A two-step purification of ATP-citrate lyase from rat liver and its use in a fluorometric assay for N-acetylglutamate synthetase.
ATP-citrate lyase (EC 4.1.3.8) was purified to homogeneity from the liver of rats maintained on a diet containing no fat and high carbohydrate. The procedure involves two steps: dye-ligand chromatography on yellow MX-6G Sepharose CL-4B and ion-exchange chromatography on DEAE-Trisacryl. The specific activity of the enzyme was 10 mumol X min-1 X mg-1 at 25 degrees C, which is equal to the highest specific activity reported to date. The yield was also the highest reported to date, being in excess of 50%, and the enzyme isolated by this procedure has little proteolytic nicking. The pure enzyme was used to establish a coupled fluorometric assay for N-acetylglutamate synthetase (amino-acid acetyltransferase, EC 2.3.1.1) based on coupling coenzyme A production to the oxidation of NADH via ATP-citrate lyase and malate dehydrogenase. The method is easy to perform compared with existing methods and enables the measurement of 100 pmol X min-1 of N-acetylglutamate synthetase activity. The method is generally applicable for measurement of enzymes which produce coenzyme A. The fluorometric method was used to measure the Km for glutamate and acetyl coenzyme A at pH 7.0 and 25 degrees C, which were 8.2 and 0.4 mM, respectively. Arginine at 1 microM gave half-maximal activation of N-acetylglutamate synthetase.
D001275:ATP Citrate (pro-S)-Lyase / Q000302:isolation & purification*; D000123:Acetyltransferases / Q000032:analysis* / Q000302:isolation & purification; D051046:Amino-Acid N-Acetyltransferase; D000818:Animals; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology*; D008297:Male; D011506:Proteins / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence
D011506:Proteins; D000123:Acetyltransferases; D051046:Amino-Acid N-Acetyltransferase; D001275:ATP Citrate (pro-S)-Lyase