Rat hepatic (Na+, K+)-ATPase: alpha-subunit isolation by immunoaffinity chromatography and structural analysis by peptide mapping.
Rat hepatic (Na+, K+)-ATPase: alpha-subunit isolation by immunoaffinity chromatography and structural analysis by peptide mapping.
复制标题
大鼠肝 (Na , K )-ATP 酶：通过免疫亲和层析分离 α 亚基并通过肽图谱进行结构分析。
DOI：
10.1021/bi00362a025
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Leffert,HL
中科院分区：
文献类型：
--
作者：
Hubert,JJ;Schenk,DB;Skelly,H;Leffert,HL
加州大学圣地亚哥分校医学系药理学部，加利福尼亚州拉霍亚 92093 收稿日期：1986 年 1 月 29 日；修订稿于 1986 年 3 月 20 日收到 摘要：大鼠肝 (Na+, K+)-ATP 酶 (EC 3.6. 1.3) 的催化 a-亚基已通过免疫亲和层析从溶解在正十二烷基八乙二醇单醚中的微粒体中分离出来。该程序采用与 Sepharose 4B 共价连接的抗催化小鼠单克隆抗体（“9-A5”），特异性阻断 [y-32P] ATP 钠泵 a 亚基的磷酸化 [Schenk, D. B., Hubert, JJ, & Leffert, HL (1984) J. Biol.化学。 259、14941-14951]。根据十二烷基硫酸钠 (Mt 92K) 中聚丙烯酰胺凝胶电泳后的表观分子量及其在纤维素涂层薄层板上的二维胰蛋白酶和胰凝乳蛋白酶肽图来判断，肝亚基实际上与纯化的大鼠、狗和人肾亚基相同。相比之下，从肽图判断，这三个物种的真正肾脏 0 亚基的结构彼此显着不同；在它们的胰凝乳蛋白酶图谱和从 9-A5-Sepharose 洗脱的假定的肝脏“0”亚基（Mr 50K 和 55K）之间没有发现可检测到的同源性。对成年大鼠肝细胞原代培养物中哇巴因敏感的 86Rb+ 摄取的其他研究揭示了在不存在或存在胰岛素、胰高血糖素和表皮生长因子等泵刺激肽的情况下具有单个拐点的抑制曲线（ID50 = 0.1 mM 哇巴因）。这些发现表明，大鼠肝细胞仅表达两种已知结构保守形式的催化亚基（肾样 a 形式）中的一种，并且如果有的话，也表达钠泵 0 亚基的结构不同形式。此外，使用 9-A5-Sepharose 的免疫亲和层析有助于从钠泵水平较低的非肾组织中分离 (Na+, K+)-ATP 酶。
Department of Medicine, Division of Pharmacology, University of California, San Diego, La Jolla, California 92093 Received January 29, 1986; Revised Manuscript Received March 20, 1986 abstract: The catalytic a-subunit of rat hepatic (Na+, K+)-ATPase (EC 3.6. 1.3) has been isolated by immunoaffinity chromatography from microsomes solubilized in n-dodecyl octaethylene glycol monoether. The procedure employs an anticatalytic mouse monoclonal antibody (“9-A5”) covalently linked to Sepharose 4B that specifically blocks phosphorylation of the sodium pump’s a-subunit from [y-32P] ATP [Schenk, D. B., Hubert, JJ, & Leffert, HL (1984) J. Biol. Chem. 259, 14941-14951]. The hepatic subunit is virtually identical with purified rat, dog, and human renala-subunits as judged by its apparent molecular weight after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Mt 92K) and its two-dimensional tryptic and chymotryptic peptide maps on cellulose-coated thin-layer plates. In contrast, the structures of authentic renal 0-subunits from the three species differ significantly from each other as judged by their peptide maps; no detectable homologies are seen between their chymotryptic maps and those of putative hepatic “0”-subunits (Mr 50K and 55K) eluted from 9-A5-Sepharose. Additional studies of ouabain-sensitive 86Rb+ uptake in primary cultures of adult rat hepatocytes reveal inhibition curves with single inflection points (ID50= 0.1 mM ouabain) in the absence or presence of pump-stimulating peptides like insulin, glucagon, and epidermal growth factor. These findings indicate that rat hepatocytes express only one of two known structurally conserved forms of catalytic subunit (the renallike a form) and, if at all, structurally divergent forms of the sodium pump’s 0-subunit. In addition, immunoaffinity chromatography with 9-A5-Sepharose facilitates the isolation of (Na+, K+)-ATPases from nonrenal tissues with low levels of sodium pumps.