Abstract
The N-acetylation of arsanilic acid was assayed in vitro by modifying a literature method for acetylation ofp-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°C, with 0.5- to 2-mgN-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 μm 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.
Footnotes
-
Send reprint requests to: Dr. Gladys S. Bayse, Department of Chemistry, P.O. Box 326, Spelman College, 350 Spelman Lane, SW, Atlanta, GA 30314. E-mail: gbayse{at}spelman.edu
-
This study was supported by National Institutes of Health/MBRS Grant 2 SO6 GM 0824–12 and RIMI Grant RR11598. LAJ, KLS and KLW were MBRS student participants. KLS and KLW are also Merck Scholars. Part of this work was presented at the National Minority Research Symposia, November 1998, New York, NY, Abstract no. B75; and November 1999, Phoenix, AZ, Abstract no. E19; another part of the work will be presented at the RIMI First Annual Research Symposium, February 2000 in Atlanta GA.
- Abbreviations used are::
- PABA
- p-aminobenzoic acid
- acetylCoA
- acetyl coenzyme A
- DTT
- dithiothreitol
- NAT
- N-acetyltransferase
- NAA
- 4-N-acetylarsanilic acid
- roxarsone
- 4-hydroxy-3-nitrobenzenearsonic acid
- acetarsone
- 4-hydroxy-3-N-acetylbenzenearsonic acid
- nitarsone
- 4-nitrobenzenearsonic acid
- Received June 9, 1999.
- Accepted December 23, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|