The availability of inorganic sulphate in blood for sulphate conjugation of drugs in rat liver in vivo. (35S)Sulphate incorporation into harmol sulphate.

1. When Na235SO4 is injected intravenously in rats, it is immediately available for sulphate conjugation of the phenolic drug harmol (7-hydroxyl-1-methyl-9H-pyrido[3,4-b]indole) in the liver. This was established by following the time course of the biliary excretion of the sulphate conjugate of harmol, and the incorporation of [35S]sulphate into harmol sulphate. 2. During the 10min immediately after injection of Na235SO4 re-distribution of [35S]sulphate took place, which resulted in a rapid initial decrease in the plasma concentration of [35S]sulphate; a concomitant decrease in the amount of [35S]sulphate incorporated into harmol sulphate was observed, indicating that the co-substrate of sulphation, adenosine 3'-phosphate 5'-sulphatophosphate, equilibrates rapidly with [35S]sulphate in plasma. 3. The results suggest that the pool size of adenosine 3'-phosphate 5'-sulphatophosphate is very small; therefore the specific radioactivity of [35S]sulphate in plasma determines the specific radioactivity incorporated into sulphate esters at any time.     

The sulphation of chondroitin sulphate in embryonic chicken cartilage

1. Whole tissue preparations and subcellular fractions from embryonic chicken cartilage were used to measure the rate of incorporation of inorganic sulphate into chondroitin sulphate in vitro. 2. In cartilage from 14-day-old embryos, [(35)S]sulphate is incorporated to an equal extent into chondroitin 4-sulphate and chondroitin 6-sulphate at a rate of 1.5nmoles of sulphate/hr./mg. dry wt. of cartilage. 3. Microsomal and soluble enzyme preparations from embryonic cartilage catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate into both chondroitin 4-sulphate and chondroitin 6-sulphate. 4. The effects of pH, ionic strength, adenosine 3'-phosphate 5'-sulphatophosphate concentration and acceptor chondroitin sulphate concentration on the soluble sulphotransferase activity were examined. These factors all influence the activity of the sulphotransferase, and pH and incubation time also influence the percentage of chondroitin 4-sulphate formed.     

The formation of exchangeable sulphite from adenosine 3''-phosphate 5''-sulphatophosphate in yeast.

A new low-molecular-weight bound sulphite was found in yeast enzyme reaction systems which convert the sulphur of 35S-labelled adenosine 3'-phosphate 5'-sulphatophosphate into exchangeable radioactive sulphite. This bound sulphite was separated from other components by paper electrophoresis and Sephadex G-25 chromatography, and shown to be a peptide with multiple thiol groups and an estimated mol.wt. of 1400. The labelled sulphur in this peptide is highly exchangeable with unlabelled sulphite, but exchangeability decreases with time and freeze-drying. The low-molecular-weight acceptor is tightly bound to enzyme B of the yeast system and, apparently, accepts the sulpho group of adenosine 3'-phosphate 5'-sulphatophosphate and is released as bound sulphite only in the presence of enzymically or chemically reduced fraction C. It is proposed that the low-molecular-weight acceptor is a carrier peptide which, after release of the reduced sulphur, becomes re-oxidized and returns to enzyme B. Fraction C appears to function as an obligatory reductant of the oxidized acceptor before it can accept another-SO-3-moiety from adenosine 3'-phosphate 5'-sulphatophosphate. These findings are consistent with mechanisms proposed for sulphate reduction in spinach and Chlorella, and suggest that fraction C is the natural thiol required in these systems. An improved column technique for the preparation of adenosine 3'-phosphate 5'-sulphatophosphate is described.     

The biosynthesis of sulphogalactosyldiacylglycerol of rat brain in vitro

Triton X-100 extracts of rat brain microsomal fraction catalyse the formation of sulphogalactosyldiacylglycerol from galactosyldiacylglycerol and adenosine 3'-phosphate 5'-sulphatophosphate. Of the various subcellular fractions of brain assayed, the microsomal fraction contained most (79%) of the adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase activity. The enzyme activity was stimulated by Triton X-100 and showed linearity with increasing time, concentrations of enzyme and added substrates. ATP and KF prolonged the linearity of the activity with time, but ATP had an overall inhibitory effect on the sulphotransferase. Both ATP and KF inhibit the degradation of adenosine 3'-phosphate 5'-sulphatophosphate, which probably causes the increased linearity of the sulphotransferase reaction with time. The enzyme preparation did not catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to either cholesterol or galabiosyldiacylglycerol (galactosylgalactosyldiacylglycerol). Significant differences between the formation of sulphogalactosyldiacylglycerol and cerebroside sulphate catalysed by the same enzyme preparation were noted. ATP and Mg(2+) strongly inhibit the formation of sulphogalactosyldiacylglycerol but equally strongly stimulate the synthesis of cerebroside sulphate. The apparent K(m) for galactosyldiacylglycerol is 200mum, and that for cerebroside is 45mum. Galactosyldiacylglycerol and cerebroside are mutually inhibitory toward the synthesis of sulphated derivatives of each. These data do not necessarily lead to the conclusion that two sulphotransferases are present, but they do indicate a possible means of controlling the synthesis of these two sulpholipids.     

Purification from rat liver of an enzyme that catalyses the sulphurylation of phenols

1. An enzyme (EC 2.8.2.1) that catalyses the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to phenols was purified approx. 2000-fold from male rat livers. 2. The purified preparation did not catalyse the sulphurylation of dehydroepiandrosterone, butan-1-ol, l-tyrosine methyl ester, 1-naphthylamine or serotonin. 3. At pH8.0 and 37 degrees C the K(m) values of the enzyme for p-nitrophenol and adenosine 3'-phosphate 5'-sulphatophosphate are 51 and 14mum respectively. The K(m) value for either substrate is independent of the concentration of the other. 4. The sulphurylation of phenol is inhibited by thiol compounds and glutathione at a concentration of 3mm caused an approx. 50% decrease in enzyme activity. 5. The K(m) of the enzyme for adenosine 3'-phosphate 5'-sulphatophosphate is unaffected by the presence of added glutathione but at a concentration of 5mm-glutathione the K(m) of the enzyme for its phenolic substrate is decreased.     

The uptake and assimilation of sulphate by Thiobacillus ferrooxidans.

Sulphate was rapidly bound by cell suspensions of Thiobacillus ferrooxidans. The binding was depressed by tetrathionate but was unaffected by Group VI anions, cysteine or methionine. Increasing uptake of sulphate was observed in cell suspensions incubated in the presence of ferrous iron. The bulk of 35S-sulphate was removed from the organisms by washing with dilute sulphuric acid and the remaining label was incorporated into cold trichloroacetic acid-soluble compounds. 35S-labelled adenosine 5'-sulphatophosphate was produced from ATP and 35S-sulphate by cell suspensions and in cell-free extracts. There was no evidence for the production of adenosine 3'-phosphate 5'-sulphatophosphate assayed by a very sensitive bioluminescence method.     

Assay of adenosine 3''-phosphate 5''-sulphatophosphate in hepatic tissues.

A fluorimetric assay, based on the ability of boiled hepatic extracts to support the sulphO-conjugation of harmol, was used to demonstrate and quantify PAdoPS (adenosine 3'-phosphate 5'-sulphatophosphate) present in liver. A stoicheiometric relationship was established between the sulphate conjugate formed and the 'active sulphate' utilized. Guinea-pig, rat, mouse and rabbit livers contain 3.3, 2.9, 0.8 and 0.5 mumol of PAdoPS/100 G wet wt. respectively.     

Kinetics and mechanism of the rat brain phenol sulphotransferase reaction.

