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

Abstract

A2/1957 influenza neuraminidase (mucopolysaccharide N-acetylneuraminylhydrolase, EC 3.2.1.18) was purified 15-fold from a recombinant virus, with about 25% overall yield of enzymic activity. Neuraminidase contained glucosamine, and a high proportion of serine and threonine. The partial specific volume was 0.713 cm3/g. Reduced neuraminidase was isotopically labeled in vitro by reaction with iodo[14C]-acetamide. 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 sedimentation-diffusion methods as 50 000-54 000, and by sodium dodecyl sulfate-acrylamide gel electrophoresis as about 50 000. Thus native neuraminidase (mol. wt. about 200 000) is probably a tetramer. Neuraminidase contained about 21 cysteine residues per subunit. These appear to be present as disulfide bonds in the native enzyme.