1.3 A structure of arylsulfatase from Pseudomonas aeruginosa establishes the catalytic mechanism of sulfate ester cleavage in the sulfatase family.
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BACKGROUND: Sulfatases constitute a family of enzymes with a highly conserved active site region including a Calpha-formylglycine that is posttranslationally generated by the oxidation of a conserved cysteine or serine residue. The crystal structures of two human arylsulfatases, ASA and ASB, along with ASA mutants and their complexes led to different proposals for the catalytic mechanism in the hydrolysis of sulfate esters. RESULTS: The crystal structure of a bacterial sulfatase from Pseudomonas aeruginosa (PAS) has been determined at 1.3 A. Fold and active site region are strikingly similar to those of the known human sulfatases. The structure allows a precise determination of the active site region, unequivocally showing the presence of a Calpha-formylglycine hydrate as the key catalytic residue. Furthermore, the cation located in the active site is unambiguously characterized as calcium by both its B value and the geometry of its coordination sphere. The active site contains a noncovalently bonded sulfate that occupies the same position as the one in para-nitrocatecholsulfate in previously studied ASA complexes. CONCLUSIONS: The structure of PAS shows that the resting state of the key catalytic residue in sulfatases is a formylglycine hydrate. These structural data establish a mechanism for sulfate ester cleavage involving an aldehyde hydrate as the functional group that initiates the reaction through a nucleophilic attack on the sulfur atom in the substrate. The alcohol is eliminated from a reaction intermediate containing pentacoordinated sulfur. Subsequent elimination of the sulfate regenerates the aldehyde, which is again hydrated. The metal cation involved in stabilizing the charge and anchoring the substrate during catalysis is established as calcium. (+info)
Effects of various compounds on the ascorbate-2-sulfate sulfohydrolase and arylsulfatase activities copurified from the liver of Charonia lampas.
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The effects of various compounds on ascorbate-2-sulfate sulfohydrolase and arylsulfatase (EC 3.1.6.1) activities in the copurified preparation from the liver of Charonia lampas were investigated. The former activity was competively inhibited by inorganic phosphate and sulfate. The latter was not affected by sulfate. Higher concentrations of sodium chloride inhibited the former and activated the latter. Neither of the activities was inhibited by sulfhydryl reagents. Both activities were competively inhibited by the other substrate. (+info)
Modulation of genotoxic enzyme activities by non-digestible oligosaccharide metabolism in in-vitro human gut bacterial ecosystems.
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Supplementation of the human diet with prebiotic substances such as inulin and non-digestible oligosaccharides (NDO), e.g., galacto-oligosaccharides (GOS), has been associated with various health benefits. However, little information is available regarding the spatial location of their metabolism in human gut bacterial ecosystems. Therefore, the present study investigated the metabolism of inulin and GOS with respect to bacterial growth, bifidobacterial stimulatory properties and anti-mutagenicity potential, in a three-stage continuous culture model of the colon which reproduces the physicochemical characteristics of the proximal (V1) and distal (V2, V3) colons. Fermentation of both carbohydrates was rapid, and occurred primarily in V1, as evidenced by acid formation. Inulin metabolism was associated with 10-fold stimulation of lactobacillus populations, together with smaller increases in bifidobacterial cell counts in V1. However, peptostreptococci, enterococci and Clostridium perfringens also increased in this fermentation vessel. In contrast, GOS was only weakly bifidogenic in V1, although these bacteria did proliferate in V2. GOS also increased lactobacilli by an order of magnitude in V1. However, overall changes in microbial populations resulting from inulin or GOS addition were minimal in V2 and V3. Potential beneficial effects of inulin metabolism included minor reductions in beta-glucosidase and beta-glucuronidase, whereas GOS strongly suppressed these enzymes, together with arylsulphatase (AS). Growth of putatively health promoting micro-organisms was not only associated with reductions in enzymes linked to genotoxicity. For example, both carbohydrates stimulated synthesis of nitroreductase and azoreductase, throughout the fermentation system, while inulin increased AS. Colonic transit time is an important factor in bacterial metabolism in the large bowel, and these data suggest that, in some circumstances, NDO fermentation will occurprincipally in the proximal colon. (+info)
M31 and macroH2A1.2 colocalise at the pseudoautosomal region during mouse meiosis.
