Inhibition of urea cycle enzymes by aspartic acid analogues

1969 ◽  
Vol 47 (3) ◽  
pp. 361-369
Author(s):  
S. M. Bayer ◽  
W. C. McMurray
Keyword(s):  
Aspartic Acid ◽  
Energy Production ◽  
Urea Cycle ◽  
Argininosuccinate Synthetase ◽  
Substrate Type ◽  
Carbamyl Phosphate ◽  
Enzyme Preparations ◽  
Carbamyl Phosphate Synthetase ◽  
Liver Homogenates

The inhibition of urea biosynthesis by analogues of aspartic acid was studied in vitro in homogenates and enzyme preparations from rat liver. Each of the analogues tested inhibited the overall utilization of citrulline for urea formation by liver homogenates. The concentrations required to give 50% inhibition were: N-allylaspartate, 0.248 M; α-methylaspartate, 0.140 M; β-methylaspartate, 0.078 M; and β-hydroxy-β-methylaspartate, 0.038 M. The β-substituted analogues partly replaced aspartate as a substrate for citrulline utilization in liver homogenates. The replacement was probably due to transamination of the analogues with oxaloacetate, since the effect was not observed when the assay mixture did not contain a substrate which could yield oxaloacetate.A study of individual enzymes of the urea cycle showed that arginase, argininosuccinase, and ornithine transcarbamylase were not greatly affected by the analogues. However, carbamyl phosphate synthetase as well as argininosuccinate synthetase were strongly inhibited, suggesting that the analogues act by some mechanism other than simple antagonism of aspartate. Part of the inhibition was related to the ability of the analogues to complex Mg2+, since increased concentrations of Mg2+ prevented the inhibition of carbamyl phosphate synthetase and reduced the inhibition of argininosuccinate synthetase by α-methylaspartate and N-allylaspartate. In addition, β-methylaspartate was found to depress oxidative and phosphorylative reactions, thus interfering with the energy production required for urea formation.Aspartic acid in concentrations comparable with those required to effect inhibition by α-methylaspartate produced a marked inhibition of citrulline utilization in liver homogenates and of purified argininosuccinate synthetase. This observation suggests that part of the inhibitions observed with the analogues are of the "substrate type".

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).

scholarly journals Synthesis of Carbamyl Phosphate Synthetase in Amphibian Liver in Vitro

1969 ◽  
Vol 244 (19) ◽  
pp. 5295-5308 ◽  
Author(s):  
G.E. Shambaugh ◽  
J.B. Balinsky ◽  
Philip P. Cohen
Keyword(s):  
Carbamyl Phosphate ◽  
Carbamyl Phosphate Synthetase

scholarly journals Activity of the carbamyl phosphate synthetase I promoter in liver nuclear extracts is dependent on a cis-acting C/EBP recognition element.

1989 ◽  
Vol 9 (7) ◽  
pp. 2928-2933 ◽  
Author(s):  
B W Howell ◽  
M Lagacé ◽  
G C Shore
Keyword(s):  
In Vitro Transcription ◽  
Proximal Promoter ◽  
Upstream Region ◽  
Carbamyl Phosphate ◽  
Cis Acting ◽  
Carbamyl Phosphate Synthetase ◽  
Recognition Element ◽  
Cis Element ◽  
Nuclear Extracts

We have identified an essential cis element in the proximal promoter region of the rat carbamyl phosphate synthetase I (CPSI) gene that is requisite for promoter activity in liver nuclear extracts. Excess synthetic oligonucleotides specifying this region abolished promoter-dependent in vitro transcription. We show that C/EBP, a nuclear factor enriched in liver but found as well in other tissues, such as gut, fat, and lung, interacts with an inverted repeat, GTTGCAAC, at the core of the essential cis element. In brain, a tissue that did not express CPSI or contain significant levels of C/EBP, a different factor was capable of binding at or near the C/EBP recognition element. Activity of the CPSI promoter in liver nuclear extracts was also dependent on sequences 5' to the C/EBP motif; presumably, factors binding to elements within this upstream region are instrumental in restricting CPSI gene expression to liver and intestinal mucosa.

scholarly journals N-Acetylglutamate and its changing role through evolution

2003 ◽  
Vol 372 (2) ◽  
pp. 279-290 ◽  
Author(s):  
Ljubica CALDOVIC ◽  
Mendel TUCHMAN
Keyword(s):  
Amino Acid ◽  
Autosomal Recessive ◽  
Urea Cycle ◽  
Evolutionary Relationship ◽  
Autosomal Recessive Disorder ◽  
The Other ◽  
Arginine Biosynthesis ◽  
Carbamyl Phosphate ◽  
Carbamyl Phosphate Synthetase ◽  
Changing Role

N-Acetylglutamate (NAG) fulfils distinct biological roles in lower and higher organisms. In prokaryotes, lower eukaryotes and plants it is the first intermediate in the biosynthesis of arginine, whereas in ureotelic (excreting nitrogen mostly in the form of urea) vertebrates, it is an essential allosteric cofactor for carbamyl phosphate synthetase I (CPSI), the first enzyme of the urea cycle. The pathway that leads from glutamate to arginine in lower organisms employs eight steps, starting with the acetylation of glutamate to form NAG. In these species, NAG can be produced by two enzymic reactions: one catalysed by NAG synthase (NAGS) and the other by ornithine acetyltransferase (OAT). In ureotelic species, NAG is produced exclusively by NAGS. In lower organisms, NAGS is feedback-inhibited by l-arginine, whereas mammalian NAGS activity is significantly enhanced by this amino acid. The NAGS genes of bacteria, fungi and mammals are more diverse than other arginine-biosynthesis and urea-cycle genes. The evolutionary relationship between the distinctly different roles of NAG and its metabolism in lower and higher organisms remains to be determined. In humans, inherited NAGS deficiency is an autosomal recessive disorder causing hyperammonaemia and a phenotype similar to CPSI deficiency. Several mutations have been recently identified in the NAGS genes of families affected with this disorder.

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.

Activity of the carbamyl phosphate synthetase I promoter in liver nuclear extracts is dependent on a cis-acting C/EBP recognition element

1989 ◽  
Vol 9 (7) ◽  
pp. 2928-2933
Author(s):  
B W Howell ◽  
M Lagacé ◽  
G C Shore
Keyword(s):  
In Vitro Transcription ◽  
Proximal Promoter ◽  
Upstream Region ◽  
Carbamyl Phosphate ◽  
Cis Acting ◽  
Carbamyl Phosphate Synthetase ◽  
Recognition Element ◽  
Cis Element ◽  
Nuclear Extracts

We have identified an essential cis element in the proximal promoter region of the rat carbamyl phosphate synthetase I (CPSI) gene that is requisite for promoter activity in liver nuclear extracts. Excess synthetic oligonucleotides specifying this region abolished promoter-dependent in vitro transcription. We show that C/EBP, a nuclear factor enriched in liver but found as well in other tissues, such as gut, fat, and lung, interacts with an inverted repeat, GTTGCAAC, at the core of the essential cis element. In brain, a tissue that did not express CPSI or contain significant levels of C/EBP, a different factor was capable of binding at or near the C/EBP recognition element. Activity of the CPSI promoter in liver nuclear extracts was also dependent on sequences 5' to the C/EBP motif; presumably, factors binding to elements within this upstream region are instrumental in restricting CPSI gene expression to liver and intestinal mucosa.

Carbamyl phosphate synthetase in the chick

1969 ◽  
Vol 47 (1) ◽  
pp. 61-63 ◽  
Author(s):  
Charles G. Maresh ◽  
Theodore H. Kwan ◽  
Sumner M. Kalman
Keyword(s):  
Enzyme Activity ◽  
Chick Embryo ◽  
Urea Cycle ◽  
Mitochondrial Fraction ◽  
Pyrimidine Biosynthesis ◽  
Developmental Pattern ◽  
Synthetase Activity ◽  
Young Chick ◽  
Carbamyl Phosphate ◽  
Carbamyl Phosphate Synthetase

Carbamyl phosphate synthetase activity is present in the liver of the chick embryo and in the young chick. This enzyme activity is not dependent upon acetylglutamate and is not present in the mitochondrial fraction. Because the urea cycle is not present in chick liver and because of the developmental pattern of the enzyme activity, we infer that this enzyme is involved in pyrimidine biosynthesis.

A STUDY ON THE "IN VITRO" SYNTHESIS OF CITRULLINE: PART I. EFFECT OF SOME N-ALKYL DERIVATIVES OF ASPARTIG ACID

1964 ◽  
Vol 42 (9) ◽  
pp. 1317-1324 ◽  
Author(s):  
Louis Berlinguet ◽  
René Charbonneau
Keyword(s):  
Aspartic Acid ◽  
Inhibitory Action ◽  
Liver Homogenate ◽  
Chelating Agent ◽  
Magnesium Ion ◽  
Carbamyl Phosphate ◽  
In Vitro Synthesis ◽  
Alkyl Derivatives ◽  
Derivatives Of

The effects of various N-alkyl derivatives of aspartic acid on the synthesis of citrulline in the presence of a particulate fraction obtained from a rat liver homogenate were studied. Even though aspartic acid has no role in this synthesis, both N-methyl and N-isopropyl aspartic acids were found to increase the synthesis of citrulline by 60%, whereas N-cyclohexyl aspartic acid decreased it by 50%. N-Allyl aspartic acid has the strongest effect which is an almost complete inhibition at low concentration of 1.2 × 10−2 M.It seems that N-allyl aspartic acid inhibits directly or indirectly the first step in the synthesis of citrulline leading to the formation of carbamyl phosphate. At various concentrations, none of the intermediates in this synthesis, except magnesium ion, can reverse the inhibition. In order that N-allyl aspartic acid retain its inhibitory action, the ω-carboxyl group has to be free and the double bond in the allyl group must be intact. From these results, it is postulated that N-allyl aspartic acid acts as a chelating agent for magnesium.

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