The specific assay of arylsulphatase C, a rat liver microsomal marker enzyme

Conditions based on previous assays with potassium p-acetylphenyl sulphate have been established for the specific assay of arylsulphatase C in rat tissues. The enzyme has optimum activity with 40mm substrate at pH8.0 in the presence of 0.1m-phosphate buffer. Under these conditions arylsulphatase C can be assayed without interference from the other arylsulphatase enzymes present and is useful as a marker for the endoplasmic reticulum in cell-fractionation studies.     

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.     

A fluorimetric method for measuring the activity of soil enzymes

Summary A fluorimetric method is described for the measurement of the activity of a range of soil enzymes. The method is based on the measurement of 4-methylumbelliferone (MUB), a fluorescent product liberated on hydrolysis of the enzyme substrate. The main advantage of the method over colorimetric techniques is that separation of MUB from the soil is unnecessary and the method is therefore suitable for routine, automated analyses. The method was used to measure the activity of β-cellobiase, β-galactosaminidase, β-glucosidase and β-xylosidase over a wide range of substrate concentration and in a range of soils. Kinetic parameters are reported for these enzymes. The method was also shown to be suitable for the assay of arylsulphatase and acid and alkaline phosphatase in soil. The technique should be applicable to a wide range of soil hydrolases, using the same assay methods.     

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.     

Micrococcus luteus endonucleases for apurinic/apyrimidinic sites in deoxyribonucleic acid. 1. Purification and general properties

Two chromatographically distinct endonucleases from Micrococcus luteus, specific for apurinic and apyrimidinic sites (AP-endonucleases A and B), have been extensively purified and characterized. Both are free from DNA glycosylase, unspecific endonuclease, and phosphatase activities. The two enzymes behave as monomeric proteins of approximately 35000 daltons. In addition to their different chromatographic properties on CM-cellulose, P-cellulose, hydroxylapatite, and DNA--Sepharose, both AP-endonucleases can be distinguished as follows: AP-endonuclease A has an isoelectric point of 4.8, shows a half-life of 4 min at 45 degrees C, reacts optimally at pH 7.5 and has a KM value of 2.3 X 10(-6) M. AP-endonuclease B has a pI of 8.8, is more stable at 45 degrees C (half-life of 10 min), and reacts optimally between pH 6.5 and pH 8.5; its KM value is 3.7 X 10(-6) M.     

Degradation of biochemical activity in soil sterilized by dry heat and gamma radiation.

The activities of three enzymes present in soil, phosphatases, urease, and decarboxylase, were monitered as indicators of the loss of biochemical information occurring when soil was sterilized by dry heat (0.08% relative humidity), gamma radiation, or a combination of both. More enzymatic activity was retained in soil sterilized by a long exposure to dry heat at relatively low temperature (8 weeks at 100.5 degrees C) than by a shorter exposure to a higher temperature (2 weeks at 124.5 degrees C). No enzymatic activity was detectable in soil sterilized by an even higher temperature (4 days at 148.5 degrees C). Soil sterilized with 7.5 Mrads of radiation retained much higher enzymatic activity than with heat sterilization. Combining sublethal doses of heat radiation effectively sterilized the soil and yielded enzymatic activities higher than those of soil sterilized by dry heat alone but lower than those of soil sterilized by radiation.     

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.     

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.     

Preparation and properties of immobilized papain and lipase.

Papain and lipase were immobilized on derivatized Sepharose 4-B. The activated agarose had a binding capacity of 1.2 micronmol amino groups/ml packed agarose or 17 mg proteins/g dry agarose. The immobilized enzyme preparations were tested for the effects of pH of assay, temperature of assay, and substrate concentrations. The effect of 6M urea on the activity of papain was also determined. Soluble forms of the enzymes were used for comparison. Immobilization of the enzymes resulted in slightly different pH and temperature optima for activities. For immobilized papain Km(app) was similar to the one observed with soluble papain. Immobilization of lipase, however, cause a decrease in Km values. The immobilized enzyme preparations were stable when stored at 4 degrees C and pH 7.5 for periods up to eight months. The soluble enzymes lost their activity within 96 hr under similar storage conditions. Immobilized papain did not lose any activity after treatment with 6M urea for 270 min, whereas soluble papain lost 81% of its activity after the urea treatment, indicating that the immobilization of papain imparted structural and conformational stability to this enzyme.     

