Purification and properties of pyruvate kinase type M 2 from rat lung

Abstract

(1) Pyruvate kinase type M 2 from rat lung has been purified 840-fold with an overall yield of 20%. The enzyme gave a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of M r = 224 000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M 2, excepts that of the chicken liver. The enzyme was crystallized. (2) The enzyme has its pH optimum at pH 6.5. The K 0.5 value for phospho enolpyruvate is 0.26 mM ( n H = 1.81) which decreases in the presence of 0.2 mM fructose 1,6-bisphosphate to 0.056 mM ( n H = 1.06). 1 μM fructose 1,6-bisphosphate activates the enzyme at 0.1 mM phospho enolpyruvate half-maximally. The K m value for ADP at 1 mM phospho enolpyruvate is 0.4 mM. The K m value for other nucleoside diphosphates increases in the order ADP<GDP<IDP<UDP. (3) No evidence for an interconversion of pyruvate kinase type M 2 from rat or chicken lung was found. The enzyme was neither a substrate for the cAMP-dependent protein kinase from rabbit muscle nor for the cAMP-independent protein kinase from chicken liver. Since pyruvate kinase type M 2 from chicken liver is inactivated by phosphorylation catalyzed by a cAMP-independent protein kinase (Eigenbrodt, E., Abdel-Fattah Mostafa, M. and Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1047–1055) we suggest that the interconvertible form of pyruvate kinase type M 2 may represent a separate form of the pyruvate kinase type M 2 family.