Improved colorimetric procedure for quantitating N-carbamoyl-β-alanine with minimum dihydrouracil interference

West, Thomas P.; Shanley, Mark S.; O’Donovan, Gerard A.

doi:10.1016/0003-2697(82)90293-7

Cited by 15 publications

(3 citation statements)

References 6 publications

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“…Dihydropyrimidinase activity was determined using a colorimetric assay for N-carbamoyl-/I-alanine (West et al, 1982). The reaction mixture contained (in 1 ml) 0.1 M-Tris/HCl buffer, pH 7.5, 10 mMMgCl,, 1 mM-dihydrouracil and cell-free extract.…”

Section: Methodsmentioning

confidence: 99%

“…The mixture was assayed over a 30 rnin period. At various intervals, 1 ml of the colour mix of West et al (1982) was added directly to the assay tubes to halt the reaction. Following colour mix addition, the tubes were capped with marbles and placed in a 70 "C water-bath under fluorescent lighting for 2 h. The absorbance of each assay tube was determined at 466 nm and activity was derived using an experimentally calculated absorption coefficient .…”

Section: Methodsmentioning

confidence: 99%

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Reductive catabolism of pyrimidine bases by Pseudomonas stutzeri

Xu¹,

West²

1992

Journal of General Microbiology

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Pyrimidine base catabolism in Pseudomonas stutzeri ATCC 17588 was investigated and was found to occur by means of the reductive pathway. Pyrimidine bases and their respective reductive pathway catabolic products could serve as nitrogen sources for growth of P. stutzeri. Activities of the three enzymes associated with the reductive pathway of pyrimidine catabolism were detected in cells of P. stutzeri. The initial enzyme of the reductive pathway, dihydropyrimidine dehydrogenase, utilized NADH as its nicotinamide cofactor. Cells grown on pyrimidine bases as nitrogen sources contained elevated dehydrogenase activity relative to those grown on ammonium sulphate as nitrogen source. Activities of the second and third reductive pathway enzymes, dihydropyrimidinase and N-carbamoyl-P-alanine amidohydrolase, respectively, were also affected by growth conditions. If pyrimidine or dihydropyrimidine bases served as nitrogen sources, increases in the levels of these enzymes were observed compared to their activities determined when the nitrogen source was ammonium sulphate.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Reductive catabolism of pyrimidine bases by Pseudomonas stutzeri

Xu¹,

West²

1992

Journal of General Microbiology

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“…The activity of purified OsDHP-T1 recombinant protein was assayed by measuring N-carbamoyl-ß-alanine using a colorimetric procedure ( West et al., 1982 ; Mejias-Torres & Zimmermann, 2002 ). Reactions were initiated by adding enzyme to concentrations of 90 - 150 nM to a solution containing 0.1 M potassium buffer, pH 8, 1 mg/mL bovine serum albumin and dihydrouracil, 37°C for 10 minutes, and then quenched followed by an incubation for 120 minutes at 70°C for color development.…”

Section: Methodsmentioning

confidence: 99%

New Insights into rice pyrimidine catabolic enzymes

Lopez

Narvaez-Ortiz²,

Rincon‐Benavides³

et al. 2023

Front. Plant Sci.

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IntroductionRice is a primary global food source, and its production is affected by abiotic stress, caused by climate change and other factors. Recently, the pyrimidine reductive catabolic pathway, catalyzed by dihydropyrimidine dehydrogenase (DHPD), dihydropyrimidinase (DHP) and β-ureidopropionase (β-UP), has emerged as a potential participant in the abiotic stress response of rice.MethodsThe rice enzymes were produced as recombinant proteins, and two were kinetically characterized. Rice dihydroorotate dehydrogenase (DHODH), an enzyme of pyrimidine biosynthesis often confused with DHPD, was also characterized. Salt-sensitive and salt-resistant rice seedlings were subjected to salt stress (24 h) and metabolites in leaves were determined by mass spectrometry.ResultsThe OsDHPD sequence was homologous to the C-terminal half of mammalian DHPD, conserving FMN and uracil binding sites, but lacked sites for Fe/S clusters, FAD, and NADPH. OsDHPD, truncated to eliminate the chloroplast targeting peptide, was soluble, but inactive. Database searches for polypeptides homologous to the N-terminal half of mammalian DHPD, that could act as co-reductants, were unsuccessful. OsDHODH exhibited kinetic parameters similar to those of other plant DHODHs. OsDHP, truncated to remove a signal sequence, exhibited a kcat/Km = 3.6 x 103 s-1M-1. Osb-UP exhibited a kcat/Km = 1.8 x 104 s-1M-1. Short-term salt exposure caused insignificant differences in the levels of the ureide intermediates dihydrouracil and ureidopropionate in leaves of salt-sensitive and salt-resistant plants. Allantoin, a ureide metabolite of purine catabolism, was found to be significantly higher in the resistant cultivar compared to one of the sensitive cultivars.DiscussionOsDHP, the first plant enzyme to be characterized, showed low kinetic efficiency, but its activity may have been affected by truncation. Osb-UP exhibited kinetic parameters in the range of enzymes of secondary metabolism. Levels of two pathway metabolites were similar in sensitive and resistant cultivars and appeared to be unaffected by short-term salt exposure.”

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Isolation and characterization of a dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis

West

1991

Arch. Microbiol.

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Improved colorimetric procedure for quantitating N-carbamoyl-β-alanine with minimum dihydrouracil interference

Cited by 15 publications

References 6 publications

Reductive catabolism of pyrimidine bases by Pseudomonas stutzeri

Reductive catabolism of pyrimidine bases by Pseudomonas stutzeri

New Insights into rice pyrimidine catabolic enzymes

Isolation and characterization of a dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis

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