A facile tritium release assay for mammalian l-dihydroorotate dehydrogenase

Thomas W. Kensler, David A. Cooney, Hiremagalur N. Jayaram, Clay Schaeffer, David D. Choie

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

Capitalizing on the high concentrations of l-aspartate transcarbamylase and l-dihydroorotase present in the high-speed supernatant from mutant BHK 21 cells resistant to N-(phosphonacetyl)-l-aspartate, l-[5,6-3H]dihydroorotic acid has been biosynthesized with these crude enzymes from l-[2,3-3H]aspartic acid and carbamyl phosphate. The product was purified by paper chromatography in solvents which separate it from both l-aspartic and N-carbamyl-l-aspartic acids. Using this substrate, it has been demonstrated that mitochondria from mouse liver catalyze the release of tritium in a time- and protein-dependent manner as a distillable form susceptible to quantitative trapping in 100% (w/v) KOH; presumably this form is 3H2O. Other subcellular fractions release only minor amounts of radioactivity. When comparisons were made of the specific activities of l-dihydroorotate dehydrogenase measured by published techniques as well as by the present tritium release assay, the results were in acceptable agreement. Because of its notable sensitivity, facility, and precision, the technique described herein can be especially recommended for use in analytical studies requiring the manipulation of large numbers of vessels.

Original languageEnglish (US)
Pages (from-to)315-319
Number of pages5
JournalAnalytical biochemistry
Volume117
Issue number2
DOIs
StatePublished - Nov 1 1981

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Kensler, T. W., Cooney, D. A., Jayaram, H. N., Schaeffer, C., & Choie, D. D. (1981). A facile tritium release assay for mammalian l-dihydroorotate dehydrogenase. Analytical biochemistry, 117(2), 315-319. https://doi.org/10.1016/0003-2697(81)90785-5