Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle

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Abstract
Ca2+-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial-Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulfate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only 1 polypeptide chain of MW 73,000. The purification procedure eliminated a contaminant containing 2 components of MW about 30,000 each. Whole casein or .alpha.1-casein was hydrolyzed with a maximum rate at 30.degree. C, pH 7.5, and with 5 mM-CaCl2, but myofibrils were susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30,000 dalton component as well as in various other higher- and lower-MW peptide fragments. Troponin T, troponin I, .alpha.-tropomyosin, some high-MW proteins (M protein, heavy chain of myosin) and 3 unidentified proteins are degraded. The number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton et al. The Ca2+-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. The proteinase had an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca2+-activated neutral proteinase.