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

L. Caimi, A. Lombardo, A. Preti, U. Wiesmann, G. Tettamanti

Research output: Contribution to journalArticlepeer-review

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 Km 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.).

Original languageEnglish
Pages (from-to)137-146
Number of pages10
JournalBBA - Enzymology
Volume571
Issue number1
DOIs
Publication statusPublished - Nov 9 1979

Keywords

  • Assay conditions
  • Fibroblast
  • Natural substrate
  • Neuraminidase

ASJC Scopus subject areas

  • Medicine(all)

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