Bovine Pyruvate Kinases: I. PURIFICATION AND CHARACTERIZATION OF THE SKELETAL MUSCLE ISOZYME

Abstract

Abstract Pyruvate kinase has been purified from bovine skeletal muscle by a procedure that includes only heat, ammonium sulfate fractionation, and chromatography on carboxymethyl Sephadex. Crystallization was accomplished by dialysis at 4° against 47% saturated ammonium sulfate. Since this simple purification scheme, with only slight modifications, has also been used to isolate turkey and rabbit muscle pyruvate kinases, it may be generally applicable to the isolation of muscle pyruvate kinase from birds and mammals. Bovine muscle pyruvate kinase prepared by the procedure described here was found to be homogeneous, as determined by disc gel electrophoresis at pH 9.5, gel electrophoresis in the presence of sodium dodecyl sulfate, sedimentation velocity and sedimentation equilibrium, isoelectric focusing, and immunodiffusion. It sediments at 9.9 S (s020,w) and has a maximum velocity of 400 µmoles per min per mg or more at 25°, or 2.4 times that value at 37°. The enzyme has maximal activity at pH 7.1, with an apparent Michaelis constant for phosphoenolpyruvate of 0.04 mm or less, and for ADP of 0.4 mm. Its isoelectric pH during electrofocusing is 8.9. Sedimentation equilibrium yielded a molecular weight of 230,000 with a range of ±3,000 in dilute phosphate buffer, and 57,000 ± 1,500 in 3.5 m guanidine hydrochloride, using a partial specific volume of 0.740 ml per g calculated from the amino acid composition. The molecular weight in guanidine hydrochloride, confirmed by gel electrophoresis in sodium dodecyl sulfate, indicates the presence of four polypeptide chains in the native enzyme. Binding studies suggest a total of four phosphoenolpyruvate binding sites, or one per subunit. Ion sensitivities and other chemical and physical parameters are very similar to those reported for rabbit muscle pyruvate kinase.