The epitope for the inhibitory antibody M7-PB-E9 contains Ser-646 and Asp-652 of the sheep Na+,K(+)-ATPase alpha-subunit.
The epitope for the inhibitory antibody M7-PB-E9 contains Ser-646 and Asp-652 of the sheep Na+,K(+)-ATPase alpha-subunit.
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抑制性抗体 M7-PB-E9 的表位包含绵羊 Na,K()-ATP 酶 α 亚基的 Ser-646 和 Asp-652。
DOI：
10.1021/bi00064a040
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
BallJr,WJ
中科院分区：
文献类型：
--
作者：
Abbott,A;BallJr,WJ
单克隆抗体M7-PB-E9与Na+，K+-ATP酶的α-亚基的结合部分抑制了与ATP竞争的酶活性（35%），而在镁的存在下，它刺激哇巴因结合的速率几倍[Ball，W. J.（1984）Biochemistry 23，2275-2281]。这些效应已被证明是由抗体诱导的酶的E1 * E2构象平衡向右移动引起的，其影响所有的酶-配体相互作用，除了与Mg 2+的相互作用[Abbott，A. J.，6 Ball，W. J.（1992）Biochemistry 31，11236-11243]。为了鉴定M7-PB-E9表位的位置，产生羔羊肾酶的蛋白水解片段，并通过Western印迹分析鉴定免疫反应性α片段。这些研究揭示了一个47-kDa的胰蛋白酶片段，其结合M7-PB-E9和a-COOH末端特异性抗血清，并且NH 2-末端测序显示其起源于Ala-590。用金黄色葡萄球菌V8蛋白酶消化产生36-kDa-COOH-末端片段，其起源于Gly-697并且不含抗体表位。因此，细胞内序列区Ala-590至Gly-697显示含有抗体表位。当确定M7-PB-E9识别来自各种物种和组织的α亚基的能力并与可用的测序数据相关联时，只有Ser-646存在于高反应性的羔羊、猪和禽类肾α 1蛋白中，并且在识别较差的爪蟾和大鼠肾和电鳐器官酶中改变（Asn）。此外，发现M7-PB-E9对大鼠α 3同种型具有高结合亲和力，对α 2具有低亲和力，其在Asp-652-*Glu处改变。因此，发现M7-PB-E9表位包含从Ser-636至Asp-652的7个氨基酸序列。该位点在酶的E1 ** E2构象转变中起着重要作用，它位于一个独特的结构域中，该结构域连接了MacLennan等人提出的高度保守的核苷酸结合和铰链结构域。[（1985）Nature 316，696]中描述的同源肌浆网Ca 2+，Mg 2 +-ATP酶。
The binding of monoclonal antibody M7-PB-E9 to the a-subunit of Na+, K+-ATPase partially inhibits enzyme activity (35%) in competition with ATP, while in the presence of magnesium it stimulates the rate of ouabain binding severalfold [Ball, W. J.(1984) Biochemistry 23, 2275-2281]. These effects have been shown to resultfrom an antibody-induced shifting of the enzyme’s Ei* E2 conformational equilibrium to the right that affects all enzyme-ligand interactions except that with Mg2+[Abbott, A. J., 6 Ball, W. J.(1992) Biochemistry 31, 11236-11243]. In order to identify the location of the M7-PB-E9 epitope, proteolytic fragments of the lamb kidney enzyme were generated and the immunoreactive a fragments were identified by Western blot analyses. These studies revealed a 47-kDa tryptic fragment, which bound both M7-PB-E9 and a-COOH terminus specific antisera and NH2-terminal sequencingshowed to originate at Ala-590. Digestion with Staphylococcus aureus V8 protease produced a 36-kDa-COOH-terminus fragment which originated at Gly-697 and did not contain the antibody epitope. Thus the intracellular sequence region Ala-590 to Gly-697 was shown to contain the antibody epitope. When M7-PB-E9’s ability to recognize the a subunits from various species and tissues was determined and correlated with available sequencing data, only Ser-646 was present in the highly reactive lamb, pig, and avian kidney al proteins and altered (Asn) in the poorly recognized Xenopus and rat kidney and Torpedo electroplax organ enzymes. In addition, M7-PB-E9 was found to have a high binding affinity for the rat a3 isoform and low affinity for a2 which is altered at Asp-652—*Glu. The M7-PB-E9 epitope therefore was found to encompass the 7 amino acid sequence from Ser-636 to Asp-652. This site plays an important role in the Ei** E2 conformational transitions undergone by theenzyme and it lies within a unique domain that links the highly conserved nucleotidebinding and hinge domains of a as proposed by MacLennan et al.[(1985) Nature 316, 696] for the homologous sarcoplasmic reticulum Ca2+, Mg2+-ATPase.