Ivanov A, Askari A, Modyanov N N
Department of Pharmacology, Medical College of Ohio, Toledo 43614, USA.
FEBS Lett. 1997 Dec 22;420(1):107-11. doi: 10.1016/s0014-5793(97)01493-2.
Chymotryptic cleavage of the Na,K-ATPase in NaCl medium abolishes ATPase activity and alters other functional parameters. The structure of this modified enzyme is uncertain since only one product of selective proteolysis, the 83-kDa fragment of the alpha-subunit (Ala267-C-terminus) has been identified previously. Here, we applied additional tryptic digestion followed by oxidative cross-linking to identify the products originating from the N-terminal part of the alpha-subunit. These fragments start at Ala72 or Thr74 and contain the transmembrane H1-H2 domain. Formation of cross-linked product between alpha-fragments containing H1-H2 and H7-H10 demonstrate that the structural integrity of the membrane moiety is preserved. We also determined that secondary cleavage of the 83-kDa fragment leads to the formation of C-terminal 48-kDa alpha-fragments with multiple N-termini at Ile582, Ser583, Met584 and Ile585.
在NaCl介质中，胰凝乳蛋白酶对钠钾ATP酶的切割会消除ATP酶活性，并改变其他功能参数。由于之前仅鉴定出选择性蛋白水解的一种产物，即α亚基的83 kDa片段（Ala267-羧基末端），这种修饰酶的结构尚不确定。在此，我们进行了额外的胰蛋白酶消化，随后进行氧化交联，以鉴定源自α亚基N端部分的产物。这些片段起始于Ala72或Thr74，并包含跨膜H1-H2结构域。含有H1-H2和H7-H10的α片段之间形成交联产物，表明膜部分的结构完整性得以保留。我们还确定，83 kDa片段的二次切割会导致形成C端48 kDa的α片段，其在Ile582、Ser583、Met584和Ile585处有多个N端。
