Recombinant rat and hamster N-acetyltransferases-1 and -2: relative rates of N-acetylation of arylamines and N,O-acyltransfer with arylhydroxamic acids

Abstract

Genes for the 290 amino acid, 33–34 kDa cytosolic acetyltransferases (NAT1^* and NAT2^*) from rat and hamster were cloned and expressed in Escherichia coli. Active clones were selected by a simple visual test for their ability to decolorize 4-aminoazobenzene in bacterial medium by acetylation. These recombinant acetyltransferases were analyzed for: (i) N-acetyltransferase, which was assayed by the rate of acetyl coenzyme A-dependent N-acetylation of 2-aminofluorene (2-AF) or 4-aminoazobenzene (AAB); (ii) arylhydroxamic acid acyltransferase, assayed by N, O-acyltransfer with N-hydroxy-N-acetyl-2-aminofluorene. Both NAT2s showed first order increases in N-acetylation rates with increasing 2-AF or AAB concentrations between 5 and 100 μM, with apparent K_m values of 22–32 and 62–138 μM respectively. Although under the same conditions the N-acetylation rates for the two NAT1s declined by >50%, below 5 μM 2-AF or AAB, the NAT rate data fit Michaelis-Menten Kinetics, and the apparent K_m values were 0.2–0.9 μM. For N, O-acetyltransferase, the apparent K_m values of the NAT1s were ∼6 μM, while the K_m values of the NAT2s were ∼20- to 70-fold higher. SDS-PAGE/Western blot analysis of the recombinant acetyltransferases gave apparent relative molecular weights (MW_r) of ∼31 kDa for both NAT1s and rat NAT2 and ∼29 kDa for hamster NAT2. Comparable MW_r values were observed for native hamster liver NAT1 and NAT2 and for rat NAT1 under the same conditions. Although we did not detect NAT2-like activity in rat liver cytosol previously, the present data show that the rat NAT2^* gene does code for a functional acetyltransferase, with properties similar to those of hamster liver NAT2. The data also indicate that at low substrate concentrations, NAT1 would apparently play the predominant role in vivo in N-acetylation and N, O-acyltransfer of aromatic amine derivatives, including their metabolic activation to DNA-reactive agents.