Some properties of rat brain phenol sulphotransferase were investigated in in vitro at pH7.4. The enzyme was purified 10-fold by chromatography on DEAE-Sephadex -50. It can be assayed with 4-hydroxy-3-methoxyphenylethylene glycol or 4-methylumbelliferone as the sulphate acceptor. The partially purified enzyme is stable for at least 1 week when stored at 4 degrees C. It is, however, additionally activated (10--20%) and stabilized by 1 mM-dithiothreitol. The activity of the enzyme does not depend on the addition of exogenous Mg2+. The pH optima for the sulphation of 4-hydroxy-3-methoxyphenylethylene glycol and 4-methylumbelliferone are 7.8 and 7.4 respectively. Substrate inhibition by the sulphate acceptor is apparent at concentrations over 0.05mM. Initial-velocity studies in the absence and presence of product and dead-end inhibitors suggested that the mechanism of the rat brain sulphotransferase reaction is sequential ordered Bi Bi with a dead-end complex of enzyme with adenosine 3',5'-biphosphate and sulphate acceptor. The sulphate donor adenosine 3'-phosphate 5'-sulphatophosphate is the first substrate that adds to the enzyme, and the sulphate acceptor is the second substrate. The dissociation constant for the complex of enzyme with sulphate donor is 21 micron. The sulphated substrate is the first product and adenosine 3',5'-biphosphate is the second product that leaves the enzyme.     

The control of sulphate reduction in Escherichia coli by O-acetyl-l-serine

1. Extracts of Escherichia coli A.T.C.C. 9723 and K(12)703 contain serine transacetylase and O-acetylserine sulphhydrase. Synthesis of the latter enzyme is repressed by growth on l-cyst(e)ine and other sulphur compounds. 2. O-Acetyl-l-serine added to cells growing on glutathione or sulphate as source of sulphur induces the enzymes that catalyse (a) the activation of sulphate to adenosine 3'-phosphate 5'-sulphatophosphate (EC 2.7.7.4 and 2.7.1.25), (b) the reduction of adenosine 3'-phosphate 5'-sulphatophosphate to sulphite and (c) the reduction of sulphite to sulphide (EC 1.8.1.2). Hydrogen sulphide is liberated from cultures growing on sulphate as source of sulphur and in the presence of O-acetylserine. 3. The cysE mutants of E. coli K(12) lack serine transacetylase. Addition of O-acetylserine permits growth on sulphate as source of sulphur; at the same time the enzymes of sulphate reduction, previously absent, are synthesized. Such mutants have no detectable intracellular cyst(e)ine when starved of sulphur. 4. These results suggest that O-acetylserine is necessary for synthesizing the enzymes of sulphate reduction in E. coli. Its action does not appear to be by interference with the repressive control exerted over these enzymes by cyst(e)ine.     

Biosynthesis of heparin. Solubilization and partial characterization of N- and O-sulphotransferases.

Assay methods were developed enabling separate determination of N- and O-sulphotransferase activities in an enzyme preparation from mouse mastocytoma. N-Desulphoheparin and chemically N-acetylated heparan sulphate were used as specific exogenous sulphate acceptors in the transfer of [35S]sulphate residues from adenosine 3'-phosphate 5'-[35S]sulphatophosphate to amino and hydroxyl groups respectively. The resulting 35S-labelled polysaccharides were isolated as their cetylpyridinium complexes on filter paper. Sulphotransferases were solubilized from a mastocytoma microsomal fraction by treatment with detergent-alkali. The pH optimum for both enzymes was about 7.5 Km with regard to adenosine 3'-phosphate 5'-sulphatophosphate was estimated to be 2 X 10(-5) M for the N-sulphotransferase and 1 X 10(-4) M for the O-sulphotransferase(s). The enzymes required bivalent cations for maximum activity, Mn2+ stimulating both the N- and O-sulphotransferase four- to five-fold, whereas Ca2+ increased the N- but not the O-sulphotransferase activity. The O-sulphotransferase was found to be more sensitive to heat-inactivation, 60% of the activity being lost after 1 min at 50 degrees C, whereas only 15% of the N-sulphotransferase activity was lost. In contrast, the N-sulphotransferase was selectively inhibited (or inactivated) by NaCl; at 0.125 M-NaCl concentration the O-sulphotransferase activity was essentially unaffected, whereas the N-sulphotransferase activity was depressed by 80%. These results strongly indicate that N- and O-sulphate-transfer reactions should be ascribed to different enzymes, or, alternatively, to separate and independent active sites on the same enzyme molecule.     

