In Neurospora the initial enzymes of pyrimidine synthesis, pyrimidine-specific carbamyl phosphate synthetase and aspartate transcarbamylase, are specified by the same locus, pyr-3. A purification technique devised for the most labile of these two enzymes, the synthetase, resulted in the copurification of aspartate transcarbamylase. The two activities remained associated throughout (NH&S04 precipitation, calcium phosphate gel adsorption, gel filtration chromatography, DEAE-cellulose chromatography, and sucrose density gradient centrifugation. An enzyme preparation estimated to be 50 pure was obtained containing pyrimidinespecific carbamyl phosphate synthetase and aspartate transcarbamylase at specific activities 50 and 36 times that found in the original derepressed extract (250 and 180 times that found in wild type crude extracts). The molecular weight of the enzyme complex was estimated to be 650,000 g per mole on the basis of its elution pattern from agaiose gel N eurospora has been shown to possess two enzymes which form carbamyl phosphate, an intermediate of both arginine and pyrimidine synthesis (Williams and Davis, 1970;. The two carbamyl phosphate synthetases are under control of separate genetic loci and can be clearly distinguished on the basis of molecular weight, feedback inhibitors, derepression conditions, and stabilization factors. Irt vivo one enzyme produces carbamyl phosphate specifically for use in the arginine pathway while the second synthesizes carbamyl phosphate specifically for utilization via the pyrimidine pathway (Davis, 1967). The syntheses of CPSp,,l and ATC (carbamoy1phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2.) are coderepressed by pyrimidine starvation. The CPSp,, activity can be completely inhibited by 5-10 X lou4 M UTP (uridine triphosphate). ATC activity is not inhibited by UTP. CPSp,, is cold labile and has much greater affinity for L-glutamine than for ammonia as a nitrogen source (Williams and Davis, 1970). . To whom reprint requests should be directed.Abbreviations used are: CPSpyr, pyrimidine-specific carbamyl phosphate synthetase; ATC, aspartate transcarbamylase.GM 19703-02). filtration columns and its sedimentation properties in sucrose density gradients. The synthetase activity of the purified complex is completely inhibited by the end product, UTP (uridine triphosphate), at 1 x 10-3 M. On sucrose density gradients the complex sediments at a value of 21 S in the absence of UTP and at 15 S in the presence of UTP. This suggests the dissociation of enzyme subunits in the presence of the feedback inhibitor; however no molecular weight change is observed, in the presence of UTP, in agarose gel filtration. Earlier studies from this laboratory have shown that under normal in viuo conditions carbamyl phosphate produced by the pyrimidine-enzyme complex is unavailable as a substrate for ornithine transcarbamylase in the arginine synthetic pathway. The observed confinement of carbamyl phosphate in pyrimidine synthesis could be explained if carbamyl phosphate existed as an...
Carbamoyl phosphate is required for arginine and pyrimidine synthesis. In the arginine pathway, it is used in the ornithine transearbamoylase (EC 2.1.2.1) reaction; in the pyrimidine pathway, it is used in the aspartate transcarbamoylase (EC 2.1.3.2) reaction. In Neurospora crassa, two pathway-specific enzymes catalyze the synthesis of carbamoyl phosphate, and two pathspecific pools of carbamoyl phosphate are maintained. Histochemical studies show-that ornithine transcarbamoylase is located in mitochondria, and, with less certainty, that aspartate transcarbamoylase is confined largely to nuclei. The enzymes that form carbamoyl phosphate are associated with the respective transcarbamoylases in the cell. Therefore, the segregation of carbamoyl phosphate pools could be accounted for by one or both organellar membranes, which demarcate two separate sites of carbamoyl phosphate metabolism in Neurospora. The alternative possibility that the enzyme complex that produces and consumes carbamoyl phosphate in the pyrimidine pathway could explain the channelling of carbamoyl phosphate, wholly or in part, is discussed.Nonrandom distribution of metabolites within cells as a factor in their metabolic fates is known as channelling. Several compounds involved in oxidative phosphorylation are confined to mitochondria, and certain enzyme aggregates appear to retain intermediates in the course of sequential reactions. The metabolism of carbamoyl phosphate (carbamoyl-P) in eukaryotes has recently been studied in relation to channelling (1-3). Carbam6yl-P is an intermediate of the arginine and pyrimidine pathways, used, respectively, by ornithine transcarbamoylase (OTCase: carbamoylphosphate: ornithine carbamoyltransferase, EC 2.1.3.1) and aspartate transcarbamoylase (ATCase: carbamoylphosphate: aspartate carbamoyltransferase, EC 2.1.3.2). Eukaryotes, in contrast to bacteria, have two enzymes for carbamoyl-P synthesis. In Neurospora crassa, both are glutamine-dependent carbamoyl-P synthetases (EC 2.7.2.5). Studies with mutants lacking one or the other of the synthetases show that one (carbamoyl-P synthetase A) has a specific role in the arginine pathway, while the other (carbamoyl-P synthetase P) functions solely in the pyrimidine pathway (1, 4, 5). These studies suggest that two discrete pools of carbamoyl-P are maintained, since mutants lacking one of the synthetases have an absolute and specific requirement for the corresponding end-product, either uridylic acid or arginine. More direct evidence that supports this view has been published (2).In mammals, it was established that citrulline synthesis (OTCase and carbamoyl-P synthetase I, which uses ammonia as an N donor) was largely a mitochondrial function (6, 7). Carbamoyl-P synthetase II and ATCase were apparently localized in the high-speed supernatant or light membrane fraction of mammalian tissues according to differential centrifugation studies (8). Recent histochemical localization of OTC in mitochondria (and possibly in the cytosol) of rat hepatocytes has been demons...
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