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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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Heterologous expression and purification of recombinant mouse Nat2 in E. coli. SDS-PAGE analysis of expression and affinity purification of mouse Nat2. Mouse Nat2 was produced in E. coli Rosetta(DE3)pLysS strain and purified by Ni-NTA affinity chromatography. The purification gel shows expression of His-Nat2 at 34 kDa. Lanes: 1, whole cells; 2, soluble fraction; 3, unbound wash; 4, 0 mM imidazole (IMZ) wash; 5, 1 mM IMZ wash; 6, 10 mM IMZ wash; 7, 20 mM IMZ wash; 8, 50 mM IMZ wash; 9, first 100 mM IMZ wash; 10, second 100 mM IMZ wash; M, low-range molecular weight markers (BioRad). Thrombin cleavage of His-Nat2. Purified His-Nat2 was incubated with thrombin (5 U/mg NAT) at 4 °C for 6 h for complete His-tag cleavage. Lanes: M, low-range molecular weight marker (BioRad); 1, His-Nat2 only (8 μg); 2, thrombin-treated, Nat2 (8 μg).
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fig1: Heterologous expression and purification of recombinant mouse Nat2 in E. coli. SDS-PAGE analysis of expression and affinity purification of mouse Nat2. Mouse Nat2 was produced in E. coli Rosetta(DE3)pLysS strain and purified by Ni-NTA affinity chromatography. The purification gel shows expression of His-Nat2 at 34 kDa. Lanes: 1, whole cells; 2, soluble fraction; 3, unbound wash; 4, 0 mM imidazole (IMZ) wash; 5, 1 mM IMZ wash; 6, 10 mM IMZ wash; 7, 20 mM IMZ wash; 8, 50 mM IMZ wash; 9, first 100 mM IMZ wash; 10, second 100 mM IMZ wash; M, low-range molecular weight markers (BioRad). Thrombin cleavage of His-Nat2. Purified His-Nat2 was incubated with thrombin (5 U/mg NAT) at 4 °C for 6 h for complete His-tag cleavage. Lanes: M, low-range molecular weight marker (BioRad); 1, His-Nat2 only (8 μg); 2, thrombin-treated, Nat2 (8 μg).

Mentions: The mouse Nat2 was cloned into E. coli expression vector pET28b(+) and expressed in E. coli Rosetta (DE3)pLysS with an N-terminal hexa-histidine tag. The recombinant mouse His-Nat2 was purified using immobilised metal affinity chromatography, using a Ni-NTA column (Novagen), and eluted (Fig. 1a). Pure Nat2 predominantly eluted in the 50 mM and 100 mM imidazole washes. The hexa-histidine tag was readily removed by thrombin digestion (Fig. 1b). The purified recombinant Nat2 was active, readily catalysing the acetylation of PABA, a well-studied arylamine substrate of mouse Nat2 [27] (Table 1). The enzyme was relatively stable, and the enzymic activity was maintained (>85%) after incubation at temperatures ranging between 4 °C and 25 °C over a 72 h period.


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)

Heterologous expression and purification of recombinant mouse Nat2 in E. coli. SDS-PAGE analysis of expression and affinity purification of mouse Nat2. Mouse Nat2 was produced in E. coli Rosetta(DE3)pLysS strain and purified by Ni-NTA affinity chromatography. The purification gel shows expression of His-Nat2 at 34 kDa. Lanes: 1, whole cells; 2, soluble fraction; 3, unbound wash; 4, 0 mM imidazole (IMZ) wash; 5, 1 mM IMZ wash; 6, 10 mM IMZ wash; 7, 20 mM IMZ wash; 8, 50 mM IMZ wash; 9, first 100 mM IMZ wash; 10, second 100 mM IMZ wash; M, low-range molecular weight markers (BioRad). Thrombin cleavage of His-Nat2. Purified His-Nat2 was incubated with thrombin (5 U/mg NAT) at 4 °C for 6 h for complete His-tag cleavage. Lanes: M, low-range molecular weight marker (BioRad); 1, His-Nat2 only (8 μg); 2, thrombin-treated, Nat2 (8 μg).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Heterologous expression and purification of recombinant mouse Nat2 in E. coli. SDS-PAGE analysis of expression and affinity purification of mouse Nat2. Mouse Nat2 was produced in E. coli Rosetta(DE3)pLysS strain and purified by Ni-NTA affinity chromatography. The purification gel shows expression of His-Nat2 at 34 kDa. Lanes: 1, whole cells; 2, soluble fraction; 3, unbound wash; 4, 0 mM imidazole (IMZ) wash; 5, 1 mM IMZ wash; 6, 10 mM IMZ wash; 7, 20 mM IMZ wash; 8, 50 mM IMZ wash; 9, first 100 mM IMZ wash; 10, second 100 mM IMZ wash; M, low-range molecular weight markers (BioRad). Thrombin cleavage of His-Nat2. Purified His-Nat2 was incubated with thrombin (5 U/mg NAT) at 4 °C for 6 h for complete His-tag cleavage. Lanes: M, low-range molecular weight marker (BioRad); 1, His-Nat2 only (8 μg); 2, thrombin-treated, Nat2 (8 μg).
Mentions: The mouse Nat2 was cloned into E. coli expression vector pET28b(+) and expressed in E. coli Rosetta (DE3)pLysS with an N-terminal hexa-histidine tag. The recombinant mouse His-Nat2 was purified using immobilised metal affinity chromatography, using a Ni-NTA column (Novagen), and eluted (Fig. 1a). Pure Nat2 predominantly eluted in the 50 mM and 100 mM imidazole washes. The hexa-histidine tag was readily removed by thrombin digestion (Fig. 1b). The purified recombinant Nat2 was active, readily catalysing the acetylation of PABA, a well-studied arylamine substrate of mouse Nat2 [27] (Table 1). The enzyme was relatively stable, and the enzymic activity was maintained (>85%) after incubation at temperatures ranging between 4 °C and 25 °C over a 72 h period.

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