Effects of autolysis on properties of mu- and m-calpain.
Although the biochemical changes that occur during autolysis of mu- and m-calpain are well characterized, there have been few studies on properties of the autolyzed calpain molecules themselves. The present study shows that both autolyzed mu- and m-calpain lose 50-55% of their proteolytic activity within 5 min during incubation at pH 7.5 in 300 mM or higher salt and at a slower rate in 100 mM salt. This loss of activity is not reversed by dialysis for 18 h against a low-ionic-strength buffer at pH 7.5. Proteolytic activity of the unautolyzed calpains is not affected by incubation for 45 min at ionic strengths up to 1000 mM. Size-exclusion chromatography shows that ionic strengths of 100 mM or above cause dissociation of the two subunits of autolyzed calpains and that the dissociated large subunits (76- or 78-kDa) aggregate to form dimers and trimers, which are proteolytically inactive. Hence, instability of autolyzed calpains is due to aggregation of dissociated heavy chains. Autolysis removes the N-terminal 19 (m-calpain) or 27 (mu-calpain) amino acids from the large subunit and approximately 90 amino acids from the N-terminus of the small subunit. These regions form contacts between the two subunits in unautolyzed calpains, and their removal leaves only contacts between domain IV in the large subunit and domain VI in the small subunit. Although many of these contacts are hydrophobic in nature, ionic-strength-induced dissociation of the two subunits in the autolyzed calpains indicates that salt bridges have an important, possibly indirect, role in the domain IV/domain VI interaction.
Li
Hongqi
H
Muscle Biology Group, University of Arizona, Tucson, AZ 85721-0038, USA.
Thompson
Valery F
VF
Goll
Darrel E
DE
D000818:Animals; D001329:Autolysis; D002154:Calpain / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D008970:Molecular Weight; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D021122:Protein Subunits; D011189:Potassium Chloride; D002154:Calpain; C449000:m-calpain; C449002:mu-calpain