Purification and characterization of bile salt sulfotransferase from human liver
Abstract
Bile salt sulfotransferase, the enzyme responsible for the formation of bile salt sulfate esters, was purified extensively from normal human liver. The purification procedure included DEAE-Sephadex chromatography, taurocholate-agarose affinity chromatography, and preparative isoelectrofocusing. The final preparation had a specific activity of 18 nmol min−1 mg protein−1, representing a 760-fold purification from the cytosol fraction with a overall yield of 15%. The human enzyme has a Mr of 67,000 and a pI of 5.2. DEAE-Sephadex chromatography of the cytosol fraction revealed only a single species of activity. The limiting Km for the sulfuryl donor, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), is 0.7 μmM. The limiting Km for the sulfuryl acceptor, glycolithocholate (GLC), is 2 μm. Reciprocal plots were intersecting. Product inhibition studies established that adenosine 3′,5′-diphosphate (PAP) was competitive with PAPS (Ki = 0.2 μM) and noncompetitive with respect to GLC. GLC sulfate was competitive with GLC (Ki = 2.2 μM) and noncompetitive with respect to PAPS. Also, 3-ketolithocholate, a dead-end inhibitor, was competitive with GLC (Ki = 0.6 μM) and noncompetitive with respect to PAPS. Iso-PAP (the 2′ isomer of PAP) was competitive with PAPS (Ki = 0.3 μM) and noncompetitive with GLC. The cumulative results of the steady-state kinetics experiments point to a random mechanism for the binding of substrates and release of products. The purified enzyme displays no activity toward estrone, testosterone, or phenol. Among the reactive substrates tested, the values are in the order GLC > 3-βOH-5-cholenic acid > glycochenodeoxycholate > glycocholate. p-Chloromercuribenzoate inactivated the enzyme. Either PAPS or GLC protected against inactivation, suggesting the presence of a sulfhydryl group at the active Site.
References (35)
- A.E. Cowen et al.
Gastroenterology
(1975) - L.J. Chen et al.
Biochim. Biophys. Acta
(1977) - S. Barnes et al.
Biochim. Biophys. Acta
(1982) - L. Loof et al.
Biochim. Biophys. Acta
(1978) - L.J. Chen et al.
Life Sci
(1978) - J.B. Adams et al.
Biochim. Biophys. Acta
(1974) - L.J. Chen
Biochim. Biophys. Acta
(1982) - L. Loof et al.
Biochim. Biophys. Acta
(1980) - J. Farley et al.
J. Biol. Chem
(1976) - F. Renosto et al.
J. Biol. Chem
(1984)
Anal. Biochem
Anal. Biochem
Anal. Biochem
J. Biol. Chem
Biochim. Biophys. Acta
Anal. Biochem
Gastroenterology
Cited by (29)
Redox switching mechanism of the adenosine 5′-phosphosulfate kinase domain (APSK2) of human PAPS synthase 2
2023, StructureAdenosine 5′-phosphosulfate kinase (APSK) catalyzes the rate-limiting biosynthetic step of the universal sulfuryl donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS). In higher eukaryotes, the APSK and ATP sulfurylase (ATPS) domains are fused in a single chain. Humans have two bifunctional PAPS synthetase isoforms: PAPSS1 with the APSK1 domain and PAPSS2 containing the APSK2 domain. APSK2 displays a distinct higher activity for PAPSS2-mediated PAPS biosynthesis during tumorigenesis. How APSK2 achieves excess PAPS production has remained unclear. APSK1 and APSK2 lack the conventional redox-regulatory element present in plant PAPSS homologs. Here we elucidate the dynamic substrate recognition mechanism of APSK2. We discover that APSK1 contains a species-specific Cys-Cys redox-regulatory element that APSK2 lacks. The absence of this element in APSK2 enhances its enzymatic activity for excess PAPS production and promotes cancer development. Our results help to understand the roles of human PAPSSs during cell development and may facilitate PAPSS2-specific drug discovery.
Sulfotransferases
2018, Comprehensive Toxicology: Third EditionSulfotransferases catalyze the formation of sulfuric acid esters, most often referred to as sulfates, from a wide range of xenobiotics and their metabolites, as well as various endogenous neurotransmitters, hormones, bile acids, carbohydrates, and proteins. This article is focused on the cytosolic sulfotransferases, a superfamily of enzymes that, depending on the specific substrates and products of the sulfation reaction, are responsible for either detoxication or metabolic activation of a wide range of drugs, toxins, carcinogens, environmental chemicals, and other xenobiotics. An overview of current aspects of the nomenclature, gene organization, regulation of expression, enzyme structures and catalytic mechanisms, specificities for substrates and inhibitors, and roles in toxicology is provided for these cytosolic sulfotransferases.
