Regulation of Assimilatory Sulfate Reduction by Cadmium in Zea mays L

Plants cultivated with Cd can produce large amounts of phytochelatins. Since these compounds contain much cysteine, these plants should have an increased rate of assimilatory sulfate reduction, the biosynthetic pathway leading to cysteine. To test this prediction, the effect of Cd on growth, sulfate assimilation in vivo and extractable activity of two enzymes of sulfate reduction, ATP-sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase were measured in maize (Zea mays L.) seedlings. For comparison, nitrate reductase activity was determined. In 9-day-old cultures, the increase in fresh and dry weight was significantly inhibited by 50 micromolar and more Cd in the roots and by 100 and 200 micromolar in the shoots. Seedlings cultivated with 50 micromolar Cd for 5 days incorporated more label from ³⁵SO₄²⁻ into higher molecular weight compounds than did controls, indicating that the predicted increase in the rate of assimilatory sulfate reduction took place. Consistent with this finding, an increased level of the extractable activity of both ATP-sulfurylase and adenosine 5′-phosphosulfate sulfotransferase was measured in the roots of these plants at 50 micromolar Cd and at higher concentrations. This effect was reversible after removal of Cd from the nutrient solution. In the leaves, a significant positive effect of Cd was detected at 5 micromolar for ATP-sulfurylase and at 5 and 20 micromolar for adenosine 5′-phosphosulfate sulfotransferase. At higher Cd concentrations, both enzyme activities were at levels below the control. Nitrate reductase (EC 1.6.6.1) activity decreased at 50 micromolar or more Cd in the roots and was similarly affected as ATP-sulfurylase activity in the primary leaves.     

Does PAPS generation determine the overall sulfate conjugation in human platelets?

The formation of 3'-phospho-adenosine-5'phosphosulfate (PAPS) depends on essentially two enzymic reactions catalysed by ATP-sulfurylase and APS-kinase (adenosine 5'-phosphosulfate-kinase). In this paper, PAPS generation by human platelets was determined by the transfer of 35sulfate from PAP35S formed in vitro to N-acetyldopamine (NADA), using the phenolsulfotransferase extracted from rat liver. A pre-requisite of this quantitative procedure was the prior inhibition of the sulfate-activating system in the latter enzyme preparation. This was accomplished by the addition of 10 mM EDTA and 14 mM pyrophosphate. The PAPS-generating system of human platelets exhibited two pH peaks with higher activity at pH 8 than pH 6. Optimal concentrations of ATP and Mg++ at 7 mM were required for the two reactions. PAPS generation so measured showed a highly significant correlation with the overall sulfate conjugation of NADA: a correlation coefficient of 0.96 was established from data obtained from 60 platelet preparations of normal subjects.     

Rapid and simple measurement of ATP-sulfurylase activity in crude plant extracts using an ATP meter for bioluminescence determination

ATP-sulfurylase (EC 2.7.7.4.) catalyzes the first step in assimilatory sulfate reduction, forming adenosine 5′-phosphosulfate (APS) and pyrophosphate from ATP and SO₄^2?. The extractable activity of ATP-sulfurylase was determined in crude extracts from Phaseolus vulgaris by measuring the formation of ATP, produced in the reverse reaction from APS and pyrophosphate, using purified luciferase and luciferin in an ATP meter. One determination can be performed per minute. The rates of ATP-sulfurylase activity determined by this method were about 25 times higher than the ones measured in the forward reaction as AP³⁵S formed from ATP and ³⁵SO₄^2?.     

Sulfate transport-deficient mutants of Chinese hamster ovary cells. Sulfation of glycosaminoglycans dependent on cysteine

We isolated 59 Chinese hamster ovary cell mutants defective in 35SO4 incorporation into glycosaminoglycans. Thirty-five mutants incorporated [6-3H]glucosamine into glycosaminoglycans normally, suggesting that they were specifically impaired in sulfate incorporation. Cell hybridization studies revealed that the 35 mutants defined a unique complementation group. Pulse-labeling one of the mutants with 35SO4 showed that it possessed a defect in a saturable, 4-acetamido-4-isothiocyanostilbene-2,2'-disulfonic acid-sensitive transport system required for sulfate uptake. Despite the dramatic reduction in 35SO4 incorporation, the mutant synthesized sulfated heparan and chondroitin chains. Incubation of the mutant with [35S]cysteine resulted in the formation of 35SO4, which was subsequently incorporated into the glycosaminoglycans. Similar results were obtained when wild-type cells were incubated in sulfate-free growth medium containing [35S]cysteine, and isotope dilution analysis indicated that about 15 microM of sulfate was derived from cysteine catabolism. We also found that the sulfate transport deficiency rendered the mutant resistant to 5 microM sodium chromate, whereas wild-type cells did not divide under these conditions. However, the mutant also did not proliferate in medium containing 5 microM chromate when grown in the presence of wild-type cells, suggesting that chromate was transported through cell-cell contacts. Since co-cultivating sulfate transport-deficient mutants with mutants defective in xylosyltransferase or galactosyltransferase I partially restored 35SO4 incorporation into glycosaminoglycans, intercellular sulfate transport occurred as well. Therefore, the availability of sulfate for glycosaminoglycan synthesis depends on sulfate uptake, turnover of sulfur-containing amino acids, and sulfate transport between cells.     

Mutants of Salmonella typhimurium responding to cysteine or methionine: their nature and possible role in the regulation of cysteine biosynthesis.

Nineteen mutants of Salmonella typhimurium responding to either cysteine or methionine (cym) have been identified amongst cysteine (cys) and methionine (met) auxotrophs. Their growth responses to known intermediates in the related pathways of cysteine and methionine biosynthesis and complementation patterns in abortive transduction tests divided the mutants into six groups. Results of conjugation, cotransduction and deletion mapping experiments substantiated these groups, each of which carried a lesion within known cys genes. Enzyme assays on cym mutants from five of the six groups confirmed their cys gene deficiencies. Growth response and enzyme assay data were not consistent with mutants being leaky cys mutants (spared by methionine). None of eight cym mutants tested were able to convert [35S]methionine into [35S]cysteine. Selenate specifically inhibits the early enzymes of cysteine synthesis. In cym mutants this inhibition was relieved by cysteine but not by methionine, indicating that cym mutants require active cys enzymes for growth on methionine. There was evidence that methionine stimulated in vivo activity of cys enzymes in a cym mutant. Resistance to inhibition by 1,2,4-triazole results in reduced levels of the O-acetyl serine sulphydrylase. In cym mutants triazole resistance gave unstable suppression of the cym phenotype. Cym mutants may result from mutation in regulatory regions common to each of the cys genes, with the precise role of methionine as yet unknown.     

Properties of the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) synthesizing systems of brain and liver

Chromatography of brain and liver 100,000g supernatants over HPLC molecular sieve columns revealed striking differences in the molecular weight distribution of ATP-sulfurylase and APS-kinase of the two tissues, pointing to different enzymic species for both enzymes in brain and liver. This was further substantiated by kinetic characterization of the two enzymes of both tissues. APS-kinase of liver is allosterically activated by ATP, while the brain enzyme is not. ATP-sulfurylase of brain is activated at high, but still physiological concentrations of ATP. Brain ATP-sulfurylase is inhibited by phenylalanine.     

Regulation of Assimilatory Sulfate Reduction by Herbicide Safeners in Zea mays L

Effects of the herbicide safeners N,N-diallyl-2,2-dichloroacetamide and 4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzooxazin (CGA 154281) on the contents in cysteine and glutathione, on the assimilation of ³⁵SO₄²⁻, and on the enzymes of assimilatory sulfate reduction were analyzed in roots and primary leaves of maize (Zea mays) seedlings. Both safeners induced an increase in cysteine and glutathione. In labeling experiments using ³⁵SO₄²⁻, roots of plants cultivated in the presence of safeners contained an increased level of radioactivity in glutathione and cysteine as compared with controls. A significant increase in uptake of sulfate was only detected in the presence of CGA 154281. One millimolar N,N-diallyl-2,2-dichloroacetamide applied to the roots for 6 days increased the activity of adenosine 5′-phosphosulfate sulfotransferase about 20- and threefold in the roots and leaves, respectively, compared with controls. CGA 154281 at 10 micromolar caused a sevenfold increase of this enzyme activity in the roots, but did not affect it significantly in the leaves. A significant increase in ATP-sulfurylase (EC 2.7.7.4) activity was only detected in the roots cultivated in the presence of 10 micromolar CGA 154281. Both safeners had no effect on the activity of sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8). The herbicide metolachlor alone or combined with the safeners induced levels of adenosine 5′-phosphosulfate sulfotransferase, which were higher than those of the appropriate controls. Taken together these results show that the herbicide safeners increased both the level of adenosine 5′-phosphosulfate sulfotransferase activity and of the thiols cysteine and glutathione. This indicates that these safeners may be involved in eliminating the previously proposed regulatory mechanism, in which increased concentrations of thiols regulate assimilatory sulfate reduction by decreasing the activities of the enzymes involved.     

Properties and Localization of the Sulfate-Activating System in Rat Brain

The formation of the sulfate donor [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) from inorganic [35S]sulfate was studied using a novel assay. The assay was based on the quantitative transfer of radioactivity from [35S]PAPS to beta-naphthol under the action of phenolsulfotransferase activity from rat brain cytosol, with the [35S]beta-naphthyl sulfate formed being isolated by polystyrene bead chromatography. This simple assay was validated by comparison of results with those derived from direct assay of [35S]PAPS isolated by either TLC or ion exchange chromatography. [35S]PAPS formation by a high-speed supernatant of rat cerebral cortex occurred with an optimal pH of approximately 7.6, varied linearly with time and protein concentration, and depended on the presence of Mg2+-ATP. The latter could not be replaced by other nucleotides such as GTP, UTP, or CTP, which at 1-5 mM concentrations inhibited the reaction. Mg2+ could not be replaced by Mn2+, which at micromolar concentrations inhibited the reaction. The apparent Km values of Mg2+-ATP (at 0.1 mM [35S]sulfate) and inorganic sulfate (at 5 mM Mg2+-ATP) were 2.7 and 0.2 mM, respectively. These kinetics parameters corresponded to those reported for purified ATP sulfurylase (EC 2.7.7.4), the enzyme responsible for the first step of PAPS synthesis in liver. The product of its reaction, [35S]adenosine 5'-phosphosulfate (APS), could not be detected after incubations, an observation implying that the action of APS kinase was not rate limiting in cerebral extracts tested under the selected experimental conditions. [35S]PAPS formation was detectable in cytosolic fractions from various brain regions, which displayed only limited differences in synthesizing activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrostatic pressure alters the time course of GTP

The effects of hydrostatic pressure on the receptor-stimulated exchange of guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on the a subunit of G proteins were studied in two congeneric marine teleost fishes that differ in their depths of distribution. The poorly hydrolyzable GTP analog [35S]guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) was used to monitor the modulation of signal transduction by the A1 adenosine receptor agonist N6-R-(phenylisopropyl)adenosine (R-PIA) in brain membranes of the scorpaenids Sebastolobus alascanus and S. altivelis. The maximal binding (Bmax) and dissociation constant (K(d)) values, determined from equilibrium binding isotherms at atmospheric pressure (5 degrees C), were similar in the two species. The Bmax values for these species are much lower than literature values for mammalian brain tissue (25 degrees C); however, the K(d) values of the teleost and mammalian G proteins are similar. The EC50 values for the A1 adenosine receptor agonist R-PIA were similar in the two species. Hydrostatic pressure of 204 atm altered the binding of [35S]GTP[S]; basal [35S]GTP[S] binding decreased 25%. The A1 adenosine receptor agonist R-PIA and the muscarinic cholinergic receptor agonist carbamyl choline stimulated [35S]GTP[S] binding at 1 and 204 atm. At atmospheric pressure the half-time (t1/2) of [35S]GTP[S] binding differed between the two species. The GTP[S] on rate (k(on)) is larger in the shallower-living S. alascanus. Increased hydrostatic pressure altered the time course, decreasing the t1/2 in both species. The pressures that elicit this change in the time course differ between the species. However, interpolating over the range of in situ pressures the species experience, the values are similar in the two species. The guanyl nucleotide binding properties of the G protein a subunits appear to be conserved at the environmental temperatures and pressures the species experience.     

Synthesis and properties of a nonhydrolyzable adenosine phosphosulfate analog

Initial activation of inorganic sulfate for subsequent synthesis of sulfated biomolecules requires the action of ATP-sulfurylase to generate adenosine 5'-phosphosulfate (APS). This activated sulfate intermediate is both chemically labile and susceptible to enzymatic degradation. Consequently, it has not proven useful as a ligand for either purification or characterization of the various APS-utilizing enzymes. For these purposes, a stable analog of APS was required. This paper describes the simple and efficient synthesis and structural confirmation of a nonhydrolyzable APS analog, beta-methylene APS, with an overall molar yield of 40-50%. The method involves nucleophilic substitution of the chlorine moiety of a 5'-chloromethylphosphonate ester of 2',3'-O-isopropylidene adenosine by a sulfite ion. We also report the initial utilization of this compound as an inhibitor in kinetic trials of both ATP-sulfurylase and APS kinase and as an affinity ligand for the purification of these two APS-utilizing enzymes from cartilaginous tissue.     

Tyramine-O-sulfate, in addition to tyrosine-O-sulfate, is produced and secreted by HepG2 human hepatoma cells, but not by 3Y1 rat embryo fibroblasts

The spent media of HepG2 human hepatoma cells and 3Y1 rat embryo fibroblasts labeled with [35S]sulfate, upon ultrafiltration, were analyzed by a two-dimensional thin-layer separation procedure. Autoradiographs of the cellulose thin-layer plate revealed the presence of tyramine-O-[35S]sulfate in addition to tyrosine-O-[35S]sulfate in spent medium from human hepatoma cells. In contrast, only tyrosine-O-[35S]sulfate was observed in spent medium of 3Y1 rat fibroblasts. Using adenosine, 3'-phosphate, 5'-phospho[35S]sulfate as the sulfate donor, sulfotransferase(s) present in HepG2 cell homogenate catalyzed the sulfation of tyramine to tyramine-O-[35S]sulfate, but not the sulfation of tyrosine to tyrosine-O-[35S]sulfate. Endogenous aromatic amino acid decarboxylase present in HepG2 homogenate was shown to catalyze the decarboxylation of [3H]tyrosine to form [3H]tyramine while attempts to use it for the decarboxylation of tyrosine-O-sulfate to form tyramine-O-sulfate were unsuccessful. These results suggest that tyramine-O-sulfate may be derived from the de novo sulfation of tyramine, instead of the decarboxylation of tyrosine-O-sulfate.     

Localization of enzymes of assimilatory sulfate reduction in pea roots

The localization of enzymes of assimilatory sulfate reduction was examined in roots of 5-d-old pea (Pisum sativum L.) seedlings. During an 8-h period, roots of intact plants incorporated more label from ³⁵SO ₄ ^2- in the nutrient solution into the amino-acid and protein fractions than shoots. Excised roots and roots of intact plants assimilated comparable amounts of radioactivity from ³⁵SO ₄ ^2- into the amino-acid and protein fractions during a 1-h period, demonstrating that roots of pea seedlings at this stage of development were not completely dependent on the shoots for reduced sulfur compounds. Indeed, these roots contained activities of ATP-sulfurylase (EC 2.7.7.4), adenosine 5-phosphosulfate sulfotransferase, sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) at levels of 50, 30, 120 and 100%, respectively, of that in shoots. Most of the extractable activity of adenosine 5-phosphosulfate sulfotransferase was detected in the first centimeter of the root tip. Using sucrose density gradients for organelle separation from this part of the root showed that almost 40% of the activity of ATP-sulfurylase, adenosine 5-phosphosulfate sulfotransferase and sulfite reductase banded with the marker enzyme for proplastids, whereas only approximately 7% of O-acetyl-l-serine sulfhydrylase activity was detected in these fractions. Because their distributions on the gradients were very similar to that of nitrite reductase, a proplastid enzyme, it is concluded that ATP-sulfurylase, adenosine 5-phosphosulfate sulfotransferase and sulfite reductase are also exclusively or almost exclusively localized in the proplastids of pea roots. O-Acetyl-l-serine sulfhydrylase is predominantly present in the cytoplasm.Abbreviation APSSTase adenosine 5-phosphosulfate sulfotransferase     

The transsulfuration pathway in Tetrahymena pyriformis.

Four enzymes necessary for the metabolism of methionine by the trans-sulfuration pathway, methionine adenosyltransferase (EC 2.5.1.6), adenosylhomocysteinase (EC 3.3.1.1), cystathionine beta-synthase (EC 4.2.1.22) and cystathionine gamma-lyase (EC 4.4.1.1) were identified in Tetrahymean pyriformis. The ability of these cells to transfer 35S from E135S]methionine to form [35S] cysteine was also observed and taken as direct evidence for the functional existence of this pathway in Tetrahymena. An intermediate in the pathway and an active methyl donor, S-adenosylmethionine, was qualitatively identified in Tetrahymena and its concentration was found to be greater in late stationary phase cells than in early stationary phase cells.     

Enzymic comparisons of the inorganic sulfur metabolism in autotrophic and heterotrophic Thiobacillus ferrooxidans.

Activities of enzymes which mediate the oxidation of thiosulfate to sulfate and the assimilation of sulfate to sulfide were assayed in various cell-free fractions of Thiobacillus ferrooxidans grown autotrophically on either ferrous iron or thiosulfate or heterotrophically on glucose. There was no activity of the thiosulfate-oxidizing enzyme in extracts of bacteria grown with ferrous iron. Comparable activities for ATP-sulfurylase (EC 2.7.7.4), ADP-sulfurylase (EC 2.7.7.5), and adenylate kinase (EC 2.7.4.3) were found in the bacteria grown autotrophically with either Fe2+ or S2O32- or heterotrophically with glucose.     

Cysteine and methionine-precursors of methyl sulphone metabolites of 2,4',5-trichlorobiphenyl in mice

In order to account for the origin of the sulphur atom in methyl sulphones derived from polychlorinated biphenyls (PCB), groups of mice were treated with 2,4'-5-trichlorobiphenyl and [35S]cysteine or [35S]methionine, respectively. Control animals received the labelled amino acids only. The radioactive substances extracted from lung tissue were characterized by partition between hexane and sulphuric acid, thin-layer radiochromatography (TLRC), gas chromatography (GC) and mass fragmentography (MF). In lung extracts of both experimental groups sulphuric acid soluble metabolites were present: Their Rf-values on TLRC were identifical with that of 4-methylsulphonyl-2,4'-5-trichlorobiphenyl and their identity was confirmed by GC and GC-MF, which also indicated the presence of a minor sulphone isomer. The study shows that both cysteine and methionine could function as donors of the sulphur atom in the methyl sulphone metabolites derived from PCB.     

Sulfate incorporation from ascorbate 2-sulfate into chondroitin sulfate by embryonic chick cartilage epiphyses.

Radioactivity was significantly incorporated from ascorbate 2-[35S]sulfate into chondroitin sulfate by embryonic chick cartilage epiphyses. The extent of incorporation was comparable with that from inorganic [35S]sulfate. The radioactive chondroitin sulfate formed from ascorbate 2-[35S]sulfate gave two radioactive disaccharides on chondroitinase-ABC [EC 4.2.2.4] digestion. The incorporation was markedly decreased by inorganic sulfate. The time course of incorporation from ascorbate 2-[35S]sulfate and inorganic [35S]sulfate into chondroitin sulfate and the constituent disaccharides suggest that the incorporation rates from the two radioactive substances are different.     

Characterization of ectonucleotidases on vascular smooth-muscle cells.

We compared the properties of the ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5'-nucleotidase, EC 3.1.3.5) in intact pig aortic smooth-muscle cells in culture with the properties that we previously investigated for ectonucleotidases of aortic endothelial cells [Cusack, Pearson & Gordon (1983) Biochem. J. 214, 975-981]. In experiments with nucleotide phosphorothioate diastereoisomers, stereoselective catabolism of adenosine 5'-[beta-thio]triphosphate, but not of adenosine 5'-[alpha-thio]triphosphate, by the triphosphatase and stereoselective catabolism of adenosine 5'-[alpha-thio]diphosphate by the diphosphatase were found, as occurs in endothelial cells. In contrast with endothelial ecto-5'-nucleotidase, the smooth-muscle-cell enzyme catabolized adenosine 5'-monophosphorothioate (AMPS) to adenosine: the affinity of the enzyme for AMPS was greater than for AMP, and Vmax for AMPS was about one-sixth that for AMP. In both cell types AMPS was an apparently competitive inhibitor of AMP catabolism by 5'-nucleotidase. The relative rates of catabolism of nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety were similar to those in endothelial cells. No ectopyrophosphatase activity was detected in smooth-muscle cells, in contrast with endothelial cells, where modest activity is present.     

Direct photoaffinity labeling of proteins with adenosine 3'-[32P]phosphate 5'-phosphosulfate. Atractyloside inhibits labeling of a Mr = 34,000 protein in an adrenal medullary Golgi fraction

Direct photoaffinity labeling with radioactively labeled adenosine 3'-phosphate 5'-phosphosulfate (PAPS) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography was used to identify PAPS binding proteins in a Golgi membrane preparation of bovine adrenal medulla. [3'-32P]PAPS was synthesized from adenosine 5'-phosphosulfate (APS) and [gamma-32P]ATP using APS kinase prepared from yeast and was purified by reverse-phase ion pair high performance liquid chromatography. Upon irradiation with UV light, [3'-32P]PAPS, as well as [35S]PAPS under conditions which minimized sulfotransferase-catalyzed incorporation of 35SO4 from [35S]PAPS into proteins, bound selectively to a 34-kDa protein of the Golgi membrane preparation. PAPS binding to the 34-kDa protein was strongly inhibited by the presence of 50 microM atractyloside. The 34-kDa PAPS binding protein therefore appears to be similar to the mitochondrial ATP/ADP translocator with regard to both molecular weight and inhibition by atractyloside of adenine nucleotide binding. Photoaffinity labeling will be useful in the purification and functional identification of the 34-kDa protein.     

A stereochemical and positional isotope exchange study of the mechanism of activation of isoleucine by isoleucyl-tRNA synthetase from Escherichia coli

Isoleucyl-tRNA synthetase from Escherichia coli catalyzes the activation of [18O2]isoleucine by adenosine 5'-[(R)-alpha-17O]triphosphate with inversion of configuration at phosphorus. Moreover, isoleucyl-tRNA synthetase does not catalyze positional isotope exchange in adenosine 5'-[beta-18O2]triphosphate in the absence of isoleucine or in the presence of the competitive inhibitor isoleucinol, which effectively eliminates the possibility of either adenylyl-enzyme or adenosine metaphosphate intermediates being involved. Together, these observations require that isoleucyl-tRNA synthetase catalyzes the activation of isoleucine by associative "in line" nucleotidyl transfer. The synthesis of adenosine 5'-[(R)-alpha-17O]diphosphate and its conversion to adenosine 5'-[(R)-alpha-17O]triphosphate is described and an explanation provided for the reported differences between the treatment of adenosine 5'-[(S)-alpha-thiodiphosphate] with cyanogen bromide and bromine in [18O]water.     

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.