Damiani E, Margreth A, Furlan A, Dahms A S, Arnn J, Sabbadini R A
J Cell Biol. 1987 Mar;104(3):461-72. doi: 10.1083/jcb.104.3.461.
Transverse tubule (TT) membranes isolated from chicken skeletal muscle possess a very active magnesium-stimulated ATPase (Mg-ATPase) activity. The Mg-ATPase has been tentatively identified as a 102-kD concanavalin A (Con A)-binding glycoprotein comprising 80% of the integral membrane protein (Okamoto, V.R., 1985, Arch. Biochem. Biophys., 237:43-54). To firmly identify the Mg-ATPase as the 102-kD TT component and to characterize the structural relationship between this protein and the closely related sarcoplasmic reticulum (SR) Ca-ATPase, polyclonal antibodies were raised against the purified SR Ca-ATPase and the TT 102-kD glycoprotein, and the immunological relationship between the two ATPases was studied by means of Western immunoblots and enzyme-linked immunosorbent assays (ELISA). Anti-chicken and anti-rabbit SR Ca-ATPase antibodies were not able to distinguish between the TT 102-kD glycoprotein and the SR Ca-ATPase. The SR Ca-ATPase and the putative 102-kD TT Mg-ATPase also possess common structural elements, as indicated by amino acid compositional and peptide mapping analyses. The two 102-kD proteins exhibit similar amino acid compositions, especially with regard to the population of charged amino acid residues. Furthermore, one-dimensional peptide maps of the two proteins, and immunoblots thereof, show striking similarities indicating that the two proteins share many common epitopes and peptide domains. Polyclonal antibodies raised against the purified TT 102-kD glycoprotein were localized by indirect immunofluorescence exclusively in the TT-rich I bands of the muscle cell. The antibodies substantially inhibit the Mg-ATPase activity of isolated TT vesicles, and Con A pretreatment could prevent antibody inhibition of TT Mg-ATPase activity. Further, the binding of antibodies to intact TT vesicles could be reduced by prior treatment with Con A. We conclude that the TT 102-kD glycoprotein is the TT Mg-ATPase and that a high degree of structural homology exists between this protein and the SR Ca-ATPase.
从鸡骨骼肌中分离出的横小管（TT）膜具有非常活跃的镁刺激ATP酶（Mg - ATP酶）活性。该Mg - ATP酶初步被鉴定为一种102kD的伴刀豆球蛋白A（Con A）结合糖蛋白，占整合膜蛋白的80%（冈本，V.R.，1985年，《生物化学与生物物理学档案》，237:43 - 54）。为了确定Mg - ATP酶就是102kD的TT成分，并描述该蛋白与密切相关的肌浆网（SR）钙ATP酶之间的结构关系，制备了针对纯化的SR钙ATP酶和TT 102kD糖蛋白的多克隆抗体，并通过蛋白质免疫印迹法和酶联免疫吸附测定法（ELISA）研究了两种ATP酶之间的免疫关系。抗鸡和抗兔SR钙ATP酶抗体无法区分TT 102kD糖蛋白和SR钙ATP酶。氨基酸组成分析和肽图谱分析表明，SR钙ATP酶和假定的102kD TT Mg - ATP酶也具有共同的结构元件。这两种102kD的蛋白表现出相似的氨基酸组成，尤其是在带电氨基酸残基的数量方面。此外，这两种蛋白的一维肽图谱及其免疫印迹显示出惊人的相似性，表明这两种蛋白具有许多共同的表位和肽结构域。针对纯化的TT 102kD糖蛋白制备的多克隆抗体通过间接免疫荧光法仅定位在肌肉细胞富含TT的I带中。这些抗体显著抑制了分离的TT囊泡的Mg - ATP酶活性，Con A预处理可以防止抗体对TT Mg - ATP酶活性的抑制。此外，用Con A预先处理可以减少抗体与完整TT囊泡的结合。我们得出结论，TT 102kD糖蛋白就是TT Mg - ATP酶，并且该蛋白与SR钙ATP酶之间存在高度的结构同源性。