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Progression through meiotic prophase is associated with dramatic changes in chromosome condensation. Two proteins that have been implicated in effecting these changes are the mammalian HP1-like protein M31 (HP1beta or MOD1) and the unusual core histone macroH2A1.2. Previous analyses of M31 and macroH2A1.2 localisation in mouse testis sections have indicated that both proteins are components of meiotic centromeric heterochromatin and of the sex body, the transcriptionally inactive domain of the X and Y chromosomes. This second observation has raised the possibility that these proteins co-operate in meiotic sex chromosome inactivation. In order to investigate the roles of M31 and macroH2A1.2 in meiosis in greater detail, we have examined their localisation patterns in surface-spread meiocytes from male and female mice. Using this approach, we report that, in addition to their previous described staining patterns, both proteins localise to a focus within the portion of the pseudoautosomal region (PAR) that contains the steroid sulphatase (Sts) gene. In light of the timing of its appearance and of its behaviour in sex-chromosomally variant mice, we suggest a role for this heterochromatin focus in preventing complete desynapsis of the terminally associated X and Y chromosomes prior to anaphase I. (+info)
Sulfur economy and cell wall biosynthesis during sulfur limitation of Chlamydomonas reinhardtii.
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We have identified two novel periplasmic/cell wall polypeptides that specifically accumulate during sulfur limitation of Chlamydomonas reinhardtii. These polypeptides, present at high levels in the extracellular polypeptide fraction from a sulfur-deprived, cell wall-minus C. reinhardtii strain, have apparent molecular masses of 76 and 88 kD and are designated Ecp76 and Ecp88. N-terminal sequences of these polypeptides facilitated the isolation of full-length Ecp76 and Ecp88 cDNAs. Ecp76 and Ecp88 polypeptides are deduced to be 583 and 595 amino acids, respectively. Their amino acid sequences are similar to each other, with features characteristic of cell wall-localized hydroxyproline-rich glycoproteins; the N terminus of each polypeptide contains a predicted signal sequence, whereas the C terminus is rich in proline, alanine, and serine. Ecp76 and Ecp88 have either no (Ecp88) or one (Ecp76) sulfur-containing amino acid and transcripts encoding these polypeptides are not detected in cultures maintained on complete medium, but accumulate when cells are deprived of sulfur. This accumulation is temporally delayed relative to the accumulation of sulfur stress-induced arylsulfatase and ATP sulfurylase transcripts. The addition of sulfate back to sulfur-starved cultures caused a rapid decline in Ecp76 and Ecp88 mRNAs (half lives < 10 min). Furthermore, the C. reinhardtii sac1 mutant, which lacks a regulatory protein critical for acclimation to sulfur limitation, does not accumulate Ecp76 or Ecp88 transcripts. These results suggest that the Ecp76 and Ecp88 genes are under SacI control, and that restructuring of the C. reinhardtii cell wall during sulfur limitation may be important for redistribution of internal and efficient utilization of environmental sulfur-containing molecules. (+info)
Ultrastructural cytochemical evidence for the activation of lysosomes in the cytocidal effect of Chlamydia psittaci.
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The cytopathic effect of the polyarthritis strain of Chlamydia psittaci was studied in cultured bovine fetal spleen cells and found to be mediated by the release of lysosomal enzymes into the host cytoplasm during the late stages of chlamydial development. Ultrastructural cytochemical analysis and cell fractionation studies of infected cells revealed a close relationship between the stage of chlamydial development, fine structural features of the host, and localization of lysosomal enzyme activities. After adsorption, chlamydiae entered the host cells by endocytosis. The endocytic vacuoles containing individual chlamydiae and later the inclusion vacuoles containing the different chlamydial developmental forms were always free from lysosomal enzyme activity. Even after extensive multiplication of chlamydiae, lysosomal enzymes remained localized within lysosomes or their precursors in the host cell. Coincident with the process of chlamydial maturation, lysosomal enzymes were released into the host cytoplasm and were always associated with disintegration of host cell constituents and lysis. The chlamydiae appeared to be protected from this lysosomal enzyme activity by the inclusion membrane. After release from the inclusion, elementary bodies maintained their fine structural features, whereas all other chlamydial developmental forms lost their ultrasturctural integrity. (+info)
Identity and activities of lysosomal enzymes in parenchymal and non-parenchymal cells from rat liver.
