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.     

Enzymic degradation of heparin A sulphamidase and a sulphoesterase from Flavobacterium heparinum

A sulphamidase and a sulphoesterase were isolated from adapted cells of Flavobacterium heparinum. These enzymes were partially purified from the ;heparinases' present in the bacterial extracts and characterized. The sulphamidase has a high specificity for glucosamine N-sulphate and glucosamine 2,6-disulphate. The activity decreases sharply with increasing molecular weight of the substrates tested. The sulphamidase and the sulphoesterase activities were distinguished from each other by their different sensitivities to concentration of phosphate ion and to temperature. The importance of these enzymes in the study of the structure of heparin is discussed.     

The intracellular distribution of arylsulphatase C and oestrogen sulphatase activities in rat kidney and spleen.

The arylsulphatase C of rat kidney and spleen is localized predominantly in the microsomal fraction. The distribution pattern is paralleled by that of oestrogen sulphatase, as in rat liver. The general usefulness of arylsulphatase C as a microsomal marker enzyme is indicated.     

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.     

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.     

Studies on the meta and para O-sulphation of the catechol compound 3,4-dihydroxybenzoic acid by rat liver sulphotransferase in vitro.

The enzymic meta and para O-sulphation of 3,4-dihydroxybenzoic acid was investigated in vitro with a dialysed high-speed supernatant from rat liver. The O-sulphated products were identified by comparison with the reference compounds. The chemical synthesis and identification of the reference O-sulphate esters is described in detail. The sulphotransferase activity of the dialysed supernatant from rat liver towards 3,4-dihydroxybenzoic acid was 580 pmol of 3-O-sulphate and 120 pmol of 4-O-sulphate formed/min per mg of protein at the optimal pH of 7.4. The meta/para ratio of O-sulphation was independent of pH, time of incubation, concentration of enzyme and presence of dithiothreitol. The O-sulphate esters of 3,4-dihydroxybenzoic acid were found to be good substrates for the arylsulphatase reaction at pH 5.6. The arylsulphatase activity of a dialysed preparation from rat liver was 4.0 nmol of 3-O- and 5.7 nmol of 4-O-sulphate ester hydrolysed/min per mg of protein, respectively. Arylsulphatase from Helix pomatia had an activity of 620 pmol of 3-O-sulphate and of 16.6 nmol of 4-O-sulphate ester hydrolysed/min per unit (mumol/h) of sulphatase.     

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.     

Improvements in the method for the electron microscopic localization of arylsulphatase activity

Summary The optimal conditions for the demonstration of arylsulphatase activity in the proximal convoluted tubule cells of the rat kidney were studied at light and electron microscopic level. 8-hydroxyquinoline sulphate, p-nitrophenyl sulphate and 2-hydroxy-5-nitrophenylsulphate were used as substrates and barium and lead as capturing ions. The effect of fixation, capturing ions, substrate concentration and pH was studied biochemically. The results of these biochemical studies were then verified histochemically. Finally a recommended method for the light and electron microscopic demonstration of arylsulphatase activity was presented.     

Phosphatidate biosynthesis in mitochondrial subfractions of rat liver

1. After conventional fractionation of rat liver homogenates in 0.88m-sucrose the mitochondrial fraction was subjected to short-term water lysis followed by separation of the resulting membrane preparations. 2. Phosphatidate formation was measured in all subcellular fractions and subfractions and was compared with the distribution of succinate dehydrogenase, monoamine oxidase, rotenone-insensitive NADH cytochrome c reductase, arylsulphatase, urate oxidase, arylesterase and glucose 6-phosphatase. 3. The results obtained indicated that mitochondria were capable of synthesizing phosphatidate, though this activity was only about one-third of the total homogenate activity. 4. Mitochondrial phosphatidate formation was located predominantly in the outer mitochondrial membrane. Although this membrane preparation was found to be significantly contaminated by the microsomal fraction, this contamination was estimated to account for not more than about 20% of the total phosphatidate formation observed in preparations of outer mitochondrial membrane.     

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.     

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.     

