Structural analysis of the products of chymotryptic cleavage of the E1 form of Na,K‐ATPase α‐subunit: identification of the N‐terminal fragments containing the transmembrane H1‐H2 domain

FEBS Letters

Modyanov, Nikolai N¹  Askari, Amir¹  Ivanov, Alexander¹ 

[1] Department of Pharmacology, Medical College of Ohio, Toledo, OH 43614, USA

关键词: Na+;    K+-ATPase;    α-Subunit;    Chymotrypsin;    Trypsin;    Cu2+-phenanthroline;    Oxidative cross-linking;   

DOI  :  10.1016/S0014-5793(97)01493-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 α-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 α-subunit. These fragments start at Ala⁷² or Thr⁷⁴ and contain the transmembrane H₁-H₂ domain. Formation of cross-linked product between α-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 α-fragments with multiple N-termini at Ile⁵⁸², Ser⁵⁸³, Met⁵⁸⁴ and Ile⁵⁸⁵.

【 授权许可】

Unknown