Purification of soluble arylsulphatases from ox brain

1. Two soluble arylsulphatases (A and B) have been extracted from ox brain by a modified Albers autolysis method and purified by acetone and ammonium sulphate precipitation and dialysis. 2. A 1600-fold purification was achieved with arylsulphatase A and 320-fold purification with arylsulphatase B. 3. The specific activity of arylsulphatase A was 266000 4-nitrocatechol units/mg. of protein N, and that of arylsulphatase B was 64600units/mg. of protein N. 4. Arylsulphatase A seems to be electrophoretically homogeneous. 5. With 3mm-dipotassium 2-hydroxy-5-nitrophenyl sulphate as substrate the optimum pH for the activity of arylsulphatase A was 4.7, and for arylsulphatase B it was 6.1 with a 60mm solution of the same substrate.     

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.     

Arylsulphatases in the rabbit oviduct: postovulatory changes tested by histochemical and biochemical procedures

Summary A histochemical and biochemical study of the activity of arylsulphatases A and B was carried out on the oviduct of female rabbits during the first days after mating. The histochemical results demonstrated that the ampullary and the isthmic epithelial cells have a positive reaction to the sulphatases during the whole of the postovulatory period tested. The enzymatic activity is mainly localized in the basal cellular cytoplasm. The biochemical results confirmed that both arylsulphatase A and B are active. Arylsulphatase A activity is more intense in the ampulla than in the isthmus and it increases during the whole of the postovulatory period; in the isthmus the activity increases up to 72 h, thereafter decreasing again. The arylsulphatase B activity is always lower than arylsulphatase A activity; maximum activity is reached between 66 to 72 h after mating. The arylsulphatase B is relatively higher in the ampulla than in the isthmus. The biological role of these enzymes is discussed in relation to the regulation of the sulphated glycoconjugates.     

Enzyme activity in intertidal sands and salt-marsh soils

Summary Arylsulphatase activity was detected and characterized in intertidal sands and salt-marsh soils. In sands, highest activity was found in the rhizosphere ofHippophae rhamnoides L; although low activity was also found near Ammophila roots and in sands lacking plant-cover. Highest arylsulphatase activities, however, occurred in salt-marsh soils underSalicornia sp. Properties of arylsulphatase were similar in nearly all respects, to those reported for soil and marine sediment arylsulphatase.Linearity between amount of sand or salt-marsh soil and cellulase and rhodanese activities were also demonstrated, but no attempt was made to further characterise these enzymes.     

Effects of acute and chronic administration of ethanol on rat brain arylsulphatase A and B

Acute (4g/kg i.p.) and chronic (Sustacal^TM diet containing 10% ethanol for 20 days) administration of ethanol to male Sprague-Dawley rats produced no change in the content or enzyme activity of brain arylsulphatase A. In contrast to the lack of effect on arylsulphatase A, the acute and chronic administration of ethanol resulted in an increase in the activity of brain arylsulphatase B (15.8% and 18.4%, respectively). However, the enhancement of the activity of arylsulphatase B was observed only in the brain homogenates which were subjected to osmotic shock. No enhancement of the arylsulphatase B activity was found in the supernatant soluble fraction after the acute and chronic administration of ethanol. Furthermore, acute and chronic ethanol administration did not alter the activities of arylsulphatase A and B in microsomes which have been suggested as sites of the synthesis of lysosomal hydrolases. In addition, 80 mM ethanol, in vitro, did not affect the activity of arylsulphatase A and B. The results of the present study suggest that the acute or chronic administration of ethanol might enhance the activity of lysosomal membrane bound arylsulphatase B via altering the lipid metabolism of lysosomal membranes.     

A rapid direct assay for the determination of the separate activities of the three arylsulphatases of Aspergillus oryzae.

1. The three arylsulphatases of Aspergillus oryzae exhibit pronounced kinetic differences and substrate specificities. Arylsulphatase I hydrolyses all substrates tested, whereas arylsulphatase III will not hydrolyse tyrosine O-sulphate or phenolphthalein disulphate. Arylsulphatase II does not hydrolyse p-nitrophenyl sulphate or phenolphthalein disulphate at appreciable rates in the absence of added phenolic compounds. Phenols such as tyramine increase the rate of hydrolysis of these substances by this enzyme 1000-fold. At pH 6.9 arylsulphatase I exhibits an apparent Km of 0.1 mM for p-nitrophenyl sulphate, whereas the Km of arylsulphatase III for this substrate is 1 mM. 2. These differences were utilized to develop an assay procedure which can be used to determine the separate activities of the three enzymes present in mixtures. This assay has potential use as a means of examining the relative activities of the three enzymes in investigations of the differences in the mechanisms regulating their synthesis.     

