Shimon M B, Goldshleger R, Karlish S J
Biochemistry Department, Weizmann Institute of Science, Rehovot 76100, Israel.
J Biol Chem. 1998 Dec 18;273(51):34190-5. doi: 10.1074/jbc.273.51.34190.
This paper describes specific Cu2+-catalyzed oxidative cleavage of alpha and beta subunits of Na,K-ATPase at the extracellular surface. Incubation of right side-out renal microsomal vesicles with Cu2+ ions, ascorbate, and H2O2 produces two major cleavages of the alpha subunit within the extracellular loop between trans-membrane segments M7 and M8 and L7/8. Minor cleavages are also detected in loops L9/10 and L5/6. In the beta subunit two cleavages are detected, one before the first S-S bridge and the other between the second and third S-S bridges. Na,K-ATPase and Rb+ occlusion are inactivated after incubation with Cu2+/ascorbate/H2O2. These observations are suggestive of a site-specific mechanism involving cleavage of peptide bonds close to a bound Cu2+ ion. This mechanism allows several inferences on subunit interactions and spatial organization. The two cleavage sites in L7/8 of the alpha subunit and two cleavage sites of the beta subunit identify interacting segments of the subunits. L7/8 is also close to L9/10 and to cation occlusion sites. Comparison of the locations of Cu2+-catalyzed cleavages with Fe2+-catalyzed cleavages (Goldshleger, R., and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596-9601) suggests division of the membrane sector into two domains comprising M1-M6 and M7-M10/Mbeta, respectively.
本文描述了铜离子（Cu2+）催化的Na，K-ATP酶α和β亚基在细胞外表面的特异性氧化裂解。将外翻的肾微粒体囊泡与Cu2+离子、抗坏血酸和过氧化氢一起孵育，会在跨膜片段M7和M8与L7/8之间的细胞外环内产生α亚基的两个主要裂解位点。在L9/10环和L5/6环中也检测到少量裂解。在β亚基中检测到两个裂解位点，一个在第一个二硫键之前，另一个在第二个和第三个二硫键之间。用Cu2+/抗坏血酸/过氧化氢孵育后，Na，K-ATP酶和铷离子（Rb+）的封闭作用失活。这些观察结果表明存在一种位点特异性机制，涉及靠近结合的Cu2+离子处肽键的裂解。该机制允许对亚基相互作用和空间组织进行一些推断。α亚基L7/8中的两个裂解位点和β亚基的两个裂解位点确定了亚基的相互作用片段。L7/8也靠近L9/10和阳离子封闭位点。将Cu2+催化裂解的位置与Fe2+催化裂解的位置进行比较（Goldshleger，R.，和Karlish，S.J.D.（1997年）美国国家科学院院刊94，9596 - 9601）表明，膜区可分为两个结构域，分别由M1 - M6和M7 - M10/Mβ组成。
