Arylsulphatase activity in the oviduct of the frogRana esculenta. II. Progesterone-induced changes following ovariectomy and hypophysectomy

Summary The effects of progesterone treatment on arylsulphatase activity were studied histochemically and biochemically in the frog oviduct under different experimental conditions. In ovariectomized animals, the hormone induced a large increase in enzyme activity, while in hypophysectomized ones there was a large decrease in this activity. These results indicate that the facilitatory and inhibitory effects of progesterone on arylsulphatase activity are influenced by the presencein situ of the gonad. Hypotheses are advanced to explain different effects of the progesterone treatment.     

Autophagy-related changes of arylsulphatases A and B in rat liver lysosomes.

The total arylsulphatase activity and the relative activities of lysosomal arylsulphatases A and B were measured in the liver of control rats and rats subjected to treatments that provoke hepatic autophagocytosis. The total liver arylsulphatase activities were increased in starved and starved glucagon-treated rats, but not in sham-operated and hepatectomized rats. Arylsulphatases A and B in the mitochondrial-lysosomal (M-L) fraction were separated by polyacrylamide-gel electrophoresis at pH 8.8; they were made visible by incubating the gels with p-nitrocatechol sulphate as substrate, and measured by quantitative densitometry. In untreated controls, arylsulphatases A and B comprised 41.4 +/- 0.5% and 58.6 +/- 0.5% of the total arylsulphatase activity respectively; the arylsulphatase A/arylsulphatase B activity ratio was 0.71. All experimental treatments produced a significant decrease in the percentage of lysosomal arylsulphatase present as the A form and an increase in that present as the B form, and the activity ratio of arylsulphatase A/arylsulphatase B declined. The magnitude of these changes increased in the following direction: starvation for 24h=sham hepatectomy less than glucagon + starvation less than subtotal hepatectomy. These results indicate that the arylsulphatase A/arylsulphatase B activity ratio in liver lysosomes of normal rats is maintained within rather narrow limits, and this ratio declines during enhanced autophagocytosis. These findings, together with observations that suggest that arylsulphatase B may be a partially degraded form of arylsulphatase A, are consistent with the view that the A form is more rapidly converted into the B form during autophagy, owing to the digestive activity of the other lysosomal hydrolases present in autophagic vacuoles.     

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections.

The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study show that the enzyme histochemical technique--except for some limitations--is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.     

Studies on the Arylsulphatase and phenol sulphotransferase activities of Aspergillus oryzae.

1. Crude extracts of Aspergillus oryzae grown under conditions of sulphur limitation possess high arylsulphatase activity. 2. This activity can be greatly enhanced by the inclusion of tyramine or a number of other phenols in the assay medium. 3. The arylsulphatase activity of these extracts can be resolved into three distinct fractions by chromatography on DEAE-cellulose. 4. The effect of tyramine is restricted to one of these fractions only. 5. Evidence is presented which indicates that this effect is the consequence of a phenol sulphotransferase activity, which shows no requirement for 3'-phosphoadenosine 5'-phosphate as a cofactor, and which will not transfer sulphate from 3'-phosphoadenosine 5'-sulphatophosphate to potential phenolic acceptors. 6. The three enzymes differ also in their molecular weights and substrate specificities.     

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.     

Localization of arylsulphatase A and B activities in rat kidney.

Very high arylsulphatase activity has been detected in rat kidney. It is the highest in renal cortex (19 U/g tissue), 3-30 times higher than in other rat organs. Histochemically, arylsulphatase B (N-acetylgalactosamine-4-sulphate sulphatase) activity is localized in large lysosomes of proximal convoluted tubules, where it accounts for over 90% of total arylsulphatase activity. This suggests that the enzyme plays an important role in the degradation of endocytosed sulphated oligosaccharides.     

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.     

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.     

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections

Summary The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study whow that the enzyme histochemical technique — except for some limitations — is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.     

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.     

Acid phosphatase and arylsulphatase activities in testes after AET or MEA treatment of adult mice.

Temporal changes of acid phosphatase (E.C. 3.1.3.2) and arylsulphatase (E.C. 3.1.6.1) activities in testes of adult Swiss mice after AET (2-amino-ethylisothiouronium Br. HBr) or MEA (cysteamine HCl) treatment, were studied. The animals were injected intraperitoneally with the S-containing substances in a single dose of 400 mg/kg body weight. The enzyme activities in crude organ homogenates were assessed every four hours during a 24-hour period. Administration of the aminothiol agents to mouse organism caused greater changes in the acid phosphatase activity than in the arylsulphatase activity, and the two chemical compounds AET and MEA given, influenced the enzyme activities in testes in a different way. Treatment of mice with AET resulted in a decrease of the acid phosphatase activity related to 1 g of fresh tissue at 16.00 and the whole organ weight at 24.00 and 16.00 as well as in a decrease of the arylsulphatase activity expressed per the whole weight of testes at 08.00. After MEA injection, the acid phosphatase activity related to 1 mg of protein, 1 g of fresh tissue and the whole organ weight was decreased at 20.00(1), and the enzyme activity expresse per 1 mg of protein and 1 g of fresh tissue was increased at 24.00, but the arylsulphatase activity related to both 1 mg of protein at 08.00, 12.00 and to the whole weight of testes at 08.00, was reduced.     

