2-Keto-4-hydroxyglutarate Aldolase of Bovine Liver: Purification, criteria of purity, and general properties

Abstract

Abstract 2-Keto-4-hydroxyglutarate aldolase, which catalyzes the reversible cleavage of 2-keto-4-hydroxyglutarate to yield pyruvate and glyoxylate, has been purified 1300-fold from bovine liver extracts. The purified enzyme appears to be homogeneous when examined by electrophoresis on starch gel or cellulose polyacetate (Sepraphore III) or by chromatography on columns of Sephadex. A molecular weight of 120,000 is obtained with a calibrated column of Sephadex G-200. Two protein bands, both of which are enzymatically active, are observed in polyacrylamide gel electrophoresis. Under certain conditions, two protein peaks are also seen in the ultracentrifuge; the individual components are estimated to have molecular weights of 113,000 and 208,000. The enzyme has a pH optimum of 8.8, is not stimulated by divalent metal ions, and is not inhibited by a number of metal chelating agents. No absolute requirement for added thiol compounds can be shown, but enzymatic activity is strongly inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, or iodoacetate.