THE EFFECTS OF TRUNCATIONS OF THE SMALL-SUBUNIT ON M-CALPAIN ACTIVITYAND HETERODIMER FORMATION

In order to study subunit interactions in calpain, the effects of smal l subunit truncations on m-calpain activity and heterodimer formation have been measured. It has been shown previously that active calpain i s formed by co-expression of the large subunit (80 kDa) of rat m-calpa in with a Delta 86 form (21 kDa) of the small subunit. cDNA for the fu ll-length 270 amino acid (28.5 kDa) rat calpain small subunit has now been cloned, both with and without an N-terminal histidine tag (NHis(1 0)). The full-length small subunit constructs yielded active calpains on coexpression with the large subunit, and the small subunit was auto lysed to 20 kDa on exposure of these calpains to Ca2+. A series of del etion mutants of the small subunit, NHis(10)-Delta 86, -Delta 99, -Del ta 107, and -Delta 116, gave active heterodimeric calpains with unchan ged specific activities, although in decreasing yield, and with a prog ressive decrease in stability. NHis(10)-Delta 125 formed a heterodimer which was inactive and unstable. Removal of 25 C-terminal residues fr om Delta 86, leaving residues 87-245, abolished both activity and hete rodimer formation. The results show that: (a) generation of active m-c alpain in Escherichia coli requires heterodimer formation; (b) small s ubunit 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 Cys(105) --> Ser-80k/De lta 86 calpain, used in order to preclude autolysis, did not dissociat e in the presence of Ca2+, a result which does not support the proposa l that Ca2+-induced dissociation is involved in calpain activation.