E-F hand structure-domain of calcium-activated neutral protease (CANP) can bind Ca2+ ions.
The cDNA fragments corresponding to the domains with four consecutive E-F hand structures in the large and small subunits of chicken and rabbit calcium-activated neutral protease (CANP) were inserted into an expression vector (pUC8 or pUC18). The resulting plasmids were used to transform E. coli, and isopropyl-1-thio-beta-D-galactoside (IPTG)-inducible expression was performed. The resulting four kinds of E-F hand structure-domains (the chicken large subunit, rabbit high- and low-calcium-requiring large subunits, and rabbit small subunit) were purified and analyzed for their calcium-binding abilities and capacities by the microscale filter assay. Most of the E-F hand structures could bind calcium and 2 or 4 mol of Ca2+ ions bound to the four consecutive E-F hand structures. The calcium-binding affinity of the E-F hand structures in the large subunit roughly corresponds to the calcium concentration required for its CANP activity.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D003001:Cloning, Molecular; D004926:Escherichia coli; D008970:Molecular Weight; D011487:Protein Conformation; D011817:Rabbits
D002147:Calmodulin; D002154:Calpain; D002118:Calcium