Recombinant NAT1 | Transfection Efficiency4-a | NAT1 Content4-b | Specific Activity | ||
---|---|---|---|---|---|
4-Aminosalicylate | 5-Aminosalicylate | 2-Aminofluorene | |||
U of β-gal/mg of cytosolic protein | U of NAT1/mg of cytosolic protein | nmol of arylamide formed/min/mg of cytosolic protein | |||
NAT1 4 | 0.57 (100)4-c | 0.87 (100) | 139 ± 35 (100) | 112 ± 23 (100) | 149 ± 16 (100) |
NAT1 10 | 0.59 (104) | 0.82 (94) | 120 ± 28 (86) | 103 ± 21 (92) | 128 ± 13 (86) |
NAT1 11 | 0.80 (140) | 0.86 (99) | 111 ± 22 (80) | 96 ± 18 (86) | 129 ± 18 (87) |
NAT1 16 | 1.40 (246) | 0.44 (51) | 53 ± 9 (38) | 46 ± 6 (41) | 58 ± 10 (39) |
None (pCR3vector) | 0 | ND4-d | ND | 3 (2) |
COS-1 cells (21 dishes per NAT1* allele) were transiently cotransfected with NAT1* recombinant construct (NAT1* coding and 3′ noncoding exons) and the reporter vector pCR3βGal. The cells were pooled, disrupted by sonication, and the cytosolic fraction isolated by differential centrifugation of the homogenate. Catalytic activity of the cytosol was determined at 37°C with reaction mixtures in 20 mM Tris-HCl buffer, pH 7.5, containing 4-aminosalicylate (0.1 mM), 5-aminosalicylate (0.05 mM), or 2-aminofluorene (0.1 mM), acetyl CoA (0.1 mM), and the additional components detailed under Experimental Procedures. The activities are the mean ± S.D. of a total of 24 values obtained as follows: a) the average of the slopes of the lines constructed from data on protein and time dependence of the reactions, respectively [18 determinations: triplicates for each of three time points (5, 10, and 15 min with mixtures containing 1 μg of cytosol), and three amounts of cytosolic protein (0.5, 1, and 3 μg in mixtures incubated for 5 min)]; and b) the average of triplicates from each of two additional experiments conducted under the previously established initial rate conditions with respect to amount of cytosolic protein and reaction time (1 μg of protein irrespective of substrate; reactions carried out for 5 min with 2-aminofluorene and for 10 min with 4- and 5-aminosalicylate).
↵4-a Transfection efficiency was ascertained by the catalytic activity of β-galactosidase at 37°C, determined from the absorbance of the reaction product (o-nitrophenol) at 420 nm. Reaction mixtures were in triplicate, and initial experiments were done to ensure the linearity of the reaction with COS-1 cell cytosolic protein and time; standard curves were generated with varying amounts of β-galactosidase (0.05–3 units).
↵4-b The numbers represent the slope of the linear segment of the curves depicted in Fig. 2. One unit (U) of immunoreactive NAT1 was artibitrarily equated to 1000 relative band density units.
↵4-c Values in parentheses are the percentage of those obtained with NAT1 4.
↵4-d ND, not detected.