The distribution of l-asparagine synthetase in the principal organs of several mammalian and avian species

1. Sulphate-dependent PP(i)-ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP(2-) and MgP(2)O(7) (2-); ATP sulphurylase activity was not correlated with the concentration of free Mg(2+). 2. Sulphate-dependent PP(i)-ATP exchange was independent of PP(i) concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PP(i)-ATP exchange was quantitatively defined by the rate equation applicable to the initial rate of a bireactant sequential mechanism under steady-state conditions. 3. Chlorate, nitrate and ADP inhibited the exchange reaction. The inhibition by chlorate and nitrate was uncompetitive with respect to ATP and competitive with respect to sulphate. The inhibition by ADP was competitive with respect to ATP and non-competitive with respect to sulphate. 4. ATP sulphurylase catalysed the synthesis of [(32)P]ATP from [(32)P]PP(i) and adenosine 5'-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PP(i) and adenosine 5'-sulphatophosphate. 5. The synthesis of ATP from PP(i) and adenosine 5'-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PP(i) and adenosine 5'-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5'-sulphatophosphate and non-competitive with respect to PP(i). It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP(2-) was the first product to react with the enzyme and MgP(2)O(7) (2-) was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5'-sulphatophosphate could not be demonstrated.     

Studies on the Arylsulphatase and phenol sulphotransferase activities of Aspergillus oryzae.

1. Crude extracts of Aspergillus oryzae grown under conditions of sulphur limitation possess high arylsulphatase activity. 2. This activity can be greatly enhanced by the inclusion of tyramine or a number of other phenols in the assay medium. 3. The arylsulphatase activity of these extracts can be resolved into three distinct fractions by chromatography on DEAE-cellulose. 4. The effect of tyramine is restricted to one of these fractions only. 5. Evidence is presented which indicates that this effect is the consequence of a phenol sulphotransferase activity, which shows no requirement for 3'-phosphoadenosine 5'-phosphate as a cofactor, and which will not transfer sulphate from 3'-phosphoadenosine 5'-sulphatophosphate to potential phenolic acceptors. 6. The three enzymes differ also in their molecular weights and substrate specificities.     

Measurement and kinetic study of the formation of adrenaline sulphate in vitro.

The sulpho-conjugation of [14C]adrenaline form inorganic sulphate and ATP or preformed adenosine 3'-phosphate 5'-sulphatophosphate was demonstrated in the high-speed supernatant prepared from the liver and small intestine of various animals. Hydrolysis with sulphatase indicated the sulphate nature of the conjugate. The overall sulphation reaction has a pH optimum of 9.0. Maximal activity was obtained with a ratio of ATP/Mg2+ of 1 at 4--6mM. Above their optimal concentrations, ATP and Mg2+, separately or in combination, were inhibitory. Dithiothreitol at 3 mM stimulated the reaction by about 30%. The Km for adrenaline, determined by the sulphotransferase reaction and by the three-step (sulphate-activating and sulphotransferase) reactions was 125 micrometer. The rate of synthesis of [14C]-adrenaline sulphate, expressed in pmol/min per mg of protein for the livers of dog, monkey, rat, guinea pig and rabbit were, respectively, 144, 77, 47, 11 and 6. The corresponding values for the small intestines of dog and monkey were 60 and 62. Brain and heart tissues showed no measurable activity.     

The Sulphation of p-hydroxyphenylpyruvic acid and related compounds by the rat liver cytosol.