Purification and some properties of arylsulphatases A and B from rabbit kidney cortex.

Arylsulphatases A and B (EC 3.1.6.1) of rabbit kidney cortex were purified 5250- and 7720-fold respectively by a multiple-column-chromatography method. The specific activity toward 4-nitrocatechol sulphate was 42mumol/min per mg for arylsulphatase A and 62 mumol/min per mg for arylsulphatase B. Each enzyme migrated as a single band on polyacrylamide-gel electrophoresis, and the enzyme activity corresponded to the band of protein on the gel. The rate of hydrolysis of ascorbic acid 2-sulphate by arylsulphatase A was three times that for cerebroside 3-sulphate. Arylsulphatase B hydrolysed UDP-N--acetylgalactosamine 4-sulphate and glucosamine 4,6-disulphate, but not galactosamine 6-sulphate.     

THE REGIONAL DISTRIBUTION, AGE DEPENDENT VARIATION AND SPECIES DIFFERENCES OF BRAIN ARYLSULPHATASES

Abstract— The relative proportions of arylsulphatase A and B were determined by the method of B aum , D odgson and S pencer (1959) in brains of various animal species and it was found that there was a considerable variation in the concentration of these two enzymes.
Arylsulphatase A and B of various animal species including rat, man, monkey, sheep and chicken were partially separated using zinc acetate fractionation procedure and gel electrophoresis. The chicken brain arylsulphatase A had a similar electrophoretic mobility to that of arylsulphatase B of other species. Further, chicken brain arylsulphatase A precipitated at a zinc acetate concentration of 0005 M, a condition under which arylsulphatase B from the brain of other species precipitated.
Kinetic properties such as K _m value and inhibitory effect of sulphite and phosphate ions indicated that chicken brain arylsulphatase A was similar to arylsulphatase A of other species.
The results on regional distribution of arylsulphatase A and B activities in monkey brain and in developing rat brain suggest a relationship between arylsulphatase A and sulphatides and arylsulphatase B and mucopolysaccharides.     

Arylsulphatase C and Estrone Sulphatase of Sheep Hypothalamus, Preoptic Area, and Midbrain: Separation by Hydrophobic Interaction Chromatography and Evidence for Differences in Their Lipid Environment

Abstract: Arylsulphatase C and estrone sulphatase activities of sheep hypothalamus-preoptic area-midbrain were examined for their susceptibility to phospholipase action. Russel's viper phospholipase A could completely inactivate estrone sulphatase without affecting arylsulphatase C. The latter was partially inactivated by S. aureus phospholipase C but not by C. welchi phospholipase C. Both arylsulphatase C and estrone sulphatase were inactivated to different extents by sodium deoxycholate, which is known to activate the intrinsic phospholipases of brain. Hydrophobic interaction chromatography on phenyl-Sepharose resulted in the differential elution of arylsulphatase C and estrone sulphatase. The results suggest that one enzyme is not responsible for arylsulphatase C and estrone sulphatase activities.     

THE PATTERNS OF ARYLSULPHATASES A AND B IN HUMAN NORMAL AND METACHROMATIC LEUCODYSTROPHY TISSUES AND THEIR RELATIONSHIP TO THE CEREBROSIDE SULPHATASE ACTIVITY

Abstract— The arylsulphatase A and B patterns of human tissues and leucocytes have been established by isoelectric focussing. Assay conditions, which enable an evaluation of these patterns as quantitatively as possible, have been studied. The dependences of the enzyme patterns on the origin of the tissues and on the storage conditions have been determined. The arylsulphatase A obtained by isoelectric focussing exhibits cerebroside sulphatase activity in the presence of detergents. A purified preparation of the arylsulphatase B likewise shows a significant, although low, cerebroside sulphatase activity. In cases of the conventional types of metachromatic leucodystrophy the arylsulphatase A activity is missing, while in an atypical form of this disease ('ML Variant' according to A ustin et al . (1965) the arylsulphatase A, B and C activities are deficient. In both forms, however, residual activities of the deficient enzymes could be detected which showed isoelectric points identical to those of the normal enzymes.
The following nomenclature is proposed: 'Variant B' for the conventional type, in which the arylsulphatase B activity is present, and 'Variant O' for the exceptional cases, in which all arylsulphatase activities are deficient. The significance of the cerebroside sulphatase activity of arylsulphatase B for a possible residual turnover of cerebroside sulphates in the conventional type of the disease is discussed.     

