Purification and characterization of sialidase fromClostridium sordellii G12

Roggentin, Peter; Berg, W.; Schauer, Roland

doi:10.1007/bf01048368

Cited by 23 publications

(16 citation statements)

References 18 publications

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“…with recombinant baculovirus, cells obtained from a 3.2 cm Petri dish were washed with PBS, resuspended in 100 gl PBS, sonicated for 1 s, and tested in various concentrations for NA activity using 0.2 gM-4-methylumbelliferyl g-D-N-acetylneuraminic acid (Sigma) as a substrate in 0.1 M-sodium acetate buffer, pH 5.5, in a total volume of 0-1 ml. The reaction was stopped after 10 rain at 37 °C by addition of 0-9 ml 0-133 M-glycine buffer, pH 10, containing 60 mMNaC1 and 40 mM-Na2CO a (Roggentin et al, 1987). The fluorescence of released 4-methylumbelliferone was determined in a Perkin Elmer (LS-5B) luminescence spectrometer using excitation at 365 nm and emission at 450 nm.…”

Section: Metabolic Labelling and Immunoprecipitation Labelling Withmentioning

confidence: 99%

N1 neuraminidase of influenza virus A/FPV/Rostock/34 has haemadsorbing activity

Hausmann

Kretzschmar

Garten

et al. 1995

Journal of General Virology

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The neuraminidase (NA) gene of influenza virus A/FPV/ Rostock/34 virus (H7N1) was cloned and expressed in Sf9 cells using a baculovirus vector. The expression product had the expected molecular mass and showed neuraminidase activity. NA expressed in St9 cells also showed haemagglutinating activity as indicated by its ability to induce haemadsorption of chicken red blood cells. Haemadsorption depended on the presence of neuraminic acid on the erythrocytes, but was not blocked by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, a specific inhibitor of the enzymatic activity. These observations suggest that the N1 neuraminidase has two distinct reactive sites: a catalytic site and a receptor binding site. This concept was confirmed by site-directed mutagenesis which showed that the haemadsorbing activity of FPV NA was abolished when amino acids located on two surface loops at the putative binding site were exchanged. Substitutions on either of the two loops were sufficient for this effect. The mutated NAs retained full neuraminidase activity. In contrast, a mutated NA lacking neuraminidase activity still had haemadsorbing activity.

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Section: Metabolic Labelling and Immunoprecipitation Labelling Withmentioning

confidence: 99%

N1 neuraminidase of influenza virus A/FPV/Rostock/34 has haemadsorbing activity

Hausmann

Kretzschmar

Garten

et al. 1995

Journal of General Virology

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“…3) might be explained by partial degradation during purification (Roggentin et al, 1988 b). Furthermore, enzymic and molecular properties of the C. sordellii sialidase (Roggentin et al, 1987) and the cloned C. perfringens sialidase (unpublished results) are similar. The lack of a hydrophobic leader sequence in the latter sialidase represents the main difference in the primary structure bFtween the two enzymes.…”

Section: Discussionmentioning

confidence: 64%

“…A triplet sequence Met-Lys-Lys represents positively charged amino acids, followed by a stretch of mostly hydrophobic residues ending with Ala at the assumed cleavage site of the leader peptide. Discounting the leader peptide, the molecular mass of the encoded protein can be calculated as 41 900 Da, which is close to the 40000 Da estimated by SDS-PAGE for the C. sordellii sialidase (Roggentin et al, 1987). Sequences showing dyad symmetry were not found downstream from the stop codon.…”

Section: R E S U L T Smentioning

confidence: 95%

“…Inhibitors like Hg2+, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, W(4-nitropheny1)oxamic acid and N-acetylneuraminic acid decreased the activity of both sialidases to the same extent. Substrate specificity and K,,, values, and the temperature and pH optima of the C. sordellii sialidase (Roggentin et al, 1987) were not changed by cloning in E. coli. One difference, however, was measured concerning the isoelectric point (PI), which was 0-3 pH units lower for the cloned enzyme as compared to the original one.…”

