Purification and some properties of an alkaline proteinase from rat skeletal muscle

Abstract

Rat skeletal muscle was homogenized in 0.05 M Tris/HCl, pH 8.5, containing 1 M KCl. Myofibrillar proteins were precipitated by addition of (NH4)2SO4 (33% saturation). The alkaline proteolytic activity that was precipitated with the myofibrillar proteins was solubilized with trypsin (conjugated to Sepharose) and further purified by affinity chromatography, ion-exchange chromatography and gel filtration. The purified enzyme migrates as a single band in polyacrylamide disc electrophoresis, and has optimum hydrolytic activity with azocasein and [14C]Hb as substrates at pH 9.4 and 9.6, respectively. Its apparent MW, as determined by gel filtration on Sephadex G-75, is 30,800. The purified alkaline proteinase is strongly inhibited by equimolar amounts of soya-bean trypsin inhibitor and ovomucoid, whereas di-isopropyl phosphorofluoridate and .alpha.-toluenesulphonyl fluoride have no effect. N-ethylmaleimide and p-chloromercuribenzoate have inhibitory effects on the enzyme activity. Bivalent metal ions (Fe2+, Co2+, Zn2+, Mg2+, Mn2+) diminish the proteolytic activity, at 1 mM concentrations. Ca2+ ions and the metal ion-chelating agent EDTA are without effect on enzyme activity. The enzyme is part of the alkaline proteolytic activity that appears to be associated with myofibrillar proteins.