The N-acetylation of arsanilic acid In vitro by mammalian enzymes.
The N-acetylation of arsanilic acid was assayed in vitro by modifying a literature method for acetylation of p-aminobenzoic acid. Conditions included final concentrations of 1.0 mM dithiothreitol, 1.0 mM EDTA, 0.45 mM acetyl coenzyme A, an acetyl coenzyme A regenerating system using bacterial phosphotransacetylase and acetyl phosphate, 5.0 mM arsanilate substrate, and 25 mM sodium/potassium phosphate buffer, pH 7.4, in a total volume of 0.5 ml. Incubation was at 37 degrees C, with 0.5- to 2-mg N-acetyltransferase enzyme protein from a preparation of guinea pig liver. The reaction was terminated by heat precipitation. The resulting supernatant was put through a 4 mm 0.45 microm polysulfone membrane syringe filter. The filtrate could then be injected directly onto the HPLC. With arsanilic acid as substrate, the product N-acetylarsanilic acid (NAA) was identified by its retention time (33 min) in the HPLC system of the laboratory. The 33-min fraction collected from the HPLC was scanned and gave the characteristic UV spectrum of NAA, with peaks at 203 and 256 nm. In addition, the product comigrated in the HPLC system with standard NAA. Under comparable assay conditions, the N-acetylation of arsanilate by the guinea pig enzyme preparation is about 24% the rate of that of the model substrate p-aminobenzoic acid. Typical activity for arsanilate acetylation was 0.5 nmol/min/mg enzyme protein. Using the same assay system and HPLC detection method, the supernatant from bacterial lysates containing recombinant human N-acetyltransferase 1 exhibited acetylation activity toward arsanilate of 720 nmol/min/mg enzyme protein.
Bayse
G S
GS
Department of Chemistry, Spelman College, Atlanta, Georgia 30314, USA. gbayse@speman.edu
Jinadu
L A
LA
Shaw
K L
KL
Wiley
K L
KL
D000107:Acetylation; D000821:Animal Feed; D000818:Animals; D001147:Arsanilic Acid / Q000493:pharmacokinetics*; D001191:Arylamine N-Acetyltransferase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D010856:Columbidae; D005503:Food Additives / Q000493:pharmacokinetics; D006168:Guinea Pigs; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008322:Mammals; D011994:Recombinant Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D005503:Food Additives; D007527:Isoenzymes; D011994:Recombinant Proteins; D001191:Arylamine N-Acetyltransferase; D001147:Arsanilic Acid