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 (α(2 → 3)sialyllactose, α(2 → 6)sialyllactose, disialyllactose), sialylglycolipids (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 K_m values were higher for sialyloligosaccharides (about 10^-3 M), lower for gangliosides (about 10^-4 M); the apparent maximum velocity was higher with α(2 → 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 α(2 → 3)sialyllactose: 400 mU ± 6 S.E.).