Rabbit skeletal muscle calcium-dependent protease requiring millimolar CA2+. Purification, subunit structure, and Ca2+-dependent autoproteolysis.
Rabbit skeletal muscle calcium-dependent protease which requires millimolar Ca2+ concentration for activity is a dimer composed of Mr = 73,000 and 30,000 subunits. The subunit structure has been confirmed by co-elution of the two polypeptide bands on Sephadex G-200 chromatography, and by cross-linking the enzyme with dimethyl suberimidate followed by sodium dodecyl sulfate-disc gel electrophoresis. Preincubation of the enzyme with p-chloromercuribenzoate prior to nondenaturing electrophoresis resulted in dissociation of the subunits. The rabbit muscle calcium-dependent protease was rapidly inactivated when incubated in the presence of 6 mM Ca2+. The half-life of protease activity at 30 degrees C was independent of protease concentrations over the range of 0.005 to 0.272 mg/ml. The rate of inactivation was not affected by a 270-fold molar excess of a substrate protein succinylated lysozyme. Protease activity also rapidly decreased (t1/2 = 8.4 min) during the assay at 37 degrees C as determined by a decrease in linearity of the time course when substrate was limiting. The rate of protease inactivation during the assay was essentially the same as that observed when the protease was incubated at 37 degrees C in the absence of substrate (t1/2 = 7.2 min). The addition of either leupeptin or the calcium-dependent protease inhibitor protein from dog heart prevented protease inactivation. The protease displayed an increase in activity during the time course of autoproteolysis at 30 degrees C when activity was measured in the presence of 0.2 mM Ca2+ instead of 5 mM Ca2+.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D004285:Dogs; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits
D046911:Macromolecular Substances; D011480:Protease Inhibitors; D010447:Peptide Hydrolases; D002118:Calcium