Catalytic and immunochemical properties of arylsulphatase A from urine, modified by potassium ferrate

Arylsulphatase A (EC 3.1.6.1.) from urine was inactivated with potassium ferrate, a strong oxidizing agent. The inhibition could be prevented by competitive inhibitors, tetraborate and orthophosphate. Tetraborate which was shown to be a powerful competitive inhibitor (determined Ki = 4 X 10(-5) M) gave more efficient protection. The partially inactivated enzyme exhibited a Km value similar to that of the unmodified arylsulphatase A, and its Vmax decreased in proportion to the loss of enzymatic activity. The partially modified enzyme did not lose its ability to catalyse hydrolysis of p-nitrocatechol sulphate according to the "anomalous kinetics" exhibited towards this substrate and characteristic for arylsulphatase A. The immunochemical properties of arylsulphatase A either fully or partially inactivated were similar to those of the native enzyme. The results allow to conclude that ferrate reacts with arylsulphatase A in its active site. Thus ferrate seems to be a very sensitive probe for amino acid residues essential for catalytic activity of arylsulphatase A.     

Arylsulphatase in echinoderm immunocompetent cells

Summary Two peaks of arylsulphatase activity were detected biochemically in coelomocyte lysate preparations of seven different Echinodermata species. Both peaks were inactivated by sulphite and sulphate ions, indicating that Type II arylsulphatase is present in the coelomocytes of the species tested. Arylsulphatase was localized histochemically in the granules of spherula cells, suggesting that in echinoderms a common cell type with granulocyte-like functions is present. The enzyme was also localized in the amoebocytes of echinoid species.     

Differential repression of arylsulphatase synthesis in Aspergillus oryzae.

1. The activities of the three arylsulphatases (arylsulphate sulphohydrolase, EC 3.1.6.1) of Aspergillus oryzae produced under a variety of repressing and non-repressing conditions were determined. 2. These enzymes exhibit different sensitivities to repression by inorganic sulphate. 3. Arylsulphatase I, but not arylsulphatases II and III, exhibits a transient de-repression in the early growth phase in sulphate media. 4. When the fungus is cultured in repressing media and subsequently transferred to non-repressing media, the synthesis of the three enzymes is non-co-ordinate. 5. Growth of the fungus in media containing choline O-sulphate or tyrosine O-sulphate as the sole source of sulphur results in complete de-repression of arylsulphatase I, But the synthesis of arylsulphatases II and III is essentially fully repressed. 6. The marked similarities between the repression characteristics of arylsulphatases II and III, contrasted with those of arylsulphatase I, indicate that the genetic locus of arylsulphatase I is distinct from that of arylsulphatases II and III, suggesting that there are distinct physiological roles for the enzyme.     

Arylsulphatase and acid phosphatase activity associated with developing and ripe spermatozoa of the musselMytilus edulis

Summary Arylsulphatase and acid phosphatase activity were demonstrated cytochemically in spermatozoa of the marine musselMytilus edulis. Reaction product resulting from arylsulphatase activity was measured using an integrating microdensitometer and found to increase with incubation time and to be variable according to the pH of the incubation medium. Two peaks in activity, at pH 4.5 and 6.0 were evident for some experimental protocols suggesting the possibility of two isoenzymes; however, studies on the ultrastructural localization of the enzyme showed no difference between sites of activity for the two pH values. Ultrastructural localization of arylsulphatase showed activity associated with the Golgi body of developing spermatids and in particular within the proacrosomal vesicles but limited to the periphery of the acrosomal vesicle which is formed with the fusion of the proacrosomal vesicles. In spawned spermatozoa arylsulphatase activity was localized in association with the axial rod and subacrosomal material; activity also occurred along the outer acrosomal membrane and within the acrosomal vesicle and also associated with the acrosomal process following the acrosome reaction. Sulphate groups were demonstrated cytochemically within the vitelline coat of oocytes in the mantle tissue. These findings suggest that arylsulphatase could be one of the lysins previously demonstrated inM. edulis spermatozoa. Acid phosphatase activity was demonstrated in spawned spermatozoa around the nuclear envelope and along the outer acrosomal mambrane.     

