A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography
| Autor: | Herbert M. Pinedo, Godefridus J. Peters, E. Laurensse, A. Leyva |
|---|---|
| Rok vydání: | 1987 |
| Předmět: |
Male
Orotic acid Oxidoreductases Acting on CH-CH Group Donors Pyrimidine Biophysics Dihydroorotate Dehydrogenase Dehydrogenase Mitochondria Liver In Vitro Techniques Biochemistry High-performance liquid chromatography Absorbance chemistry.chemical_compound Mice medicine Animals Leukemia L1210 Molecular Biology Chromatography High Pressure Liquid chemistry.chemical_classification Detection limit Chromatography biology Chemistry Rats Inbred Strains Cell Biology Chromatography Ion Exchange Enzyme assay Rats Kinetics Enzyme biology.protein Spectrophotometry Ultraviolet Oxidoreductases medicine.drug |
| Zdroj: | Analytical biochemistry. 161(1) |
| ISSN: | 0003-2697 |
| Popis: | 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 preparation. Orotic acid was quantified with high-performance liquid chromatography 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 per 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 mixture. 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. With both assays kinetic parameters of the enzyme were determined. In the high concentration range (80–1000 μ m ) both K m and V max values were comparable. With the HPLC assay the concentration range was extended down to 12 μ m and initial rates could be determined. The apparent K m was about 12 μ m . The HPLC assay is also suitable for use in the study of inhibition of DHO dehydrogenase. |
| Databáze: | OpenAIRE |
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