Assay of brain particulate neuraminidase III. Preparation of the enzyme devoid of endogenous substrates

G. Tettamanti, A. Preti, A. Lombardo, M. Gasparini, V. Zambotti

Research output: Contribution to journalArticlepeer-review

Abstract

1. 1.|The crude preparation of brain particulate neuraminidase (mucopolysaccharide N-acetylneuraminylhydrolase, EC 3.2.1.18) (0-105 000 × g pellet, prepared from rabbit brain homogenate in isotonic sucrose) was depleted from endogenous (intrinsic) substrates by autolysis performed under the following conditions: 0.18 M sodium acetate buffer, 0.3% Triton X-100, final pH 4.2, 25°, 5 h incubation. 2. 2.|After this treatment only 54% protein, 51% sialoglycoproteins, 89% gangliosides, 75% total and lipid-bound phosphorus remained particulate. Neuraminidase remained firmly particulate and fully active (97% recovery). Thus, the 105 000 × g sediment obtained after the above treatment is the enzyme preparation devoid of endogenous substrates. 3. 3.|The basic properties of the enzyme present in the preparation devoid of endogenous substrates were the same observed in the starting crude preparation. The Km for disialoganglioside GD1a was 4.8·10-6 M; the vmax 1.6 nmoles released N-acetylneuraminic acid per min. 4. 4.|The examined properties of particulate neuraminidase depleted from endogenous substrates by our procedure were very similar to those of the enzyme prepared according to Z. Leibovitz and S. Gatt (Biochim. Biophys. Acta, 152 (1968) 136). However, the final enzyme recovery was 5 times higher with our procedure.

Original languageEnglish
Pages (from-to)228-237
Number of pages10
JournalBBA - Enzymology
Volume258
Issue number1
DOIs
Publication statusPublished - Jan 20 1972

ASJC Scopus subject areas

  • Medicine(all)

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