Heat stability of Bacillus cereus enzymes within spores and in extracts.

Inactivation rates for nine enzymes extracted from Bacillus cereus spores were measured at several temperatures, and the temperature at which each enzyme had a half-life of 10 min (inactivation temperature) was determined. Inactivation temperatures ranged from 47 degrees C for glucose 6-phosphate dehydrogenase to 70 degrees C for leucine dehydrogenase, showing that spore enzymes were not unusually heat stable. Enzymes extracted from vegetative cells of B. cereus had heat stabilities similar to the respective enzymes from spores. When spores were heated and the enzymes were subsequently extracted and assayed, inactivation temperatures for enzymes within the spore ranged from 86 degrees C for glucose 6-phosphate dehydrogenase to 96 degrees C for aldolase. The internal environment of the spore raised the inactivation temperature of most enzymes by approximately 38 degrees C. Loss of dipicolinic acid from spores was initially slow compared with enzyme inactivation but increased rapidly with longer heating. Viability loss was faster than loss of most enzyme activities and faster than dipicolinic acid release.     

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.     

Effects of AET and MEA on the lysosomal hydrolase activities in the mouse organs.

The adult male Swiss mice were injected intraperitoneally with AET (2-aminoethylisothiouronium Br.HBr) or MEA (cysteamine HCl), in a toxic dose of 400 mg/kg body weight. The acid phosphatase (E.C. 3.1.3.2) and arylsulphatase (E. C. 3.1.6.1) activities in crude homogenates of liver and kidneys were assessed every fourth hour throughout a 24-h period. Different patterns of temporal changes in the acid phosphatase and arylsulphatase activities in liver and kidneys expressed in nkat per 1 mg of protein, 1 g of fresh tissue and per the whole organ weight, were found. The extent and timing of the alterations in the activity of each of the lysosomal hydrolases were dependent on the particular organ chosen and aminothiol compound given.     

Chemical,microbial and physical properties of manufactured soils produced by co-composting municipal green waste with coal fly ash

Increasing proportions of coal fly ash were co-composted with municipal green waste to produce manufactured soil for landscaping use. Only the 100% green waste treatment reached a thermophilic composting phase (?50 °C) which lasted for 6 days. The 25% and 50% ash treatments reached 36–38 °C over the same period while little or no self-heating occurred in the 75% and 100% ash treatments. Composted green waste had a low bulk density and high total and macro-porosity. Addition of 25% ash to green waste resulted in a 75% increase in available water holding capacity. As the proportions of added ash in the composts increased, the organic C, soluble C, microbial biomass C, basal respiration and activities of β-glucosidase, L-asparaginase, alkali phosphatase and arylsulphatase enzymes in the composted products all decreased. It could be concluded that addition of fly ash to green waste at a proportion higher than 25% did not improve the quality parameters of manufactured soil.     

Soil textural class plays a major role in evaluating the effects of land use on soil quality indicators

