The effects of truncations of the small subunit on m-calpain activity and heterodimer formation.
In order to study subunit interactions in calpain, the effects of small subunit truncations on m-calpain activity and heterodimer formation have been measured. It has been shown previously that active calpain is formed by co-expression of the large subunit (80 kDa) of rat m-calpain with a delta 86 form (21 kDa) of the small subunit. cDNA for the full-length 270 amino acid (28.5 kDa) rat calpain small subunit has now been cloned, both with and without an N-terminal histidine tag (NHis10). The full-length small subunit constructs yielded active calpains on co-expression with the large subunit, and the small subunit was autolysed to 20 kDa on exposure of these calpains to Ca2+. A series of deletion mutants of the small subunit, NHis10-delta 86, -delta 99, -delta 107, and -delta 116, gave active heterodimeric calpains with unchanged specific activities, although in decreasing yield, and with a progressive decrease in stability. NHis10-delta 125 formed a heterodimer which was inactive and unstable. Removal of 25 C-terminal residues from delta 86, leaving residues 87-245, abolished both activity and heterodimer formation. The results show that: (a) generation of active m-calpain in Escherichia coli requires heterodimer formation; (b) small subunit residues between 94 and 116 contribute to the stability of the active heterodimer but do not directly affect the catalytic mechanism; (c) residues in the region 245-270 are essential for subunit binding. Finally, it was shown that an inactive mutant Cys103-->Ser-80k/delta 86 calpain, used in order to preclude autolysis, did not dissociate in the presence of Ca2+, a result which does not support the proposal that Ca(2+)-induced dissociation is involved in calpain activation.
Elce
J S
JS
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Davies
P L
PL
Hegadorn
C
C
Maurice
D H
DH
Arthur
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D001329:Autolysis / Q000235:genetics; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D003001:Cloning, Molecular; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006868:Hydrolysis; D008168:Lung / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D051381:Rats; D017384:Sequence Deletion
D002154:Calpain; D002118:Calcium