外髓质钠钾ATP酶主动转运钠钾的分子基础。

Molecular basis for active Na,K-transport by Na,K-ATPase from outer renal medulla.

作者信息

Jørgensen P L

出版信息

Biochem Soc Symp. 1985;50:59-79.

Abstract

Active transcellular transport of NaCl in thick ascending limb of Henle's loop (TAL) consists of primary active transport of Na+ and secondary active transport of Cl-. The Na,K-pump maintains a low electrochemical potential for Na+ in the cytoplasm. The Na,K,Cl-cotransport system couples the entry of Cl- across the lumen membrane to entry of Na+ along its gradient. In mammalian kidney the TAL is concentrated in the red outer medulla and this tissue has exceedingly high concentrations of Na,K-pump sites, comparable with those in salt glands of birds and fishes. With the red outer medulla as starting material we developed procedures for purification of the Na,K-pump in membrane-bound form. The pump protein can be immobilized in two-dimensional membrane crystals by stabilization of the E2 form with vanadate or phosphate. The minimum functional protein unit of both membrane-bound and soluble Na,K-ATPase is an alpha beta-dimer (alpha-subunit Mr 84,000-106,000 and beta-subunit Mr 32,000-38,000 plus carbohydrate). Binding data, proteolytic digestion and fluorescence analysis show that both Na,K-induced and ATP-dependent conformational transitions of the alpha-subunit can be demonstrated in preparations consisting of soluble and fully active alpha beta-units. This is consistent with the notion that a single alpha beta-unit can catalyse the whole series of intermediary reactions and cation transport in the membrane. The Na,K,Cl-cotransport system can be identified by assaying bumethanide-sensitive ion fluxes in membrane vesicles from the outer renal medulla. Using equilibrium binding with [3H]bumethanide as an assay the cotransporter has been partially purified to capacities of 600-1,000 pmol/mg of protein. A polypeptide of Mr 34,000 is covalently labelled with [3H]bumethanide by direct photolysis. The [3H]bumethanide binding protein is not extracted by nonionic detergent except at high ionic strength, suggesting that the Na,K,Cl-cotransport protein is associated with cytoskeleton components. This association may be important for control of the entry of NaCl into the cytoplasm and for cellular regulation of the rate of active transport of NaCl across the tubule cells in the TAL.

摘要

在髓袢升支粗段（TAL）中，NaCl的主动跨细胞转运包括Na⁺的原发性主动转运和Cl⁻的继发性主动转运。Na⁺-K⁺泵维持细胞质中Na⁺的低电化学势。Na⁺-K⁺-Cl⁻共转运系统将Cl⁻跨管腔膜的进入与Na⁺沿其梯度的进入相偶联。在哺乳动物肾脏中，TAL集中在肾外髓质外层，该组织中Na⁺-K⁺泵位点的浓度极高，与鸟类和鱼类盐腺中的浓度相当。以肾外髓质外层为起始材料，我们开发了纯化膜结合形式的Na⁺-K⁺泵的方法。通过用钒酸盐或磷酸盐稳定E2形式，泵蛋白可以固定在二维膜晶体中。膜结合型和可溶性Na⁺-K⁺-ATP酶的最小功能蛋白单位是αβ二聚体（α亚基Mr 84,000 - 106,000，β亚基Mr 32,000 - 38,000加碳水化合物）。结合数据、蛋白水解消化和荧光分析表明，在由可溶性且完全活性的αβ单位组成的制剂中，可以证明α亚基的Na⁺-K⁺诱导的和ATP依赖性的构象转变。这与单个αβ单位可以催化膜中的整个系列中间反应和阳离子转运的观点一致。Na⁺-K⁺-Cl⁻共转运系统可以通过测定来自肾外髓质的膜囊泡中对布美他尼敏感的离子通量来鉴定。使用与[³H]布美他尼的平衡结合作为测定方法，共转运体已部分纯化至600 - 1,000 pmol/mg蛋白质的容量。一种Mr 34,000的多肽通过直接光解与[³H]布美他尼共价标记。除了在高离子强度下，[³H]布美他尼结合蛋白不会被非离子去污剂提取，这表明Na⁺-K⁺-Cl⁻共转运蛋白与细胞骨架成分相关。这种关联对于控制NaCl进入细胞质以及对TAL中小管细胞跨膜主动转运NaCl速率的细胞调节可能很重要。