Assay of ornithine carbamoyltransferase activity in human liver using carbon-labeled ornithine and thin-layer chromatography.
Ornithine carbamoyltransferase (EC 2.1.3.3) activity in human liver homogenates has been measured using 14C-labeled ornithine and unlabeled carbamoyl phosphate. A thin-layer chromatographic (TLC) procedure is used to separate the radioactive substrate and product, ornithine and citrulline, respectively, and the regions of the chromatogram corresponding to ornithine and citrulline are cut out and counted in a liquid scintillation spectrophotometer. The method has the following advantages: (1) the radioactive substrate ornithine is more stable in solution than carbamoyl phosphate, (2) 14C-labeled ornithine is available in higher specific activity than carbamoyl phosphate, (3) all radioactivity may be accounted for by using the TLC system, (4) the developed thin-layer chromatogram is stable indefinitely, (5) in contrast to colorimetric assays, other compounds in the raction mixture do not interfere with the citrulline determination, and (6) most importantly, the rate of the enzyme reaction at various time intervals can be determined by taking aliquots from the same incubation tube.
D002021:Buffers; D002250:Carbon Radioisotopes; D002855:Chromatography, Thin Layer; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008722:Methods; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism*; D013997:Time Factors
D002021:Buffers; D002250:Carbon Radioisotopes; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase