Cortas N, Elstein D, Markowitz D, Edelman I S
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
Biochim Biophys Acta. 1991 Nov 18;1070(1):223-8. doi: 10.1016/0005-2736(91)90168-8.
Three isoforms of the alpha subunit of Na,K-ATPase, alpha 1, alpha 2, and alpha 3 have been characterized at the DNA, mRNA and protein levels. In admixtures, isoforms migrate as doublets (i.e. alpha 1 and another band originally designated alpha +, comprising alpha 2 + alpha 3) when analyzed by SDS-PAGE. As deduced from cDNA sequences their masses range from 111.7 to 112.6 kDa. With conventional protein standards, however, SDS-PAGE yields nominal masses of 85-105 kDa. In this system, the presence of a doublet that reacted with a polyclonal anti-Na,K-ATPase antibody in the kidney was interpreted as indicating two molecular or conformational species of the kidney alpha sub-unit (Siegel, G.J. and Desmond, T.J. (1989) J. Biol. Chem. 264, 4751-4754). We report that Na,K-ATPase purified from dog, guinea pig and rat kidney medulla or from rat brain, can yield two distinct bands when analyzed by SDS-PAGE or STS-PAGE, migrating between 85 and 105 kDa. An additional band migrating at 117 and 120 kDa appears often in enzyme purified from rat and guinea pig kidney medulla. The apparent molecular weights and relative intensities of these bands vary with temperature and duration of incubation during sample preparation. N-terminal sequencing and monospecific antibody probes revealed that the two distinct bands obtained from the kidney enzyme consist only of the alpha 1 isoform. The band appearing at 117-120 kDa also contains only the alpha 1 N-terminal sequence. In contrast, as reported earlier (Sweadner, K.J. (1979) J. Biol. Chem. 254, 6060-6067), the doublet seen in brain preparations consists of alpha 1 and alpha 2 or (alpha 2 + alpha 3). We conclude that monospecific antibody probes or N-terminal sequencing must be used to identify Na,K-ATPase isoforms by SDS- or STS-PAGE. In addition, gel conditions that may affect the mobilities of the isoforms are discussed.
钠钾ATP酶α亚基的三种同工型，即α1、α2和α3，已在DNA、mRNA和蛋白质水平上得到表征。在混合物中，当通过SDS-PAGE分析时，同工型以双峰形式迁移（即α1和另一条最初命名为α+的条带，包括α2+α3）。根据cDNA序列推断，它们的质量范围为111.7至112.6 kDa。然而，使用传统蛋白质标准品时，SDS-PAGE得到的标称质量为85-105 kDa。在该系统中，肾脏中与多克隆抗钠钾ATP酶抗体反应的双峰的存在被解释为表明肾脏α亚基的两种分子或构象形式（Siegel, G.J.和Desmond, T.J. (1989) J. Biol. Chem. 264, 4751-4754）。我们报告，从狗、豚鼠和大鼠肾髓质或大鼠脑中纯化的钠钾ATP酶，通过SDS-PAGE或STS-PAGE分析时可产生两条不同的条带，迁移在85至105 kDa之间。在从大鼠和豚鼠肾髓质纯化的酶中经常出现一条迁移在117和120 kDa的额外条带。这些条带的表观分子量和相对强度随样品制备过程中的温度和孵育时间而变化。N端测序和单特异性抗体探针显示，从肾脏酶获得的两条不同条带仅由α1同工型组成。出现在117-120 kDa的条带也仅包含α1 N端序列。相比之下，如先前报道（Sweadner, K.J. (1979) J. Biol. Chem. 254, 6060-6)，在脑制备物中看到的双峰由α1和α2或（α2+α3）组成。我们得出结论，必须使用单特异性抗体探针或N端测序通过SDS-或STS-PAGE来鉴定钠钾ATP酶同工型。此外，还讨论了可能影响同工型迁移率的凝胶条件。
