An enzymatic technique for measuring N-phosphonacetyl-L-aspartic acid in tissues.
An enzymatic technique is presented for measuring N-phosphonacetyl-L-aspartic acid (PALA; NSC-224131) in biologic specimens. Tightly bound PALA is quantitatively detached from its target enzyme, L-aspartic acid transcarbamylase (ATCase), by heating at 95 degrees C for 5 minutes. Denatured proteins are removed by centrifugation. PALA in the supernatant fluid is quantitated by exposing intact splenic ATCase to representative aliquots or subdilutions of the resultant supernatant in the presence of L-[4-14C]aspartic acid and carbamyl phosphate. After 30 minutes' incubation at 37 degrees C, unreacted L-[4-14 C]aspartic acid is dissipated enzymatically and newly formed [4-14C]carbamyl-L-aspartic acid is quantitated by scintillation spectrometry. The percentage inhibition of ATCase responds in a linear way to the logarithm of the concentration of PALA between 0.10 and 1.00 micrometer. The PALA concentration of an unknown is determined indirectly by matching the percentage inhibition caused by the unknown to the inhibition caused by a known series of standard concentrations of PALA over the linear range. This assay is sensitive, adequately reproducible despite the use of an unpurified enzyme, and notably facile. It can be used to measure PALA in plasma, urine, tissues, and tumors of subjects treated with this new oncolytic drug.
D000818:Animals; D000970:Antineoplastic Agents / Q000032:analysis* / Q000494:pharmacology; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000494:pharmacology; D001681:Biological Assay; D004926:Escherichia coli / Q000201:enzymology; D008722:Methods; D051379:Mice; D009943:Organophosphorus Compounds / Q000032:analysis*; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000032:analysis*; D013154:Spleen / Q000201:enzymology
D000970:Antineoplastic Agents; D009943:Organophosphorus Compounds; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid