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

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 K_m for disialoganglioside GD1a was 4.8·10^-6 M; the v_max 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.