A radiochemical assay for beta-ureidopropionase using radiolabeled N-carbamyl-beta-alanine obtained via hydrolysis of [2-(14)C]5, 6-dihydrouracil.
A radiochemical assay was developed to measure the activity of beta-ureidopropionase in human liver homogenates which is based on the detection of the reaction product (14)CO(2) by liquid scintillation counting. Radiolabeled N-carbamyl-beta-alanine was prepared within 15 min by a simple hydrolysis of [2-(14)C]5, 6-dihydrouracil under alkaline conditions at 37 degrees C. The enzymatic reaction proved to be linear with time up to at least 3.5 h and protein concentrations up to at least 1 mg/ml. Human beta-ureidopropionase obeyed Michaelis-Menten kinetics with an apparent Km for N-carbamyl-beta-alanine of 15.5 +/- 1.9 microM. The assay proved to be very accurate and sensitive with an intraassay coefficient of variation of 2% and a detection limit of 28 pmol for the product CO(2).
Academic Medical Center, University of Amsterdam, Emma Children's Hospital and Department of Clinical Chemistry, Amsterdam, 1100 DE, The Netherlands. a.b.vanKuilenburg@amc.uva.nl
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Van Gennip
D000581:Amidohydrolases / Q000032:analysis* / Q000378:metabolism; D002245:Carbon Dioxide / Q000032:analysis; D002250:Carbon Radioisotopes; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011865:Radioisotope Dilution Technique; D015203:Reproducibility of Results; D012588:Scintillation Counting / Q000379:methods; D012680:Sensitivity and Specificity; D014498:Uracil / Q000031:analogs & derivatives* / Q000737:chemistry; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism
D002250:Carbon Radioisotopes; C007419:dihydrouracil; D015091:beta-Alanine; D002245:Carbon Dioxide; C035122:N-carbamoyl-beta-alanine; D014498:Uracil; D000581:Amidohydrolases; C021590:beta-ureidopropionase