Cytosol preparations of rat liver and kidney were examined for their ability to transfer sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to p-hydroxyphenylpyruvic acid. Little activity towards this substrate was observed, and the main product detected in the reaction mixtures was identified as p-hydroxybenzyl alcohol O-sulphate. This was not formed from p-hydroxybenzaldehyde, a spontaneous oxidation product of p-hydroxyphenylpyruvic acid, by sulphation followed by a rapid enzyme-catalysed reduction of the intermediate phydroxybenzaldehyde O-sulphate. This product was formed mainly by this sequence of reactions, but the reverse, reduction followed by sulphation, also appeared possible. p-Hydroxybenzyl alcohol itself was very readily sulphated by both preparations, and the liver also produced a sulpho-conjugate of homogentisic acid. These observations are important in calculating the turnover of L-tyrosine O-sulphate in the mammalian system, and establish that p-hydroxyphenylpyruvic acid O-sulphate is an end product of its metabolism, rather than an intermediate in its synthesis by reversed transamination.     

Purification and steady-state kinetics of adenosine 5''-pyrophosphate sulphurylase from baker''s yeast.

ADP sulphurylase (EC 2.7.7.5) was purified by chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was assayed by measuring the incorporation of [32P]Pi into ADP in the presence of the substrate for the reverse reaction, adenosine 5'-sulphatophosphate. In the concentration ranges investigated, by using initial-velocity, product-inhibition and isotope-exchange studies, the data were consistent with a Ping Pong reaction mechanism, with Km for adenosine 5'-sulphatophosphate of 1.20 +/- 0.08 mM and a Km for Pi of 4.95 +/- 0.15 mM. Competitive substrate inhibition by Pi (Ki = 11.7 +/- 0.3 mM) was found. ADP sulphurylase catalyses a sulphate-independent Pi-ADP exchange reaction, the kinetics of which are consistent with the kinetics of the overall reaction, inconsistent with the assay of Burnell & Anderson [(1973) Biochem. J. 133, 417-428], which is based on a sulphate-dependent Pi-ADP exchange reaction.     

Purification and properties of arylsulphatase A from chicken brain

1. Chicken brain arylsulphatase A was purified 2000-fold, with overall recovery 14%, by using ammonium sulphate fractionation, ethanol precipitation, Sephadex G-200 gel filtration and DEAE-Sephadex column chromatography. 2. The purified preparation was free from beta-glucuronidase, beta-galactosidase, acid phosphatase, inorganic pyrophosphatase and adenosine 3'-phosphate 5'-sulphatophosphate sulphohydrolase activities. 3. Polyacrylamide-gel electrophoresis indicated that the purified preparation was not homogeneous. 4. Chicken brain arylsulphatase was markedly inhibited by carbonyl reagents in the presence of traces of Cu(2+) in the system. Other metal ions such as Fe(2+) and Zn(2+), were inactive. 5. Ascorbic acid alone had no effect on enzyme activity but enhances the inhibition by Cu(2+). 6. Chicken brain arylsulphatase A resembled arylsulphatase A of other animal species in its kinetic properties such as K(m) value, anomalous time-activity relationship and the inhibitory effect of phosphate, sulphite and sulphate ions. However, its electrophoretic mobility, behaviour under zinc acetate fractionation and stimulation by Ag(+) were similar to arylsulphatase B of other animal species. Thus, this enzyme did not correspond to either arylsulphatase A or arylsulphatase B but properties of both. 7. The purified enzyme preparation can degrade cerebroside 3-sulphate.     

Localization of a sulphate-activating system within Euglena mitochondria.

Intact mitochondria, obtained from Euglena gracilis Klebs var. bacillaris Cori mutant W10BSmL, which lacks plastids, and purified on Percoll density gradients, form adenosine 3'-phosphate 5'-phosphosulphate from sulphate. The optimal conditions include addition of 17 mM-Tricine/KOH, pH 7.6, 18 mM-MgCl2, 250 mM-sucrose, 5.66 mM-sodium ADP (or 0.94 mM-sodium ATP), 1 mM-K2SO4, carrier-free 35SO4(2-) (32.1 microCi) and 1.0 mg of mitochondrial protein in a total volume of 2.65 ml and incubation at 30 degrees C. Experiments with the inhibitor of adenylate kinase P1, P5-di(adenosine 5'-)pentaphosphate indicate that ATP is the preferred substrate for sulphate activation; ADP is utilized by conversion into ATP via adenylate kinase. ATP sulphurylase, adenylylsulphate kinase (APS kinase) and inorganic pyrophosphatase constitute the sulphate-activating system; ADP sulphurylase is undetectable. Fractionation of Euglena mitochondria with digitonin and centrifugation allowed the separation of outer-membrane vesicles and mitoplasts as judged by electron microscopy and selected enzymic markers. The detergent-labile association of the sulphate-activating system with the mitoplasts (similar to that of adenylate kinase), the fact that most of the adenosine 3'-phosphate 5'-phosphosulphate formed by intact mitochondria is found in the surrounding medium, and the ease with which nucleotide substrates reach the activating system in intact organelles, suggest that the enzymes of sulphate activation are located on the outer surface of the mitochondrial inner membrane.     

