Autolysis, Ca2+ requirement, and heterodimer stability in m-calpain.
The roles of N-terminal autolysis of the large (80 kDa) and small (28 kDa) subunits in activation of rat m-calpain, in lowering its Ca2+ requirement, and in reducing its stability have been investigated with heterodimeric recombinant calpains containing modified subunits. Both autolysis and [Ca2+]0.5 were influenced by the ionic strength of the buffers, which accounts for the wide variations in previous reports. Autolysis of the small subunit (from 28 to 20 kDa) was complete within 1 min but did not alter either the Ca2+ requirement ([Ca2+]0.5) or the stability of the enzyme. Autolysis of the NHis10-80k large subunit at Ala9-Lys10 is visible on gels, was complete within 1 min, and caused a drop in [Ca2+]0.5 from 364 to 187 microM. The lower value of [Ca2+]0.5 is therefore a property of the Delta9-80k large subunit. Autolysis at Ala9-Lys10 of the unmodified 80-kDa large subunit is not detectable on gels but was assayed by means of the fall in [Ca2+]0.5. This autolysis was complete in 3.5 min and was inhibited by high [NaCl]. The autolysis product of these calpains, which is essentially identical to that of natural m-calpain, was unstable in buffers of high ionic strength. Calpain in which the large subunit autolysis site had been mutated was fully active but did not undergo a drop in [Ca2+]0.5, showing that m-calpain is active prior to autolysis. The main physiological importance of autolysis of calpain is probably to generate an active but unstable enzyme, thus limiting the in vivo duration of calpain activity.
Elce
J S
JS
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada. jse@post.queensu.ca
Hegadorn
C
C
Arthur
J S
JS
D000818:Animals; D001329:Autolysis; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D019281:Dimerization; D004795:Enzyme Stability; D009154:Mutation; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion
D002364:Caseins; D011994:Recombinant Proteins; D002154:Calpain; D002118:Calcium