A Radiochemical Assay for β-Ureidopropionase Using Radiolabeled N-Carbamyl-β-alanine Obtained via Hydrolysis of [2-14C]5,6-Dihydrouracil
✍ Scribed by A.B.P. Van Kuilenburg; H. Van Lenthe; A.H. Van Gennip
- Book ID
- 102559138
- Publisher
- Elsevier Science
- Year
- 1999
- Tongue
- English
- Weight
- 63 KB
- Volume
- 272
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A radiochemical assay was developed to measure the activity of -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--alanine was prepared within 15 min by a simple hydrolysis of [2-14 C]5,6-dihydrouracil under alkaline conditions at 37°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 -ureidopropionase obeyed Michaelis-Menten kinetics with an apparent K m for N-carbamyl--alanine of 15.5 ؎ 1.9 M. 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 .