The development of arylsulphatase in the small intestine of the rat

1. Arylsulphatase activity was measured in stomach, proximal and distal third of small intestine, colon, liver and kidney of foetal and neonatal Sprague-Dawley rats and Swiss mice, with nitrocatechol sulphate as substrate. 2. The specific activity in the distal small intestine, but not in the stomach, proximal small intestine or colon, increased about fourfold between 5 and 16 days after birth in both conventional and germ-free rats. 3. No comparable increase occurred in the distal small intestine of the mouse. 4. The specific activity of acid phosphatase in the distal small intestine of the rat rose only slightly when the arylsulphatase activity increased. 5. The pH optimum and Michaelis constant of arylsulphatase activity of the distal small intestine were similar for 1-day-old, 9-day-old and adult rats. 6. When extracts of distal small intestine of 1-day-old and 9-day-old rats were incubated together, the arylsulphatase activities were additive.     

Purification and characterization of a type II phosphatidylinositol 4-kinase from rat spleen and comparison with a PtdIns 4-kinase from lymphocytes.

A PtdIns 4-kinase from rat spleen particulate fraction was purified to homogeneity and its molecular properties were compared with a PtdIns 4-kinase from splenic lymphocytes. The enzyme activity was solubilized from spleen particulate fraction with Triton X-100 and chromatographed sequentially on phosphocellulose, DEAE-sephacel, heparin acrylamide and hydroxyapatite columns. The purified enzyme preparation showed a 55 kDa band on SDS-PAGE with silver staining. Renaturation of the enzyme activity from SDS-PAGE showed that it comigrated with the 55 kDa protein. Characterization of the enzyme showed that it was a type II PtdIns 4-kinase. Polyclonal antibodies raised against PtdIns 4-kinase inhibited the enzyme activity in in vitro assays. Analysis of adult rat tissue particulate fractions on immunoblots showed restricted immunoreactivity among PtdIns 4-kinases. However, the immunoreactivity is conserved in lymphoid tissues from mouse to human, suggesting that lymphoid tissue has a distinct PtdIns 4-kinase. Activation of rat splenocytes with Con A showed two fold increase in PtdIns 4-kinase activity. Comparison of PtdIns 4-kinases from spleen and splenic lymphocytes showed identical chromatographic behaviour, molecular mass, immunoreactivity, K(m) values for PtdIns and inhibition by adenosine.     

Influence of heparin on the removal of serum lipoprotein lipase by the perfused liver of the rat

Isolated rat livers were perfused with whole rat blood containing postheparin lipoprotein lipase (LPL) activity. LPL activity disappeared rapidly from the perfusate; the extraction ratio (portal vein-hepatic vein difference) was 0.70 for all time periods studied. Control experiments established that the disappearance of LPL was not due to non-specific inactivation in the apparatus or to the release of an inhibitory by the liver. The addition of heparin to the perfusate in suitable concentration (4 units/ml) almost completely blocked the disappearance of LPL activity from the perfusate. In addition to the perfusion experiments, we studied the effect of heparin on LPL activity when added to the LPL assay system. When heparin was added to the assay system containing fresh postheparin serum from rats, it stimulated LPL activity by about 70%. When heparin was added to postheparin serum which had been perfused through the liver, it stimulated LPL activity over 200%, but it did not restore LPL to its preperfusion value. These observations are compatible with a two-step inactivation system for LPL by the liver. The first step may involve a dissociation of a heparin-apoenzyme complex followed by destruction of the heparin. The second step may involve the removal of the apoenzyme of LPL.     

Localization of fragments binding nuclear proteins in the promotor region of the human alpha-1-antitrypsin gene]

Human and mice nuclear extracts from livers and mice spleen extract were analysed in an attempt to find any proteins capable of binding to the human alpha 1-antitrypsin gene promoter. The nuclei of all studied tissues contain such proteins. The proteins were partially purified on DEAE-trisacryl, heparin sepharose and phosphocellulose columns. The multiple sites for liver nuclear proteins binding to the human alpha 1-antitrypsin gene promoter were found by the DNAse I footprinting technique.     

