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.
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