1 Introduction N [1] N [2] [2] NAT [3–12] [13] [14] NAT1 [13] [15–17] [5] [14] in vivo [18,19] Nat2 [20] [13,21] In order to provide a firm foundation for establishing such a model, we have generated pure recombinant mouse Nat2 and investigated its activity with a wide range of substrates and the effects of a range of potential inhibitors, including endogenous and exogenous steroids. 2 Materials and methods 2.1 Chemicals All chemicals were purchased from Sigma–Aldrich and all molecular biology reagents were purchased from Promega, unless otherwise stated. 2.2 Nat2 Nat2 [22] Nat2 Escherichia coli β d g 15 15 E. coli g [23] 15 15 [24] 2.3 Enzymic assays 2.3.1 Acetylation of arylamines [25] g N,N 2.3.2 Hydrolysis of AcCoA (free CoA production) [2,26] ® l 2.4 NMR spectroscopy 1 15 15 2 2 2 1 t 2 t 1 1 N 2 3 Results 3.1 Nat2 Nat2 E. coli E. coli Fig. 1 Fig. 1 [27] Table 1 3.2 Substrate specificity of mouse Nat2 [2] Fig. 2 Supplementary Table 1 Fig. 2 [22,27,28] Fig. 2 [28] [29] [2] Fig. 2 [2] 3.3 Mouse Nat2 inhibition by steroidogenic compounds NAT1 [15–17] [14] Table 2 Supplementary Fig. 1 50 Table 3 β 3.4 Structure of mouse Nat2 [30] S M. smegmatis Fig. 3 [31] Fig. 3 Mycobacterium marinum [32] [33] Fig. 4 3.5 NMR studies 15 1 15 Fig. 5 1 N 15 [34] 1 Fig. 6 Table 4 132 Fig. 7 67 68 1 N 15 1 N 1 N 107 107 [35] 3.6 Interaction of mouse Nat2 with exogenous steroid inhibitors 1 Figs. 8 and 9 Table 4 Fig. 9 N 67 132 Fig. 9 50 107 4 Discussion Nat2 Nat2 [36] [37] in vivo [38] [39] [40] [41] [41–44] [45] [46] [20] Appendix A Supplementary data doi:10.1016/j.bcp.2007.12.012 Appendix A Supplementary data 2 2