Section: Codon Usagementioning

confidence: 98%

“…A sialidase of Clostridium sordellii G 12, an isolate from human gas gangrene, has been purified (Roggentin et al, 1987). This enzyme was used to start investigations on the molecular biology of bacterial sialidases.…”

Section: Introductionmentioning

confidence: 99%

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Cloning, Sequencing and Expression of a Sialidase Gene from Clostridium sordellii G12

Rothe

Roggentin²,

Frank³

et al. 1989

Microbiology

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A 4.3 kb XbaI restriction fragment of DNA from Clostridium sordellii G12 hybridized with a synthetic oligonucleotide representing the N-terminus of the sialidase protein secreted by C. sordellii. This cloned fragment was shown to encode only part of the sialidase protein. The sialidase gene of C. sordellii was completed by a 0.7 kb RsaI restriction fragment overlapping one end of the XbaI fragment. After combining the two fragments and transformation of Escherichia coli, a clone that expressed sialidase was obtained. The nucleotide sequence of the sialidase gene of C. sordellii G12 was determined. The sequence of the 18 N-terminal amino acids of the purified extracellular enzyme perfectly matched the predicted amino acid sequence near the beginning of the structural gene. The amino acid sequence derived from the complete gene corresponds to a protein with a molecular mass of 44735 Da. Upstream from the putative ATG initiation codon, ribosomal-binding site and promoter-like consensus sequences were found. The encoded protein has a leader sequence of 27 amino acids. The enzyme expressed in E. coli has similar properties to the enzyme isolated from C . sordellii, except for small differences in size and isoelectric point. Significant homology (70%) was found with a sialidase gene from C. perfringens.

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Characterization of sialidase from bloodstream forms ofTrypanosoma vivax

Buratai

Nok

Ibrahim

et al. 2005

Cell Biochem. Funct.

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Sialidase (EC: 3.2.1.18) from Trypanosoma vivax (Agari Strain) was isolated from bloodstream forms of the parasite and purified to apparent electrophoretic homogeneity. The enzyme was purified 77-fold with a yield of 32% and co-eluted as a 66-kDa protein from a Sephadex G 110 column. The T. vivax sialidase was optimally active at 37 degrees C with an activation energy (E(a)) of 26.2 kJ mole(-1). The pH activity profile was broad with optimal activity at 6.5. The enzyme was activated by dithiothreitol and strongly inhibited by para-hydroxy mercuricbenzoate thus implicating a sulfhydryl group as a possible active site residue of the enzyme. Theenzyme hydrolysed Neu5Ac2,3lac and fetuin. It was inactive towards Neu5Ac2,6lac, colomic acid and the gangliosides GM1, and GDI. Initial velocity studies, for the determination of kinetic constants with fetuin as substrate gave a V(max) of 142.86 micromol h(-1) mg(-1) and a K(M) of 0.45 mM. The K(M) and V(max) with Neu5Ac-2,3lac were 0.17 mM and 840 micromole h(-1) mg(-1) respectively. The T. vivax sialidase was inhibited competitively by both 2,3 dideoxy neuraminic acid (Neu5Ac2,3en) and para-hydroxy oxamic acid. When ghost RBCs were used as substrates, the enzyme desialylated the RBCs from camel, goat, and zebu bull. The RBCs from dog, mouse and ndama bull were resistant to hydrolysis.

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Purification and characterization of sialidase fromClostridium sordellii G12

Cited by 23 publications

References 18 publications

N1 neuraminidase of influenza virus A/FPV/Rostock/34 has haemadsorbing activity

N1 neuraminidase of influenza virus A/FPV/Rostock/34 has haemadsorbing activity

Cloning, Sequencing and Expression of a Sialidase Gene from Clostridium sordellii G12

Characterization of sialidase from bloodstream forms ofTrypanosoma vivax

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