Digestion of mu- and m-calpain by trypsin and chymotrypsin.
Proteolytic digestion by trypsin and chymotrypsin was used to probe conformation and domain structure of the mu- and m-calpain molecules in the presence and the absence of Ca(2+). Both calpains have a compact structure in the absence of Ca(2+); incubation with either protease for 120 min results in only three or four major fragments. A 24-kDa fragment was produced by removal of the Gly-rich area in domain V of the 28-kDa subunit. The other fragments were from the 80-kDa subunit. Except for trypsin digestion of m-calpain, the region between amino acids 245 and 265 (human sequence) was very susceptible to cleavage by both proteases in the absence of Ca(2+); this region is in domain II (IIb of the crystallographic structure). Although no proteolytically active fragments could be isolated from either tryptic or chymotryptic digests, the calpain molecule can remain assembled in a proteolytically active complex even after the 80-kDa subunit has been completely degraded. The results suggest that interaction among different regions of the entire calpain molecule is required for its full proteolytic activity. In the presence of 1 mM Ca(2+), both calpains are degraded to fragments less than 40-kDa in less than 5 min. The C-terminal ends of both subunits, from amino acids 503 to 506 to the end of the 80-kDa subunit and from amino acids 85 to 88 to the end of the 28-kDa subunit, were resistant to degradation by either protease in the presence or in the absence of Ca(2+). Hence, this part of the calpain molecule is in a compact structure that does not change significantly in the presence of Ca(2+).
Thompson
Valery F
VF
Muscle Biology Group, University of Arizona, Tucson 85721, USA. valery.thompson@arizona.edu
Lawson
Kathryn R
KR
Barlow
James
J
Goll
Darrel E
DE
D002154:Calpain / Q000737:chemistry* / Q000378:metabolism; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism
D010455:Peptides; D002918:Chymotrypsin; D014357:Trypsin; D002154:Calpain; C449000:m-calpain; C449002:mu-calpain