Differences in uncoupled sodium efflux between red blood cells and kidney Na,K-ATPase are not based on differences in the cDNA for the alpha subunit.
Differences in uncoupled sodium efflux between red blood cells and kidney Na,K-ATPase are not based on differences in the cDNA for the alpha subunit.
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红细胞和肾脏 Na,K-ATP 酶之间非偶联钠流出的差异并非基于 α 亚基 cDNA 的差异。
DOI：
10.1111/j.1749-6632.1997.tb52235.x
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发表时间：
1997
影响因子：
5.2
通讯作者：
Hoffman,JF
中科院分区：
文献类型：
--
作者：
Stengelin,M;Hoffman,JF
方法用高效滤器(RCZL，PALL)过滤100或500ml的健康献血员血中的白细胞，在生理盐水中洗涤6次。信使RNA被提取出来，如前所述。3裂解红细胞，沉淀核糖核蛋白，提取总RNA，分离m-RNA。典型的产量是每500毫升血液中250μg总核糖核酸。信使RNA与上标II(Gibco BRL)用寡核苷酸或随机六核苷酸或两者的混合反转录。在一些制剂中，用RNase H处理单链cDNA，并去掉引物(QIAick，Qiagen)。每次聚合酶链式反应使用约5 ng的单链cDNA.通常，使用以下PCR条件：94℃变性1分钟，55-65℃退火1分钟，72℃延伸1分钟，40个扩增循环。表2显示了所使用的引物。扩增产物在琼脂糖凝胶上分析，切下适当的条带，提取DNA(QIAick)，亚克隆(pGEM，Promega)，测序(Sequenase II，amersham)。结果以网织红细胞单链α为模板，从α亚单位高度保守的区域(T Abe2)扩增。对10多个不同聚合酶链式反应的产物进行了测序。只发现了与Na，K-α酶没有同源性的ATP1亚型或序列。我们还使用了针对α2、α3和α4异构体的引物进行聚合酶链式反应，在我们的制备中没有发现这些异构体的证据。因此，我们得出结论，只有α-1亚型在红细胞中大量表达。
METHODSBlood (100 or 500 ml) from healthy human donors was filtered with a high performance filter (RCZL, Pall) to remove leukocytes and washed six times in saline solution. Messenger RNA was extracted, as previously described. 3 Briefly, red cells were lysed, ribonucleoproteins precipitated, total RNA extracted, and m-RNA isolated (Oligotex, Qiagen). A typical yield was 250 μg of total RNA per 500 ml blood. Messenger RNA was reverse transcribed with Superscript II (Gibco BRL) using either oligo-dT or random hexanucleotide primers or a mixture of both. In some preparations, single-stranded cDNA was treated with RNase H, and primers were removed (QIAquick, Qiagen). Approximately 5 ng of single-stranded cDNA were used per PCR reaction. Typically, the following PCR conditions were used: denaturation for 1 minute at 94 C, annealing for 1 minute at 55–65 C, and extension for 1 minute at 72 C, 40 amplification cycles. T able 2 shows the primers used. PCR products were analyzed on agarose gel, appropriate bands excised, and DNA extracted (QIAquick), subcloned (pGEM, Promega), and sequenced (Sequenase II, Amersham).RESULTSTo detect any α isoform, reticulocyte single-stranded cDNA was used as a template for PCR with primers from highly conserved areas of the α subunit (T able 2). Products from more than 10 different PCR reactions were sequenced. Only the α 1 isoform or sequences with no homology to the Na, K-ATPase were found. We also used PCR with primers specific for the α2, α3, and α4 isoforms and found no evidence of these isforms in our preparation. We therefore conclude that only the α 1 isoform is expressed in significant amounts in red blood cells.