胃H⁺/K⁺转运ATP酶β亚基的特性分析
Characterization of a beta subunit of the gastric H+/K(+)-transporting ATPase.
作者信息
Reuben M A, Lasater L S, Sachs G
机构信息
Center for Ulcer Research and Education, Wadsworth Veterans Administration Hospital, Los Angeles, CA.
出版信息
Proc Natl Acad Sci U S A. 1990 Sep;87(17):6767-71. doi: 10.1073/pnas.87.17.6767.
The catalytic subunit of the H+/K(+)-transporting ATPase (EC 3.6.1.3) has 62% identity to the alpha, or catalytic subunit, of the Na+/K(+)-transporting ATPase (EC 3.6.1.37); however, a homologous beta subunit was unknown until recently. Removal of the carbohydrate from purified hog H+/K(+)ATPase vesicles reveals a 35-kDa peptide that, when fragmented with protease V8, gives sequences homologous to both beta 1 and beta 2 subunits of the Na+/K(+)-ATPase. cDNA clones for a beta subunit of the gastric H+/K(+)-ATPase were isolated from a rabbit stomach cDNA library by using degenerate 17-mer oligonucleotide probes made to the protease V8-treated peptides. An open reading frame (54-926) encodes a predicted 291-amino acid peptide with Mr = 33,320, which exhibits 31% and 44% homologies to the Na+/K+)-ATPase beta 1 and Na+/K(+)-ATPase beta 2 proteins, respectively. A Kyte-Doolittle hydropathy plot predicts a single N-terminal transmembrane domain with a small hydrophobic region near the C terminus. The presumed extracytosolic domain contains seven potential N-linked glycosylation sites and six out of nine cysteines. Northern (RNA) blot analysis of stomach RNA with the rabbit H+/K(+)-ATPase beta probe identifies a single mRNA of 1.3-1.5 kilobases, similar in concentration to the alpha subunit mRNA. The presence of a defined gastric H+/K(+)-ATPase beta subunit extends the homology between H+/K(+)-ATPase and the Na+/K(+)-ATPase subclass of phosphoenzyme transport ATPases and distinguishes them from the monomeric Ca2+ and proton pump subclasses.
H⁺/K⁺转运ATP酶（EC 3.6.1.3）的催化亚基与Na⁺/K⁺转运ATP酶（EC 3.6.1.37）的α亚基或催化亚基有62%的同一性；然而，直到最近同源的β亚基还不为人知。从纯化的猪H⁺/K⁺ATP酶囊泡中去除碳水化合物后可发现一个35 kDa的肽段，用蛋白酶V8切割该肽段后得到的序列与Na⁺/K⁺ATP酶的β1和β2亚基均同源。通过使用针对蛋白酶V8处理肽段设计的简并17聚体寡核苷酸探针，从兔胃cDNA文库中分离出胃H⁺/K⁺ATP酶β亚基的cDNA克隆。一个开放阅读框（54 - 926）编码一个预测的291个氨基酸的肽段，其Mr = 33320，分别与Na⁺/K⁺ATP酶β1和Na⁺/K⁺ATP酶β2蛋白有31%和44%的同源性。Kyte - Doolittle亲水性图谱预测有一个单一的N端跨膜结构域，在C端附近有一个小的疏水区域。推测的胞外结构域包含七个潜在的N - 连接糖基化位点和九个半胱氨酸中的六个。用兔H⁺/K⁺ATP酶β探针进行胃RNA的Northern（RNA）印迹分析，可鉴定出一个1.3 - 1.5千碱基的单一mRNA，其浓度与α亚基mRNA相似。明确的胃H⁺/K⁺ATP酶β亚基的存在扩展了H⁺/K⁺ATP酶与磷酸酶转运ATP酶的Na⁺/K⁺ATP酶亚类之间的同源性，并将它们与单体Ca²⁺和质子泵亚类区分开来。
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