Hydrolytic and autolytic behavior of two forms of calcium-activated neutral protease (CANP).
Some endogenous substrates were incubated with two forms of calcium-activated neutral protease (CANP) with high (muCANP) and low (mCANP) sensitivities to calcium ions. In addition to analyses of the processes of their degradation, changes in the molecular properties of these CANPs were also examined. Among the tested substrate proteins, the myosin heavy chain of rabbit skeletal muscle myofibrils and spectrin or band 3 protein of human erythrocyte membranes were degraded relatively rapidly. So far as these proteins were concerned, a higher degradation velocity was observed for muCANP than for mCANP. Vimentin from ascites tumor cells was degraded most rapidly and no difference was observed in degradation velocity between muCANP and mCANP. In all cases, muCANP and mCANP produced different proteolytic peptide fragments, suggesting the different substrate-specificities of these CANPs. The degradation of substrates always accompanied the autodigestion of CANPs, and the small subunits of both CANPs were degraded in the early stage of the autodigestion. The large subunit of muCANP (79K) was converted to a 76K polypeptide via a 77K polypeptide as an intermediate. The autodigested muCANP with 76K polypeptide retained sufficient protease activity and, moreover, its calcium-sensitivity was higher than that of intact muCANP. The possibility is thus proposed that restricted autodigestion is a necessary activation step for the appearance of activity of muCANP. No such transition was observed for mCANP.
D000818:Animals; D002154:Calpain / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D004910:Erythrocyte Membrane; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental; D008565:Membrane Proteins / Q000097:blood; D051379:Mice; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D013379:Substrate Specificity; D014746:Vimentin / Q000378:metabolism
D007527:Isoenzymes; D008565:Membrane Proteins; D014746:Vimentin; D002154:Calpain