Purification and kinetic characterization of a dopamine-sulfating form of phenol sulfotransferase from human brain

The kinetic and biochemical properties of a purified, monoamine-sulfating form of phenol sulfotransferase (M-PST) from human brain are described. M-PST activity was separated and purified from phenol-sulfating activity by anion-exchange chromatography on DEAE-cellulose and subsequently purified on AffiGel Blue and Sephacryl S-200, routinely giving a final purification of over 20 000-fold, with approximately a 3% yield. The molecular weight of the active species, as estimated by gel filtration chromatography, was 250 000. The purified enzyme was inhibited by NaCl (50% at 325 mM) and showed an optimum for dopamine sulfation at pH 7.0. Of the monoamine substrates examined, 4-methoxytyramine was the most extensively sulfated at 20 microM, while at higher substrate concentrations (200 microM), tyramine was the apparent preferred substrate. Kinetic analysis demonstrated that sulfation by M-PST proceeds via an ordered, bisubstrate reaction mechanism, where 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is the leading substrate. True Km values for dopamine and PAPS were 2.9 and 0.35 microM, respectively. The product inhibitor 3'-phosphoadenosine 5'-phosphate possessed a Ki of 0.07 microM, while the dead-end inhibitor ATP exhibited a Ki of 170 microM.     

Purification and characterization of bile salt sulfotransferase from human liver

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 microM. The limiting Km for the sulfuryl acceptor, glycolithocholate (GLC), is 2 microM. Reciprocal plots were intersecting. Product inhibition studies established that adenosine 3',5'-diphosphate (PAP) was competitive with PAPS (Ki = 0.2 microM) and noncompetitive with respect to GLC. GLC sulfate was competitive with GLC (Ki = 2.2 microM) and noncompetitive with respect to PAPS. Also, 3-ketolithocholate, a dead-end inhibitor, was competitive with GLC (Ki = 0.6 microM) and noncompetitive with respect to PAPS. Iso-PAP (the 2' isomer of PAP) was competitive with PAPS (Ki = 0.3 microM) 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 Vmax/Km values are in the order GLC greater than 3-beta OH-5-cholenic acid greater than glycochenodeoxycholate greater than glycocholate. p-Chloromercuribenzoate inactivated the enzyme. Either PAPS or GLC protected against inactivation, suggesting the presence of a sulfhydryl group at the active site.     

Localization and characterization of phenol sulfotransferase in human platelets

Phenol sulfotransferase was localized as a soluble enzyme in platelets from human blood. The enzyme was found to esterify a variety of endogenous phenolic biogenic amines including tyramine, dopamine, norepinephrine and 5-hydroxytryptamine as well as phenol. Of the substrates tested dopamine was found to be most rapidly conjugated when present at a concentration of 30 μM while tyramine was found to be the best substrate at a concentration of 100 μM. The Km value for tyramine was 59 μM and tyramine concentrations of 400 μM or greater resulted in apparent substrate inhibition. The possible clinical implications of of these findings are discussed.     

Purification and characterization of galactocerebroside sulfotransferase from rat kidney

Galactocerebroside sulfotransferase (EC 2.8.2.11) was purified to apparent homogeneity from rat kidneys. The purified protein is stable at -20 degrees C, and has an estimated molecular weight of 64,000 and a pI of 5.1. In contrast to other known sulfotransferases, the enzyme appears not to require divalent metal ions for activity. The Km for the donor, 3'-phosphoadenosine 5'-phosphosulfate, is 5.2 microM. Structural studies on this "active" sulfate donor show the requirement of a phosphate group at the 3' position of the ribose moiety. Modification of the amino group at either the 6 or 8 position on the purine ring renders the corresponding compounds poor substrates. Both galactosylceramide and lactosylceramide are effective acceptors for this enzyme, while galactosylsphingosine and galactosylglycerolipids are sulfated only poorly, suggesting that the in vivo sulfation of these glycolipids is carried out by different sulfotransferases. The active site of the enzyme contains arginine residues which appear to be important in binding the sulfate donor. The enzyme protein is hydrophobic and binds 0.17 mg [3H]Triton X-100/mg protein. The purified enzyme contains bound lipids, consisting primarily of cholesterol and phosphatidylcholine. The lipid environment affects the activity of the enzyme which, in turn, regulates the sulfation of glycolipids.     

