Detail of "5988-19-2"

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A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography

A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography. Peters, G. J.; Laurensse, E.; Leyva, A.; Pinedo, H. M. (Dep. Oncol., Free Univ. Hosp., Amsterdam 1007 MB, Neth.). Anal. Biochem., 161(1), 32-8 (English) 1987. CODEN: ANBCA2. ISSN: 0003-2697. DOCUMENT TYPE: Journal CA Section: 7 (Enzymes) A new method to assay the mitochondrial pyrimidine de novo enzyme, dihydroorotate (DHO) dehydrogenase, which catalyzes the dehydrogenation of DHO, with orotic acid as the product was developed. The assay was optimized using a rat liver mitochondrial prepn. Orotic acid was quantified by HPLC using an anion-exchange column (Partisil-SAX) with UV detection at 280 nm. Isocratic elution with low phosphate buffer at pH 4.0 was used. The detection limit was 20 pmol/injection, which is comparable to previously described radiometric assays. The HPLC assay was compared with a spectrophotometric assay measuring orotic acid formation in a deproteinized reaction mixt. Absorbance was measured at the optimal wavelength for orotic acid, 278.5 nm. This assay is less sensitive and less specific than the HPLC assay, which can also detect UMP which might be formed from orotic acid in whole homogenates. Kinetic parameters of the enzyme were detd. with both assays. In the high concn. range (80-1000 mM), both Km and Vmax values were comparable.In this experiment, several chemicals are used like 5988-19-2 and 9029-03-2 With the HPLC assay the concn. range was extended to 312 mM and initial rates could be detd. The apparent Km was ~12 mM. The HPLC assay is also suitable for use in the study of inhibition of DHO dehydrogenase. .

Mechanistic studies with deuterated dihydroorotates on the dihydroorotate oxidase from Crithidia fasciculata

Mechanistic studies with deuterated dihydroorotates on the dihydroorotate oxidase from Crithidia fasciculata. Pascal, Robert A., Jr.Several substances are used for example 90242-05-0 and 5988-19-2 which are their cas registry numbers.; Walsh, Christopher T. (Dep. Chem., Massachusetts Inst. Technol., Cambridge, MA 02139, USA). Biochemistry, 23(12), 2745-52 (English) 1984. CODEN: BICHAW. ISSN: 0006-2960. DOCUMENT TYPE: Journal CA Section: 7 (Enzymes) Deuterium-labeled dihydroorotates bearing 1, 2, or 3 deuteriums at the pair of C4 and C5 positions were synthesized in high isotopic and chiral purity and characterized by NMR and mass spectroscopy. These substrates were used with FMN-contg. biosynthetic dihydroorotate oxidase from C. fasciculata to probe its reaction stereochem. and mechanism. At pH 6.0, (4RS)-[5,5-2H2]dihydroorotate showed a Vmax isotope effect (DV) of 2.83; since (4S,5R)-[5-2H]dihydroorotate showed a DV of ￡1.1, a secondary effect, the overall stereochem. of desatn. was anti as previously reported for the degradative orotate reductase from Clostridium oroticium. (4RS)-[4-2H]Dihydroorotate showed a DV of 2.97, indicating that removal of C4-H was also partially rate-limiting at pH 6.0. When trideuterio(4RS)-[4,5,5-2H3]dihydroorotate was treated, a DV of 8.0, a value close to the product of the sep. isotope effects at the 4- and 5S-positions, was obsd. At this pH then, both C-H cleavage steps are partly rate-limiting in catalysis. Under anaerobic conditions without an electron acceptor, the enzyme catalyzes the preferential exchange of the 5S H atom with solvent protons. The aggregate isotope effects on Vmax (DV) and on Vmax/Km (D(V/K)) were analyzed and suggest a stepwise rather than a concerted mechanism for this biosynthetic desatn. in pyrimidine biosynthesis. .