Arylsulphatase A activity and sulphatide concentration in the female rabbit oviduct are under physiological hormonal influence

Summary Oviduct samples of female rabbits in different phases of the reproductive cycle were analysed to establish the role of sex steroid hormones in the regulation of sulphatide concentration and arylsulphatase A activity. In addition to biochemical procedures, histochemical techniques were used to localize both enzyme activity and the natural substrate. The plasma concentrations of progesterone and 17β-oestradiol were determined by radioimmunoassay (RIA). The findings show that the parameters examined undergo considerable changes during the different phases of the reproductive cycle. Oestrogens exert an inducing action on arylsulphatase A activity, while progesterone inhibits it. Fluctuations of the catabolic arylsulphatase activity condition the sulphatide concentration, which reaches maximum values at the eighth post-ovulatory day when progesterone dominance is consolidated. In this phase of the reproductive cycle, thin-layer chromatography confirms the presence not only of larger quantities of sulphatides, but also of all other lipid fractions.     

Studies on the heparin sulphamidase activity from rat spleen. Intracellular distribution and characterization of the enzyme

A sulphamidase activity present in rat spleen capable of hydrolysing N-[(35)S]sulphated heparin was studied. This activity was associated with the lysosomal fraction. Studies in vivo showed that the rat is capable of significantly desulphating heparin. Lysosomes in all the major tissues can effectively accumulate heparin. The heparin sulphamidase and arylsulphatase activities from rat spleen were separated by isoelectric focusing. Heparin sulphamidase was a distinct entity from all the arylsulphatase activities.     

The minor anionic form of arylsulphatase B (arylsulphatase Bm) of monkey brain. Purification and phosphoprotein nature

The anionic form of arylsulphatase B (arylsulphatase Bm) was purified to apparent homogeneity from monkey brain through steps involving chromatography on diethylaminoethyl-cellulose, Blue-Sepharose, Biogel HTP and finally Biogel P-300 gel filtration. The molecular weight of the purified enzyme as deduced by gel filtration on Biogel P-300 and by sodium dodecylsulphate gel electrophoresis was ∼ 30,000.Escherichia coli alkaline phosphatase treatment of arylsulphatase Bm resulted in the conversion of upto 84% of the enzyme into a less charged form of enzyme, that could not bind to diethylaminoethyl cellulose. Potassium phosphate an inhibitor of alkaline phosphatase prevented this conversion. Upon acid hydrolysis the purified enzyme yielded approximately 7.0 mol of inorganic phosphate per mol of protein.Vibrio cholerae neuraminidase treatment did not alter the charge on arylsulphatase Bm.     

Arylsulphatase activity and cerebroside sulphates in the frog oviduct during the reproductive cycle

Summary The presence of arylsulphatase A and cerebroside sulphates in different tracts ofRana esculenta oviduct during different phases of the reproductive cycle were investigated by histochemical and biochemical procedures. The results indicate that enzyme activity shows seasonal fluctuations connected with the phase of the sexual cycle. The concentrations of cerebroside sulphates (the natural substrates of arylsulphatase A) is related to the activity of this hydrolytic enzyme. The role of arylsulphatase A activity in regulating the substrate concentration, and particularly that of sulphatides, is discussed.     

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.     

Studies of the oestrogen sulphatase and arylsulphatase C activities of rat liver

Detailed studies on the hydrolysis of p-acetylphenyl sulphate and oestrone sulphate by rat liver preparations strongly indicate that arylsulphatase C and oestrogen sulphatase are the same enzyme. Liver is the richest source of both enzymes, which have identical intracellular distributions, being localized mainly in the microsomal fraction. Low oestrogen sulphatase and arylsulphatase C activities were present in foetal liver and these increased at a similar rate after birth. The activities of the enzymes in an ethionine-induced hepatoma were similarly low. Results of heat inactivation, mixed-substrate and competitive-inhibition experiments employing liver microsomal fractions were also consistent with one enzyme being involved. Oestradiol-17beta 3-sulphate was also hydrolysed by microsomal preparations and activity towards both this substrate and oestrone sulphate was inhibited by oestrone and oestradiol-17beta. The physiological significance of this inhibition is discussed.     

Differential stabilities of soil enzymes. Assay and properties of phosphatase and arylsulphatase.

Methods have been refined for the assay of phosphatase and arylsulphatase activities in soil, based on the chromogenic p-nitrophenyl ester substrates. Basic assay conditions have been defined, and pH optima and kinetic parameters have been determined. The enzymes follow Michaelis-Menten kinetics; this conclusion is based on three methods of analysis of data determined over a wide range of substrate concentrations. The enzyme activities are very stable to storage of wet soil for up to 4 weeks at soil temperatures and above. For example, phosphatase had a half-life of approximately 2 weeks at 50 degrees C; arylsulphatase was rather less stable. Both enzymes retained 80% of activity after incubation with pronase for 1 week at 25 degrees C. On the basis of this work and studies on other soil enzymes, it is concluded that remarkable stability is a general feature of soil enzymes.     

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.