Kinetic studies on the sialidase of three influenza B and three influenza A virus strains

Abstract

Sialidase of influenza virus type A has been extensively studied through structural and kinetic approaches. However, sialidase of influenza virus type B has been less investigated. In this work, we have studied the activity and some properties (optimal pH, KM, Vmax, thermal stability) of sialidase in three influenza virus strains of type B (circulating in the period 1983-86) and also the activity and properties of sialidase from three virus strains of type A circulating at the same period of time. The results show that the activity and the Vmax was always higher for sialidase of type A viruses relative to those values of type B. Differences were also found for optimal pH and, in some cases, for thermal stability of the sialidase between strains belonging to the influenza viruses type A and B. However, the behaviour for the sialidase in all strains was very similar towards two competitive inhibitors. Thus, it could be suggested that the evolution pattern of the sialidase of both types of influenza viruses determines some modifications which result in a higher efficiency for sialidase of some strains of influenza virus type A, but maintaining in the two types of viruses a similar behaviour towards competitive inhibitors.

Site‐directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design

“…The lysate was then subjected to 10% polyacrylamide gel electrophoresis and transferred to Immobilon-P TM transfer membrane using the BioRad trans-blot apparatus. The membrane was blocked with 5 % dry milk in NaCl/P i and developed using polyclonal goat anti-B/Lee NA serum followed by biotin labeled anti-goat secondary antibody (Southern Biotechnology Associates Inc.) and 35 S-labeled Streptavidin (Amersham). The amount of expressed NA protein was determined using a phosphorimager (model PSI P90, Molecular Dynamics) and compared to a band of purified B/Lee/40 NA heads (68 ng).…”

“…The bands were quantified by phosphorimager and the ratio of protein immunoprecipitated with monoclonal versus polyclonal antibodies was determined. This gives the ratio of 35 S-labeled native NA to 35 S-labeled native ϩ unfolded NA, which can then be applied to total protein obtained by Western blots to give the amount of total native protein.…”

“…4) show that the low activities of mutant NAs do not approach wild-type levels up to 50 h post-transfection, indicating that the inactivity of Y409F and the low activities of the other mutants are not due to a delay in protein synthesis or folding. Rapid turnover by degradation is excluded by the West- 35 S-labeled streptavidin used for detection. The bands were quantified by phosphorimager.…”

Sialidase Activity in Rimantadine-Resistant and -Sensitive Influenza A Viruses

“…The same behav ior was observed for both viral variants. One unit of enzyme was defined as the amount of enzyme which hydrolyzes 1 pmol of substrate/min under the assay conditions [11], hibitors, and the assay of amantadine hydro chloride as an eventual effector of the sialidase activity in vitro. Km was virtually the same for both variants, as was the hydrolysis ratio using three natural substrates, i.e.…”

Specificity of the N1 and N2 sialidase subtypes of human influenza A virus for natural and synthetic gangliosides

“…Sialylated glycoproteins and gangliosides are the cellular receptors for influenza viruses (Suzuki et al, 1985a(Suzuki et al, , 1986(Suzuki et al, , 1989(Suzuki et al, , 1992Suzuki, 1994;Xu et al, 1994). The substrate specificity of various influenza virus sialidases have been studied previously (Corfield et al, 1982;Cabezas et al, 1989;Xu et al, 1995), and the linkage specificity of N2 sialidase can shift from Neu5Acα2-3Gal to Neu5Acα2-6Gal (Baum and Paulson, 1991). The specificity of the molecular species of sialic acid, the position at which sialic acid was attached, and the sialyl sugar chain structure, which includes the ceramide moiety of gangliosides remain unknown.…”