Isolation and characterization of a bovine gene encoding phenol sulfotransferase

Phenol sulfotransferases (PST) esterify hydroxylated aromatic compounds with sulfate, and therefore play a role in the metabolism of xenobiotics. In this investigation, a bovine PST gene of 2372 bp was amplified from genomic DNA. Sequence overlap with the cognate cDNA revealed seven exons, with all introns containing GT/AG splicing donor/acceptor sites. This product was subcloned into an SV40-based expression vector and transfected into WI-26 human lung fibroblasts. Immunoblot analyses revealed production of the anticipated 32-kDa protein, and the active enzyme displayed steady-state kinetic properties consistent with the enzyme characterized in bovine lung (apparent K_m=6.5 μM for 2-naphthol).     

Purification and characterization of N-acetylglucosaminyl sulfotransferase from chick corneas

Sulfation is an important conjugation pathway in deactivating thyroid hormones, keeping the proper hormonal balance, and increasing the rate of thyroid hormone metabolism. We have identified, cloned, and characterized a sulfotransferase (SULT) that is capable of thyroid hormone conjugation in the dog. This enzyme, designated cSULT1B1, displays a strong identity (>84%) to the human ST1B2 enzyme. However, cSULT1B1 displays less identity, about 73%, to mouse and rat orthologs. In addition, the canine enzyme is three amino acids shorter than the rodent ones but has the same length as the human ortholog, 296 amino acids. The bacterial expressed and partial purified cSULT1B1 enzyme sulfates p-nitrophenol and 1-naphtol, but not dopamine. The thyroid hormones 3,3'-diiodothyronine and 3,5,3'-triiodothyronine are efficiently sulfated. 3,3',5'-Triiodothyronine is sulfated to lesser degree while sulfation of 3,5'-diiodothyronine and 3,3',5,5'-tetraiodothyronine cannot be detected. The cSULT1B1 is found in the colon (highest level), kidney and small intestine in dogs, but surprisingly not in the male dog liver although low levels of immunoreactivity were detected in the female dog liver. The male dog expresses more of SULT1B1 enzyme in the lower part of the small intestine while the female dog displays an opposite pattern of expression. These results describe the cloning and characterization of a canine thyroid hormone sulfating enzyme that is more closely related to the human ortholog than to the rodent thyroid sulfating enzymes.     

Purification and characterization of tripeptidylpeptidase-II from post-mortem human brain

A soluble tripeptidylaminopeptidase has been isolated from human post-mortem cerebral cortex by anion exchange, hydrophobic interaction and size-exclusion chromatography. From gel filtration studies the active enzyme can exist in both high molecular weight (Mr>10⁶) and smaller forms. The enzyme hydrolyses Ala-Ala-Phe-7-amino-4-methylcoumarin with a pH optimum of around 7.5 and K_m of 148 M. It did not hydrolyse N-succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin, aminoacyl- or dipeptidyl-7-amino-methylcoumarins and was not inhibited by bestatin. The enzyme was inhibited by phenylmethylsulphonyl-fluoride, 3,4-dichloroisocoumarin, N-hydroxymercuriphenyl-sulphonic acid and N-ethylmaleimide showing that its activity is serine and cysteine dependent. The purified enzyme released tripeptides from several naturally occurring neuropeptides with quite broad specificity. Cholecystokinin octapeptide, angiotensin III and neurokinin A were the most rapidly hydrolysed. Peptides with Pro residues arount the point of cleavage were not hydrolysed.     

Purification and characterization of aldehyde dehydrogenase from human brain

Aldehyde dehydrogenase (EC 1.2.1.3) has been purified from human brain; this constitutes the first purification to homogeneity from the brain of any mammalian species. Of the three isozymes purified two are mitochondrial in origin (Peak I and Peak II) and one is cytoplasmic (Peak III). By comparison of properties, the cytoplasmic Peak III enzyme could be identified as the same as the liver cytoplasmic E1 isozyme (N.J. Greenfield and R. Pietruszko (1977) Biochim. Biophys. Acta 483, 35-45). The Peak I and Peak II enzymes resemble the liver mitochondrial E2 isozyme, but both have properties that differ from those of the liver enzyme. The Peak I enzyme is extremely sensitive to disulfiram while the Peak II enzyme is totally insensitive; liver mitochondrial E2 isozyme is partially sensitive to disulfiram. The specific activity is 0.3 mumol/mg/min for the Peak I and 3.0 mumol/mg/min for the Peak II enzyme; the specific activity of the liver mitochondrial E2 isozyme is 1.6 mumol/min/mg under the same conditions. The Peak I enzyme is also inhibited by acetaldehyde at low concentrations, while the Peak II enzyme and the liver mitochondrial E2 isozyme are not inhibited under the same conditions. The precise relationship of brain Peak I and II enzymes to the liver E2 isozyme is not clear but it cannot be excluded at the present time that the two brain mitochondrial enzymes are brain specific.     

