Purification and characterization of pig kidney pyruvate kinase (type A).

Abstract

Pyruvate kinase type A was purified from pig kidney with a yield of 9%. The final enzyme fraction had a specific activity of 500 units/mg of protein. The enzyme appeared to be homogeneous on polyacrylamide gel electrophoresis in detergent and in ultracentrifugation experiments. The molecular weight of the enzyme was found to be 210,000 with the use of ultracentrifugation and 249,000 at gel chromatography. The sedimentation coefficient (S degrees 20, w) was calculated to be 9.8 S. For the reduced and alkylated pyruvate kinase, a molecular weight of 60,000 was found with the use of several methods. The Stokes radius for the enzyme was calculated to be 56 A. No NH2-terminal amino acid was detected in the enzyme, and the only findings in carbohydrate analyses of the kidney pyruvate kinase were trace amounts of glucose. The isoelectric point of the enzyme was estimated to be pH 5.6. Pig kidney pyruvate kinase type A was not phosphorylated on incubation with ATP and cyclic 3':5'-AMP-dependent protein kinase. The amino acid compositions of pig kidney and pig muscle pyruvate kinases were very similar and differed clearly from that of pig liver pyruvate kinase.