Optimal conditions for the assay of fibroblast neuraminidase with different natural substrates

Abstract 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.).