Improved bacterial expression of the human P form phenolsulfotransferase. Applications to drug metabolism.

Bacterial expression of human phenol phenolsulfotransferase (P-PST) has provided the opportunity to understand better the catalytic properties and biological role of this enzyme. However, as the yield of pure protein from the currently used expression system was low, we subcloned the P-PST c-DNA into pET-15b, a vector containing an oligohistidine domain, for improved expression. The fusion protein, His-P-PST, was isolated from the bacterial cytosol in a single affinity chromatography step, using a Ni2+ agarose column. The yield of His-P-PST from the pET-15b vector was improved 12-fold, compared with P-PST from the original vector. The purity was > 99%, as established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry scanning. The enzyme was stable for at least 3 weeks when stored in 20% glycerol at -80 degrees C. A very rapid deterioration of the enzyme during 37 degrees C incubations was effectively prevented by the addition of bovine serum albumin. The sulfonation of several substrates was very similar for His-P-PST and P-PST, with Vmax/KM values (first order rate constants) for the high-affinity substrate p-nitrophenol of 143 +/- 27 and 120 +/- 25 ml min-1 microgram-1 PST [mean +/- SE; not significant (NS)], respectively, and for the low-affinity substrate acetaminophen of 0.21 +/- 0.11 and 0.14 +/- 0.07 ml min-1 microgram-1 PST (NS). The Vmax/KM for the sulfonation of the isoproterenol enantiomers showed a (+)/(-)-enantiomer ratio of 6.2 for His-P-PST and 7.4 for P-PST. Interestingly, 3- to 10-fold higher apparent KM values were obtained for these substrates with the crude human liver cytosol, compared with the recombinant P-PSTs, suggested to be due to endogenous or dietary P-PST inhibitors in the liver. In addition, the inhibition of acetaminophen sulfonation by quercetin was very similar for His-P-PST and P-PST, with IC50 values of 0.10 +/- 0.03 and 0.05 +/- 0.01 microM (NS), respectively. The additional amino acid residues in the His-P-PST, compared with the recombinant P-PST, thus did not significantly alter the catalytic properties. This bacterial expression system should lend itself to routine use in studies of the metabolism of drugs and environmental chemicals.

Stereoselective sulphate conjugation of salbutamol by human lung and bronchial epithelial cells.

1. The metabolism of (+)-, (-)- and (+/-)-salbutamol by sulphoconjugation was determined in vitro using human lung cytosol and bronchial epithelial BEAS-2B cell homogenate. 2. For the lungs the intrinsic clearance (Vmax/Km) value for the pharmacologically active (-)-salbutamol (0.49 +/- 0.32 ml min-1 g-1 protein) exceeded that of (+)-salbutamol (0.046 +/- 0.028 ml min-1 g-1 protein) by 11-fold. This was mainly due to a difference in Km value, which was 16 times higher for (+)-salbutamol (1300 +/- 170 microM) than for (-)-salbutamol (83 +/- 12 microM). 3. The stereoselectivity of sulphoconjugation of salbutamol was very similar in the BEAS-2B cells, although the absolute activity was considerably lower. 4. The enzyme catalyzing this reaction both in the lungs and in the BEAS-2B cells was the monoamine (M) form phenolsulphotransferase. 5. These observations emphasize that the smooth muscle of the bronchi most likely are exposed to considerably higher concentrations of the potentially toxic (+)-enantiomer than of the bronchodilating (-)-enantiomer during therapy with (+/-)-salbutamol.

Flavonoids, potent inhibitors of the human P-form phenolsulfotransferase. Potential role in drug metabolism and chemoprevention.

The common dietary constituent quercetin was a potent inhibitor of sulfoconjugation of acetaminophen and minoxidil by human liver cytosol, partially purified P-form phenolsulfotransferase (PST), and recombinant P-form PST, with IC50 values of 0.025-0.095 microM. Quercetin inhibition of acetaminophen was noncompetitive with respect to acceptor substrate, with a Ki value of 0.067 microM. A number of other flavonoids, such as fisetin, galangin, myricetin, kaempferol, chrysin, and apigenin, were also potent inhibitors of P-form PST-mediated sulfation, with IC50 values < 1 microM. Studies of structural analogs indicated the flavonoid 7-hydroxyl group as particularly important for potent inhibition. Potential human metabolites of quercetin were poor inhibitors. Curcumin, genistein, and ellagic acid (other polyphenolic natural products) were also inhibitors of P-form PST, with IC50 values of 0.38-34.8 microM. Quercetin was also shown to inhibit sulfoconjugation by the human hepatoma cell line Hep G2. Although less potent in this intact cell system (IC50 2-5 microM), quercetin was still more potent than 2,6-dichloro-4-nitrophenol, the classical P-form PST inhibitor that has been shown to be an inhibitor also in vivo. These observations suggest the potential for clinically important drug interactions, as well as a possible role for flavonoids as chemopreventive agents in sulfation-induced carcinogenesis.

Quercetin, a potent and specific inhibitor of the human P-form phenosulfotransferase.

The natural product quercetin was a potent inhibitor of the human P-form phenolsulfo-transferase with an IC50 value of 0.10 +/- 0.03 microM (mean +/- SEM; N = 5), which was three to four orders of magnitude more potent than its inhibition of other human sulfotransferases. The inhibition was noncompetitive with a Ki value of 0.10 microM. The potency and mechanism of this inhibition appear similar to those of the current standard P-form inhibitor, 2,6-dichloro-4-nitrophenol. Among other flavonoids examined, kaempferol was found to have an IC50 value of 0.39 +/- 0.07 microM, naringenin 10.6 +/- 1.6 microM and naringin 265 +/- 90 microM (N = 3). These observations suggest the potential for clinically important pharmacologic and toxicologic interactions by flavonoid-containing foods and beverages.