Arylsulphatases in human brain: separation, purification, and certain properties of the two soluble arylsulphatases

Abstract— Arylsulphatases (aryl-sulphate sulphohydrolases; E.C. 3.1.6.1) in the soluble subcellular fraction (105000g, 2 h) of human brain were partially purified by ammonium sulphate fractionation, ion exchange chromatography, and Sephadex gel filtration. Potassium-4-methylumbelliferone-sulphatase (MUS-sulphatase) adsorbed on DEAE-cellulose was purified approximately 700-fold over activity in the soluble fraction and the unadsorbed MUS-sulphatase was similarly purified approximately 600-fold. The arylsulphatase adsorbed to DEAE-cellulose exhibited a K_m value for MUS of 12.5 mM and a pH optimum of 5.7, whereas the unadsorbed arylsulphatase exhibited a K_m value for MUS of 8.3 mM and a pH optimum of 5.4. The molecular weights of the two enzymes were approximately 109,600 and 51,300, respectively. Sulphate (0.5 mM) showed pronounced mixed inhibition only of the unadsorbed arylsulphatase. Ag⁺ ions (0.25 mM) showed 96 per cent inhibition of the adsorbed arylsulphatase, whereas an activation of the unadsorbed arylsulphatase was observed.     

Pyroglutamyl [correction of Pyroglutamil] -peptidase I activity in the cortex of the cat brain during development.

Thyrotropin Releasing Hormone (TRH) is a principal regulator of thyroid system function. However, significant concentrations of TRH were found throughout the central nervous system, the cortex being one of the areas most richly endowed with thyroliberin. Research concerning the functional role of this brain peptide is performed, in part, by studying peptidase enzymes which may be involved in the inactivation of the peptide. The pGlu-His bond is cleaved by two pyroGlu-peptidases: I (soluble) and II (membrane-bound). In the present investigation, developmental activity of the soluble form is described in the cortices of the cat brain. The selected maturation stages were 15 and 30 days postnatal. The cortices were the frontal, parietal, area 17 and areas 18 and 19 as a whole, distinguishing brain hemispheres in all cases. PyroGlu-aminopeptidase I activity increased significantly with age in all the brain regions except area 17. It is suggested that this enzyme activity plays a part in the neurochemical changes that take place during brain maturation.     

Purification and partial characterization of arylsulphatase C from human placental microsomes

Arylsulphatase C (EC 3.1.6.1) has been purified 300-fold from human placental microsomes using a four step procedure involving solubilization with Triton X-100, chromatography on hydroxyapatite, column chromatofocussing and ion-exchange chromatography on DEAE-Sepharose. The purified enzyme is electrophoretically homogeneous and has a molecular weight of 440 000 as determined by polyacrylamide gradient gel electrophoresis. On analysis of the preparation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate a polypeptide of molecular weight 74 000 was observed, suggesting that the enzyme as purified may be a hexamer. The behaviour of the enzyme during chromatofocussing indicates the enzyme has a pI of 6.56. Steroid sulphatase, as measured by activity towards dehydroepiandrosterone sulphate, co-purifies with arylsulphatase C suggesting that both activities are due to a single enzyme.     

Quantitative Studies of White Matter : II. Enzymes involved in triose phosphate metabolism

Methods for measurement of glyceraldehyde-P dehydrogenase, triose-P isomerase, fructose 1,6-diphosphate aldolase, and the DPN-linked and flavin-linked α-glycero-P dehydrogenases in small amounts of tissue have been worked out. These enzymes have been measured in ten tracts in rabbit central nervous system. The activities of all the enzymes measured, except the flavin-linked α-glycero-P dehydrogenase, are present in larger amounts in lightly myelinated than in heavily myelinated tracts, but are relatively low in fibrillar layer of olfactory bulb, which is unmyelinated. Aldolase, like P-fructokinase (measured previously), is especially low in fibrillar layer. Taken together with relatively high 6-P-gluconate dehydrogenase activity found earlier this supports the hypothesis that the pentose-P shunt is particularly active in this tract. The activity of DPN-linked α-glycero-P dehydrogenase is inversely proportional to the lipid content of the myelinated tracts, which suggests that its primary role is not related to lipid synthesis in adult brain. The activities of flavin-linked α-glycero-P dehydrogenase are unrelated to those of the DPN-linked enzyme, which is contrary to expectation if the two enzymes function as partners in the "α-glycerophosphate shuttle."     

Relative distribution of arylsulphatases A and B in rat liver parenchymal and other cells

The relative activities of arylsulphatases A and B were measured in rat liver parenchymal and non-parenchymal cells, in peritoneal macrophages and in a number of rat tissues. Although absolute values cannot be obtained, it was shown that the arylsulphatase B/arylsulphatase A activity ratio is much higher in non-parenchymal cells than in parenchymal cells. The ratios in adrenals, brain and testis are very similar to each other but differ from those found in spleen, kidney and liver. These ratio variations may be caused by alterations in the activity of the B enzyme rather than the A enzyme. The relatively high B enzyme/A enzyme ratios in all rat tissues explains why the method devised for the independent assay of human arylsulphatases A and B cannot be employed with rat tissues.     

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.