。小泡与谷胱甘肽马来酰亚胺、对氯汞苯甲酸或对氯汞苯磺酸（膜不透性试剂）反应，可阻止巯基与N-乙基马来酰亚胺的进一步反应。这一结果表明，所有与N-乙基马来酰亚胺反应的巯基都在小泡的外侧。由于半胱氨酸12位于ATP酶的亲水性NH2末端部分，标记结果表明ATP酶的NH2末端在膜的细胞质一侧。这些结果与早期的观察结果（Reithmeier，R.A.F.，de Leon，S.，和MacLennan，D.H.（1980）J.Biol.Chem.255，11839 - 11846）一致，即（Ca2+ + Mg2+）-ATP酶在合成时没有NH2末端信号序列。
