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

Biochemistry ◽  
1970 ◽  
Vol 9 (22) ◽  
pp. 4329-4335 ◽  
Author(s):  
Larry Gale Williams ◽  
Sharon Bernhardt ◽  
Rowland Hallowell Davis
Keyword(s):  
Neurospora Crassa ◽  
Aspartate Transcarbamylase ◽  
Carbamyl Phosphate ◽  
Carbamyl Phosphate Synthetase

scholarly journals Adenovirus Preterminal Protein Binds to the CAD Enzyme at Active Sites of Viral DNA Replication on the Nuclear Matrix

1998 ◽  
Vol 72 (4) ◽  
pp. 2896-2904 ◽  
Author(s):  
Peter C. Angeletti ◽  
Jeffrey A. Engler
Keyword(s):  
Western Blotting ◽  
Nuclear Matrix ◽  
Active Sites ◽  
Binding Activity ◽  
Aspartate Transcarbamylase ◽  
Viral Dna ◽  
Carbamyl Phosphate ◽  
Carbamyl Phosphate Synthetase ◽  
Cell Extracts

ABSTRACT Adenovirus (Ad) replicative complexes form at discrete sites on the nuclear matrix (NM) via an interaction mediated by the precursor of the terminal protein (pTP). The identities of cellular proteins involved in these complexes have remained obscure. We present evidence that pTP binds to a multifunctional pyrimidine biosynthesis enzyme found at replication domains on the NM. Far-Western blotting identified proteins of 150 and 240 kDa that had pTP binding activity. Amino acid sequencing of the 150-kDa band revealed sequence identity to carbamyl phosphate synthetase I (CPS I) and a high degree of homology to the related trifunctional enzyme known as CAD (for carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase). Western blotting with an antibody directed against CAD detected a 240-kDa band that comigrated with that detected by pTP far-Western blotting. Binding experiments showed that a pTP-CAD complex was immunoprecipitable from cell extracts in which pTP was expressed by a vaccinia virus recombinant. Additionally, in vitro-translated epitope-tagged pTP and CAD were immunoprecipitable as a complex, indicating the occurrence of a protein-protein interaction. Confocal fluorescence microscopy of Ad-infected NM showed that pTP and CAD colocalized in nuclear foci. Both pTP and CAD were confirmed to colocalize with active sites of replication detected by bromodeoxyuridine incorporation. These data support the concept that the pTP-CAD interaction may allow anchorage of Ad replication complexes in the proximity of required cellular factors and may help to segregate replicated and unreplicated viral DNA.

scholarly journals Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa

Reproduction ◽  
2002 ◽  
pp. 757-768 ◽  
Author(s):  
EA Carrey ◽  
C Dietz ◽  
DM Glubb ◽  
M Loffler ◽  
JM Lucocq ◽  
...  
Keyword(s):  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Dihydroorotate Dehydrogenase ◽  
Aspartate Transcarbamylase ◽  
Carbamyl Phosphate ◽  
Pyrimidine Nucleotides ◽  
Carbamyl Phosphate Synthetase ◽  
De Novo Biosynthesis ◽  
Ump Synthase ◽  
Mammalian Spermatozoa

Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis.

VARIATION WITH AGE OF THE ENZYMES OF THE UREA CYCLE AND ASPARTATE TRANSCARBAMYLASE IN RAT LIVER

1967 ◽  
Vol 45 (9) ◽  
pp. 1427-1432 ◽  
Author(s):  
R. Charbonneau ◽  
A. Roberge ◽  
L. Berlinguet
Keyword(s):  
Urea Cycle ◽  
Protein Biosynthesis ◽  
Adult Life ◽  
Limiting Factor ◽  
Argininosuccinate Synthetase ◽  
Aspartate Transcarbamylase ◽  
Carbamyl Phosphate ◽  
Carbamyl Phosphate Synthetase ◽  
Metabolic Activities ◽  
Very High

The activities of aspartate transcarbamylase and of five enzymes involved in the urea cycle were determined in the liver of rats from the embryonic state to adulthood. Aspartate transcarbamylase activity is very high in the embryo and at birth. It remains high until the rat reaches a body weight of 50 g, after which there is a rapid decrease which levels off to a plateau at adulthood. The enzymatic activities of carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinase, and arginase are very low at the embryonic stage. The activity of these enzymes increases gradually with age until a plateau is reached, except for argininosuccinase which also increases in young animals but decreases in adult life. Of these enzymes, argininosuccinate synthetase always has the lowest activity and seems to be the limiting factor in the synthesis of urea. These results indicate that the biosynthesis of pyrimidines and urea vary inversely at different ages that correspond to different metabolic activities of the animals. Thus, an inverse relation is established between the two pathways from carbamyl phosphate, leading to protein biosynthesis (formation of RNA from orotic acid) and to protein catabolism (formation of urea).

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