RECOMBINANT RAT AND HAMSTER N-ACETYLTRANSFERASE-1 AND N-ACETYLTRANSFERASE-2 - RELATIVE RATES OF N-ACETYLATION OF ARYLAMINES AND N,O-ACYLTRANSFER WITH ARYLHYDROXAMIC ACIDS

Genes for the 290 amino acid, 33-34 kDa cytosolic acetyltransferases ( NAT1 and NAT2*) from rat and hamster were cloned and expressed in Esc herichia 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 coenzy me A-dependent N-acetylation of 2-aminofluorene (2-AF) or 4-aminoazobe nzene (AAB); (ii) arylhydroxamic acid acyltransferase, assayed by N,O- acyltransfer with N-hydroxy-N-acetyl-2-aminofluorene. Both NAT2s showe d first order increases in N-acetylation rates with increasing 2-AF or AAB concentrations between 5 and 100 mu M, with apparent K-m values o f 22-32 and 62-138 mu M respectively. Although under the same conditio ns the N-acetylation rates for the two NAT1s declined by > 50%, below 5 mu M 2-AF or AAB, the NAT rate data fit Michaelis-Menten kinetics, a nd the apparent K-m values were 0.2-0.9 mu M. For N,O-acyltransferase, the apparent K-m values of the NAT1s were similar to 6 mu M, while th e K-m values of the NAT2s were similar to 20- to 70-fold higher, SDS-P AGE/Western blot analysis of the recombinant acetyltransferases gave a pparent relative molecular weights (MW(r)) of similar to 31 kDa for bo th NAT1s and rat NAT2 and similar to 29 kDa for hamster NAT2, Comparab le MW(r) values were observed for native hamster liver NAT1 and NAT2 a nd for rat NAT1 under the same conditions, Although we did not detect NAT2-like activity in rat liver cytosol previously, the present data s how that the rat NAT2 gene does code for a functional acetyltransfera se, with properties similar to those of hamster liver NAT2, The data a lso indicate that at low substrate concentrations, NAT1 would apparent ly play the predominant role in vivo in N-acetylation and N,O-acyltran sfer of aromatic amine derivatives, including their metabolic activati on to DNA-reactive agents.