Measurement of aspartate carbamoyltransferase activity by high performance liquid chromatography

J. L. Grem, J. C. Drake, C. J. Allegra

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

We developed an assay which permits measurement of aspartate carbamoyltransferase (ACTase) activity. Cytosol from human peripheral blood mononuclear cells was used as the enzyme source. Using [14C]carbamoyl phosphate as the radiolabeled substrate, the formation of [14C]carbamoyl aspartate was quantitated by high performance liquid chromatography (HPLC) using an anion-exchange column with UV detection at 200-280 nm and an on-line liquid scintillation detector. A gradient method from an initially low concentration of ammonium phosphate, 1 mM (pH 3.0), to a higher concentration, 38 mM (pH 4.5), was used. The apparent K(m) values of carbamoyl phosphate and aspartate were 58 μM and 1.9 mM, respectively. ACTase inhibition by N-(phosphonacetyl)-1-aspartate (PALA) was consistent with a competitive model with respect to carbamoyl phosphate. The assay conditions were optimized to permit measurement of ACTase activity prior to and following therapy with PALA; ACTase was inhibited in a dose-dependent manner. This HPLC method permits direct quantitation of both the product of the reaction and the initial integrity of the substrate, [14C]carbamoyl phosphate, which is unstable in aqueous solutions.

Original languageEnglish (US)
Pages (from-to)545-554
Number of pages10
JournalAnti-Cancer Drugs
Volume4
Issue number5
DOIs
StatePublished - Jan 1 1993

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Aspartate Carbamoyltransferase
Carbamyl Phosphate
Aspartic Acid
High Pressure Liquid Chromatography
Cytosol
Anions
Blood Cells
Enzymes

Keywords

  • Aspartate carbamoyltransferase
  • Biochemical monitoring
  • HPLC
  • N-(phosphonoacetyl)-1-aspartate

ASJC Scopus subject areas

  • Oncology
  • Pharmacology
  • Pharmacology (medical)
  • Cancer Research

Cite this

Measurement of aspartate carbamoyltransferase activity by high performance liquid chromatography. / Grem, J. L.; Drake, J. C.; Allegra, C. J.

In: Anti-Cancer Drugs, Vol. 4, No. 5, 01.01.1993, p. 545-554.

Research output: Contribution to journalArticle

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