ADP‐insensitive phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+‐ATPase has a compact conformation resistant to proteinase K, V8 protease and trypsin

FEBS Letters

Daiho, Takashi²  Toyoshima, Chikashi¹  Danko, Stefania²  Suzuki, Hiroshi²  Yamasaki, Kazuo²  Kamidochi, Mika² 

[1] Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan;Department of Biochemistry, Asahikawa Medical College, Midorigaokahigashi, Asahikawa 078-8510, Japan

关键词: Ca2+-ATPase;    P-type ion transporting ATPase;    Ca2+ pump;    Sarcoplasmic reticulum;    Phosphorylated intermediate;    Proteolysis;    SR;    sarcoplasmic reticulum;    E1P;    ADP-sensitive phosphoenzyme;    E2P;    ADP-insensitive phosphoenzyme;    prtK;    proteinase K;    TG;    thapsigargin;    MOPS;    3-(N-morpholino)propanesulfonic acid;    MES;    2-(N-morpholino)ethanesulfonic acid;   

DOI  :  10.1016/S0014-5793(01)02111-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Sarcoplasmic reticulum Ca²⁺-ATPase was digested with proteinase K, V8 protease and trypsin in the absence of Ca²⁺. Unphosphorylated enzyme was rapidly degraded. In contrast, ADP-insensitive phosphoenzyme formed with P_i and phosphorylated state analogues produced by the binding of F⁻ or orthovanadate, were almost completely resistant to the proteolysis except for tryptic cleavage at the T1 site (Arg⁵⁰⁵). The results indicate that the phosphoenzyme and its analogues have a very compact form in the cytoplasmic region, being consistent with large domain motions (gathering of three cytoplasmic domains). Results further show that the structure of the enzyme with bound decavanadate is very similar to ADP-insensitive phosphoenzyme. Thapsigargin did not affect the changes in digestion time course induced by the formation of the phosphorylated state analogues.

【 授权许可】

Unknown