Rat Liver Pyruvate Carboxylase

Abstract

Abstract Pyruvate carboxylase purified to near homogeneity from rat liver mitochondria sedimented as a single peak in the ultracentrifuge, s020,w = 15 S, and migrated as a single band on sodium dodecyl sulfate, acrylamide gels. The protomer molecular weight was 1.3 x 105; this unit dissociated further to a mixture of three or four different polypeptide chains. The native enzyme contained both biotin and tightly bound manganese. In addition to substrates the reaction required the presence of acetyl-CoA, magnesium in excess of MgATP, and a monovalent cation (preferably K+, NH4+, or Rb+). A discussion of the mechanistic function of the cations is presented based on experiments in which the substrates were varied at fixed levels of the metal activators. Variation of acetyl-CoA resulted in sigmoid saturation curves that were marked functions of pH, temperature, and ionic strength. Other aspects of the reaction are discussed preparatory to a kinetic study of the over-all mechanism.