Sulfotransferases
2010, Comprehensive Toxicology, Second EditionSulfotransferases catalyze the formation of sulfuric acid esters, most often referred to as sulfates, from a wide range of xenobiotics and their metabolites, as well as various endogenous neurotransmitters, hormones, bile acids, carbohydrates, and proteins. This chapter is focused on the cytosolic sulfotransferases, a superfamily of enzymes that, depending on the specific substrates and products of the sulfation reaction, are responsible for either detoxication or metabolic activation of a wide range of drugs, toxins, carcinogens, environmental chemicals, and other xenobiotics. An overview of current aspects of the nomenclature, gene organization, regulation of expression, enzyme structures and catalytic mechanisms, specificities for substrates and inhibitors, and roles in toxicology is provided for these cytosolic sulfotransferases.
The constitutive androstane receptor and pregnane X receptor function coordinately to prevent bile acid-induced hepatotoxicity
2004, Journal of Biological ChemistryA double null mouse line (2XENKO) lacking the xenobiotic receptors CAR (constitutive androstane receptor) (NR1I3) and PXR (pregnane X receptor) (NR1I2) was generated to study their functions in response to potentially toxic xenobiotic and endobiotic stimuli. Like the single knockouts, the 2XENKO mice are viable and fertile and show no overt phenotypes under normal conditions. As expected, they are completely insensitive to broad range xenobiotic inducers able to activate both receptors, such as clotrimazole and dieldrin. Comparisons of the single and double knockouts reveal specific roles for the two receptors. Thus, PXR does not contribute to the process of acetaminophen hepatotoxicity mediated by CAR, but both receptors contribute to the protective response to the hydrophobic bile acid lithocholic acid (LCA). As previously observed with PXR (Xie, W., Radominska-Pandya, A., Shi, Y., Simon, C. M., Nelson, M. C., Ong, E. S., Waxman, D. J., and Evans, R. M. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3375-3380), pharmacologic activation of CAR induces multiple LCA detoxifying enzymes and provides strong protection against LCA toxicity. Comparison of their responses to LCA treatment demonstrates that CAR predominantly mediates induction of the cytochrome p450 CYP3A11 and the multidrug resistance-associated protein 3 transporter, whereas PXR is the major regulator of the Na+-dependent organic anion transporter 2. These differential responses may account for the significant sensitivity of the CAR knockouts, but not the PXR knockouts, to an acute LCA dose. Because this sensitivity is not further increased in the 2XENKO mice, CAR may play a primary role in acute responses to this toxic endobiotic. These results define a central role for CAR in LCA detoxification and show that CAR and PXR function coordinately to regulate both xenobiotic and bile acid metabolism.
5β-Scymnol sulfotransferases from the liver of two Australian ray species
1998, Comparative Biochemistry and Physiology - B Biochemistry and Molecular BiologyThe tissue distribution of sterol sulfotransferase activity was investigated within the ray species Trygonorrhina fasciata and Trygonoptera species. The liver and kidney cytosolic fractions of each species were found to contain an enzyme that catalyzed the transfer of the sulfonate group from 3′-phosphoadenosine 5′-phosphosulfate to the elasmobranch bile alcohol 5β-scymnol. The liver is the major site of bile salt biosynthesis and detoxication, with the kidney possibly playing a secondary role. The two ray species liver sulfotransferases were partially purified by column chromatography and characterized. The enzymic properties of the partially purified liver sulfotransferases from the T. fasciata and Trygonoptera sp. ray species were similar in character. They followed Michaelis–Menten kinetics and were inhibited by the substrate 5β-scymnol and the reaction product adenosine-3′,5′-diphosphate. The enzymes were activated by the addition of magnesium ions, but had no absolute requirement for these ions; however, they were found to require sulfhydryl groups for activity. The steroid alcohols 5β-scymnol and 5α-cyprinol were both substrates for the sulfation reaction and the enzymes showed pH optimums of about 6.5, with a range of 5.5–7.5 and a temperature range from 22 to 37°C. This is the first report concerning sulfotransferase activity in ray species.
5β-Scymnol sulfotransferase isolated from the tissues of an Australian shark species
1998, Comparative Biochemistry and Physiology - B Biochemistry and Molecular BiologyThe enzyme system involved in the sulfation of 5β-scymnol [(24R)-5β-cholestane-3α,7α,12α,24,26,27-hexol] has been investigated in the shark species Heterodontus portusjacksoni. The liver enzyme was partially purified by column chromatography and chemically characterized. In its partially purified form the enzyme showed typical Michaelis–Menten kinetics for 5β-scymnol and the sulfonate donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) with an apparent Km of 3.04 μM for 5β-scymnol and 4.35 μM for PAPS. The reaction product adenosine-3′,5′-diphosphate and the substrates tested all inhibited the liver enzyme at concentrations above 10 μM. Substrate specificity of the enzyme was investigated using 5α-cyprinol, dehydroepiandrosterone, 17β-estradiol and testosterone. Only 5α-cyprinol was as efficiently sulfated as 5β-scymnol and suggests that the presence of the chiral alcohol group at C-24 is not essential for binding of the C27 bile steroids in the active site of the enzyme. A survey of tissue cytosolic fractions revealed that sterol sulfotransferase activity was present in the liver, kidney and testis; however, it was absent from the spleen, pancreas, brain, duodenum, heart and ovary.