Examination of human tissue cytosols for expression of sulfotransferase isoform 1A2 (SULT1A2) using a SULT1A2-specific antibody.

Sulfotransferase isoform 1A2 (SULT1A2) is a member of the cytosolic sulfotransferase family of phase II detoxification enzymes. Studies with recombinant enzymes have shown that SULT1A2 can catalyze the bioactivation of several procarcinogens, indicating a potential role in chemical carcinogenesis. However, previous studies have suggested that the SULT1A2 transcript has a splicing defect that might prevent it from becoming translated into protein; therefore, we sought to determine the expression of SULT1A2 in tissues. An antibody directed against a region of human SULT1A2 that differs from other known sulfotransferase isoforms was developed and used to screen a large number of cytosolic fractions from various tissues. Although the SULT1A2 antibody recognized recombinant SULT1A2 and did not cross-react with other SULT isoforms, the expression of SULT1A2 was not detected in any tissue examined. These studies suggest that if SULT1A2 is expressed as protein, the levels are very low and that SULT1A2 probably does not play a physiological role in chemical carcinogenesis.

Design, production and characterization of antibodies discriminating between the phenol- and monoamine-sulphating forms of human phenol sulphotransferase.

1. Phenol sulphotransferases (PSTs) are important enzymes in xenobiotic and endobiotic detoxication, and a key component of the body's chemical defence mechanism. 2. Human phenol-(P-PST) and monoamine-(M-PST) sulphating forms of PST share 93% amino acid sequence identity, and to date the various antibodies produced against PSTs all recognize both enzymes. 3. We have identified two peptides based on the cDNA-derived amino acid sequences of human P-PST and M-PST, which elicited for the first time antibodies capable of discriminating between these highly homologous enzymes. 4. These antibodies represent valuable tools for studying the expression, distribution and function of human phenol sulphotransferases.

Preparation and characterization of anti-peptide antibodies directed against human phenol and hydroxysteroid sulphotransferases.

Sulphotransferases (STs) catalyze the sulphation and, in general, detoxication of a large number of xenobiotics and endogenous compounds. A total of six synthetic peptides derived from the cDNA-derived amino acid sequences of the human phenol-sulphating form of phenosulphotransferase (P-PST) and human hydroxysteroid sulphotransferase (HST)--three from each sequence--were separately conjugated to the carrier protein keyhole limpet hemocyanin, and used to immunize rabbits. One successful antibody preparation was produced from among the P-PST peptides, and two from the HST peptides. On immunoblot analysis following SDS/PAGE, the anti-P-PST antibodies recognized two major forms of phenol ST in man, P-PST and the monoamine-sulphating form of PST, M-PST, and the two antibody preparations against HST recognized the human HST. These experiments demonstrate that it is possible to design specific antibodies against human sulphotransferases based on their amino acid sequences.

Heterologous systems for expression of mammalian sulfotransferases.

This paper describes the use of both mammalian and bacterial expression systems as tools to study the structural and functional relationships of proteins encoded by cDNAs to both rat and human aryl sulfotransferases. In particular, we describe the use of the mammalian COS cell system for functional expression studies, and the use of Escherichia coli for the expression and purification of a sulfotransferase fusion protein suitable as an antigen for the generation of sulfotransferase antibodies.

Identification of a new adult human liver sulfotransferase with specificity for endogenous and xenobiotic estrogens.

We present evidence for the existence of a previously undescribed sulfotransferase isoenzyme in adult human liver which sulfates endogenous and xenobiotic estrogens (estrone and 17 alpha-ethinylestradiol). The enzyme was resolved from other sulfotransferases using anion exchange chromatography and further purified by gel filtration and affinity chromatography. Using antibodies against rat liver estrogen and hydroxysteroid sulfotransferases, it was shown that the adult human estrogen sulfotransferase has a subunit molecular weight of 33kDa, is immunologically related to its rat counterpart and also to the human phenolsulfotransferase enzyme family, but not to human hydroxysteroid sulfotransferase.

Phenol sulfotransferase expression in the airways: enzymological and immunohistochemical demonstration.

