The sulphatase of ox liver. XIX. On the nature of the polymeric forms of sulphatase A present in dilute solutions.

Weight-average elution volumes of sulphatase A (an arylsulphate sulphohydrolase, EC 3.1.6.1) from Sephadex G-200 have been determined as functions of protein concentration, pH, ionic strength and temperature. The results are used to calculate the apparent association equilibrium constants for tetramer formation and the associated standard-state thermodynamic parameters. While the apparent association constant decreased from 10(28) to 10(21) M-3 on increasing the pH from 4.5 to 5.6 at ionic strength 0.1, at any particular pH value studied it was relatively insensitive to temperature variation so that deltaH is close to zero and tetramer formation in solution is associated with a positive entropy change. At pH 5.0, increasing the ionic strength from 0.1 to 2 decreased the association constant by a factor of 100. Methylumbelliferone sulphate has no effect on the association of sulphatase A. The equilibrium results are used to define the degree of association of sulphatase A likely to encountered in experiments designed to elucidate its kinetic properties. In the liver lysosome, the tetramer is probably the dominant species. The monomer and tetramer of sulphatase A have similar, or identical, specific activities with nitrocatechol sulphate and 4-methylumbelliferone sulphate as substrates. With nitrocatechol sulphate, sulphatase A shows Michaelis kinetics under conditions where the monomer is the dominant species and non-Michaelis kinetics where the tetramer is dominant. There is apparently a negative cooperativity between the monomer units in the tetramer. In 2 mM sodium taurodeoxycholate and 0.035 M MnCl2, but not in 0.1 M NaCl, the tetramer shows Michaelis kinetics. This is not due to dissociation of the tetramer. The critical micellar concentration of sodium taurodeoxycholate is about 0.8 mM in both 0.1 M NaCl and 0.035 M McCl2 but the aggregation number is greater in the latter.     

Comparison of the cerebroside sulphatase and the arylsulphatase activity of human sulphatase A in the absence of activators.

A cerebroside sulphatase (cerebroside-3-sulphate 3 sulphohydrolase, EC 3.1.6.8) assay based on radio thin-layer chromatography is described. The substrate was labelled by the catalytic addition of tritium to cerebroside sulphate. Using this assay the cerebroside sulphatase activity of sulphatase A (Aryl-sulphate sulphohydrolase, EC 3.1.6.1) from human liver and kidney in the absence of activators was investigated. The pH optimum of this reaction depends on the buffer concentration, being pH 4.5 at 50 mM and 5.3 at 10 mM sodium formate. With the latter concentration the apparent Km for cerebroside sulphate is 0.06 mM; SO2-4 and nitrocatechol sulphate inhibit noncompetitively with a Ki of 4.51 mM for Na2SO4 and 0.43 mM for nitrocatechol sulphate. The cerebroside sulphatase activity of sulphatase A is highly dependent on the ionic strength. The optimum sodium formate concentration is 10 mM, and the cerebroside suophatase activity decreases rapidly with increasing buffer concentration. The same concentration dependence is observed in the inhibitory effect of cerebroside sulphate on the arylsulphatase reaction. The inhibition decreases at increasing buffer concentrations, becoming an activation at 70 mM sodium formate. The progress curve of the cerebroside sulphatase reaction shows a deviation from linearity similar to that of the arylsulphatase reaction. Investigation of the effect of preincubation with cerebroside sulphate on the arylsulphatase activity of the enzyme shows that cerebroside sluphatase activity and inactivation of the enzyme by cerebroside sulphate occur simultaneously. These observations are interpreted as supporting the assumption that cerebroside suophate and arylsulphates are degraded at an identical active site on the same enzyme. Differences in the properties of the cerebroside sulphatase and the arylsulphatase reaction of the enzyme may be attributed to the differences in the physiocochemical state of the two substrates.     

