Loss of enzyme activity in a site-directed mutant of influenza neuraminidase compared to expressed wild-type protein
Identifieur interne : 002364 ( Main/Exploration ); précédent : 002363; suivant : 002365Loss of enzyme activity in a site-directed mutant of influenza neuraminidase compared to expressed wild-type protein
Auteurs : Michael R. Lentz [États-Unis] ; Gillian M. Air [États-Unis]Source :
- Virology [ 0042-6822 ] ; 1986.
English descriptors
- Teeft :
- Academic press, Acid substitution, Active site, Agno gene, Antigenic, Catalytic reaction, Cell biol, Cell surface, Chemical modification, Codon, Cold spring harbor laboratory, Crystal structure, Enzymatic properties, Enzyme, Enzyme activity, Eric hunter, Expression vector, Fetuin, Further evidence, Glycoprotein, Helper virus, High levels, Immunofluorescence, Indirect immunofluorescence, Influenza, Influenza virus, Influenza virus neuraminidase, Initiator codon, Internal fluorescence, Klenow fragment, Lactose, Late promoter, Late replacement vector, Laver, Lentz, Monoclonal antibodies, Monoclone pool, Mutagenesis, Mutagenic primer, Mutant, Mutant protein, Mutant proteins, Mutation, Neuraminidase, Neuraminidase enzyme activity, Neuraminidase gene, Nucleic acids, Oligonucleotidedirected mutagenesis, Primer, Promoter, Rabbit antiserum, Recombinant, Recombinant vector, Rous sarcoma virus, Sali site, Same manner, Sazi site, Sialic acid, Simian virus, Sodium phosphate, Substrate binding, Substrate binding pocket, Substrate specificity, Svnawt, Tryptophan, Unfixed cells, Uninfected cells, Unpublished results, Vector pqps, Viral, Viral envelope, Viral neuraminidase, Virology, Virus, Virus particles, Virus stocks.
Abstract
Abstract: Full-length double-stranded DNA copies of the neuraminidase (NA) gene of influenza virus A/Tokyo/3/67 (N2) and a mutant generated in vitro by site-specific, oligonucleotide directed mutagenesis with a substitution of leucine for tryptophan at position 178 were cloned into an SV40 late replacement expression vector. Indirect immunofluorescence of cells infected with these recombinant vectors showed the presence of NA protein in the cytoplasm and on the surface of infected cells. Cells expressing the wild-type protein showed neuraminidase enzyme activity for both fetuin, a sialated glycoprotein (mol wt = 50,000) and N-acetylneuraminyl lactose, a trisaccharide (mol wt = 600). This enzyme activity was inhibited by 44% toward N-acetylneuraminyl lactose and by 98% toward fetuin by adding anti-NA antibody before substrate. In contrast, cells expressing the mutant NA had no detectable enzyme activity for either substrate. The conserved nature of the tryptophan at position 178 in all known NA strains, its location in the substrate binding pocket in the three-dimensional structure and the lack of activity of the mutant protein indicate that this residue is essential for enzyme activity.
Url:
DOI: 10.1016/0042-6822(86)90404-6
Affiliations:
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Indirect immunofluorescence of cells infected with these recombinant vectors showed the presence of NA protein in the cytoplasm and on the surface of infected cells. Cells expressing the wild-type protein showed neuraminidase enzyme activity for both fetuin, a sialated glycoprotein (mol wt = 50,000) and N-acetylneuraminyl lactose, a trisaccharide (mol wt = 600). This enzyme activity was inhibited by 44% toward N-acetylneuraminyl lactose and by 98% toward fetuin by adding anti-NA antibody before substrate. In contrast, cells expressing the mutant NA had no detectable enzyme activity for either substrate. The conserved nature of the tryptophan at position 178 in all known NA strains, its location in the substrate binding pocket in the three-dimensional structure and the lack of activity of the mutant protein indicate that this residue is essential for enzyme activity.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country></list><tree><country name="États-Unis"><noRegion><name sortKey="Lentz, Michael R" sort="Lentz, Michael R" uniqKey="Lentz M" first="Michael R." last="Lentz">Michael R. Lentz</name></noRegion><name sortKey="Air, Gillian M" sort="Air, Gillian M" uniqKey="Air G" first="Gillian M." last="Air">Gillian M. Air</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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