ABSTRACT
Cytosolic sulfotransferases (STs) are generally thought to be involved in detoxification of xenobiotics, as well as homeostasis of endogenous compounds such as thyroid/steroid hormones and catecholamine hormones/neurotransmitters. We report here the identification and characterization of a zebrafish estrogen-sulfating cytosolic ST. The zebrafish ST was bacterially expressed, purified, and examined for enzymatic activities using a variety of endogenous compounds as substrates. Results showed that the enzyme displayed much higher activities toward two endogenous estrogens, estrone (E(1)) and 17beta-estradiol (E(2)), in comparison with thyroid hormones, 3,3',5-triiodothyronine (T(3)) and thyroxine (T(4)), dopamine, dihydroxyphenylalanine (Dopa), and dehydroepiandrosterone (DHEA). The kinetic parameters, K(m), and V(max), with estrogens and thyroid hormones as substrates were determined. The calculated V(max)/K(m) for E(1), E(2), T(3), and T(4) were, respectively, 31.6, 16.7, 1.5, and 0.8 nmol min(-1) mg(-1) microM(-1), indicating clearly the estrogens being preferred physiological substrates for the enzyme. The inhibitory effects of isoflavone phytoestrogens on the sulfation of E(2) by this zebrafish ST were examined. The IC(50) determined for quercetin, genistein, and daidzein were 0.7, 2.5, and 8 microM, respectively. Kinetic analyses revealed that the mechanism underlying the inhibition by these isoflavones to be of the competitive type.
Subject(s)
Cytosol/enzymology , Estrogens/metabolism , Isoflavones/pharmacology , Plant Preparations/pharmacology , Sulfotransferases/metabolism , Zebrafish Proteins/metabolism , Animals , Cytosol/metabolism , Dose-Response Relationship, Drug , Genistein/metabolism , Genistein/pharmacology , Glutathione Transferase/metabolism , Humans , Isoflavones/antagonists & inhibitors , Isoflavones/metabolism , Phytoestrogens , Plant Preparations/antagonists & inhibitors , Plant Preparations/metabolism , Quercetin/metabolism , Quercetin/pharmacology , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Sulfotransferases/classification , Sulfotransferases/genetics , Sulfotransferases/isolation & purification , Thyroid Hormones/metabolism , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/isolation & purificationABSTRACT
Environmental estrogen-like chemicals are increasingly recognized as a potential hazardous factor for wildlife as well as humans. We have recently embarked on developing a zebrafish model for investigating the role of sulfonation in the metabolism and adverse functioning of environmental estrogens. Here, we report on a systematic investigation of the sulfonation of representative environmental estrogens (bisphenol A, 4-n-octylphenol, 4-n-nolylphenol, diethylstilbestrol, and 17 alpha-ethynylestradiol) by zebrafish cytosolic sulfotransferases (STs). Of the seven enzymes tested, four zebrafish STs (designated ZF ST #2, ZF ST #3, ZF ST #4, and ZF DHEA ST) exhibited differential sulfonating activities toward the five environmental estrogens tested, with ZF ST #3 being more highly active than the other three. It was further demonstrated that bisphenol A, 4-n-octylphenol, and 4-n-nonylphenol exerted concentration-dependent inhibition of the sulfonation of 17 beta-estradiol, implying a potential role of these environmental estrogens in interfering with the sulfonation, and possibly homeostasis, of endogenous estrogens. Kinetic studies revealed that the mechanism underlying the inhibition by bisphenol A or 4-n-nonylphenol to be of the competitive type.
Subject(s)
Cytosol/enzymology , Estrogens/metabolism , Sulfotransferases/chemistry , Animals , Benzhydryl Compounds , Cytosol/metabolism , DNA, Complementary/metabolism , Diethylstilbestrol/chemistry , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Environment , Estradiol Congeners/chemistry , Estrogens, Non-Steroidal/chemistry , Ethinyl Estradiol/chemistry , Kinetics , Phenols/chemistry , Plasmids/metabolism , Protein Isoforms , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , ZebrafishABSTRACT
Kinetic studies were performed to dissect the mechanism underlying the remarkable Mn(2+) stimulation of the Dopa/tyrosine-sulfating activity of the human monoamine (M)-form phenol sulfotransferase (PST). The activities and the stimulation by Mn(2+) are highly stereospecific for the d-form enantiomers of tyrosine and Dopa. Analysis of the kinetic results strongly suggests that tyrosine-Mn(2+) and tyrosine-Mn(2+)-tyrosine complexes are obligatory substrates, whereas Dopa-Mn(2+) complexes may be better substrates than Dopa alone. This activation of the Dopa/tyrosine-sulfating activity of M-form PST by Mn(2+) via complex formation between Mn(2+) and the substrate is the first reported case of a regulatory mechanism in this important class of enzymes. Our previous studies using point-mutated M-form PSTs established that the Mn(2+) (in the substrate-Mn(2+) complex) exerts its stimulatory effect by binding predominantly to the Asp-86 residue at the active site. We present here further studies using dopamine as substrate to bolster this conclusion. The possible physiological implications of this rather unusual specificity for the d-amino acid and its derivatives and the stimulation by Mn(2+) are discussed in the context of protective and detoxification mechanisms that may operate in neurodegenerative processes in the brain. The Mn(2+) stimulation of the activity of M-form PST toward d-enantiomers of Dopa/tyrosine may have implications for other substrates (including chiral drugs) and for the other cytosolic sulfotransferases that are involved in the regulation of endogenous metabolites as well as in detoxification.
