Organization of cytoplasmic domains of sarcoplasmic reticulum Ca2+‐ATPase in E1P and E1ATP states: a limited proteolysis study

FEBS Letters

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

[1] Institute of Molecular and Cellular Biosciences, The 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;    AMPPCP;    adenosine 5′-(β;    γ-methylene)triphosphate;    AMPPNP;    adenosine 5′-(β;    γ-imido)triphosphate;    TNP;    2′;    3′-O-(2;    4;    6-trinitrocyclohexadienylidene);    NEM;    N-ethylmaleimide;   

DOI  :  10.1016/S0014-5793(01)02801-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

In order to characterize the domain organization of sarcoplasmic reticulum Ca²⁺-ATPase in different physiological states, limited proteolysis using three proteases (proteinase K (prtK), V8 and trypsin) was conducted systematically and quantitatively. The differences between E₂ and E₂P were examined in our previous study and E₂P was characterized by the complete resistance to all three proteases (except for trypsin attack at the very top of the molecule (T1 site)). The same strategies were employed in this study for E₁ATP, E₁PADP and E₁P states. Because of the transient nature of these states, they were either stabilized by non-hydrolyzable analogues or made predominant by adjusting buffer conditions. Aluminum fluoride (without ADP) was found to stabilize E₁P. All these states were characterized by strong (E₁ATP) to complete (E₁PADP and E₁P) resistance to prtK and to V8 but only weak resistance to trypsin at the T2 site. Because prtK and V8 primarily attack the loops connecting the A domain to the transmembrane helices whereas the trypsin T2 site (Arg¹⁹⁸) is located on the outermost loop in the A domain, these results lead us to propose that the A domain undergoes a large amount of rotation between E₁P and E₂P. Combined with previous results, we demonstrated that four states can be clearly distinguished by the susceptibility to three proteases, which will be very useful for establishing the conditions for structural studies.

【 授权许可】

Unknown