McDonough A A, Hiatt A, Edelman I S
J Membr Biol. 1982;69(1):13-22. doi: 10.1007/BF01871237.
Antibodies have been produced, in three rabbits, to Na/K-ATPase purified from guinea pig renal outer medulla. Each rabbit produced antibodies to both the alpha (catalytic) and the beta (glycoprotein) subunits of Na/K-ATPase. The titers of the anti-alpha and anti-beta antibodies varied with time and between rabbits. None of the antisera inhibited Na/K-ATPase activity under various preincubation conditions. A method is presented for separating small amounts of anti-alpha subunit from anti-beta subunit antibodies. There was no cross-reactivity of antibodies to one subunit with the other subunit. The alpha subunit of the Na/K-ATPase was cleaved into a 41,000-dalton peptide (that contains the ATP phosphorylating site) and a 58,000-dalton hydrophobic peptide as described by Castro and Farley (Castro, J., Farley, R.A., 1979, J. Biol. Chem. 254: 2221-2228). Anti-alpha antibodies from all of the rabbits reacted with both proteolytic fragments. The anti-guinea pig Na/K-ATPase antisera (pooled) cross-reacted with the alpha subunit of Na/K-ATPase from human, cow, dog, rabbit, rat, mouse, turtle, and toad; and with the beta subunit from human, rat, and mouse. The loci of cross-reactivity were investigated using partially purified canine kidney Na/K-ATPase cleaved with trypsin as described above. The anti-sera from rabbits 1 and 2 cross-reacted with the 41,000-dalton peptide from the dog but very little with the 58,000-dalton peptide. No cross-reactivity was observed with antiserum from rabbit 3 to either fragment. Guinea pig kidney RNA was translated in a rabbit reticulocyte lysate system followed by immunoprecipitation with the antisera. The molecular weight of the cell-free synthesized alpha chain was 96,000 daltons. Its identity was established with purified anti-alpha antibodies and by immunocompetition with purified Na/K-ATPase and Ca-ATPase. Translation of the beta subunit was not detected in this system.
已在三只兔子体内产生了针对从豚鼠肾外髓质纯化的钠钾 - ATP酶的抗体。每只兔子都产生了针对钠钾 - ATP酶的α（催化）亚基和β（糖蛋白）亚基的抗体。抗α和抗β抗体的效价随时间以及兔子个体的不同而变化。在各种预温育条件下，没有一种抗血清能抑制钠钾 - ATP酶的活性。本文介绍了一种从抗β亚基抗体中分离少量抗α亚基抗体的方法。针对一个亚基的抗体与另一个亚基没有交叉反应。如Castro和Farley（Castro, J., Farley, R.A., 1979, J. Biol. Chem. 254: 2221 - 2228）所述，钠钾 - ATP酶的α亚基被裂解为一个41,000道尔顿的肽段（包含ATP磷酸化位点）和一个58,000道尔顿的疏水肽段。所有兔子的抗α抗体都与这两个蛋白水解片段发生反应。抗豚鼠钠钾 - ATP酶抗血清（混合）与人、牛、狗、兔子、大鼠、小鼠、乌龟和蟾蜍的钠钾 - ATP酶的α亚基发生交叉反应；并与人、大鼠和小鼠的β亚基发生交叉反应。使用如上述用胰蛋白酶裂解的部分纯化的犬肾钠钾 - ATP酶研究交叉反应位点。兔子1和兔子2的抗血清与来自狗的41,000道尔顿肽段发生交叉反应，但与58,000道尔顿肽段的交叉反应很少。未观察到兔子3的抗血清与任何一个片段发生交叉反应。豚鼠肾RNA在兔网织红细胞裂解物系统中进行翻译，然后用抗血清进行免疫沉淀。无细胞合成的α链的分子量为96,000道尔顿。通过纯化的抗α抗体以及与纯化的钠钾 - ATP酶和钙 - ATP酶的免疫竞争确定了其身份。在该系统中未检测到β亚基的翻译。
