Determination of molecular weight and molecular structure of rat-liver pyruvate carboxylase

The molecular weight of pyruvate carboxylase isolated from pigeon and rat liver mitochondria was examined using analytical ultracentrifugation and electron microscopy. The enzyme molecule appeared as a tetramer with the four subunits arranged at the corners of a square. Sedimentation studies in the analytical ultracentrifuge, extrapolated to infinite dilution, showed the tetramer to have a molecular weight Mc=0r of 280 000 and an So20,w of 12.7 S. The tetramer could be dissociated into trimers and dimers of lower specific enzymic activity by storage at 4 degrees C or incubation at -- 20 degrees C at low protein concentrations. The isolated trimers and dimers had a molecular weight Mc=0r of 210 000 and 140 000, respectively, and an So20,w of 10.85 S and 7.55 S, respectively. Incubation with 2 M urea at 20 degrees C yielded enzymically inactive subunits (Mc=0r = 70 000; So20,w = 4.95 S). The molecular weights (for pyruvate carboxylase and its subunits), as calculated from the subunit diameter observed in the electron microscope, were consistent with the values obtained from sedimentation studies.