Degradation of the heparin matrix of mast cell granules by cultured fibroblasts

The ability of cultured rat fibroblasts to phagocytose rat peritoneal mast cell granules has been previously demonstrated by light and electron microscopy. To determine if the heparin matrix of ingested granules could be degraded by fibroblasts after phagocytosis, the heparin within peritoneal mast cells was labeled with [35S]sulfate in vivo. The 35S-labeled rat peritoneal mast cells were purified and their granules were isolated and shown to contain [35S]heparin proteoglycan. Incubation of [35S]heparin proteoglycan-containing granules with cultured rat fibroblasts revealed internalization of radioactivity by the fibroblasts over the first 24 hr consistent with phagocytosis of the granules by these fibroblasts. The [35S]heparin proteoglycan internalized by the fibroblasts was shown to decrease in size over 72 hr indicating that the fibroblasts were capable of degrading the heparin within the ingested granules. Degradation of [35S]heparin proteoglycan within the fibroblast was accompanied by the appearance of free [35S]sulfate in the extracellular compartment. Similar findings were obtained using cultured human fibroblasts. These data demonstrate for the first time that both rat and human fibroblasts are not only capable of ingesting mast cell granules but also of degrading mast cell granule heparin proteoglycan. This ingestion and degradation of mast cell granules by fibroblasts may represent an important mechanism in the regulation of the biologic expression of heparin and other granule-associated mediators in immediate hypersensitivity reactions.     

The suramin-treated rat as a model of mucopolysaccharidosis. Variation in the reversibility of biochemical and morphological changes among different organs

We have examined the reversibility of the biochemical and pathological changes induced in the spleen, kidney and lung of the suramin-treated rat which we have previously proposed as a useful model of the human condition, mucopolysaccharidosis (MPS). Rats were injected with a single intravenous dose of suramin (250 mg/kg) and allowed to survive for periods of up to 6 months. The organs were examined for suramin content, pathological changes, biochemical storage of glycosaminoglycans (GAGs) and for the blockage of the relevant hydrolytic enzymes. The extent and rate of suramin accumulation and the retention of the drug varied considerably between organs with the greatest concentration of suramin (4,000 micrograms/g) occurring in the kidney 2 weeks after injection. Suramin persisted at gradually decreasing levels in all organs for the duration of the experiment, remaining at the highest level (1,150 micrograms/g) in the kidney. The concentration of GAGs peaked 10-18 days after administration of the drug, in all organs. Within 6 months the level had returned to normal in the liver, spleen and lung, but remained elevated in the kidney. The activities of beta-glucuronidase and acid phosphatase were decreased in all organs at diminishing levels throughout the experiment. There was a significant increase in the activity of arylsulphatase B, except in the kidney, where the predominant effect was a reduction of activity. Recovery from the morphological changes was evident in all organs except the lung within 6 months of suramin administration. The reversibility of the biochemical and pathological changes in the various tissues is discussed and compared with the earlier results described for the liver (Rees et al. 1986) and the implications of using suramin for the treatment of human trypanosomiasis, onchocerciasis and AIDS are considered.     

Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1)

Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.     

Arylsulphatase activity in the oviduct of the frog Rana esculenta. I. Oestradiol-induced changes following ovariectomy and hypophysectomy

Summary The effects of oestradiol treatment on arylsulphatase activity in the frog oviduct are reported. Oestradiol-induced changes were also investigated in ovariectomized and hypophysectomized animals. Under all the experimental conditions, hormonal treatment causes an increase in enzyme activity. This can be observed biochemically and also histochemically on frozen sections. Hypotheses are advanced to explain fluctuations in arylsulphatase activity.     

Occurrence of NI and NII type protein kinases in the nuclei from various tissues of the rat

Cyclic nucleotide-independent protein kinases that preferentially phosphorylated casein and phosvitin as substrate were surveyed in various tissue nuclei of the rat. Enzymes were extracted from the isolated nuclei of liver, kidney, spleen, brain, heart, or testis tissue with a buffer solution containing 0.4 m NaCl, and analyzed by DEAE-Sephadex, phosphocellulose, and Bio-Gel A-1.5m column chromatographies. The chromatographic study together with characterization of the enzymes demonstrated that all the tissues contained in their cell nuclei commonly two protein kinases, the NI and NII types, and that these were exclusively found as main nuclear casein kinases. NII enzyme activity was stimulated by polyamines and strongly inhibited by heparin. By contrast, the NI enzymes were little influenced by these compounds. We interpret the present results as suggesting that NI and NII type protein kinases may be found in the cell nuclei from many tissues of rat, and have distinct functions in the cell nuclei.