A site-directed antibody recognizes a component of the ouabain-binding domain of the alpha 1 subunit of rat Na+,K(+)-ATPase.
A site-directed antibody recognizes a component of the ouabain-binding domain of the alpha 1 subunit of rat Na+,K(+)-ATPase.
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定点抗体识别大鼠 Na ,K( )-ATP 酶 α 1 亚基的哇巴因结合结构域的成分。
DOI：
10.1139/o93-077
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
O'Neil,RG
中科院分区：
文献类型：
--
作者：
Scully,RR;Pressley,TA;O'Neil,RG
抗体（AbE1）是针对来自大鼠Na+，K+- atp酶α1亚基的乌阿巴因结合位点的一个成分的寡肽而产生的。用1 μM abe1预孵育部分纯化的大鼠肾脏Na+，K+- atp酶，可部分抑制该酶，降低其对1 mM瓦苦因的敏感性。针对不相关蛋白的抗体无效。出乎意料的是，在大鼠肾脏免疫印迹上，160千道尔顿（kDa）的双偶体AbE1检测到的蛋白质量大于α1亚基的97千道尔顿。根据Laemmli的样品缓冲液配方（缓冲液L，一种低混沌性缓冲液），用热（85°C）缓冲液制备的裂解物中，abe1总是结合到160kda的双联态上。相比之下，无论温度如何，用含有更高浓度洗涤剂和还原剂的缓冲液（Laemmli的“均质”缓冲液，缓冲液H，一种高混沌性缓冲液）制备的裂解物的印迹上，abe1都不能结合任何蛋白质。然而，在均质缓冲液中产生的裂解物中存在a，通过存在97 kda的条带被证实，这些条带被另外两个识别α1亚基的位点定向抗体检测到。用样品缓冲液L制备的匀浆检测，其中一个抗体结合在160 kDa的条带上，表明α1是AbE1结合条带的一个组分。结果表明，abe1能够识别Na+，K+- atp酶的w - abain结合位点的一个组分，但只有在α1亚基结合成更高分子质量复合物的条件下才能识别。关键词：同工酶，乌阿巴因受体，定点抗体
Antibodies (AbE1) were raised against an oligopeptide derived from a component of the ouabain-binding site of the α1subunit of rat Na+,K+-ATPase. Preincubation of partially purified Na+,K+-ATPase from rat kidney with 1 μM AbE1partially inhibited the enzyme and reduced its sensitivity to 1 mM ouabain. Antibodies against an unrelated protein were ineffectual. Unexpectedly, the masses of the proteins detected on immunoblots of rat kidney by AbE1, a 160 kilodalton (kDa) doublet, was greater than the 97 kDa of the α1subunit. In lysates prepared with hot (85 °C) buffer constituted according to Laemmli's formulation for sample buffer (buffer L, a low chaotropic buffer), AbE1always bound to the 160-kDa doublet. In contrast, AbE1did not bind any proteins on blots of lysates that were prepared with buffer that contained greater concentrations of detergent and reducing agent (Laemmli's "homogenization" buffer, buffer H, a high chaotropic buffer), regardless of temperature. However, the presence of a, in lysates produced in homogenization buffer was confirmed by the presence of 97-kDa bands that were detected with two additional site-directed antibodies that recognize the α1subunit. When homogenates prepared with sample buffer L were examined, one of these antibodies bound to bands of 160 kDa, indicating that α1is a component of the bands bound by AbE1. It is concluded that AbE1recognizes a component of the ouabain-binding site of Na+,K+-ATPase, but only under conditions in which the α1subunit was associated into a higher molecular mass complex.Key words: isoenzyme, ouabain receptor, site-directed antibody.
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DOI：
--
发表时间：
1982
期刊：
Journal of the National Cancer Institute
影响因子：
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作者：
Biswas,C;Bloch,KJ;Gross,J
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DOI：
10.1016/s0174-173x(83)80004-1
发表时间：
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期刊：
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作者：
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Eisen,AZ
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影响因子：
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作者：
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DOI：
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发表时间：
1984
期刊：
Invasion and Metastasis
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作者：
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