A novel 14-kDa V-ATPase subunit in the tobacco hornworm midgut.
A novel 14-kDa V-ATPase subunit in the tobacco hornworm midgut.
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DOI：
10.1016/s0021-9258(17)41926-0
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发表时间：
1994-02
期刊：
影响因子：
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通讯作者：
Ralph Gräf;Alexandra Lepier;William R. Harvey;Helmut Wieczorek
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文献类型：
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作者：
Ralph Gräf;Alexandra Lepier;William R. Harvey;Helmut Wieczorek
通过用抗V-ATP酶全酶血清进行鸟枪筛选，分离编码计算分子量为13，839 Da的亲水性蛋白的cDNA克隆。推导的氨基酸序列与其他已知蛋白质没有显着的同源性。Southern印迹显示，只有一个基因编码的14 kDa的蛋白质的存在。通过对重组蛋白的亲和纯化的单特异性抗体证明了高度纯化的杯状细胞顶端膜中存在14 kDa蛋白，并在相同程度上抑制ATP依赖的质子转运以及V-ATP酶活性。因此，14-kDa蛋白质被证明是V-ATP酶全酶的一部分。单特异性抗体与ATP酶的结合似乎需要酶的ATP依赖性构象变化，因为仅当抗体结合步骤期间存在ATP时才发生抑制。14-kDa的亚基可以从膜上剥离处理与离液剂KI，确认它是可溶性复合物的V-ATP酶的一部分。在免疫印迹中，14 kDa特异性抗体与几种异源性V-ATP酶无交叉反应。
A cDNA clone encoding a hydrophilic protein with a calculated molecular mass of 13,839 Da was isolated by shotgun screening with an anti-V-ATPase holoenzyme serum. The deduced amino acid sequence showed no significant homology to any other known protein. Southern blots revealed the existence of only one gene encoding the 14-kDa protein. Monospecific antibodies purified by affinity to the recombinant protein demonstrated the presence of a 14-kDa protein in the highly purified goblet cell apical membrane and inhibited ATP-dependent proton transport as well as V-ATPase activity to the same extent. Thus, the 14-kDa protein was shown to be a part of the V-ATPase holoenzyme. Binding of the monospecific antibodies to the ATPase seemed to require an ATP-dependent conformational change of the enzyme, since inhibition only occurred when ATP was present during the antibody binding step. The 14-kDa subunit could be stripped from the membrane by treatment with the chaotropic agent KI, confirming it to be part of the soluble complex of the V-ATPase. In immunoblots, the 14-kDa-specific antibodies showed no cross-reaction with several xenic V-ATPases.