Pyruvate carboxylase from Aspergillus nidulans

Publisher Website
Google Scholar
Abstract
1. A method is described for purification of pyruvate carboxylase from Aspergillus nidulans by affinity chromatography on monomeric avidin‐Sepharose. The purified enzyme is homogeneous as judged by electrophoretic and immunochemical analysis. The sub‐unit Mr determined by electrophoresis in the presence of sodium dodecyl sulphate is 133000 ± 5000.2. Electron microscopic analysis of purified A. nidulans pyruvate carboxylase after negative staining with uranyl acetate reveals the presence of molecules showing rhomboid and triangular projections as well as a projection showing two intensity maxima. A cleft running along the longitudinal axis of the sub‐unit is observed in the rhomboid and triangular projections. Interconversion between all three projections can be obtained in a tilt series.3. Significantly better preservation of molecular structure is obtained if A. nidulans pyruvate carboxylase is prepared for electron microscopy in the presence of acetyl‐CoA, 2‐oxoglutarate or as the enzyme‐avidin complex. l‐Aspartate has no significant effect when added alone but markedly decreases the enhanced preservation given by acetyl‐CoA. No marked alterations in molecular dimensions are caused by any of these additions.4. l‐Aspartate, but not 2‐oxoglutarate, enhances the rate of inactivation observed on incubation of A. nidulans pyruvate carboxylase at 4°C in the presence of 0.5 M KCl.5. Addition of l‐aspartate in low concentrations enhances the effectiveness of inhibition by 2‐oxoglutarate by causing a decrease in the value of [I]0.5 without affecting the Hill coefficient h or the extent of activity observed at saturating 2‐oxoglutarate concentrations. Conversely addition of low concentrations of 2‐oxoglutarate decreases the concentration of l‐aspartate required to give 50% inhibition but also causes a fall in h and an absolute increase in the extent of activity observed in the presence of saturating l‐aspartate concentrations.6. The data are consistent with the proposal that A. nidulans pyruvate carboxylase is a tetrameric molecule in which the four sub‐units are located at the corners of a tetrahedron. Metabolites which regulate the activity of the enzyme do not cause major alterations in this molecular structure but may alter its stability. The data presented also provide further evidence supporting the postulate that l‐aspartate and 2‐oxoglutarate occupy distinct regulatory sites on A. nidulans pyruvate carboxylase.