Studies on casein. 1. Purification and properties of phosphoprotein phosphatase

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Abstract
An improved method for the preparation of phosphoprotein phosphatase from ox spleen is described. The enzyme was purified 60-fold with a yield of 21%. Frozen spleen was homogenized with cold 0.5 M NaCl in 0.05 M sodium acetate-acetic acid buffer, pH 5.9. The supernatant extract was cooled to 0[degree] C and shaken with cold butanol. The emulsion separated into 3 layers on centrifugation. The bottom layer containing the enzyme was collected and the intermediate layer was extracted with buffered NaCl. The combined extracts were cooled to 2[degree] and the fraction that precipitated on standing at -15[degree] between 20-50% acetone was collected. The precipitate was dialyzed at 2[degree] for 48 hours against distilled water. A precipitate settled and was dissolved in 0.5 M NaCl - 0.05 M sodium acetate buffer, pH 5.9 and centrifuged to remove some insoluble material. The yellowish supernatant contained the phosphoprotein phosphatase activity. The enzyme preparation was free from proteolytic activity and had no action on [beta]-glycerol phosphate. The enzyme was active on caseins, phenyl phosphate and inorganic pyrophosphate. Hence it cannot be described as a specific phosphoprotein phos-phatase. The pH optimum with caseins and phenyl phosphate was about pH 5.5. The enzyme was stable over a wide range of pH and at temperatures below 70[degree].