Fluorescent staining of sialidases in polyacrylamide gel electrophoresis and ultrathin-layer isoelectric focusing

Berg, W.; Gutschker-Gdaniec, Gabriele; Schauer, Roland

doi:10.1016/0003-2697(85)90371-9

Cited by 34 publications

(10 citation statements)

References 9 publications

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“…Native PAGE and SDS-PAGE [26,27] analysis of the stage V preparation and material from subsequent purification steps was performed in order to determine the purity of the enzyme and to estimate the mol. wt of the sialidase.…”

Section: Native Page and Sds-pagementioning

confidence: 99%

“…The purified sialidase (10 ìg in the presence of SDS) was resolved at 100 V for 4 h. Sialidase was located in the gel with 4-MU-NeuNAc [26]. Briefly, the gel was incubated in 0.2 M sodium phosphate buffer (pH 7.0) containing Triton X-100 10% v=v at 378C for 2 h before spreading 500 ìl of 100 ìM 4-MU-NeuNAc over the surface of the gel.…”

Section: Native Page and Sds-pagementioning

confidence: 99%

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Isolation and characterisation of sialidase from a strain of Streptococcus oralis

Byers¹,

Tarelli²,

Homer³

et al. 2000

Journal of Medical Microbiology

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Streptococcus oralis, the most virulent of the viridans streptococci, produces a sialidase and this exo-glycosidase has been implicated in the disease process of a number of pathogens. The sialidase of S. oralis strain AR3 was purified in order to understand the characteristics of this putative virulence determinant. The enzyme isolated as a high mol. wt aggregate (c. 325 kDa) was purified 4520-fold from late exponential phase cultures by a combination of ultrafiltration, ammonium sulphate precipitation, ionexchange and gel filtration chromatography. The sialidase component had a mol. wt of 144 kDa as determined by SDS-PAGE analysis. The purified sialidase released Nacetylneuraminic acid from a range of sialoglycoconjugates including human AE 1 -acid glycoprotein, bovine submaxillary mucin, colominic acid and sialyl-AE2,3-and sialyl-AE2,6-lactose. Also, N-glycolylneuraminic acid was cleaved from bovine submaxillary mucin. The sialidase had a K m of 11.8 ìM for AE 1 -acid glycoprotein, was active over a broad pH range with a pH optimum of 6.0 and cleaved AE2,3-, AE2,6-and AE2-8-sialyl glycosidic linkages with a marked preference for AE2,3-linkages. The enzyme was competitively inhibited by the sialic acid derivative, 2,3-dehydro-N-acetylneuraminic acid, with a K IC of 1.2 ìM. The characteristics of the purified sialidase would support a nutritional role for this enzyme that may be significant in the proliferation of this organism in the oral cavity and at extra-oral sites in association with life-threatening infections.

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Section: Native Page and Sds-pagementioning

confidence: 99%

Section: Native Page and Sds-pagementioning

confidence: 99%

Isolation and characterisation of sialidase from a strain of Streptococcus oralis

Byers¹,

Tarelli²,

Homer³

et al. 2000

Journal of Medical Microbiology

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“…If cells are pretreated with detergents for allowing the substrate to enter cells, the activity of intracellular sialidase isoenzymes may be detected by this method. Furthermore, this method would be applicable to detection of sialidase that is separated on acrylamide gel, as previously reported with 4MU-Neu5Ac as the substrate (Berg et al 1985). The present method should be a useful tool in the analysis of localization and stage-specific expression of mammalian sialidase.…”

Section: Resultsmentioning

confidence: 90%

Fluorescent cytochemical detection of sialidase activity using 5-bromo-4-chloroindol-3-yl-α-D-N-acetylneuraminic acid as the substrate

