Purification and Properies of a New Alkaline Protease of Rat Skeletal Muscle

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Purification and properties of a new alkaline protease of rat skeletal muscle have been reported. The purification procedure of the enzyme is as follows: skeletal muscle tissue was extracted successively with Hasselbach-Schneider solution, 5M urea solution and 2% sodium deoxycholate solution. After then, the enzyme was extracted from the residue with 1.1M potassium iodide solution. This enzyme solution was treated with n-butanol, and dialyzed against water. The enzyme precipitated during dialysis was collected and dissolved in 1.1M potassium iodide solution. The enzyme solution was fractionated with acetone, and chromatographed on Sephadex G-200. The final preparation showed over 20000 times of purity.
The optimum pH range of the enzyme activity is 9.5_??_10.5, and the maximum reaction rate occurs at 47_??_57°C. The enzyme is stable below 47°C at pH 7.3. At 37°C, the enzyme is stable during 30min at least, in the pH range of 5.5-10.0. Below pH 5.0, it is relatively labile. Hg²⁺, Ca²⁺, Mg²⁺, Mn²⁺, C0²⁺, and Zn²⁺ scarcely affect the enzyme activity at the concentration of 1mM. Ethylenediaminetetraacetate shows little effect on the activity at the concentration of 10mM, and iodoacetamide, 2, 4-dinitrophenol, p-chloro-mercuribenzoate show the similar effect at the concentration of 1mM. Diisopropyl-fluro-phosphate inhibits the enzyme activity. From the results obtained, this enzyme is presumed to be responsible for the activity of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range.