A.B. Roy

The sulphatase of ox liver: XV. Changes in the properties of sulphatase A in the presence of substrate

Abstract Enzyme with modified kinetic properties has been isolated from reaction mixtures of sulphatase A (arylsulphate sulphohydrolase, EC 3.1.6.1) and its substrate, either nitrocatechol sulphate or 4-nitrophenyl sulphate. A method is described for the determination of the proportion of the modified enzyme in these preparations. The position of the equilibrium between the native and modified forms is invariant with enzyme concentration and with pH between pH 5 and 7. Sulphate displaces the equilibrium in favour of the native enzyme primarily through the formation of a native enzyme-sulphate complex. The modified enzyme has the same sedimentation coefficients as native sulphatase A at pH 5 and 7.5 and does not contain any detectable 4-nitrocatechol or nitrocatechol sulphate. The apparently inactive modified enzyme is strongly activated by SO 4 2- , but only slightly activated by 4-nitrocatechol. Phosphate and pyrophosphate, inhibitors of the native enzyme, also activate the modified enzyme. The low activity of the modified enzyme was found to be due to a powerful substrate inhibition which is decreased in the presence of SO 4 2- . A reaction mechanism which explains the anomalous kinetics of sulphatase A has been proposed.

The sulphatase of ox liver. XVI. A comparison of the arylsulphatase and cerebroside sulphatase activities of sulphatase A.

Abstract The initial velocities for the hydrolyses of cerebroside sulphate and nitrocatechol sulphate by the sulphatase A of ox liver have been studied under similar conditions in the pH-stat. Cerebroside sulphatase activity requires the presence of sodium taurodeoxycholate and MnCl 2 (or certain other salts) in the reaction mixture. The MnCl 2 lowers the critical micellar concentration of the inhibitory ionic form of the bile salt and allows the formation of mixed micelles of taurodeoxycholate and cerebroside sulphate. The K m varies with taurodeoxycholate concentration and is 0.07 mM cerebroside sulphate a 2 mM bile salt. The reaction is noncompetitively inhibited by K 2 SO 4 with a K i of 0.47 mM. It is 50% inhibited by 25 mM hydroxylamine · HCl. The kinetics of the reaction are consistent with a uni-ter mechanism in which the substrate is a mixed micelle of taurodeoxycholate and cerebroside sulphate, and the products are released in the order cerebroside, sulphate and taurodeoxycholate. The arylsulphatase activity is inhibited by ionic taurodeoxycholate. Salts lessen this inhibition by lowering the critical micellar concentration but they also activate the enzyme. At pH 4.5 in the presence of taurodeoxycholate and MnCl 2 the properties of the arylsulphatase are not significantly different from those at the optimum pH of 5.6. The arylsulphatase is competitively inhibited by K 2 SO 4 with a K i of 0.15 mM. Explanations of the apparent differences between the arylsulphatase and cerebroside sulphatase activities of sulphatase A are given and it is concluded that both are due to a single site on the protein. It has been shown that certain cations form complexes with 4-nitrocatechol: this reaction is accompanied by the liberation of H + and by an increased absorption at 430 nm.

The sulphatase of ox liver. XI. The isoelectric focussing of a purified preparation of sulphatase B.

Abstract A method is described for the preparation of sulphatase B from ox liver: two fractions, sulphatases Bα and Bβ, have each been purified about 2000-fold and obtained in a yield of about 5%. Both fractions have molecular weights of about 25 000 and they are indistinguishable kinetically. The identity of sulphatase B with the arylsulphatase which hydrolyses p -nitrophenyl sulphate only in the presence of Cl − has been confirmed. The purified enzymes have been examined by the technique of isoelectric focussing and it has been shown that during this procedure the sulphatases B aggregate to mixtures of polymers having molecular weights of up to at least 300 000. The aggregation is probably due to electrostatic interaction because it is reversed by increasing the ionic strength to 0.1.

he sulphatase of ox liver XVII. Sulphatase A as a glycoprotein

Abstract The glycoprotein nature of sulphatase A has been confirmed. The monomer of sulphatase A (mol. wt 107 000) contains eight molecules of glactose, 14 of mannose, 18 of glucosamine and eight of sialic acid together with traces of focuse and glucose. The latter may be contaminant. Treatment of sulphatase A with neuraminidase quantitatively removes the sialic acid showing that this must be in the terminal position of the carbohydrate. The desialylated enzyme retains the properties of native sulphatase A apart from a slight change in charge and it is quite distinct from sulphatase B. The desialylated enzyme still hydrolyses cerebroside sulphate. The implications of these findings in the biochenmistry of metachromatic leucodystrophy are considered.

l-ascorbic acid 2-sulphate: A substrate for mammalian arylsulphatase

Abstract Ascorbic acid 2-sulphage has a stability in acid comparable to that of phenyl sulphate and is rather more acid-labile than simple carbohydrate sulphates. At its optimum pH of 4.8 sulphatase A(aryl-sulphate sulphohydrolase EC 3.1.6.1.) hydrolyses ascorbic acid sulphate with a specific activity of 90 μmol/mg per min (150 μmol/mg per min with nitrocatechol sulphate at pH 5.6). At pH 4.8 the kinetics are non-Michaelis. At pH 5.6 Michaelis kinetics are obeyed and K m is 21 mM ascorbic acid 2-sulphate. K 2 SO 4 is a competitive inhibitor with a K i of 0.2 and 0.6 mM at pH 4.8 and 5.6, respectively. Sulphatase A is converted into a substrate-modified form during its hydrolysis of ascorbic acid sulphate. Sulphatase B also hydrolyses ascorbic acid 2-sulphate. At pH 4.8 and in the presence of 0.15 M NaCl the specific activity is 0.92 μmol/mg per min (90 μmol/mg per min for nitrocatechol sulphate at pH 5.6). In the absence of NaCl the activity is greatly decreased. K m is 8 mM. K 2 SO 4 is a competitive inhibitor with a K i of 0.1 mM. Ascorbic acid is not hydrolysed at a detectable rate by the arylsulphatases of the mollusc Dicathais orbita or of Aerobacter aerogenes .