Purification and characterization of a new form of glutathione S-transferase from human erythrocytes

Presence of a new form of glutathione S-transferase has been demonstrated in human erythrocytes. using two different affinity ligands this enzyme has been separated from the previously characterized glutathione S-transferases ?. The new enzyme is highly basic with a pI of > 10. The new enzyme which represents less than 5 percent of glutathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene as substrate and about 10 percent of total glutathione S-transferase protein of erythrocytes has different amino acid composition, substrate specificities, and immunological characteristics from those of the major erythrocyte glutathione S-transferase ?. Immunological properties of the new enzyme indicate that this form may be different from other glutathione S-transferases of human tissues.     

Purification and characterization of cholesterol sulfotransferase from rat skin.

Previous work has demonstrated that the activity of the enzyme cholesterol sulfotransferase is rapidly and dramatically increased upon squamous differentiation of a variety of epithelial cells in culture, including epidermal keratinocytes. As a step toward understanding the molecular mechanisms underlying this differentiation-related change, we now report the partial purification and characterization of this enzyme activity from rat skin. Supernatant solutions from rat skin homogenates were subjected to a series of column chromatography steps including anion exchange, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The purification procedure resulted in cholesterol sulfotransferase activity purified 2,700-fold with a 11% recovery. The most purified preparation yielded a major Coomassie blue-stained band on denaturing polyacrylamide gel electrophoresis of an apparent molecular weight (MW) of 40,000 Da. Photoaffinity labeling with the donor substrate, 3'-phosphoadenosine-5'-phospho-[35S]-sulfate resulted in a single radiolabeled protein band on denaturing polyacrylamide gel electrophoresis, again of apparent MW 40,000 Da, strongly suggesting that the major Coomassie blue-stained band in the most purified preparation is the cholesterol sulfotransferase protein. Among 3beta-hydroxysteroids with a A5 double bond that were tested, each served as a substrate, while androgens, estrogens, corticosteroids, p-nitrophenol and DOPA did not serve as substrates. Apparent Michaelis constants for the 3beta-hydroxysteroid substrates ranged from 0.6 to 8 microM.     

Purification and activation of brain sulfotransferase.

Galactosylceramide sulfotransferase (EC 2.8.2.11) catalyzes the biosynthesis of sulfatide from galactocerebroside and adenosine 3'-phosphate 5'-phosphosulfate (PAPS). This enzyme is developmentally controlled, reaching a maximum activity in the brains of mice corresponding to that of maximum myelination. The product, sulfatide, is an important component of myelin. This transferase from mouse brain has been purified 2600-fold using a combination of pyridoxal 5'-phosphate- and ATP-ligated columns. The purified enzyme yielded a single band following SDS-polyacrylamide gel electrophoresis with an apparent M(r) of 31,000. The entire purification procedure can be completed in 1 day. The pH optimum for the enzyme is 7.0. The Km for PAPS is 1.2 x 10(-6) M, and the Km for cerebroside is 2.6 x 10(-5) M. Cerebroside concentrations > 80 pmol/ml are inhibitory. Enzyme preparations were associated with several lipids. Vitamin K+P(i) activated purified preparations of the sulfotransferase and maintained enzyme activity during storage at -80 degrees C.     

Purification and characterization of tyrosylprotein sulfotransferase.

Tyrosylprotein sulfotransferase (TPST) is a Golgi membrane enzyme involved in the post-translational modification of secretory and membrane proteins. Here we describe the 140,000-fold purification of this enzyme from bovine adrenal medulla to apparent homogeneity and determine its substrate specificity. The key step in the purification was affinity chromatography on a substrate peptide to which the enzyme bound in the presence of nucleotide cosubstrate. TPST is a 54-50 kd integral membrane glycoprotein. The presence of sialic acid strongly suggests that within the Golgi complex, TPST is localized in the trans-most subcompartment. TPST was found to specifically sulfate tyrosine residues adjacent to acidic amino acids. These results define a major determinant for the specificity of protein sulfation in the trans Golgi.     

