title: Measurement of aspartate carbamoyltransferase activity by high performance liquid chromatography.
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 Km values of carbamoyl phosphate and aspartate were 58 microM and 1.9 mM, respectively. ACTase inhibition by N-(phosphonacetyl)-l-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.
authors.affiliation: NCI-Navy Medical Oncology Program, National Naval Medical Center, Bethesda, MD 20889-5105.
authors.forename: J L
authors.initials: JL
authors.lastname: Grem
authors.forename: J C
authors.initials: JC
authors.lastname: Drake
authors.forename: C J
authors.initials: CJ
authors.lastname: Allegra
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000737:chemistry; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D000964:Antimetabolites, Antineoplastic; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid