Activation of calpain I and calpain II: a comparative study using terbium as a fluorescent probe for calcium-binding sites.
The present study demonstrates the activation of calpain I and calpain II by micromolar levels of terbium and has utilized the enhancement in the fluorescence of protein-bound terbium to study and compare the calcium binding sites of the two enzymes. Calpain I and calpain II were isolated from bovine erythrocytes and brain, respectively. While the rates of activation of calpain I by terbium and calcium are comparable, the rate of activation of calpain II was much greater in the presence of terbium than in the presence of calcium. Binding of terbium ions to calpains was monitored by the enhanced terbium fluorescence and by the changes in the intrinsic protein fluorescence of calpains. Stoichiometric titrations indicated that calpain I and calpain II bound four and six molar equivalents of terbium ion, respectively. During the titration, the intrinsic protein fluorescence of calpain II was successively quenched whereas that of calpain I showed an abrupt drop just prior to the saturation. The association constants (Ka) increased from 10(5) to 10(7) M-1 for calpain I and from 10(4) to 10(6) M-1 for calpain II with addition of increasing molar equivalents of terbium. Titration of enzymatic activities with calcium showed that the activation of calpain I required fewer molar equivalents of metal ions than were necessary for the activation of calpain II, in agreement with stoichiometric titration with terbium.
Zimmerman
U J
UJ
Department of Pathology, University of Pennsylvania Medical School, Philadelphia 19104.
Schlaepfer
W W
WW
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D002417:Cattle; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000494:pharmacology*
D002135:Calcium-Binding Proteins; D013725:Terbium; D002154:Calpain; D002118:Calcium