Lysosomal aryl sulphatase in the liver

Summary Lysosomal aryl sulphatase activity in rat liver is demonstrated by a modification of the existing processes of fixation, incubation and processing. The choice and concentration of the fixative, duration of fixation and thickness of liver slices are found to be important factors in maintaining the levels of enzyme activity. Reliable and reproducible results are obtained by fixing thin liver slices (1 mm) for 18–24 h, in 2% glutaraldehyde buffered to pH 7.4 by 0.1M cacodylate buffer and incubating sections inHopsu et al. (1967) medium using (160 mg) nitrocatechol sulphate as substrate. Aryl sulphatase activity is localised in discrete pericanalicular granules recognised as lysosomes, which stain less intensely than acid phosphatase by the lead method.Supported by a grant from the Nuffield Foundation.     

L-ascorbic acid 2-sulphate. A substrate for mammalian arylsulphatases.

Ascorbic acid 2-sulphate 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 mumol/mg per min (150 mumol/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 Km 12 21 mM ascorbic acid 2-sulphate. K2SO4 is a competitive inhibitor with a Ki 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 mumol/mg per min (90 mumol/mg per min for nitrocatechol sulphate at pH 5.6). In the absence of NaCl the activity is greatly decreased. Km is 8 mM. K2SO4 is a competitive inhibitor with a Ki 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.?     

The sulphatase of ox liver. XXV. Sulphatase A as a hysteretic enzyme

The kinetic behaviour of the system native--substrate-modified sulphatase A (arylsulphate sulphohydrolase, EC 3.1.6.1) has been investigated and it has been shown that the progress curve of the complete reaction, including both the inactivation and reactivation stages, can be treated as that of a simple hysteretic system in which the substrate-modified enzyme is activated by a product of the reaction. It has been concluded that the early suggestions that the modification of sulphatase A was accompanied by the exposure of a second ligand-binding site are incorrect. It has been shown that, in the absence of sulphate, the rate of reversion of substrate-modified to native sulphatase A is increased by 4-nitrocatechol but not by the same concentration of 2-nitrophenol. A detailed reaction sequence is proposed. This explains the kinetic behaviour of sulphatase A with nitrocatechol sulphate or 2-nitrophenyl sulphate as substrate and in the presence or absence of sulphate.     

Lysosomal aryl sulphatase in pulmonary alveolar cells

Summary Lysosomal aryl sulphatase has been localised in the lung at the electron microscopic level using a nitrocatechol sulphate barium chloride medium. Variations in fixative concentration and incubation time were found to be important in minimising lysosomal leakage. The distribution of aryl sulphatase in the lung corresponded closely to that of acid phosphatase. Large amounts were found in alveolar macrophages and small quantities in the type II alveolar epithelial cell. In the latter cell the enzyme was found in the lamellar vacuoles thought to represent the site of surfactant production. The significance of this in regard to the function of these organelles is discussed.     

Measurement of the arylsulphatase of Patella vulgata with 4-methylumbelliferone sulphate

1. The preparation, purification, chemical and spectral properties of potassium 4-methylumbelliferone sulphate are described. 2. The use of 4-methylumbelliferone sulphate as a substrate for the arylsulphatase of Patella vulgata is presented with specific reference to the fluorimetric assay procedure used with this substrate. 3. 4-Methylumbelliferone sulphate is compared with the previously used synthetic sulphatase substrates nitrocatechol sulphate and p-nitrophenyl sulphate with respect to K_m, V_max. and sensitivity in the assay of arylsulphatase. 4. 4-Methylumbelliferone sulphate was strongly inhibited by phosphate. Sulphate, a less potent inhibitor, appeared to be of the competitive type with some anomalous characteristics.     