Purification and kinetic characterization of a fructosyltransferase from Aspergillus aculeatus

A fructosyltransferase present in Pectinex Ultra SP-L, a commercial enzyme preparation from Aspergillus aculeatus, was purified to 107-fold and further characterised. The enzyme was a dimeric glycoprotein (20% (w/w) carbohydrate content) with a molecular mass of around 135 kDa for the dimer. Optimal activity/stability was found in the pH range 5.0-7.0 and at 60 degrees C. It was stable or slightly activated (upto 1.4-fold) in the presence of reducing agents, such as dithiothreitol and 2-mercaptoethanol, and detergents, such as sodium dodecylsulphate and Tween 80. The enzyme was able to transfer fructosyl groups from sucrose as donor producing the corresponding series of fructooligosaccharides: 1-kestose, nystose and fructosylnystose. Using sucrose as substrate, the k(cat) and K(m) values for transfructosylating activity were 1.62+/-0.09 x 10(4)s(-1) and 0.53+/-0.05 M, whereas for hydrolytic activity the corresponding values were 775+/-25s(-1) and 27+/-3 mM. At elevated sucrose concentrations, the fructosyltransferase from A. aculeatus showed a high transferase/hydrolase ratio that confers it a great potential for the industrial production of prebiotic fructooligosaccharides.     

Substrate specificity of phenol sulfotransferase from primary cultures of bovine brain microvessel endothelium

The substrate specificity of the thermostable phenol sulfotransferase (PST) from primary cultures of brain microvessel endothelial cell monolayers was characterized. Selected catecholamines, catecholamine metabolites, and p-nitrophenol at 5, 50, and 500 M were used as substrates in PST assays of cytosol extracts. Endogenous catecholamines, epinephrine, norepinephrine, and dopamine, exhibited no detectable activity as substrates (500 M) compared to 500 M p-nitrophenol as substrate (1.8 pmol/mg/min specific activity) for the PST. In contrast, 500 M of either deaminated or 3-O-methylated metabolites of catecholamines exhibited intermediate (1.0 pmol/mg/min specific activity) to low (0.2 pmol/mg/min specific activity) activity, respectively, as substrates compared to p-nitrophenol as substrate for the PST. Additionally, 500 M of metabolites of catecholamines that were both deaminated and 3-0-methylated exhibited high activity (>3.0 pmol/mg/min specific activity) as substrates compared top-nitrophenol as substrate for the PST. Qualitatively similar results were observed at lower substrate concentrations. Therefore, results from this study suggest a potential role for PST as part of the enzymatic blood-brain barrier in regulating transendothelial passage of endogenous catecholamines between the blood and the brain.     

Identification and characterization of tyrosylprotein sulfotransferase from human saliva

Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands previously (William et al. Arch Biochem Biophys 338: 90-96). Tyrosylprotein sulfotransferase catalyses the sulfation of a variety of secretory and membrane proteins and is believed to be present only in the cell. In the present study, this enzyme was identified for the first time in human saliva. Analysis of human saliva and parotid saliva for the presence of tyrosylprotein sulfotransferase revealed tyrosine sulfating activity displayed by both whole saliva and parotid saliva at pH optimum of 6.8. In contrast to tyrosylprotein sulfotransferase isolated from submandibular salivary glands, salivary enzyme does not require the presence of Triton X-100, NaF and 5'AMP for maximal activity. Similar to the submandibular TPST, the enzyme from saliva also required MnCl2 for its activity. Maximum TPST activity was observed at 20 mM MnCl2. The enzyme from saliva was immunoprecipitated and purified by immunoaffinity column using anti-TPST antibody. Affinity purified salivary TPST showed a single band of 50-54 kDa. This study is the first report characterizing a tyrosylprotein sulfotransferase in a secretory fluid.     

Purification and characterization of N-hydroxy-2-acetylaminofluorene sulfotransferase from rat liver.

