Jones R F, Land S J, King C M
Molecular and Chemical Carcinogenesis Program, Karmanos Cancer Institute, Wayne State University College of Medicine, Detroit, MI 48201, USA.
Carcinogenesis. 1996 Aug;17(8):1729-33. doi: 10.1093/carcin/17.8.1729.
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 microM, with apparent K(m) values of 22-32 and 62-138 microM respectively. Although under the same conditions the N-acetylation rates for the two NAT1s declined by > 50%, below 5 microM 2-AF or AAB, the NAT rate data fit Michaelis-Menten kinetics, and the apparent K(m) values were 0.2-0.9 microM. For N,O-acyltransferase, the apparent K(m) values of the NAT1s were approximately 6 microM, while the K(m) values of the NAT2s were approximately 20- to 70-fold higher. SDS-PAGE/Western blot analysis of the recombinant acetyltransferases gave apparent relative molecular weights (MWr) of approximately 31 kDa for both NAT1s and rat NAT2 and approximately 29 kDa for hamster NAT2. Comparable MWr 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.
克隆了大鼠和仓鼠中编码290个氨基酸、33 - 34 kDa胞质乙酰转移酶（NAT1和NAT2）的基因，并在大肠杆菌中表达。通过一项简单的视觉测试选择活性克隆，该测试依据它们在细菌培养基中通过乙酰化使4-氨基偶氮苯脱色的能力。对这些重组乙酰转移酶进行了以下分析：（i）N-乙酰转移酶，通过2-氨基芴（2-AF）或4-氨基偶氮苯（AAB）的乙酰辅酶A依赖性N-乙酰化速率进行测定；（ii）芳基异羟肟酸酰基转移酶，通过与N-羟基-N-乙酰-2-氨基芴进行N,O-酰基转移来测定。两种NAT2在2-AF或AAB浓度在5至100 microM之间增加时，N-乙酰化速率呈一级增加，其表观K(m)值分别为22 - 32 microM和62 - 138 microM。尽管在相同条件下，当2-AF或AAB浓度低于5 microM时，两种NAT1的N-乙酰化速率下降超过50%，但NAT速率数据符合米氏动力学，其表观K(m)值为0.2 - 0.9 microM。对于N,O-酰基转移酶，NAT1s的表观K(m)值约为6 microM，而NAT2s的K(m)值高约20至70倍。对重组乙酰转移酶进行SDS-PAGE/蛋白质免疫印迹分析，结果显示两种NAT1和大鼠NAT2的表观相对分子量（MWr）约为31 kDa，仓鼠NAT2的表观相对分子量约为29 kDa。在相同条件下，天然仓鼠肝脏NAT1和NAT2以及大鼠NAT1也观察到了类似的MWr值。尽管我们之前在大鼠肝脏胞质溶胶中未检测到NAT2样活性，但目前的数据表明大鼠NAT2*基因确实编码一种功能性乙酰转移酶，其特性与仓鼠肝脏NAT2相似。数据还表明，在低底物浓度下，NAT1在体内芳香胺衍生物的N-乙酰化和N,O-酰基转移中，包括其代谢活化为DNA反应性试剂的过程中，显然将发挥主要作用。
