. 2017 Apr 25;58(6):1114–1131. doi: 10.1194/jlr.M074302

TABLE 3.

SULT1 substrate screen of column-purified recombinant His-mL-STL enzyme using barium precipitation method

	Specific activity (pmol/min/mg), mean ± SEM
	Vary pH at 50 μM substrate concentration			Vary substrate concentration at pH 5.5
Compounds	pH 5.5	pH 6.5	pH 7.5	50 μM	100 μM
Phenols
Phenol	2.28 ± 3.31	2.24 ± 0.94^ns	2.71 ± 0.43^ns	3.93 ± 0.77	8.12 ± 6.77^ns
2-Ethylphenol	7.59 ± 2.48	0.74 ± 2.21^ns	2.28 ± 0.70^ns	2.51 ± 4.64	1.31 ± 2.00^ns
4-Ethylphenol	4.36 ± 0.07	1.55 ± 2.31^ns	3.17 ± 1.77^ns	1.97 ± 6.81	3.18 ± 2.51^ns
4-Isoprophylphenol	5.73 ± 0.27	9.19 ± 7.37^ns	3.20 ± 0.05^ns	−0.95 ± 0.12	3.99 ± 0.08^ns
2-Prophylphenol	7.39 ± 3.36	11.04 ± 7.87^ns	0.42 ± 0.42^ns	−4.48 ± 0.02	0.00 ± 3.40^ns
4-Nitrophenol	4.06 ± 3.55	8.17 ± 3.17^ns	5.63 ± 5.11^ns	1.45 ± 2.69	2.45 ± 2.23^ns
1-Naphthol	1.21 ± 3.77	5.67 ± 3.17^ns	2.28 ± 0.01^ns	−1.18 ± 1.48	5.50 ± 9.13^ns
2-Naphthol	2.55 ± 0.67	8.55 ± 7.18^ns	11.71 ± 9.78^ns	0.51 ± 0.71	1.41 ± 6.41^ns
Acetaminophen	8.26 ± 0.27	8.85 ± 3.41^ns	2.69 ± 1.29^ns	13.07 ± 5.30	4.85 ± 9.10^ns
p-Cresol	3.36 ± 1.49	1.98 ± 1.20^ns	9.26 ± 10.14^ns	12.42 ± 5.06	8.53 ± 2.18^ns
4-Nitrocatechol	−5.92 ± 4.04	3.82 ± 4.50^ns	−1.38 ± 0.33^ns	−6.45 ± 0.71	7.70 ± 1.12^ns
Resveratrol	1.27 ± 1.46	2.86 ± 6.95^ns	12.62 ± 7.19^ns	3.04 ± 7.54	−0.62 ± 7.89^ns
Vanillin	4.76 ± 1.90	8.34 ± 6.30^ns	0.81 ± 1.33^ns	20.60 ± 15.57	36.69 ± 28.44^ns
Catecholamines
Dopamine hydrochloride	0.24 ± 2.80	4.21 ± 2.17^ns	0.47 ± 0.82^ns	6.68 ± 5.66	2.41 ± 0.37^ns
Isoprenaline hydrochloride	−11.98 ± 10.27	−0.91 ± 2.73^ns	−5.44 ± 2.46^ns	−9.80 ± 5.92	3.87 ± 4.55^ns
Salbutamol	1.49 ± 2.60	6.87 ± 6.42^ns	−1.48 ± 4.46^ns	9.04 ± 5.57	−2.36 ± 3.26^ns
(±)-Epinephrine	−8.46 ± 11.53	−5.84 ± 3.34^ns	−3.09 ± 0.98^ns	−8.76 ± 11.21	2.69 ± 2.92^ns
(−)-Norepinephrine	−13.38 ± 9.11	−1.62 ± 1.16^ns	−7.62 ± 2.71^ns	−10.64 ± 10.02	−2.43 ± 2.34^ns
Estrogens
Estrone	−1.55 ± 2.72	0.95 ± 2.91^ns	8.58 ± 8.93^ns	−3.54 ± 4.97	−2.56 ± 1.53^ns
β-Estradiol	−0.46 ± 0.46	1.13 ± 2.05^ns	10.12 ± 12.92^ns	5.77 ± 6.38	4.49 ± 1.18^ns
17α-Ethynylestradiol	11.53 ± 0.61	10.37 ± 0.99^ns	16.10 ± 9.79^ns	16.02 ± 2.38	0.58 ± 7.13^ns
2-Hydroxyestradiol	−0.84 ± 1.72	3.23 ± 3.13^ns	1.63 ± 1.87^ns	2.05 ± 5.51	−3.43 ± 0.20^ns
Amine
2-Naphthylamine	83.04 ± 2.48	37.31 ± 11.30***	9.24 ± 7.06***	88.95 ± 15.71	256.39 ± 101.81***
Tyrosine/derivatives
D-Tyrosine	−3.16 ± 2.98	2.11 ± 1.43^ns	−5.53 ± 0.60^ns	2.14 ± 1.33	2.10 ± 5.96^ns
3-Nitro-L-tyrosine	−8.89 ± 12.81	−9.31 ± 9.76^ns	4.32 ± 7.30^ns	−10.44 ± 12.48	4.80 ± 4.81^ns
Thyroid hormones
3,5-Diiodo-L-thyronine	−11.57 ± 5.25	−2.57 ± 1.20^ns	7.31 ± 3.63^ns	−3.67 ± 21.24	10.37 ± 0.38^ns
3,3′,5-Triiodo-L-thyronine	−1.46 ± 8.63	−1.58 ± 1.60^ns	11.22 ± 10.87^ns	15.94 ± 11.04	2.18 ± 1.73^ns
3,3′,5′-Triiodo-L-thyronine	−2.05 ± 2.90	3.11 ± 0.30^ns	13.02 ± 11.62^ns	8.73 ± 9.55	−2.09 ± 0.04^ns
Others
Minoxidil	5.51 ± 5.41	0.96 ± 0.06^ns	−0.36 ± 4.39^ns	15.13 ± 7.98	−0.87 ± 7.22^ns
4-Phenyl-1,2,3,6-tetrahydropyridine hydrochloride	1.48 ± 0.46	18.30 ± 4.19^ns	8.97 ± 7.15^ns	−1.56 ± 2.79	−1.83 ± 3.19^ns

Each reaction mixture (150 μl) comprised 100 mM sodium acetate buffer (pH 5.5) or 100 mM sodium phosphate buffer (pH 6.5 or 7.5), 2.5 mM MgCl₂, 20 μg of purified His-mL-STL protein, 50 or 100 μM substrate, and 50 μM PAPS containing 0.015 μCi [³⁵S]PAPS. The reaction mixture was incubated at 37°C for 1 h, and the reaction was terminated by the addition of barium acetate. The unreacted [³⁵S]PAPS was precipitated by barium hydroxide and zinc sulfate. Specific activity was calculated from the scintillation counting of the [³⁵S]sulfonated product. Data are mean ± SEM of two batches of purified proteins, each performed in duplicate. Differences between compounds and pH at fixed 50 μM substrate concentration as well as differences between compounds and substrate concentrations at pH 5.5 were analyzed by two-way ANOVA followed by Bonferroni post hoc test. *** P < 0.001; ns, nonsignificant at P > 0.05 level.