Optimal conditions for the assay of fibroblast neuraminidase with different natural substrates.
A method for the assay of neuraminidase in human cultured fibroblasts has been worked out. The substrates, all naturally occurring, were: sialyloligosaccharides (alpha(2 lead to 3)sialyllactose, alpha(2 leads to 6)sialyllactose, disialyllactose), sialylglycoplipids (disialogangliosides GD1a and GD1b), sialylglycoproteins and sialylglycopeptides (ovine submaxillary glycoprotein and its pronase-glycopeptides). The method was based on the determination of the enzymically liberated N-acetylneuraminic acid (NeuAc) by a chromatographic-colorimetric microprocedure. The enzyme acted on sialyloligosaccharides and, in the presence of Triton X-100, on gangliosides, while it did not appreciably affect sialylglycoproteins and sialylglycopeptides. The optimum pH was 4.0 for all tested substrates; the Km values were higher for sialyloligosaccharides (about 10(-3) M), lower for gangliosides (about 10(-4) M); the apparent maximum velocity was higher with alpha(2 leads to 3)sialyllactose (400 mU/mg protein); the reaction rate was linear with time for up to 2 h, and with up to 0.6 mg of enzymic protein. The assay method proved to be sufficiently sensitive (3-4 nmol liberated NeuAc), simple, and reproducible (mean activity on pooled fibroblasts with alpha(2 leads to 3)sialyllactose: 400 mU +/- 6 S.E.).
D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D005732:Gangliosides / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009439:Neuraminidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis; D012795:Sialoglycoproteins / Q000378:metabolism; D012867:Skin / Q000166:cytology / Q000201:enzymology*; D013379:Substrate Specificity
D005732:Gangliosides; D009844:Oligosaccharides; D012794:Sialic Acids; D012795:Sialoglycoproteins; D009439:Neuraminidase