The Effect of Ca^<2+> on the assembly and transport of Na, K-ATPase

Ca^<2>对Na,K-ATP酶组装和转运的影响

• 批准号: 05670059
• 负责人: OMORI Koichiro
• 金额: $ 1.34万
• 依托单位: Kansai Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670059/
• 关键词: Na, K-ATPase; Thapsigargin; BAPTA-AM; A23187; Retinal pigment epithelial cell; Muscle spindle; Polycystic kidney; Thyroid hormone; thapsigargin; Na,K‐ATPase; HeLa細胞

Na, K-ATPase consists of the alpha- and beta-subunits. The alpha-subunit contains the binding site for ouabain and plays as the catalytic subunit, whereas the beta-subunit is a glycosylated membrane protein and its function is not yet completely known. Both subunits are synthesized concurrently and assembled on the ER immediately after polypeptide synthesis. Recently, several studies have suggested the functional roles of the beta-subunit in the maturation of the alpha-subunit on the ER and/or the transport of this subunit to the plasma membrane. On the bases of these findings, we tried to elucidate the mechanisms of the assembly of both subunits and the transport of the enzyme.1. We investigated the effects of Ca^<2+> on the assembly and transport of the enzyme using a HeLa cell clone permanently expressing N-terminal half of the beta-subunit (beta_N) which is confined to the ER in the cells. Thapsigargin, BAPTA-AM and A23187 treatments or low Ca^<2+> medium treatment could not move the beta_N from the ER to the plasma membrane. Interestingly, A23187 treatment caused the change of oligosaccharide processing in the beta-subunit, converting the molecular weight of the subunit from 5.5 K to 4.5 K.In addition, we detected beta・beta_N complex in A23187 treated cells, immediately after polypeptide synthesis. Elucidation of the functional significance of these findings is now in progress.2. We analyzed the distribution of the enzyme in developing retinal pigment epithelial cells, muscle spindles and polycystic kidney, immunohistochemically.3. We investigated the effects of T_3 on the gene expression and enzyme activity of Na, K-ATPase in rat liver. T_3 treatment increased the level of mRNA encoding alpha-and beta-subunits 3-4-fold, and both the amount and activity of the enzyme 2-fold.

Na，K-ATP酶由α亚基和β亚基组成。α-亚基含有哇巴因的结合位点，并作为催化亚基发挥作用，而β-亚基是糖基化的膜蛋白，其功能尚未完全了解。两个亚基同时合成，并在多肽合成后立即组装在ER上。最近，一些研究表明，β-亚基的功能作用，在成熟的α-亚基的ER和/或该亚基的运输到质膜。在此基础上，我们试图阐明这两种亚基的组装和酶的转运机制.我们使用一个永久表达β亚基（β_N）N端一半的HeLa细胞克隆研究了Ca^<2+>对酶组装和转运的影响，β亚基（β_N）仅限于细胞中的ER。Thapsigargin、BAPTA-AM和A23187处理或低Ca^2+处理均不能使β_N从内质网向质膜移动。有趣的是，A23187处理引起β亚基中寡糖加工的变化，将亚基的分子量从5.5 K转换为4.5 K。此外，我们在A23187处理的细胞中检测到β·β_N复合物，多肽合成后立即。这些发现的功能意义的阐明正在进行中。通过组织化学方法分析了该酶在发育中的视网膜色素上皮细胞、肌梭和多囊肾中的分布.本文研究了T_3对大鼠肝脏Na，K-ATP酶基因表达和酶活性的影响。T_3处理使α-和β-亚基的mRNA水平增加3-4倍，酶的量和活性增加2倍。

项目成果

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SUGASAWA.K.et al: "Immunocytochemical analysses of distributions of Na,K-ATPase and GLUT1,insulin and transferrin receptors in the developing retinal pigment epithelial cells." Cell Struct. Funct.19. 21-28 (1994)

SUGASAWA.K.等人：“对发育中的视网膜色素上皮细胞中Na、K-ATP酶和GLUT1、胰岛素和转铁蛋白受体的分布进行免疫细胞化学分析。”

Yamamoto, A.et al.: "Immunocytochemical localization of Na, K-ATPase in rat muscle spindles." Cell Struct.Funct.19. 179-187 (1994)

Yamamoto, A. 等人：“大鼠肌梭中 Na、K-ATP 酶的免疫细胞化学定位。”

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