Determination of Molecular Weight and Molecular Structurof Rat-Liver Pyruvate Carboxylase

Abstract

The molecular wieght of pyruvate carboxylase [EC 6.4.1.1] isolated form pigeon and rat liver mitochondria was examined using analytical ultracentrifugation and EM. The enzyme molecule appeared as a tetramer with the 4 subunits arranged at the corners of a square. Sedimentation studies in the analytical ultracentrifuge, extrapolated to infinite dilution, showed the tetramer to have a MW .**GRAPHIC**. of 280,000 and an .**GRAPHIC**. of 12.7 S. The tetramer could be dissociated into trimers and dimers of lower specific enzymic activity by storage at 4.degree. C or incubation at -20.degree. C at low protein concentrations. The isolated trimers and dimers had a molecular weight .**GRAPHIC**. of 210,000 and 140,000, respectively, and an .**GRAPHIC**. of 10.85 S and 7.55 S, respectively. Incubation with 2 M urea at 20.degree. C yielded enzymically inactive subunits .**GRAPHIC**. = 70000; .**GRAPHIC**. = 4.95 S). The molecular weights for pyruvate carboxylase and its subunits, as calculated from the subunit diameter observed in the EM, were consistent with the values obtained from sedimentation studies.