A microtiter plate assay for aspartate transcarbamylase.
A discontinuous, colorimetric method for the assay of aspartate transcarbamylase has been adapted for use with 96-well microtiter plates. The method is based on that of L.M. Prescott and M.E. Jones (1969 Anal. Biochem. 32, 408-419) for the detection of ureido compounds, using monoxime and antipyrine. The enzymatic reaction is carried out in a volume of 150 microliters and is stopped by the addition of 100 microliters of a color mix. After development, the absorbance at 460 nm is directly proportional to the quantity of N-carbamyl-L-aspartate up to at least 0.125 mumol and to the quantity of Escherichia coli aspartate transcarbamylase up to about 7 ng. Kinetic parameters obtained from saturation curves for L-aspartate in 50 mM Tris-acetate, pH 8.0, are indistinguishable from those previously obtained: Vmax = 26,225 mumol h-1 mg-1; S0.5 = 14.7 mmol liter-1; hill constant = 2.5.
Else
A J
AJ
Laboratoire d'Enzymologie, CNRS, Gif-sur-Yvette, France.
Hervé
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D001221:Aspartate Carbamoyltransferase / Q000032:analysis*; D001426:Bacterial Proteins / Q000032:analysis*; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D008832:Microchemistry
D001426:Bacterial Proteins; D001221:Aspartate Carbamoyltransferase