Kinetic studies of the phenol sulphotransferase reaction

The kinetics of the reversible sulphotransferase-catalysed reaction between p-nitrophenol and 3′-phosphoadenylyl sulphate to give p-nitrophenyl sulphate and adenosine 3′,5′-diphosphate have been investigated using a partially purified sulphotransferase from guinea-pig liver. From a study of the initial velocities of the reactions in the forward and reverse directions at different substrate concentrations, and of the inhibitory effects of the products on the forward reaction, it was concluded that the mechanism was that of a rapid equilibrium random bi bi reaction upon which was superimposed the formation of a dead-end complex of the enzyme, p-nitrophenol and adenosine 3′,5′-diphosphate. Studies of the specificity of the enzyme have been made. Attempts to detect a transfer reaction between p-nitrophenyl sulphate and 2-naphthol with adenosine 3′,5′-diphosphate as a cofactor were unsuccessful.

Some properties of the sulphatase B of ox brain

A purified preparation of sulphatase B has been obtained from ox brain. The preparation is homogeneous with respect to sedimentation coefficient (s∘20,w, 4.4 S) and to molecular weight (60 000) at pH 5 and pH 7.5. The apparent diffusion coefficient (D∗20) is 6.6·10−7 cm2·s−1 at those pH values and the calculated frictional ratio is 1.2, showing the protein to be a globular one. At pH 7.4 E2801%nm is 17.4. This preparation has been fractionated by ion-exchange chromatography into 7 incompletely resolved fractions which presumably differ in net charge. The sulphatases B contain rather large amounts of proline and their amino acid composition resembles that of sulphatase A except for their containing larger amounts of lysine and arginine.

The sulphatase of ox liver: XIV. The subunit structure of sulphatase A

Abstract The molecule of sulphatase A (mol. wt. 107 000) remains essentially intact in 8 M urea at pH 7.5 and 8.1 and only small amounts of material of mol. wt. 55 000 are present. In 6 mM dithiothreitol and 8 M urea at pH 8.1 dissociation to material of mol. wt. 55 000 is complete and small amounts of material of mol. wt. 27 000 are also produced. Under both conditions the enzymic activity is irreversibly destroyed. Removal of the denaturants of pH 7.5 gives heterogeneous samples in which material of mol. wt. 200 000 predominates. Treatment of sulphatase A with sodium dodecyl sulphate at pH 5 also produces protein units of mol. wt. 50 000. These are presumed to be distinct from those produced by the action of dithiothreitol and urea. It is suggested that the molecule of native sulphatase A is built up from four units of mol. wt. 27 000. These are linked in pairs by disulphide bridges and these in turn are linked in pairs by hydrogen bonds and/or hydrophobic interactions.

Purification and properties of the ATP sulphurylase of rat liver

ATP sulphurylase (ATP:sulphate adenylyltransferase, EC 2.7.7.4) as been purified about 2500-fold from rat liver. It was free of ATPase, inorganic pyrophosphatase, adenosine phosphosulphate kinase and ADP sulphurylase activities. The enzyme was homogeneous to chromatography on Sepharose 4B and to density-gradient sedimentation; it was not homogeneous to acrylamide gel electrophoresis nor to sedimentation in the ultracentrifuge. Possible reasons for this heterogeneity are considered. The molecular weight of the enzyme is 410 000 as measured by chromatography on Sepharose 4B. The v is 0.80, suggesting that ATP sulphurylase is a lipoprotein. The enzyme activity is associated with a pigment having a lambdamax of 410 nm. Studies of the forward, reverse and ATP-PPi exchange reactions catalysed by ATP sulphurylase have shown that these are sequential bi-bi reactions, with ATP being the first substrate bound and adenosine phosphosulphate the last product released. The results are incompatible with previous suggestions that the ATP sulphurylase of rat liver catalysed a bi-bi ping-pong reaction.

The type II arylsulphatases of the red kangaroo

Abstract The separation of two type II arylsulphatases (aryl-sulphate sulphohydrolases, EC 3.1.6.1) from the liver of the red kangaroo, Megaleia rufa, is described. The two enzymes have many properties in common with sulphatases A and B from ox liver and have been similarly named. Neither of the enzymes has been obtained in a homogeneous form but the sulphatase A cannot be grossly impure. It has a molecular weight of 100 000, an isoelectric point of 5.1–5.4, and does not polymerise as does ox sulphatase A. Its kinetic properties are analogous to those of the ox enzyme. The sulphatase B of kangaroo liver has a molecular weight of 45 000 and an isoelectric point of 7. Kinetically it also resembles the corresponding ox enzyme. No evidence has been obtained for the existence of a sulphatase C in kangaroo liver and a steroid sulphatase, if it be present, occurs in only very small amounts. The results suggest that sulphatases A and B had developed quite early in the evolution of the mammals.