The preparation and properties of 14C-carboxamido-methylated subunits from A2/1957 influenza neuraminidase

Abstract

A2/I957 influenza neuraminidase (mucopolysaccharide N-acetylneuraminylhydrolase, EC 3.2.1.18) was purified i5-fold from a recombinant virus, with about 25~/o overall yield of enzymic activity. Neuraminidase contained glucosamine, and a high proportion of serine and threonine. The partial specific volume was o.713 cm3/g. Reduced neuraminidase was isotopically labeled in vitro by reaction with iodoE14Clacetamide. When carboxamidomethylated in the absence of urea, enzymically inactive labeled material was obtained with a maximum size similar to native neuraminidase. When carboxamidomethylated in the presence of 6 M urea, labeled, dissociated subunits were obtained that did not associate or regain enzymic activity on removal of urea. The molecular weight of dissociated subunits was determined by sedimentationdiffusion methods as 50 000-54 ooo, and by sodium dodecyl sulfate-acrylamide gel electrophoresis as about 50 ooo. Thus native neuraminidase (tool. wt. about 200 ooo) is probably a tetramer. Neuraminidase contained about 21 cysteine residues per subunit. These appear to be present as disulfide bonds in the native enzyme.

Preparation-conditioned changes of the antigenicity of influenza virus neuraminidases

“…The molecular weights of subunits present in fraction SuB were determined by use of equilibrium ultraeentrifugation procedures (16 to 24 hours with 9000 to 12,000 rpm at I0 ° C) and calculated according to MAZZONE (41). The resulting corrected V value of 0.718 agreed well with the V value of 0.713 calculated by KENDAL et al (28) for isolated neuraminidase. The resulting corrected V value of 0.718 agreed well with the V value of 0.713 calculated by KENDAL et al (28) for isolated neuraminidase.…”

The presence of two neuraminidases in an influenza virus

“…Similarly, one neuraminidase protein was reported for Lee and Be1 viruses (Laver and Baker 1972). Kendal and Eckert (1972) obtained neuraminidase as a single protein when X-7 (F,) influenza virus was treated with nagarse protease and suggested that the claims of two neuraminidases were due either to "an experimental artifact" or to "an impurity . "…”

“…1970;Webster 1970;Kendal and Eckert 1972;Gregoriades 1972;Lazdins et al 1972). This controversy has not been resolved (Bucher and Palese 1975).…”

Structural Domains and Organizational Conformation Involved in the Sorting and Transport of Influenza Virus Transmembrane Proteins

“…The mutation at residue 203 in ts60 is located in J3-2S4, a strand implicated in subunit-subunit interactions (103) . Because the tetramer ic form of NA appears to be required for enzymatic activity (16,48) , this mutation may have affected oligomer formation. Improper folding and oligomerization may also be responsible for the transport block, although the requirement of oligomerization for intracellular transport of NA remains to be demonstrated.…”