Farley R A, Miller R P, Kudrow A
Biochim Biophys Acta. 1986 Sep 5;873(1):136-42. doi: 10.1016/0167-4838(86)90199-8.
Although the animal cell (Na+ + K+)-ATPase is composed of two polypeptide subunits, alpha and beta, very little is known about the beta subunit. In order to obtain information about the structure of this polypeptide, the beta subunit has been investigated using proteolytic fragmentation, chemical modification of carbohydrate residues, and immunoblot analysis. The sialic acid moieties on the oligosaccharide groups on the beta subunit of (Na+ + K+)-ATPase were labeled with NaB3H4 after oxidation by sodium periodate, or the penultimate galactose residues on the oligosaccharides were similarly labeled after removal of sialic acid with neuraminidase and oxidation by galactose oxidase. All of the carbohydrate residues of the protein are located on regions of the beta subunit that are found on the non-cytoplasmic surface of the membrane. Cleavage of the galactose oxidase-treated, NaB3H4-labeled beta subunit by chymotrypsin at an extracellular site produced labeled fragments of 40 and 18 kDa, indicating multiple glycosylation sites along the polypeptide. Neither the 40 kDa fragment nor the 18 kDa fragment was released from the membrane by chymotrypsin digestion alone, but after cleavage the 40 kDa fragment could be removed from the membrane by treatment with 0.1 M NaOH. This indicates that the 40 kDa fragment does not span the lipid bilayer. The 40 kDa fragment and the 18 kDa fragment are also linked by at least one disulfide bond. The 18 kDa fragment also contains all of the binding sites found on the (Na+ + K+)-ATPase for anti-beta subunit antibodies. Both the 40 kDa fragment and the 18 kDa fragment were also generated using papain or trypsin to cleave the beta subunit. These data indicate that the beta subunit of (Na+ + K+)-ATPase contains multiple sites of glycosylation, that it inserts into the cell membrane near only one end of the polypeptide, and that one region of the polypeptide is particularly sensitive to proteolytic cleavage relative to the rest of the polypeptide.
尽管动物细胞的（Na⁺ + K⁺）-ATP酶由α和β两个多肽亚基组成，但对β亚基的了解却非常少。为了获取有关该多肽结构的信息，已使用蛋白水解片段化、碳水化合物残基的化学修饰以及免疫印迹分析对β亚基进行了研究。在高碘酸钠氧化后，用NaB₃H₄标记（Na⁺ + K⁺）-ATP酶β亚基上寡糖基团的唾液酸部分，或者在用神经氨酸酶去除唾液酸并用半乳糖氧化酶氧化后，类似地标记寡糖上的倒数第二个半乳糖残基。该蛋白质的所有碳水化合物残基都位于β亚基在膜非细胞质表面的区域。用胰凝乳蛋白酶在细胞外位点切割经半乳糖氧化酶处理并用NaB₃H₄标记的β亚基，产生了40 kDa和18 kDa的标记片段，表明沿多肽存在多个糖基化位点。单独用胰凝乳蛋白酶消化时，40 kDa片段和18 kDa片段都不会从膜上释放出来，但切割后，用0.1 M NaOH处理可以将40 kDa片段从膜上除去。这表明40 kDa片段不跨越脂质双层。40 kDa片段和18 kDa片段还通过至少一个二硫键相连。18 kDa片段还包含（Na⁺ + K⁺）-ATP酶上所有针对抗β亚基抗体的结合位点。使用木瓜蛋白酶或胰蛋白酶切割β亚基也产生了40 kDa片段和18 kDa片段。这些数据表明，（Na⁺ + K⁺）-ATP酶的β亚基包含多个糖基化位点，它仅在多肽的一端附近插入细胞膜，并且相对于多肽的其余部分，多肽的一个区域对蛋白水解切割特别敏感。
