Treuheit M J, Vaghy P L, Kirley T L
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
J Biol Chem. 1992 Jun 15;267(17):11777-82.
There exists a Mg(2+)-ATPase in transverse tubules which has properties that are very different from other ATPases located in skeletal muscle cells. Several groups have suggested that a 100-kDa glycoprotein is the molecular entity responsible for this Mg(2+)-ATPase activity. In this study we have extended the methods utilized in the purification of this integral membrane glycoprotein. Although the apparent pI (isoelectric point) of this protein is pH 5, most of the net charge is due to the presence of sialic acid on the associated glycan chains. After these residues are removed, the behavior of this protein on an anion exchange column changes dramatically, allowing it to be further purified. Using a combination of the earlier procedures (Kirley, T.L. (1988) J. Biol. Chem. 263, 12682-12689 and Kirley, T. L. (1991) Biochem. J. 278, 375-400.) and the one reported here, purification to specific activities of approximately 400,000 mumol of ATP hydrolyzed/mg of protein/h were obtained and all traces of the 100-kDa protein were removed. The digitonin-solubilized Mg(2+)-ATPase appears to be a dimer of two identical 67-kDa subunits as assessed by high performance size exclusion chromatography. A single diffuse protein band is observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at approximately 67 kDa, which reduced to a single tight protein band at 52 kDa after deglycosylation with the following unique N-terminal amino acid sequence: Ala-Lys-Lys-Val-Leu-Pro-Leu-Leu-Leu-Pro- Pro-Leu-Val-X-Ala-Ala-Leu-Gly-Leu-Ala-X-Phe. Therefore, the Mg(2+)-ATPase appears to be an enzyme of very high specific activity, present in small quantities in transverse tubule membranes and unrelated to the known classes of ATPases present in skeletal muscle. The data reported here on the orientation of the transverse-tubule membrane preparations are consistent with the very recent report (Saborido, A., Moro, G., and Megias A. (1991) J. Biol. Chem. 266, 23490-23498) that the Mg(2+)-ATPase is an ecto-enzyme.
横管中存在一种Mg(2+)-ATP酶，其性质与骨骼肌细胞中的其他ATP酶有很大不同。几个研究小组认为，一种100 kDa的糖蛋白是负责这种Mg(2+)-ATP酶活性的分子实体。在本研究中，我们扩展了用于纯化这种整合膜糖蛋白的方法。尽管该蛋白的表观pI（等电点）为pH 5，但大部分净电荷是由于相关聚糖链上存在唾液酸。去除这些残基后，该蛋白在阴离子交换柱上的行为发生了显著变化，从而使其能够进一步纯化。结合早期的方法（Kirley, T.L. (1988) J. Biol. Chem. 263, 12682-12689和Kirley, T. L. (1991) Biochem. J. 278, 375-400.）以及本文报道的方法，获得了约400,000 μmol ATP水解/mg蛋白/h的比活性纯化产物，并且所有100 kDa蛋白的痕迹都被去除。通过高效尺寸排阻色谱评估，洋地黄皂苷溶解的Mg(2+)-ATP酶似乎是由两个相同的67 kDa亚基组成的二聚体。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上观察到一条约67 kDa的单一弥散蛋白带，在用以下独特的N端氨基酸序列：Ala-Lys-Lys-Val-Leu-Pro-Leu-Leu-Leu-Pro-Pro-Leu-Val-X-Ala-Ala-Leu-Gly-Leu-Ala-X-Phe去糖基化后，该带减少为一条52 kDa的单一紧密蛋白带。因此，Mg(2+)-ATP酶似乎是一种比活性非常高的酶，在横管膜中含量很少，并且与骨骼肌中已知的ATP酶类别无关。本文报道的关于横管膜制剂取向的数据与最近的一份报告（Saborido, A., Moro, G., and Megias A. (1991) J. Biol. Chem. 266, 23490-23498）一致，即Mg(2+)-ATP酶是一种外切酶。
