Purification and properties of the pyruvate kinase isozyme M1 from the pig brain

Abstract

There are four pyruvate kinase isozymes in vertebrate tissues, designated as L, M1, M2, and R. Although pyruvate kinases have been purified and characterized from pig liver, muscle, kidney, and heart, the brain isozyme has not. The aim of this work was to purify, characterize and make an isozymic designation for the pig brain pyruvate kinase. Purification was accomplished by chromatography on phosphocellulose, Sephadex G200, and blue-dextran agarose columns. The molecular weight of the native enzyme was determined by sucrose density centrifugation. The degree of purity, and subunit molecular weight were determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was estimated by the rapid isoelectric focusing method in sucrose gradients. The pH optimum, and kinetics in the presence and absence of fructose-1,6-diphosphate were determined spectrophotometrically. The purification scheme used resulted in a 382-fold purification of pig brain pyruvate kinase, and a final specific activity of 191 Units/mg protein. As estimated by scanning of the sodium dodecyl sulfate polyacrylamide gels, the purification scheme also resulted in a preparation that was of at least 98% purity. Pig brain pyruvate kinase has a native molecular weight of approx. 230,000, and a subunit molecular weight of approx. 60,000. The pI was determined to be approximately 8.0, while the pH optimum was estimated at pH 7.4. Fructose-1,6-diphosphate had no effect on either the Km for phospho(enol)pyruvate, or the Vmax of the reaction. Pig brain pyruvate kinase is similar in native and subunit molecular weights to the other pyruvate kinases from pig tissues. Like other vertebrate pyruvate kinases, pig brain pyruvate kinase is a tetramer. The pH optimum of 7.4 excludes pig brain pyruvate kinase as a type L isozyme, but cannot distinguish between M1 and M2 isozymes. An isoelectric point of 8.0, as well as hyperbolic kinetics and the lack of inactivation by fructose-1,6-diphosphate, however, are consistent with the pyruvate kinase from pig brain being categorized as a type M1 isozyme.