Copurification of pyrimidine-specific carbamyl phosphate synthetase and aspartate transcarbamylase of Neurospora crassa

Abstract

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...

Pyrimidine Biosynthesis during Development of Rat Cerebellum

“…The parallel behavior of these enzymes is consistent with, and may be accounted for, by the observation of Hoogenraad et al [14] that the two activities can be copurified from hematopoietic mouse spleen. Similar copurification of activities of CPS and ATC from baker's yeast was reported by Lue and Kaplan [19,20], and from Neurospora by Williams et al [25]. Genetic evidence that the CPS and ATC of yeast and Neurospora are present as a single complex has been presented [8,15,26] and supports the suggestion that CPS and ATC of eukaryotes are present as a bifunctional complex.…”

Construction of a cDNA to the hamster CAD gene and its application toward defining the domain for aspartate transcarbamylase.

“…The expression of ATCase from such a small portion (28%) of the 7.9-kb mRNA supports the notion that the ATCase domain is encoded by a cassette of genetic information which is independent of the information encoding the CPSase and DHOase domains. Digestion of CAD protein with proteases (11,25) (23,46), to the trifunctional situation seen in mammals and Drosophila melanogaster (6). A possible fourth domain may be the allosteric regulatory region which affects the CPSase activity.…”

Carbamoyl Phosphate Compartmentation in Neurospora : Histochemical Localization of Aspartate and Ornithine Transcarbamoylases

“…Carbamoyl-P synthetase A is in mitochondria with OTCase (2). Carbamoyl-P synthetase P of Neurospora (18), yeast (19), and rat (20) is associated with ATCase in an enzyme aggregate, and is therefore in the same locations as ATCase. The metabolism of carbamoyl-P in the arginine pathway, therefore, proceeds at a different site in a cell from carbamoyl-P metabolism in the pyrimidine pathway, and this separation correlates well with the observed channelling of carbamoyl-P pools in Neurospora.…”

“…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.…”