Measurement of aspartate carbamoyltransferase activity by high performance liquid chromatography.
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.
Grem
J L
JL
NCI-Navy Medical Oncology Program, National Naval Medical Center, Bethesda, MD 20889-5105.
Drake
J C
JC
Allegra
C J
CJ
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
D000964:Antimetabolites, Antineoplastic; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid