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.     

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.     

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.     

Adenosine 5′-triphosphate sulphurylase from Saccharomyces cerevisiae

1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at -15 degrees C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP(2-) complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the K(m) of each substrate was determined at infinite concentration of the other (K(m) MgATP(2-), 0.07mm; MoO(4) (2-), 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5'-[(35)S]sulphatophosphate and SO(4) (2-), (35)SO(4) (2-) and adenosine 5'-sulphatophosphate, occurred only in the presence of either MgATP(2-) or PP(i). This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO(3) (2-), S(2)O(3) (2-) (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO(4) (2-) was produced by SO(4) (2-) (K(i)=2.0mm) and non-competitive inhibition by sulphide (K(i)=3.4mm). 8. Adenosine 5'-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity-substrate curves with both MgATP(2-) and MoO(4) (2-) to sigmoidal forms.     

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.     

Incorporation of sulphate by chick-embryo corium. Nature and products of the process in vitro.

1. The incorporation of sulphate into the trichloroacetic acid-precipitable fraction of 9-day chick-embryo corium, incubated in Krebs-Ringer phosphate buffer, pH7, is dependent on the sulphate concentration of the medium. Uptake of sulphate is linear with time for 3.5-4hr. and is maximal at 37.5 degrees in the presence of air or oxygen. d-Glucose stimulates the incorporation of sulphate but l-glutamine has no effect. 2. Incorporation of sulphate by the chick corium is enzymic and apparently involves the synthesis of active sulphate (adenosine 3'-phosphate 5'-sulphatophosphate) and the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to acceptor glycosaminoglycuronoglycan. This proposal on the nature of the process is suggested by the similarity between the energy of activation calculated for sulphate-incorporation in the chick-corium preparation and the energy requirement reported for sulphate-activation with purified yeast enzymes. 3. The 9-day chick-embryo corium is composed principally of fibroblasts; there are no histologically demonstrable mast cells. The young fibroblast is apparently responsible for the incorporation of sulphate into glycosaminoglycuronoglycans tentatively identified as chondroitin sulphate(s), heparan sulphate and heparin-like material.     

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.     

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.     

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.     

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 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.     

Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue

1. ATP sulphurylase was purified up to 1000-fold from spinach leaf tissue. Activity was measured by sulphate-dependent [(32)P]PP(i)-ATP exchange. The enzyme was separated from Mg(2+)-requiring alkaline pyrophosphatase (which interferes with the PP(i)-ATP-exchange assay) and from other PP(i)-ATP-exchange activities. No ADP sulphurylase activity was detected. 2. Sulphate was the only form of inorganic sulphur that catalysed PP(i)-ATP exchange; K(m) (sulphate) was 3.1mm, K(m) (ATP) was 0.35mm and the pH optimum was 7.5-9.0. The enzyme was insensitive to thiol-group reagents and required either Mg(2+) or Co(2+) for activity. 3. The enzyme catalysed [(32)P]PP(i)-dATP exchange; K(m) (dATP) was 0.84mm and V (dATP) was 30% of V (ATP). Competition between ATP and dATP was demonstrated. 4. Selenate catalysed [(32)P]PP(i)-ATP exchange and competed with sulphate; K(m) (selenate) was 1.0mm and V (selenate) was 30% of V (sulphate). No AMP was formed with selenate as substrate. Molybdate did not catalyse PP(i)-ATP exchange, but AMP was formed. 5. Synthesis of adenosine 5'-[(35)S]sulphatophosphate was demonstrated by coupling purified ATP sulphurylase and Mg(2+)-dependent alkaline pyrophosphatase (also prepared from spinach) with [(35)S]sulphate and ATP as substrates; adenosine 5'-sulphatophosphate was not synthesized in the absence of pyrophosphatase. Some parameters of the coupled system are reported.     

Thiol-dependent changes in the properties of rat liver sulphotransferases

1. Two enzymes (A and B) which catalyse the sulphation of p-nitrophenol and l-tyrosine methyl ester have been isolated from female rat livers. One of these enzymes (A) also catalyses the sulphation of dehydroepiandrosterone. 2. The K(m) values for the sulphation of p-nitrophenol and l-tyrosine methyl ester by enzyme B at pH7.5 are 1.5mum and 2.9mm respectively. 3. Enzyme B is oxidized on keeping at 0 degrees C when the K(m) and V(max.) values for the sulphation of p-nitrophenol are increased approx. 200-fold and fourfold respectively. This oxidized preparation of enzyme B fails to catalyse the sulphation of l-tyrosine methyl ester. 4. When the oxidized form of enzyme B is kept at 0 degrees C and low ionic strength then further forms of p-nitrophenol sulphotransferase are produced having even lower affinities for the sulphate acceptor. 5. The K(m) value for adenosine 3'-phosphate 5'[(35)S]-sulphatophosphate is not affected during storage of the enzyme under these conditions. 6. Prolonged storage of enzyme B at low ionic strength leads to a considerable degree of polymerization of p-nitrophenol sulphotransferase and l-tyrosine methyl ester sulphotransferase. 7. The changes in the kinetic properties and molecular size of enzyme B during storage are reversed by dithiothreitol.     

Structure and properties of recombinant human pyridoxine 5'-phosphate oxidase

Pyridoxine 5'-phosphate oxidase catalyzes the terminal step in the synthesis of pyridoxal 5'-phosphate. The cDNA for the human enzyme has been cloned and expressed in Escherichia coli. The purified human enzyme is a homodimer that exhibits a low catalytic rate constant of approximately 0.2 sec(-1) and K(m) values in the low micromolar range for both pyridoxine 5'phosphate and pyridoxamine 5'-phosphate. Pyridoxal 5'-phosphate is an effective product inhibitor. The three-dimensional fold of the human enzyme is very similar to those of the E. coli and yeast enzymes. The human and E. coli enzymes share 39% sequence identity, but the binding sites for the tightly bound FMN and substrate are highly conserved. As observed with the E. coli enzyme, the human enzyme binds one molecule of pyridoxal 5'-phosphate tightly on each subunit.     

Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources

Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione-Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4 degrees C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide ;maps' display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The k(cat.) and K(m) values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a k(cat.)/K(m) value near 10(7)s(-1).m(-1), which is close to the theoretical diffusion-controlled rate of enzyme-substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.     

A novel target of lithium therapy

Phosphatases converting 3'-phosphoadenosine 5'-phosphate (PAP) into adenosine 5'-phosphate are of fundamental importance in living cells as the accumulation of PAP is toxic to several cellular systems. These enzymes are lithium-sensitive and we have characterized a human PAP phosphatase as a potential target of lithium therapy. A cDNA encoding a human enzyme was identified by data base screening, expressed in Escherichia coli and the 33 kDa protein purified to homogeneity. The enzyme exhibits high affinity for PAP (K(m)<1 microM) and is sensitive to subtherapeutic concentrations of lithium (IC(50)=0.3 mM). The human enzyme also hydrolyzes inositol-1, 4-bisphosphate with high affinity (K(m)=0.4 microM), therefore it can be considered as a dual specificity enzyme with high affinity (microM range) for both PAP and inositol-1,4-bisphosphate. Hydrolysis of inositol-1,4-bisphosphate was also inhibited by lithium (IC(50)=0.6 mM). Thus, we present experimental evidence for a novel target of lithium therapy, which could explain some of the side effects of this therapy.     

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.     

3',5'-Cyclic nucleotide phosphodiesterase activity of the sulphatase A of ox liver

The sulphatase A (aryl-sulphate sulphohydrolase, EC 3.1.6.1) of ox liver hydrolyses adenosine 3',5'-monophosphate (cyclic AMP) to adenosine 5'-phosphate at an optimum pH of approx. 4.3, close that for the hydrolysis of cerebroside sulphate, a physiological substrate for sulphatase A. The Km is 11.6 mM for cyclic AMP. On polyacrylamide gel electrophoresis sulphatase A migrates as a single protein band which coincides with both the arylsulphatase and phosphodiesterase activities, suggesting that these are due to a single protein. Cyclic AMP competitively inhibits the arylsulphatase activity of sulphatase A, showing that both activities are associated with a single active site on the enzyme. sulphatase A also hydrolyses guanosine 3',5'-monophosphate, but not uridine 3',5'-monophosphate nor adenosine 2',3'-monophosphate.     

A novel phosphodiesterase from cultured tobacco cells.

A novel phosphodiesterase was purified from cultured tobacco cells to a state which appeared homogeneous on polyacrylamide gel electrophoresis. The enzyme hydrolyzed various phosphodiester and pyrophosphate bonds, including p-nitrophenyl thymidine 5'-phosphate, p-nitrophenyl thymidine 3'-phosphate, cyclic nucleotides, ATP, NAD+, inorganic pyrophosphate, dinucleotides, and poly(adenosine diphosphate ribose), which is a polymer synthesized from NAD+. However, it did not hydrolyze highly polymerized polynucleotides. The molecular weight of the native enzyme was estimated as 270 000 to 280 000 by gel filtration on Sephadex G-200 and Bio-Gel A-5m. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was composed of subunits with molecular weights calculated to be 75 000. The enzyme did not require divalent cations for activity being fully active in the presence of ethylenediaminetetraacetic acid. The pH optimum for the enzyme was approximately 6 with p-ni-trophenyl thymidine 5'-phosphate or adenosine cyclic 3',5'monophosphate, and 5.3 with NAD+. Double reciprocal plots of the initial velocity against the concentration of p-nitrophenyl thymidine 5'-phosphate gave two apparent Km values of 0.17 and 1.3 mM, suggesting the presence of at least two active sites.