Characterization of sialidase from an influenza A (H3N2) virus strain: kinetic parameters and substrate specificity.
Neuraminidase (NA) of influenza A (H3N2) viruses was characterized after purification by gel filtration and proteolytic treatment, using the X-31 variant strain that is a reassortment between the influenza A/Victoria/3/75 (responsible for the 1975 pandemic) and the influenza A/PR/8/34 virus samples, as a model. In the purification process, NA heads, that is the spike responsible for the virus sialidase activity, were purified by filtration through a Bio-Gel polyacrylamide column. The enzyme activity was determined by periodic acid/thiobarbituric acid assay and high-performance thin-layer chromatography. The sialidase showed preference for the alpha-2,3-linkage over the alpha-2,6-linkage of sialyllactoses (K(m) of 1.8 and 5.2 x 10(-4)M, respectively) at pH 5.2. The enzyme acted on natural and synthetic substrates at different hydrolysis rates, as well as on human erythrocytes (A group, Rh+) and yeast (CANDIDA ALBICANS) cells. The active NA produced by gel filtration was characterized by different parameters of its sialidase activity, also showing to be a suitable tool for the identification of natural sialocompounds and for the screening of antisialidase drugs to treat influenza virus infections.
França de Barros
José
J
Departamento de Virologia, Instituto de Microbiologia Prof Paulo de Góes (IMPPG), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
Sales Alviano
Daniela
D
da Silva
Maria Helena
MH
Dutra Wigg
Marcia
M
Sales Alviano
Celuta
C
Schauer
Roland
R
dos Santos Silva Couceiro
José Nelson
JN
D002850:Chromatography, Gel; D003460:Crystallization; D006801:Humans; D066298:In Vitro Techniques; D053122:Influenza A Virus, H3N2 Subtype; D009980:Influenza A virus / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D009439:Neuraminidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011402:Pronase; D020836:Protein Structure, Quaternary; D013045:Species Specificity; D013379:Substrate Specificity
D009439:Neuraminidase; D011402:Pronase