Phenol (aryl) sulfotransferases (PSTs) provide a conjugative pathway that detoxifies hydroxylated aromatic xenobiotics by esterification with sulfate. Both human and bovine airways have been reported to use this pathway, and in this investigation the bovine system is examined. PST activity in tracheal through fourth generation bronchial mucosal cytosols was 0.1-0.35 nmol/mg protein/min. Activity was generally greater in more distal bronchi and in parenchymal extracts, which contained 0.6-3 nmol/mg/min PST activity. Comparison of the PST activities of bronchial and parenchymal cytosols indicated similar pH activity profiles, although steady state kinetic measurements revealed different Km values for the acceptor substrate 2-naphthol (13.7 microM for bronchial, 31.3 microM for parenchymal). Anion exchange chromatography indicated two PST isoforms being expressed in different ratios. Immunoblot analysis with mouse anti-bovine PST revealed a closely spaced doublet at 32 kDa in both bronchial mucosal and parenchymal cytosolic extracts; however, this doublet was unequally stained in parenchymal extracts. Immunohistochemical analyses revealed faint positive staining of the tracheobronchial epithelium. Greatest immunostaining was observed in the nonciliated secretory epithelial cells of the bronchioles, whereas surrounding smooth muscle, endothelial cells, and alveoli were immunonegative. These results are consistent with the known locations of other detoxification enzymes within the airways.

Purification, immunochemical characterization, and immunohistochemical localization of rat hepatic aryl sulfotransferase IV.

Aryl sulfotransferases catalyze the formation of sulfuric acid esters from a diverse group of endogenous and xenobiotic organic chemicals. The isoenzyme of aryl sulfotransferase in livers of male Sprague-Dawley rats that exhibits the most varied substrate specificity is aryl sulfotransferase IV. A new method for the purification to homogeneity of aryl sulfotransferase IV was developed that, when compared with previously described procedures, provided a greater than 10-fold increase in total yield of enzyme/g of tissue. Homogeneous aryl sulfotransferase IV was used to prepare polyclonal antibodies in male New Zealand White rabbits. Results of immunochemical analyses demonstrated that these antibodies reacted with only a single protein in rat hepatic 100,000 x g supernatant fractions and, further, that the immunoreactive protein had the isoelectric point and subunit molecular mass characteristic of aryl sulfotransferase IV. Immunohistochemical analyses demonstrated that aryl sulfotransferase IV is present in hepatocytes throughout the liver, although centrilobular cells contain a significantly greater (p less than 0.01) amount of aryl sulfotransferase IV than do either midzonal or periportal cells, in which similar levels of the enzyme are found.

Purification and immunochemical characterization of a rat liver sulphotransferase conjugating paracetamol.

Paracetamol sulphotransferase (ST) was purified 250-fold from male rat liver, and the pure enzyme used to elicit antibodies in rabbit. The enzyme was active towards paracetamol at pH 9.0, as well as towards several commonly used drugs, and formed sulphates at both O- and N-atoms. Comparison of the substrate specificity of paracetamol ST with that of aryl sulphotransferases isolated by other workers suggested that we have purified a previously unknown isoenzyme of rat liver ST, although the difficulties of characterization of STs based on their substrate specificities is noted. The antibody preparation recognized only one polypeptide (Mr = 35,000) on immunoblot analysis of rabbit liver cytosol, corresponding to purified paracetamol ST. Analysis of the tissue distribution of this protein demonstrated that its expression was restricted to the liver, as was the enzyme activity. The observed sex difference in paracetamol ST (males greater than females) was determined by immunoblot analysis to be the result of reduced enzyme protein levels in females. In human liver cytosol, the antibody recognized two polypeptides, probably corresponding to M- and P-phenol STs, suggesting significant sequence similarity between rat and human phenol sulphotransferases.

Immunological characterization of human phenol sulfotransferase.

The immunological characterization of the different forms of phenol sulfotransferase (PST) in a variety of human and nonhuman tissues is described. Immunoblotting techniques revealed that polyclonal antibodies raised to human platelet MII-PST reacted with polypeptides of 32 and 34 kDa from human platelet 100,000 x g supernatant solution. Immunoblot analysis of platelet 100,000 x g supernatant solution that was fractionated over a DEAE-cellulose column indicated a close correspondence of P-PST activity, as measured by phenol sulfation, and M-PST activity, as assessed by dopamine sulfation, with the 32 and 34 kDa polypeptides, respectively. Examination of various human tissues revealed the presence of immunologically detectable levels of P-PST in liver and adrenal gland whereas both M- and P-PST were detected in placenta at a 1/10,000 dilution of the antisera. Under these conditions, PST was undetectable in human frontal cortex, pituitary gland, kidney, lung, and jejunum. Further evaluation of human liver samples from four individuals indicated a strong correlation (r = 0.94) between the amount of 32-kDa immunoreactive protein and P-PST activity. Analysis of liver samples from several animal species (monkey, rat, mouse, guinea pig, and frog) revealed the presence of immunoreactive proteins of various molecular masses, suggesting that considerable homology may exist between human and nonhuman forms of PST.