m‐Calpain subunits remain associated in the presence of calcium

FEBS Letters

Croall, Dorothy E¹  Arthur, J.Simon C²  Dutt, Previn²  Elce, John S² 

[1] Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, ME 04469-5735, USA;Department of Biochemistry, Queen's University, and The Protein Engineering Network of Centres of Excellence, Kingston, Ont. K7L 3N6, Canada

关键词: m-Calpain;    Autolysis;    Subunit dissociation;    EF-hand;    2ME;    2-mercaptoethanol;    BSA;    bovine serum albumin;    C105S;    the m-calpain large subunit active site Cys105Ser mutation;    EDTA;    ethylenediamine tetraacetic acid;    EGTA;    ethylene glycol-bis(β-aminoethylether)-N;    N;    N′;    N′-tetraacetic acid;    His-tag;    N-terminal His10- and C-terminal -His6 sequences;    NTA;    nitrilo-triacetic acid;    Z-LLY-CHN2;    N-carbobenzoxy-l-leucyl-l-leucyl-l-tyrosyl-diazomethane;   

DOI  :  10.1016/S0014-5793(98)01167-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The hypothesis that calpain subunits dissociate in the presence of Ca²⁺ has been tested by methods which avoid interference by Ca²⁺-induced aggregation and large subunit autolysis. Inactive Cys¹⁰⁵Ser-m-calpain, bound either to Ni-NTA-agarose or to immobilized casein, after incubation with Ca²⁺, could be recovered in high yield as a heterodimer. Natural bovine m-calpain, after irreversible inhibition with Z-LLY-CHN₂, also bound to immobilized casein and was eluted as a heterodimer. The Ca²⁺ requirements of calpain containing a small subunit with EF-hand mutations were higher, both before and after autolysis, than those of wild-type calpain. In mixtures of wild-type and mutant enzymes, subunit exchange did not occur in the presence of Ca²⁺. The results demonstrate that the subunits in both natural and recombinant m-calpain, in the given experimental conditions, remain associated in the presence of Ca²⁺ both before and after autolysis.

【 授权许可】

Unknown