Cytosolic sialidase from pig brain: a 'protein complex' containing catalytic and protective units

Bruno Venerando, Amelia Fiorilli, Lucia Di Francesco, Anna Chiarini, Eugenio Monti, Daniela Zizioli, Guido Tettamanti

Research output: Contribution to journalArticle

Abstract

Pig brain cytosolic sialidase purified to homogeneity, showed a single protein band on SDS-PAGE under non-reducing conditions, and three bands using reducing conditions, suggesting a complex of different units. The sialidase complex (molecular mass, Mr, 180 kDa) was resolved into a catalytic unit (Mr 30 kDa), active but very labile upon storage at 4°C and freezing and thawing, and two protective units (66 kDa and 42 kDa), inactive, but capable to stabilize the catalytic unit. Recombination of the catalytic and protective units (optimal ratio, 1:1, by weight) gave rise to a stable active complex. Using GD1a1 as substrate, the catalytic unit showed a Michaelis-Menten kinetics, and the complex a sigmoid-shaped kinetics, whereas a Michaelis-Menten kinetics was exhibited with MU-NeuAc in both cases. The apparent Vmax and Km values of the catalytic unit for MU-NeuAc and GD1a were 105.1 and 110.0 mU/mg protein, and 4.2.10-5 and 1.6.10-5 M, respectively. The model we propose for cytosolic sialidase complex is one of each protective units and 2-3 catalytic units. The sialidase complex and protective units did not display any β-d-galactosidase, β-d-N-acetylglucosaminidase, α-l-fucosidase, α-d-glucosidase and carboxypeptidase activities.

Original languageEnglish
Pages (from-to)229-237
Number of pages9
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1208
Issue number2
DOIs
Publication statusPublished - Oct 19 1994

Keywords

  • Brain
  • Cytosolic sialidase
  • Enzyme kinetics
  • Enzyme purification
  • Ganglioside

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Structural Biology

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