Ivanov A, Zhao H, Modyanov N N
Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614 USA.
Biochemistry. 2000 Aug 15;39(32):9778-85. doi: 10.1021/bi001004j.
Spatial relationships among the transmembrane (TM) segments of alpha- and beta-subunits of the Na,K-ATPase molecule have been investigated using oxidative induction of disulfide bonds. The catalytic alpha-subunit contains 10 TM alpha-helices (H1-H10) with 9 Cys residues located within or close to the membrane moiety. There is one Cys residue in the single TM segment of beta-subunit (Hbeta). Previously, the cross-linking products containing the beta-subunit and two fragments of alpha-subunit (the N-terminal containing H1-H2 helices and the C-terminal containing H7-H10 helices) have been identified in experiments with membrane-bound or detergent-solubilized preparations of the membrane moiety of trypsin-digested Na,K-ATPase [Sarvazyan, N. A., Modyanov, N. N., and Askari, A. (1995) J. Biol. Chem. 270, 26528-26532 and Sarvazyan, N. A., Ivanov, A., Modyanov, N. N., and Askari, A. (1997) J. Biol. Chem. 272, 7855-7858]. Here, we have shown that Cu(2+)-phenanthroline treatment of digitonin-solubilized preparation provides the most efficient formation of intersubunit cross-linked product that is predominantly a dimer of beta-subunit and a 22-kDa C-terminal alpha-fragment containing H7-H10 helices. This cross-linked product was isolated and subjected to CNBr cleavage. The resulting fragments were electrophoretically separated and sequenced. A 17-kDa peptide composed of Ile853-Met942 alpha-fragment and Ala5-Met56 beta-fragment was identified as a product of intersubunit disulfide cross-link between Cys44 of Hbeta and either Cys911 or Cys930, located in H8. This provides the first direct experimental evidence of the juxtaposition of Hbeta and H8 within the Na,K-ATPase molecule. The second detected cross-linked product was composed of alpha-fragments Lys947-Met963 and Tyr974-Tyr1016 linked by induced disulfide bridge between Cys964 (H9) and Cys983 (H10). The spatial proximity of these Cys residues defines the mutual orientation of H9 and H10 helices of alpha-subunit.
利用二硫键的氧化诱导作用，对钠钾ATP酶分子α亚基和β亚基跨膜（TM）片段之间的空间关系进行了研究。催化性α亚基包含10个TMα螺旋（H1-H10），其中9个半胱氨酸残基位于膜部分或靠近膜部分。β亚基的单个TM片段（Hβ）中有一个半胱氨酸残基。此前，在对胰蛋白酶消化的钠钾ATP酶膜部分进行膜结合或去污剂增溶制剂的实验中，已鉴定出含有β亚基和α亚基两个片段（包含H1-H2螺旋的N端和包含H7-H10螺旋的C端）的交联产物[萨尔瓦齐扬，N.A.，莫迪亚诺夫，N.N.，和阿斯卡里，A.（1995）《生物化学杂志》270，26528-26532以及萨尔瓦齐扬，N.A.，伊万诺夫，A.，莫迪亚诺夫，N.N.，和阿斯卡里，A.（1997）《生物化学杂志》272，7855-7858]。在此，我们表明，用铜（II）-菲咯啉处理洋地黄皂苷增溶制剂能最有效地形成亚基间交联产物，该产物主要是β亚基的二聚体和一个包含H7-H10螺旋且分子量为22 kDa的C端α片段。分离出该交联产物并进行溴化氰裂解。对所得片段进行电泳分离和测序。由Ile853-Met942α片段和Ala5-Met56β片段组成的一个17 kDa肽被鉴定为Hβ的Cys44与位于H8中的Cys911或Cys930之间亚基间二硫键交联的产物。这为钠钾ATP酶分子内Hβ和H8并列提供了首个直接实验证据。检测到的第二个交联产物由通过Cys964（H9）和Cys983（H10）之间诱导形成的二硫键相连的α片段Lys947-Met963和Tyr974-Tyr1016组成。这些半胱氨酸残基的空间接近度确定了α亚基H9和H10螺旋的相互取向。