Saito

Hagita

Iwabuchi³

et al. 2002

Histochem Cell Biol

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A novel fluorescent cytochemical method for sialidase activity was developed using 5-bromo-4-chloroindol-3-yl-alpha- D- N-acetylneuraminic acid (X-Neu5Ac) as the substrate. Intact nuclei were isolated from porcine liver and incubated at 37 degrees C for 3 h with 1 mM X-Neu5Ac at pH 4.8. The nuclei were stained with blue color that was derived from the oxidized compound of the reaction product X (5-bromo-4-chloro-3-hydroxyindole). A specific sialidase inhibitor, 2,3-dehydro-2-deoxy- N-acetylneuraminic acid, suppressed the staining in a dose-dependent manner. Despite the specificity of the cytochemical reaction, the staining was too weak to analyze the staining distribution and pattern of individual nuclei. To attain more sensitive detection of sialidase activity, the nuclei were incubated with X-Neu5Ac in the presence of Fast Red Violet LB. Individual nuclei of porcine liver were clearly stained with fluorescence that was produced by the conjugated compound of product X with Fast Red Violet LB. This fluorescent cytochemical method was also employed successfully for detection of sialidase activity of intact GOTO neuroblastoma cells in culture. The present method should provide a useful tool for investigating the localization and stage-specific expression of sialidase activity in tissues and cells.

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“…Gene detection strategy E. coli clones bearing chromosomal DNA fragments of A. viscosus inserted in pUC-vectors were assayed for sialidase production by spraying the colonies with a O.lmM aqueous solution of the fluorogenic substrate MU-Neu5Ac' 19 '. Positive colonies were visualized by a blue-white fluorescence under U.V.…”

Section: Bacterial Strains and Cultivationmentioning

confidence: 99%

Cloning, Sequencing and Expression of the Sialidase Gene fromActinomyces viscosusDSM 43798

Henningsen

Roggentin²,

Schauer³

1991

Biological Chemistry Hoppe-Seyler

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Chromosomal DNA from Actinomyces viscosus was digested with restriction endonucleases and the fragments ligated with pUC-vectors were used to transform Escherichia coli cells. Clones bearing the required sialidase gene were detected by spraying the colonies with the fluorogenic sialidase substrate MU-Neu5Ac.The identity of the cloned sialidase was confirmed after the 5700-fold enrichment and comparison with the purified enzyme of A. viscosus. Both sialidases were identical with regard to molecular mass, substrate specificity tested with sialyllactoses, and the inhibition of their activity by heterologous antisialidase antibodies. The sequenced insert (EMBL accession number X62276) revealed a mol% G + C of 68.2, typical for A.viscosus. An open reading frame of 2739 bp follows a sequence with dyad symmetry and an AG-rich region, and codes for 913 amino acids representing a molecular mass of 113 kDa.The conserved amino acid sequence [Ser-X-Asp-X-Gly-X-Thr-Trp] typical for bacterial sialidases was found at five positions in the predicted amino acid sequence. The gene of this enzyme is expressed by E. coli, despite the low relatedness of both species. Klonierung, Sequenzanalyse und Expression des Sialidase-G ens aus Actinomyces viscosus DSM 43798Zusammenfassung: Chromosomale DNA von Actinomyces viscosus wurde mit Restriktionsendonucleasen verdaut und die Fragmente in pUC-Vektoren ligiert, welche zur Transformation von E. co/z-Zellen verwendet wurden. Die gesuchten Klone, die das Sialidase-Gen enthielten, wurden anhand der Enzymaktivität mit dem fluorogenen Sialidasesubstrat MUNeuSAcdetektiert.Die Identität der klonierten Sialidase wurde nach 5 700-facher Anreicherung durch Vergleich mit dem angereicherten Enzym von Actinomyces viscosus bestätigt. Beide Sialidasen waren identisch bezüglich der Molekularmasse, der Substratspezifität getestet

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Fluorescent staining of sialidases in polyacrylamide gel electrophoresis and ultrathin-layer isoelectric focusing

Cited by 34 publications

References 9 publications

Isolation and characterisation of sialidase from a strain of Streptococcus oralis

Isolation and characterisation of sialidase from a strain of Streptococcus oralis

Fluorescent cytochemical detection of sialidase activity using 5-bromo-4-chloroindol-3-yl-α-D-N-acetylneuraminic acid as the substrate

Cloning, Sequencing and Expression of the Sialidase Gene fromActinomyces viscosusDSM 43798

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