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1. Intact parenchymal and non-parenchymal cells were isolated from rat liver. The parenchymal cells were purified by differential centrifugation, while non-parenchymal cells were obtained free of parenchymal cell contamination by preferentially destroying the parenchymal cells with the aid of pronase (0.25%). 2. The ability to isolate pure intact parenchymal and non-parenchymal cells permitted the characterization and measurement of specific activities of various lysosomal enzymes, representing the main functional hydrolytic activities of the lysosomes in these distinct cell types. 3. Lysosomal enzymes catalysing the hydrolysis of the terminal carbohydrate moiety of glycoproteins and glycolipids were not particularly enriched in the non-parenchymal cells as compared to parenchymal cells. The ratio of the specific activities of non-parenchymal cells over parenchymal cells varied between 0.7 for N-acetyl-beta-D-hexoseaminidase to 2.1 for alpha-glucosidase. This suggests no specific role of the non-parenchymal cells in the hydrolysis of terminal carbohydrate moieties of glycoproteins and glycolipids. 4. The enzymes acid phosphatase and aryl sulphatase, representing the phosphate and sulphate hydrolyzing activities, were enriched in the non-paranchymal cells as compared to the parenchymal cells by a factor of 2.5. 5. The most important peptidase cathepsin D, representing protein breakdown capacity, is enriched in the non-parenchymal cells as compared to parenchymal cells by a factor 6.0, suggesting a possible specific function of non-parenchymal cells in protein breakdown. 6. The most enriched lysosomal enzyme, representing lipid hydrolysis, is acid lipase, which is enriched in the non-parenchymal cells with a factor of 10. 7. The distribution of lysosomal enzymes between parenchymal and non-parenchymal cells suggests different functional roles of the lysosomes in these cell types. It can be concluded that the non-parenchymal cells possess a set of lysosomal enzymes which makes them extremely suitable for a phagocytic and antimicrobial function in the liver. (+info)
Deletion pattern of the STS gene in X-linked ichthyosis in a Mexican population.
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BACKGROUND: X-linked ichthyosis (XLI) is an inherited disorder due to steroid sulfatase deficiency (STS). Most XLI patients (>90%) have complete deletion of the STS gene and flanking sequences. The presence of low copy number repeats (G1.3 and CRI-S232) on either side of the STS gene seems to play a role in the high frequency of these interstitial deletions. In the present study, we analyzed 80 Mexican patients with XLI and complete deletion of the STS gene. MATERIALS AND METHODS: STS activity was measured in the leukocytes using 7-[(3)H]-dehydroepiandrosterone sulfate as a substrate. Amplification of the regions telomeric-DXS89, DXS996, DXS1139, DXS1130, 5' STS, 3' STS, DXS1131, DXS1133, DXS237, DXS1132, DXF22S1, DXS278, DXS1134-centromeric was performed through PCR. RESULTS: No STS activity was detected in the XLI patients (0.00 pmoles/mg protein/h). We observed 3 different patterns of deletion. The first two groups included 25 and 32 patients, respectively, in which homologous sequences were involved. These subjects showed the 5' STS deletion at the sequence DXS1139, corresponding to the probe CRI-S232A2. The group of 32 patients presented the 3' STS rupture site at the sequence DXF22S1 (probe G1.3) and the remaining 25 patients had the 3' STS breakpoint at the sequence DXS278 (probe CRI-S232B2). The third group included 23 patients with the breakpoints at several regions on either side of the STS gene. No implication of the homologous sequences were observed in this group. CONCLUSION: These data indicate that more complex mechanisms, apart from homologous recombination, are occurring in the genesis of the breakpoints of the STS gene of XLI Mexican patients. (+info)