N-Hydroxy-2-acetylaminofluorene (N-OH-2-AAF) sulfotransferase is an enzyme that catalyzes the sulfate transfer from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to N-OH-2-AAF to form a highly reactive product acetylaminofluorene N-sulfate. It has been purified about 2000-fold with a yield of over 12% from adult Sprague-Dawley male rat livers by an eight-step procedure. The final preparation was homogeneous on analytrical disc gel electrophoresis. The purified enzyme had activity toward p-nitrophenol with an approximately 1600-fold increase in specific activity over the crude homogenate, but it had almost no detectable activity toward steroids such as estrone, beta-estradiol, testosterone, dehydroisoandrosterone, and corticosterone. There was also very little sulfation activity toward serotonin and L-tyrosine methyl ester. The optimal pH for the enzyme activity is approximately 6.3 when measured in sodium phosphate buffer. Mg2+ at 6 to 9 mM could increase the enzyme activity up to 30%. Mn2+ activated the enzyme only slightly at very low concentrations. Zn2+, Co2+, Cu2+, and Ni2+ were all strongly inhibitory, but Ca2+ had very little effect. Thiol compounds were found to have a stabilizing effect and thiol-blocking reagents were potent inhibitors for this enzyme. The pure enzyme was very unstable especially in diluet solutions. The isoelectric point (pl) of the enzyme is 5.66 +/- 0.07. The molecular weight of the native enzyme was 68,000 +/- 500 as estimated by Sephadex G-100 and G-200 gel filtrations. A single component with molecular weight of 38,250 +/- 1,350 was observed on sodium dodecyl sulfate gel electrophoresis in the absence and presence of 2-mercaptoethanol. Comparison of the enzyme activity in mail and female rat livers at each stage of purification revealed that there was only a trace amount of N-OH-2-AAF sulfotransferase present in the female rat liver.     

Purification and characterization of hepatic and intestinal phenol sulfotransferase with high affinity for benzo[a]pyrene phenols from channel catfish, Ictalurus punctatus

Cytosol from channel catfish liver and intestinal mucosa has high sulfotransferase activity with low concentrations of 3-, 7-, or 9-hydroxybenzo[a]pyrene. To further investigate this conjugation pathway, sulfotransferase activity toward 9-hydroxybenzo[a]pyrene was isolated from catfish intestinal and hepatic cytosol by chromatography on anion exchange and PAP-agarose affinity columns. SDS-PAGE of the active fractions showed that one major band with molecular size of about 41,000 Da was isolated from intestine, while two bands of about 41,000 and 31,000 Da were obtained from liver. Antibodies against human phenol-sulfating sulfotransferase cross-reacted strongly with the 41,000-Da bands from liver and intestine, but weakly with the hepatic 31,000-Da protein. N-Terminal sequence information could not be obtained from the pure proteins. Following digestion, an internal sequence of 20 amino acid residues was obtained from the hepatic 41,000-Da protein, which matched a sequence found in several mammalian sulfotransferases. No fish sulfotransferase sequences were available for comparison. The identity of the hepatic 31,000-Da protein was not established. The purified 41,000-Da proteins had very high activities with 3-, 7-, or 9-hydroxybenzo[a]pyrene, with K(m) values in the 40-100 nM range and V(max) 125-300 nmol/min/mg of protein. Substrate inhibition was observed when the concentrations of hydroxylated benzo[a]pyrenes were above 0.5 microM. As well as benzo[a]pyrene phenols, the purified 41,000-Da sulfotransferases catalyzed sulfation of 2-naphthol, 4-nitrophenol, 4-methylumbelliferone, 7-(hydroxymethyl)-12-methylbenz[a]anthracene, dehydroepiandrosterone, estrone, and 17beta-estradiol. Phenolic compounds were the preferred substrates for the purified enzymes.     

Sulfation of hypertensive and hypotensive drugs by monkey brain phenol sulfotransferase

The substrate specificity and affinity of two forms of phenol sulfotransferase (PST) from Rhesus macaque brain cortex were studied. Catecholamines, their methylated metabolites (normetanephrine, metanephrine) and methylated precursor, -methylDOPA, were examined as substrates for both the cationic (PST I) and the anionic (PST II) forms of the enzyme. Sulfation of hypertensive drugs (phenylephrine, octopamine, metaraminol), hypotensive drugs (-methylDOPA, minoxidil), and related agents without a free hydroxy group on the benzene ring were also studied. Results indicated that both PST forms sulfated -methylDOPA and minoxidil, but only PST II transferred the sulfate group to catecholamines and most of the adrenergic agents examined.