A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme.
The purified Ca2+-activated protease (CAF) isolated from porcine skeletal muscle and capable of removing Z-disks from intact myofibrils is optimally active on either myofibril or casein substrates at pH 7.5 and in the presence of 1 mM Ca2+ and at least 2 mM 2-mercaptoethanol. No CAF activity is detected when 1 mM Mg2+, Mn2+, Ba2+, Co2+, Ni2+, and Fe2+ are added singly. When added with 1 mM Ca2+, Co2+, Cu2+, Ni2+, and Fe2+ inhibit, whereas Mg2+, Mn2+, and Ba2+ have no effect on CAF activity. CAF is irreversibly inhibited by iodoacetate but is unaffected by soybean trypsin inhibitor. S0/20,W=5.90 S, and sedimentation equilibrium molecular weight - 112 000 for purified CAF. Because purified CAF migrates as two polypeptide chains with molecular weights of 80 000 and 30 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CAF molecule must consist of one each of these two polypeptide chains. Approximate molecular dimensions of 38 X 220 A can be calculated for CAF from calibrated gel permeation column data or from S0/20,W and the molecular weight. Amino acid composition and physical properties of purified CAF distinguish it from the known catheptic enzymes and from other proteases found in blood or in granulocytes. Purified CAF removes Z-disks the 400-A periodicity associated with troponin in the I band and partly degrades M lines but causes no other ultrastructurally detectable effects when incubated with myofibrils. These results agree with the earlier finding that purified CAF degrades troponin, tropomyosin, and C-protein but has no effect on myosin, actin, or alpha-actinin, and suggest that CAF may have a physiological role in disassembly of intact myofibrils during metabolic turnover of myofibrillar proteins.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008854:Microscopy, Electron; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000187:drug effects / Q000201:enzymology*; D009210:Myofibrils / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010447:Peptide Hydrolases / Q000378:metabolism*; D011487:Protein Conformation; D013552:Swine; D013696:Temperature
D000596:Amino Acids; D009124:Muscle Proteins; D008623:Mercaptoethanol; D010447:Peptide Hydrolases; D002118:Calcium