Purification and properties of N-acetylgalactosamine 6-sulphate sulphatase from human placenta

1. N-Acetylgalactosamine 6-sulphate sulphatase was purified about 20000-fold from the soluble extract of human placenta with N-acetylgalactosamine 6-sulphate-glucuronic acid-N-acetyl[1-(3)H]galactosaminitol 6-sulphate as substrate in the activity assay. The enzyme appears to be a glycoprotein with a mol.wt. of about 100000 as determined by gel filtration. On gel electrophoresis in the presence of sodium dodecyl sulphate the major protein band had a mol.wt. of 78000. Variable charge heterogeneity was observed in several enzyme preparations. 2. The purified enzyme released up to one sulphate molecule from the disulphated trisaccharide. It was active towards N-acetylgalactosamine 6-sulphate and exhibited no measurable N-acetylglucosamine 6-sulphate sulphatase or any other known lysosomal sulphatase activity. Hydrolysis of [1-(3)H]galactitol 6-sulphate was achieved by incubation neither with a crude nor with a purified enzyme preparation. Chondroitin 6-sulphate and keratan sulphate, as well as heparin and heparan sulphate, served as competitive inhibitors of the enzyme. 3. Purified N-acetylgalactosamine 6-sulphate sulphatase activity was optimal at pH4.9 and 4.4 when assayed in 0.02m-sodium acetate buffer and at pH4.2 and 5.2 in 0.1m-sodium acetate buffer. A single pH-optimum at pH4.8 was observed for the crude enzyme and for the purified enzyme after mild periodate treatment. The sulphatase activity was inhibited by a variety of anions and cations and activated by thiol-specific and thiol reagents.     

Enzymic studies of sulphatases in tissues of the normal human and in metachromatic leukodystrophy with multiple sulphatase deficiencies: arylsulphatases A, B and C, cerebroside sulphatase, psychosine sulphatase and steroid sulphatases

Abstract— Several sulphatases (arylsulphatases A, B and C, cholesterol sulphatase, dehydroepiandroster-one sulphatase, cerebroside sulphatase and psychosine sulphatase) were deficient in various tissues from two patients with a variant form of metachromatic leukodystrophy. Deficient activities of cerebroside sulphatase and psychosine sulphatase, using physiological substrates, in tissues from metachromatic leukodystrophy with multiple sulphatase deficiencies provided another example that these enzymes may be identical to arylsulphatase A. β-Galactosidase activity was reduced to about 30-50 per cent of normal in brain and liver. Other lysosomal enzyme activities were found to be normal or elevated five to eight times. Arylsulphatase B isolated from the liver of one patient was abnormal, with respect to pi (70) and enzyme kinetics. In mixing experiments with normal enzymes the reduced activities of arylsulphatases A. B and C, cerebroside sulphatase and steroid sulphatases were shown not to be due to the presence of endogenous inhibitors. No arylsulphatase A or B activity in the brain specimen from the patient with multiple sulphatase deficiencies could be detected on isoelectric focussing. In normal brain tissue arylsulphatase A had a pi of 4-6-4-8 while arylsulphatase B had a pi of 7-8 and 8-1. When 4-methylumbelliferyl sulphate was used as a substrate the elution patterns of normal brain and liver arylsulphatase B were more heterogeneous and showed more variation than that when p-nitrocatechol sulphate was used. Arylsulphatase C and steroid sulphatases (cholesterol sulphatase, dehydroepiandrosterone sulphatase and oes-trone sulphatase I were solubilized by the addition of lysolecithin and Triton X-100 and subjected to isoelectric focussing. The pi of cholesterol sulphatase, oestrone sulphatase and arylsulphatase C was 6-8, and the elution patterns of the activities of these enzymes were similar. The pattern of dehydroepiandrosterone sulphatase was more heterogeneous and two major peaks were observed at pi 6 5 and 70. Residual enzyme activities of arylsulphatase C and steroid sulphatases from the brain of the patient with multiple sulphatase activities were not detectable by isoelectric focussing. Simultaneous deficiencies of arylsulphatase C and steroid sulphatases plus isoelectric focussing findings in tissues suggest that these enzymes are closely related in regard to their function. The nature of the genetic defect in metachromatic leukodystrophy with multiple sulphatase deficiencies is discussed.     

Assay and subcellular localization of the arylsulphatases in rat brain

—The properties and subcellular localization of type I (nitrophenyl) and type II (nitrocatechol) arylsulphatases were investigated in brain tissue of the rat, and optimal assay conditions were established. Sulphate, phosphate and sulphite ions inhibited the nitrocatechol sulphatases; nitrophenyl sulphatase was inhibited only by sulphite. The presence of latent enzyme activity was demonstrated for the nitrocatechol sulphatases, beta-glucuronidase, and beta-glycerophosphatase in rat and mouse brain homogenates. These hydrolases were highly sensitive to mechanical and osmotic damage; and Triton X-100 was very effective in releasing their latent (bound) activities, a finding suggestive of a lysosomal localization. Activity of nitrophenyl sulphatase was unaffected by osmotic changes or Triton X-100, characteristics suggesting a membranous association for this enzyme. Total activity of nitrophenyl sulphatase was approximately twice as great in canine gray matter as in canine white matter; the converse obtained for beta-glucuronidase activity. Values for total enzymic activity of the nitrocatechol sulphatases in canine white and gray matter were similar. Fractionation of homogenates from rat brain by differential centrifugations and separation of crude mitochondrial fractions by sucrose density gradient centrifugations revealed the following: (1) most of the nitrocatechol sulphatase activity (93 per cent) and all of the nitrophenyl sulphatase activity were sedimentable; (2) crude mitochondrial fractions exhibited the highest relative specific activity (RSA = 1·38) for the nitrocatechol sulphatases, whereas microsomal fractions displayed the highest RSA for nitrophenyl sulphatase (1·89); (3) the lightest fraction (A + B) and the densest fraction (E) from the sucrose density gradient contained most of the activity for both the type I and type II arylsulphatases, whereas the RSA of cytochrome oxidase was greatest in the intermediate density regions (fractions C and D); (4) the highest RSA for beta-glucuronidase and beta-glycerophosphatase occurred in gradient fraction C; (5) appreciable activity of beta-glycerophosphatase was found in a nerve ending fraction (M₃). It is suggested that the hydrolases in heterogeneous tissue like brain might be associated with lysosomal particles of differing enzyme compositions and varying populations, and that the data on distribution lend credence to the concept of bimodal and possible trimodal particle affinity for the hydrolases of brain tissues.     

A kinetic study of the subunit dissociation and reassembly of rabbit muscle phosphofructokinase.

The kinetics of dissociation and reassembly of rabbit skeletal muscle phosphofructokinase has been studied using fluorescence, stopped-flow fluorescence and enzyme activity measurements. The dissociation of the fully active tetramer in 0.8 M guanidine hydrochloride (0.1 M potassium phosphate, pH 8.0) occurs in three kinetic phases as measured by changes in the protein fluorescence emission intensity: dissociation of tetramer to dimer with a relaxation time of a few milliseconds; dissociation of dimer to monomer with a relaxation time of a few seconds; and a conformational change of the monomer with a relaxation time of a few minutes. All three phases exhibit first-order kinetics; ATP (0.05 mM) retards the second step but does not influence the rate of the other two processes. The rate of the second process increases with decreasing temperature; this may be due to the involvement of hydrophobic interactions in the stabilization of the dimeric enzyme. A further unfolding of the monomer polypeptide chain occurs at higher guanidine concentrations, and the relaxation time associated with this process was found to be 83 ms in 2.5 M guanidine, 0.1 M potassium phosphate (pH 8.0) at 23 degrees C. The phosphofructokinase monomers were reassembled from 0.8 M guanidine chloride by 1:10 dilution of the guanidine hydrochloride concentration and yielded a protein with 70-94% of the original activity, depending on the protein concentration. The reactivation process follows second-order kinetics; ATP (5 mM) increases the rate of reactivation without altering the reaction order, while fructose 6-phosphate does not influence the rate of reaction. The rate-determining step is probably the association of monomers to form the dimer.     

Structure of the histone tetramer (H3-H4)2: 1. Peculiarities of optical absorption,fluorescence and light scattering in response to changes in ionic strength and pH of the medium

Using the methods of spectrophotometry, spectrofluorimetry, light scattering and gel filtration, it was shown that, at pH 5.6 and 7.4 and various ionic strengths, the histone tetramer (H3-H4)₂ may have several structural states with different packing of the polypeptide chains of histones H3 and H4. Two structural changes of the tetramer (H3-H4)₂ at pH 7.4 in the ranges 0.1–0.3 m and 0.7–0.9 m NaCl were observed. In the high ionic strength solution, the tetramer (H3-H4)₂ had a more compact structure at pH 7.4 than at pH 5.6. At pH 3.0 destruction of the histone tetramer (H3-H4)₂ and formation of non-specific aggregates took place.     

Regulation of choline sulphatase synthesis and activity in Aspergillus nidulans

1. Choline O-sulphate is taken up from the growth medium to the same extent by sulphur-deficient and sulphur-sufficient mycelia of Aspergillus nidulans, but hydrolysis of the transported sulphate ester in vivo only occurs in the sulphur-deficient mycelia. 2. Choline sulphatase activity could not be detected in vitro in sulphur-sufficient mycelia of wild-type and sulphur mutants of A. nidulans, but after sulphur starvation all strains showed appreciable activity of this enzyme. 3. Optimum activity of choline sulphatase in an ultrasonically treated preparation of sulphur-deficient mycelia was at pH7.5. The optimum substrate concentration was in excess of 25mm and K(m) was 0.035m. The enzyme was completely inhibited by 10mm-SO(3) (2-), PO(4) (3-), CN(-) and cysteine. 4. Growth of sulphur-deficient mycelia on various sulphur sources resulted in a decrease of choline sulphatase activity in vitro. The decrease appeared to be due to a repression of choline sulphatase synthesis rather than to inhibition of activity. De-repression by growth on a sulphur-deficient medium was prevented by cycloheximide. Unlike the choline sulphatase of bacteria the fungal enzyme did not need to be substrate-induced. 5. By using sulphur mutants the identity of the co-repressor was limited to S(2)O(3) (2-), cysteine-S-sulphonate, cysteine or compounds derived directly from them. Circumstantial evidence suggests that the co-repressor is cysteine. 6. Inhibition of choline sulphatase activity in vivo was demonstrated with cysteine as the sulphur source for growth.     

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

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.     

Borate buffer inhibition of peroxidatic intermediate formation in the deutero-ferriheme-hydrogen peroxide system

The rate of formation of peroxidatically active reaction intermediate(s) via oxidation of the iron(III)-porphyrin complex, deuteroferriheme, with hydrogen peroxide decreases with increasing borate content of mixed borate-carbonate buffer solutions. Studies at pH = 9.25 in 0.035 M borate buffer and 0.035 M carbonate buffer suggest borate to function as an uncompetitive inhibitor. A comparison of slopes and intercepts of double reciprocal plots for inhibited and uninhbited reactions allows calculation of selected parameters for the deuteroferriheme-H₂O₂ reaction at pH = 9.25 in terms of a typical enzymatic stoichiometric mechanism for heme activity. This includes the Michaelis constant (K_m = 8.1 × 10^?5 M) and the first-order rate constant for conversion of heme-substrate complex to intermediate(s) (k₃ = 7.4 sec^?1). A tentative mechanistic model involving reversible interaction of borate inhibitor with heme-substrate complex is considered, and pseudo-first-order rate constants calculated on the basis of this scheme are in reasonable agreement with those obtained experimentally. It is suggested that comparable inhibitory action may be responsible for some previously reported cases of decreased catalase enzyme activity in borate buffer solutions     

The regulation of aryl sulphatase in Aspergillus nidulans

The aryl sulphatase of Aspergillus nidulans is derepressed in a medium that contains low amounts of inorganic sulphate or sulphur-containing amino acids. Isotopic labelling experiments show that the enzyme molecules are made de novo. However, once derepressed, the formation of the enzyme becomes progressively insensitive to cycloheximide. It is suggested that active sulphatase is made in two steps, one sensitive to cycloheximide and the other insensitive. Both of these steps appear to be regulated by sulphur metabolites.     

The activator of cerebroside sulphatase. Purification from human liver and identification as a protein.

1) A heat-stable activator of human sulphatase A (cerebroside sulphatase) was purified from human liver. It is required for the enzymatic degradation of cerebroside sulphates (sulphatides) in buffers (ionic strength greater than or equal 0.2) with osmolarity in the physiological range. 2) The purification steps involve extraction, acetone precipitation, heat treatment, isoelectric focusing and gel filtration. 3) Based on the definition of a specific activator unit, the purification of the final preparation was approximately 2000-fold over the acetone precipitation and several thousand-fold in the overall procedure. 4) The purified activator migrated as a single protein band when subjected to gel electrophoresis. Its effect was abolished after treatement with pronase E. The apparent molecular weight as determined by gel filtration was 21 500 +/- 1500; the isoelectric point was 4.3. 5) The activating effect of this protein factor and of taurodeoxycholate on cerebroside sulphatase activity was compared on a weight and molar basis.     

The effect of the interdependence of protein and bile salt concentrations on the measurement of cerebroside sulphate sulphatase activity in human leucocyte and fibroblast extracts

The previously observed differences in properties of human leucocyte and fibroblast cerebroside sulphate sulphatase (cerebroside-3-sulphate 3-sulphohydrolase, EC 3.1.6.8) measured in vitro have been found to be due to subtle differences in incubation conditions. Maximum enzyme activity was observed with either crude sodium taurocholate or with pure sodium taurodeoxycholate. The optimum bile salt concentration of the enzyme in leucocyte or fibroblast extracts, but not the pure ox liver enzyme, was critically dependent on protein concentration. At low concentrations of the latter (less than 0.1 mg/ml), maximum activity was observed at taurocholate concentrations less than 0.5 mg/ml; at protein concentrations greater than 0.20 mg/ml substantially more bile acid (more than 1.3 mg/ml) was required to stimulate maximum activity. Addition of Triton X-100 or bovine serum albumin to the incubation mixtures increased the optimum taurocholate concentration. The dependence of the bile salt optimum on protein concentration appears to be related to the binding of the lipid substrate to membranous protein present in the tissue extracts. Release of the bound lipid is effected either by increasing the bile salt concentration or by adding Triton X-100. In the presence of excess bile salt human leucocyte, fibroblast and liver cerebroside sulphate sulphatase activity is stimulated by Triton at low protein concentrations; under identical conditions the pure or crude ox-liver enzyme is substatially inhibited. Our data also show that cerebroside sulphate sulphatase activity measured in extracts from leucocytes and fibroblasts, the tissues normally used to effect a diagnosis of metachromatic leucodystrophy, is the result of a complex interaction of bile salt, protein, Triton X-100 and probably the substrate itself. Any slight alteration in any of those factors, without a corresponding change in any or all of the others, can have a marked effect on the measured enzyme activity, and may lead to errors in the diagnosis of metachromatic leucodystrophy.     

SILICON UPTAKE BY ALGAE WITH NO KNOWN Si REQUIREMENT. II. STRONG pH DEPENDENCE OF UPTAKE KINETIC PARAMETERS IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

Silicon uptake kinetics of the diatom Phaeodactylum tricornutum (Bohlin) were examined at pH 8.8 ± 0.1 and pH 9.1 ± 0.1. Uptake follows hyperbolic saturation kinetics at both pH's, but at the higher pH the half-saturation constant for uptake is 11.8 μM, as opposed to 54.8 μM at the lower pH. When the uptake rate is examined as a function of the calculated concentration of the monovalent conjugate base, SiO(OH)₃^?, the half-saturation constant for uptake is 6.6 μM at either pH.