Initiation of chondroitin sulphate synthesis by beta-D-galactosides. Substrates for galactosyltransferase II.

beta-Galactosides were found to initiate chondroitin sulphate chain synthesis in chick-embryo cartilage in vitro and thereby relieve inhibition by cycloheximide of [3H]-acetate incorporation into chondroitin sulphate. beta-Galactosides with an apolar aglycan group such as phenyl O-beta-galactoside were active, whereas those with a charged or polar aglycan group such as pyridine 3-O-beta-galactoside or those with sulphur instead of oxygen in the glycosidic linkage (phenyl beta-thiogalactoside) were not. beta-Galactosides also serve as substrates for microsomal galactosyltransferase activity from chick-embryo cartilage. Phenyl O-beta-galactoside and pyridine 3-O-beta-galactoside were effective substrates for this enzyme, but phenyl S-beta-thiogalactoside and pyridine 2-S-beta-thiogalactoside were only slightly active. This galactosyltransferase was shown to be a separate enzyme from galactosyltransferase I, which catalyses transfer of galactose from UDP-galactose to beta-xylosides. It is proposed that the enzyme catalysing this reaction is galactosyltransferase II, responsible for transfer of the second galactose residue of the chondroitin sulphate linkage oligosaccharide. No transfer of glucuronic acid from UDP-glucuronic acid to beta-galactosides, catalysed by the microsomal preparation could be detected.     

Adenosine 5′-pyrophosphate sulphurylase in baker''s yeast

ADP sulphurylase from baker's yeast was purified and its properties were studied. The enzyme is very heat-labile and its activity shows linear kinetics over narrow ranges of time and protein concentration. It is not activated by metals and is inhibited by thiol-reactive compounds. The enzyme, which replaces inorganic sulphate in adenosine 5'-sulphatophosphate with P(i) to yield ADP, also catalyses an exchange of P(i) into ADP. Kinetic studies show that the enzyme has a high affinity for adenosine 5'-sulphatophosphate, although concentrations in excess of 1.0mm are inhibitory. However, the kinetics for P(i) are more complex and the enzyme is not inhibited by P(i) up to 20.0mm.     

The effect of substrate concentration and pH on the enzymic sulphation of l-tyrosyl derivatives

1. The kinetics of the enzymic transfer of sulphate from adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate to derivatives of l-tyrosine were investigated with a partially purified enzyme preparation from rat liver. 2. At pH7.5 and 37 degrees C the K(m) values for l-tyrosine methyl ester and adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate are 0.3mm and 8nm respectively. The K(m) value for either substrate is independent of the concentration of the other. The available data are consistent with the sulphation reaction proceeding according to a rapid-equilibrium random Bi Bi mechanism. 3. From the effect of pH on the K(m) and V(max.) values for l-tyrosine methyl ester, tyramine and N-acetyl-l-tyrosine ethyl ester it is concluded that the enzyme is specific for substrate molecules with a free and unprotonated amino group and an un-ionized hydroxyl group. 4. The only ionizing group that can be positively attributed to the enzyme appears to influence the binding of adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate and has an apparent pK value of approx. 9.5. It is suggested that this group may be an essential thiol. 5. The enzyme is inhibited by iodoacetamide at pH7.5 and 30 degrees C and this inhibition is prevented by the presence of adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate but not by l-tyrosine methyl ester.