Hiatt A, McDonough A A, Edelman I S
J Biol Chem. 1984 Feb 25;259(4):2629-35.
Guinea pig kidney poly(A+) RNA was translated in reticulocyte lysates and wheat germ extracts. Antibodies to the holoenzyme (Na/K-ATPase) immunoprecipitated only a 96,000-dalton product which was identified as the alpha subunit with a molecular weight that was indistinguishable from that of mature alpha subunit. To explore the possibility that the primary translational product is integrated as such into membranes, guinea pig kidney poly(A+) RNA was translated in reticulocyte lysates in the presence of dog pancreas microsomes; two immunoprecipitated products were detected, the 96,000-dalton alpha subunit and a 135,000-dalton new component that was integrated into the microsomal membrane since it was completely resistant to extraction with alkali. Addition of purified alpha subunit inhibited the binding of antibody to the 135,000-dalton product and extraction with urea-sodium dodecyl sulfate recovered the 96,000-dalton product, implying that the 135,000-dalton product was an alpha-chi dimer. Translation of size-fractionated poly(A+) RNA yielded evidence that the 135,000-dalton product is encoded in two separate mRNAs. The integration in vitro of the alpha subunit is, therefore, dependent on the co-translational integration into the membranes of a smaller peptide (35,000 to 40,000 daltons) which is presumably the beta subunit. Evidence was also obtained that this mechanism is present in vivo by isolation of mRNA alpha from free polysomes, as well as detection of the cytosolic form of the alpha subunit in pulse-chase experiments in MDCK cells.
豚鼠肾多聚腺苷酸（poly(A+)）RNA在网织红细胞裂解物和小麦胚芽提取物中进行翻译。针对全酶（钠/钾-ATP酶）的抗体免疫沉淀出仅一种96,000道尔顿的产物，该产物被鉴定为α亚基，其分子量与成熟α亚基的分子量无法区分。为了探究初级翻译产物是否以这种形式整合到膜中，豚鼠肾多聚腺苷酸（poly(A+)）RNA在犬胰腺微粒体存在的情况下于网织红细胞裂解物中进行翻译；检测到两种免疫沉淀产物，96,000道尔顿的α亚基和一种135,000道尔顿的新组分，该新组分整合到微粒体膜中，因为它完全耐受碱提取。添加纯化的α亚基抑制了抗体与135,000道尔顿产物的结合，并且用尿素-十二烷基硫酸钠提取回收了96,000道尔顿的产物，这意味着135,000道尔顿的产物是α-χ二聚体。对大小分级的多聚腺苷酸（poly(A+)）RNA进行翻译得到的证据表明，135,000道尔顿的产物由两种单独的mRNA编码。因此，α亚基在体外的整合依赖于与一种较小肽（35,000至40,000道尔顿）共翻译整合到膜中，该较小肽大概是β亚基。通过从游离多核糖体中分离mRNAα以及在MDCK细胞的脉冲追踪实验中检测α亚基的胞质形式，也获得了这种机制在体内存在的证据。
