Binding and aggregation of human mu-calpain by terbium ion.
Human mu-calpain is activated maximally by 100-200 microM Ca2+. Both the 80 kDa and 29 kDa subunits of mu-calpain have a EF-hand type calcium-binding domain. It is known that trivalent terbium ion (Tb3+) mimics Ca2+ in many biological systems. We found that Tb3+ alone transiently activated calpain. However, in the presence of Ca2+, Tb3+ inhibited mu-calpain with an IC50 of about 100 microM. As high as 10 mM Ca2+ did not significantly shift the IC50 of Tb3+. Preincubating mu-calpain by Ca2+ (before Tb3+ and substrate were added) did not diminish the inhibition by Tb3+. On the other hand, pretreating mu-calpain with Tb3+ produced that Tb3+ has a slow dissociation rate for the calcium-binding sites when compared to Ca2+. Electrophoretic analysis revealed that terbium ion transiently activated mu-calpain followed by the aggregation of the proteinase.
Department of Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI 48105, USA.
K J
KJ
Raser
M
M
Buroker-Kilgore
K K
KK
Wang
D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D003956:Dialysis; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004912:Erythrocytes / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D011485:Protein Binding; D013725:Terbium / Q000378:metabolism / Q000494:pharmacology*
D002364:Caseins; D013725:Terbium; D004533:Egtazic Acid; D002154:Calpain; D002118:Calcium