Multi-enzyme complex of glutamine-dependent carbamoyl-phosphate synthetase with aspartate carbamoyltransferase and dihydroorotase from rat ascites-hepatoma cells. Purification, molecular properties and limited proteolysis.

Abstract

Glutamine‐dependent carbamoyl‐phosphate synthetase, the first enzyme of de novo pyrimidine nucleotide biosynthesis, was purified 1300‐fold from Yoshida ascites hepatoma cells (AH 13) as a multi‐enzyme complex with aspartate carbamoyltransferase and dihydroorotase, using dimethyl sulfoxide, glycerol and dithiothreitol as enzyme stabilizers. The ratios of the three enzyme activities were initially 1:65:9.2 and were essentially constant throughout the purification, and the final fraction had ratios of 1:37:11 (dihydroorotase assayed in the forward direction). The purified complex had a specific activity of the synthetase of 0.51 μmol × min−1× (mg protein)−1. It was essentially homogeneous on agarose/acrylamide composite gel electrophoresis, analytical ultra‐centrifugation, and electrofocusing; it had an apparent pI of 5.5. The complex had a sedimentation coefficient (s20, w) of 24.6 S in the presence of 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol, and its molecular weight was estimated to be 870000 by sedimentation equilibrium studies.On polyacrylamide gel electrophoresis in sodium dodecyl sulfate the complex migrated as a single protein band with a molecular weight of 210000, indicating that the complex is composed of four to five subunits of similar size. Sucrose gradient centrifugation in the presence of 1.5 M sodium chloride partially dissociated the three enzyme activities. Limited proteolysis of the complex with pancreatic elastase caused its dissociation and made separable the three activities. The synthetase was recovered as a 197000‐molecular‐weight fragment and the carbamoyltransferase as a 145000‐molecular‐weight fragment. Dihydroorotase was initially recovered as a 197000‐molecular‐weight fragment and was further converted to a 43000‐molecular‐weight fragment. It appears difficult to reconcile these results with the view that the three enzyme activities are located on the same polypeptide chain.