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Mouse N-acetyltransferase type 2, the homologue of human N-acetyltransferase type 1.

Kawamura A, Westwood I, Wakefield L, Long H, Zhang N, Walters K, Redfield C, Sim E - Biochem. Pharmacol. (2008)

Bottom Line: In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids.We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2.We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.

ABSTRACT
There is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

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Comparison of the substrate specific activity profiles of mouse Nat2, human NAT1 and human NAT2. The substrate specific activity profiles for mouse Nat2 (grey bars), human NAT1 (cross-hatched bars) and human NAT2 (black bars) are shown. The activity of mouse Nat2 was determined by measuring the rate of CoA production, as described in Section 2. Purified mouse Nat2 (0.125 μg) was incubated with arylamine substrate (each at 500 μM) and AcCoA (400 μM) in assay buffer (20 mM Tris–HCl (pH 8.0)) containing 0.5% (v/v) DMSO at 25 °C. The specific activities were determined from the linear initial rates of reaction. All measurements were performed in triplicate and are expressed as mean ± standard deviation relative to the most rapidly acetylated substrate. The human NAT1 and NAT2 data are taken from [2]. The abbreviations are defined in Section 2.
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fig2: Comparison of the substrate specific activity profiles of mouse Nat2, human NAT1 and human NAT2. The substrate specific activity profiles for mouse Nat2 (grey bars), human NAT1 (cross-hatched bars) and human NAT2 (black bars) are shown. The activity of mouse Nat2 was determined by measuring the rate of CoA production, as described in Section 2. Purified mouse Nat2 (0.125 μg) was incubated with arylamine substrate (each at 500 μM) and AcCoA (400 μM) in assay buffer (20 mM Tris–HCl (pH 8.0)) containing 0.5% (v/v) DMSO at 25 °C. The specific activities were determined from the linear initial rates of reaction. All measurements were performed in triplicate and are expressed as mean ± standard deviation relative to the most rapidly acetylated substrate. The human NAT1 and NAT2 data are taken from [2]. The abbreviations are defined in Section 2.

Mentions: Mouse Nat2 shows high specific activities with a broad range of arylamines, including PABA, 5AS, 4AS and 2AF (Fig. 2 and Supplementary Table 1). In contrast, mouse Nat2 shows low specific activities against arylhydrazine substrates such as HDZ, INH, and PHZ, and against arylamine drugs procainamide and sulfamethazine (Fig. 2). This overall trend is in agreement with the substrate selectivity observed in previous studies with impure recombinant mouse Nat2 using a subset of these compounds [22,27,28], although it is much less than the activity observed towards these substrates with human NAT2 (Fig. 2). There was low, yet measurable activity using arylhydrazine substrates with purified mouse Nat2, which has not been observed in previous studies using recombinant mouse Nat2 expressed in a transient expression system [28]. Pure recombinant mouse Nat2 also readily acetylates 2-aminofluorene, the substrate which has been used to identify mouse strains carrying an active form of mouse Nat2—the fast acetylating C57Bl/6 strain, rather than the inactive form as is found in the slow acetylating A/J strain which is unstable and likely to be subject to rapid degradation [29].


Mouse N-acetyltransferase type 2, the homologue of human N-acetyltransferase type 1.

Kawamura A, Westwood I, Wakefield L, Long H, Zhang N, Walters K, Redfield C, Sim E - Biochem. Pharmacol. (2008)

Comparison of the substrate specific activity profiles of mouse Nat2, human NAT1 and human NAT2. The substrate specific activity profiles for mouse Nat2 (grey bars), human NAT1 (cross-hatched bars) and human NAT2 (black bars) are shown. The activity of mouse Nat2 was determined by measuring the rate of CoA production, as described in Section 2. Purified mouse Nat2 (0.125 μg) was incubated with arylamine substrate (each at 500 μM) and AcCoA (400 μM) in assay buffer (20 mM Tris–HCl (pH 8.0)) containing 0.5% (v/v) DMSO at 25 °C. The specific activities were determined from the linear initial rates of reaction. All measurements were performed in triplicate and are expressed as mean ± standard deviation relative to the most rapidly acetylated substrate. The human NAT1 and NAT2 data are taken from [2]. The abbreviations are defined in Section 2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2279149&req=5

fig2: Comparison of the substrate specific activity profiles of mouse Nat2, human NAT1 and human NAT2. The substrate specific activity profiles for mouse Nat2 (grey bars), human NAT1 (cross-hatched bars) and human NAT2 (black bars) are shown. The activity of mouse Nat2 was determined by measuring the rate of CoA production, as described in Section 2. Purified mouse Nat2 (0.125 μg) was incubated with arylamine substrate (each at 500 μM) and AcCoA (400 μM) in assay buffer (20 mM Tris–HCl (pH 8.0)) containing 0.5% (v/v) DMSO at 25 °C. The specific activities were determined from the linear initial rates of reaction. All measurements were performed in triplicate and are expressed as mean ± standard deviation relative to the most rapidly acetylated substrate. The human NAT1 and NAT2 data are taken from [2]. The abbreviations are defined in Section 2.
Mentions: Mouse Nat2 shows high specific activities with a broad range of arylamines, including PABA, 5AS, 4AS and 2AF (Fig. 2 and Supplementary Table 1). In contrast, mouse Nat2 shows low specific activities against arylhydrazine substrates such as HDZ, INH, and PHZ, and against arylamine drugs procainamide and sulfamethazine (Fig. 2). This overall trend is in agreement with the substrate selectivity observed in previous studies with impure recombinant mouse Nat2 using a subset of these compounds [22,27,28], although it is much less than the activity observed towards these substrates with human NAT2 (Fig. 2). There was low, yet measurable activity using arylhydrazine substrates with purified mouse Nat2, which has not been observed in previous studies using recombinant mouse Nat2 expressed in a transient expression system [28]. Pure recombinant mouse Nat2 also readily acetylates 2-aminofluorene, the substrate which has been used to identify mouse strains carrying an active form of mouse Nat2—the fast acetylating C57Bl/6 strain, rather than the inactive form as is found in the slow acetylating A/J strain which is unstable and likely to be subject to rapid degradation [29].

Bottom Line: In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids.We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2.We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.

ABSTRACT
There is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

Show MeSH
Related in: MedlinePlus