Inappropriate land use that negatively affects ecological processes and soil quality is generally considered to be the primary cause of soil degradation in tropical agroecosystems. We hypothesized that in addition to land use, soil textural class also has an important effect on ecological processes and soil quality. To test our hypothesis, effects of land use change on soil organic fractions as well as microbial and biochemical indicators were quantified for clayey and sandy-clay-loam soils within the native Cerrado biome, pasture (Brachiaria brizantha) and sugarcane (Saccharum officinarum) agroecosystems in southwestern Brazil (Minas Gerais state). Labile carbon, humic substances, soil microbial respiration (SMR), microbial biomass carbon (MBC), metabolic quotient (qCO₂), hydrolysis of fluorescein diacetate (FDA), beta-glucosidase, urease, phosphatase and arylsulphatase activities were measured for each sample. Labile carbon concentrations were not affected by land use but were lower in sandy-clay-loam soil than clayey soil. Humic substances were at the highest concentrations in the native Cerrado and the lowest in sugarcane agroecosystems. Sandy-clay-loam soil had lower humic acid concentrations than clayey soil. Soil microbial indicators (SMR, MBC and FDA) showed lower values in pasture and sugarcane agroecosystems than in the native Cerrado. FDA was a more sensitive microbial indicator than SMR and MBC for detecting land use and textural class differences. The qCO₂ indices were greater in sugarcane systems than in either pasture or native Cerrado systems. The activity of exocellular hydrolytic enzymes, such as beta-glucosidase, urease, phosphatase and arylsulphatase, was smaller in sugarcane and pasture agroecosystems than in native Cerrado ecosystems. Within the same land use, the activity of these enzymes was always greater in clayey soil than in sandy-clay-loam soil, indicating a higher impact of land uses on enzyme activities in clayey soils. Results for the measured indicators support the hypothesis that soil textural class plays a major role in assessing differences between land use systems in the Brazilian Cerrado biome.     

Stability test of six enzymes for internal quality control

The daily quality control for the determination of the catalytic activity concentrations of enzymes is an important aspect in clinical chemistry. Instead of the expensive, commercially available control sera, we have looked for a simple, reliable and cheap method for the quality control of enzyme determinations. Commercially available enzymes were suspended in an albumin solution and ampoules were filled with 1.0 ml of these various solutions. The ampoules were stored at 4 degrees C or -20 degrees C. Once a week, during 10 months, catalytic activities of these enzyme-albumin solutions were determined together with the same activities in freshly reconstituted control sera. Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine kinase and gamma-glutamyltransferase were determined at 30 degrees C according to well-described methods. alpha-Amylase was determined with the Phadebas method at 37 degrees C. Except for creatine kinase, the stability and reliability of these enzyme solutions are fully comparable with control sera during the experimental period. The catalytic activity concentration of creatine kinase decreased slowly during the 10 months. The enzyme solutions react in the same manner as commercial test sera on changes in the reaction conditions for the enzyme determinations. The conclusion seems justified that these enzyme solutions can be used for the daily quality control of the enzyme determinations instead of control sera.     

Kinetic characteristics of soluble and brush border alkaline phosphatase and sucrase activities in developing rat intestine: effect of hormones

Suckling rat intestine contains 35 and 65% of the cytosolic and membrane-bound alkaline phosphatase (AP) activities. The corresponding values for sucrase were 20 and 80% respectively. The amount of the soluble enzymes was reduced to 7-11% in adult rat intestine. Administration of cortisone, thyroxine or insulin to suckling animals induced adult type distribution of the enzymes. There were apparent differences in kinetic characteristics of soluble and brush border enzymes, but the kinetic properties of the normally developed and hormone-induced AP and sucrase were essentially similar. This suggested identical nature of these enzymes under these conditions. A biphasic Arrhenius plot was obtained for AP in weaned and hormone injected pups with a break point around 18 degrees C, while the soluble enzyme yielded a monophasic curve (Ea = 8-11 kcal/mole). Arrhenius plot for sucrase was monophasic in the suckling, hormone-injected and adult rat intestine (Ea = 8.3-15.1 kcal/mole). Membrane-bound enzymes were generally labile, while soluble enzyme activities were stable to heat treatment (sucrase at 50 degrees C and AP at 60 degrees C) in various experimental groups.     

