Comparison of low and high calcium requiring forms of the calcium-activated neutral protease (CANP) from rabbit skeletal muscle.
Two distinct calcium-dependent neutral proteases (CANPs) with different sensitivities to calcium ions were purified concurrently by almost the same procedures from rabbit skeletal muscle and their enzymatic properties were compared (sensitivity to various divalent metal ions, the pH dependency and heat-stability of the activity, and the hydrolytic activity towards various substrates). They were further compared chemically in terms of the state of thiol groups, the amino acid compositions of subunits and the peptide fragments by digestion with S. aureus V8 protease. The low calcium requiring form of CANP (microCANP) was more sensitive to other divalent metal ions such as Sr2+ and Ba2+ than the high calcium requiring form of CANP (mCANP). The comparison of the pH dependency of these CANP activities showed that microCANP was active in a broader pH range than mCANP and the former was more heat-stable than the latter. Both CANPs had similar affinity to various substrates, but the hydrolytic velocity was several times higher with microCANP than with mCANP. Although they were inhibited by thiol protease inhibitors to the same extent, the states of thiol groups in them were quite different. The thiol group involved in the catalytic activity of the enzyme was exposed without adding Ca2+ in microCANP, whereas the group in mCANP became exposed only when sufficient Ca2+ was added. The large subunits of these two CANPs were different in their amino acid compositions and in the peptide fragment patterns produced by S. aureus V8 protease but the small subunits were indistinguishable from each other. These results led us to conclude that these two CANPs are quite different in nature and are not in a simple relationship, i.e., one of them is not derived from the other by autolysis or modification.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002154:Calpain; D002413:Cations, Divalent; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits; D013379:Substrate Specificity
D002413:Cations, Divalent; D007527:Isoenzymes; D004228:Dithionitrobenzoic Acid; D010450:Endopeptidases; D002154:Calpain; D002118:Calcium