Electron cytochemistry of developing and mature eosinophils in the bone marrow of the fowl and the duck

Synopsis Enzyme cytochemical studies have been carried out on eosinophils in the fowl and the duck. Peroxidase was found in all regions of the Golgi apparatus, the rough endoplasmic reticulum and the perinuclear cisternae of the early cells. In fowl eosinophil granules irregular deposits of peroxidase and arylsulphatase final reaction product were found, but the acid phosphatase deposits were even. In the duck in contrast, peroxidase was demonstrated in the external part of the granule only. Acid phosphatase and arylsulphatase were found in both the interna and the externa of the duck eosinophil granules. An ammoniacal silver nitrate reaction for the presence of the histone arginine was also studied. Silver deposits were found occupying all regions of the granules of eosinophils from both species of bird.The presence of the hydrolytic enzymes acid phosphatase and arylsulphatase in avian eosinophil granules supports the theory that these structures are lysosomal in nature and that they correspond with mammalian eosinophils in this respect.     

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.     

长期施肥对东北黑土酶活性的影响

在中国科学院海伦农业生态实验站长期定位试验基础上,研究了长期施用化肥和有机肥对黑土0～20 cm和20～40 cm土层土壤脲酶、磷酸酶、过氧化氢酶和转化酶活性及土壤全碳和全氮的影响.结果表明：长期施用化肥和有机肥均不同程度地提高了0～20 cm和20～40 cm土层土壤磷酸酶、脲酶和转化酶活性,特别是化肥和有机肥配合施用显著增加了该3种土壤酶的活性,增幅分别为43.6%～113.2%、25.9%～79.5%、14.7%～134.4%(0～20 cm)和56.1%～127.2%、14.5%～113.8%、16.2%～207.2%(20～40 cm),长期施用化肥对土壤过氧化氢酶活性影响不大.随着土层深度的增加,土壤磷酸酶、脲酶和转化酶活性均有所降低;长期施用氮肥对土壤脲酶、施用磷肥对土壤磷酸酶有明显的促进作用;长期施肥对土壤全C、全N含量及C/N也有明显影响.     

The effects of the continuous administration of N,N-dimethyl-4-phenylazoaniline (DAB) on the activities and the inducibilities of some drug-metabolizing enzymes in rat liver.

(1) The effect of feeding a relatively low-protein diet containing 0.06% DAB for 29 weeks on the activity of DAB-azoreductase, nitroreductase (p-nitrobenzoic acid), N-oxidase (N,N-dimethylaniline), N-demethylase (DAB), cytochrome P-450, NADPH-cytochrome c reductase, beta-glucuronidase and arylsulphatase A were studied. Rapid decreases occurred in the activities of the first six enzymes, reaching minimal values at between 4 and 8 weeks. Activities then increased in all cases to control or nearly control levels. This rate of increase was least for cytochrome P-450. At 4 weeks azoreductase activity with the chemotherapeutic agent CB10-252 (I) as substrate was significantly higher than in control rats. Early increases occurred in the activities of beta-glucuronidase and arylsulphatase A and the activity of the latter never dropped below the control level. (2) An investigation was made of the differential effects of dye feeding on some of the enzyme activities in the two major liver lobes and differences were found. (3) The effect of phenobarbital (PB) pretreatment on the DAB-fed rats was studied at 4-week intervals. The activities of DAB-azoreductase and of nitroreductase increased throughout the whole period, while the activities of the lysosomal enzymes were decreased. (4) After feeding DAB for 4 weeks the effect of PB and 3-methylcholanthrene (MC) on the activities of DAB-azoreductase, CB10-252-azoreductase and components of the azoreductases-cytochrome P-450, NADPH-cytochrome c reductase, the CO-CB10-252-azoreductase was not induced by PB or MC, and CO did not inhibit its reduction. Its reduction depended only slightly on NADH. CO caused a greater relative decrease in the activity of DAB-azoreductase in dye-fed animals and also in animals following PB and MC pretreatment, implying a greater role of cytochrome P-450 in dye-fed animals.