Studies on brain membrane-bound neuraminidase: I. General properties of the enzyme prepared from calf brain

Abstract

1. 1. A crude preparation of membrane-bound neuraminidase (mucopolysaccharide N-acetylneuraminylhydrolase, EC 3.2.1.18), obtained from calf brain, was used. This preparation was free from the soluble and lysosomal neuraminidases and depleted of endogenous substrates. 2. 2. The enzyme showed: (a) optimum pH 4.0 for gangliosides GD1a, GD1b, GT1b and GQ1; 3.8 for ovine submaxillary mucin, ovine submaxillary mucin-sialoglycopeptides and brain sialoglycopeptides; 3.1 for sialyllactose (C-3 isomer); (b) apparent higher affinity for gangliosides ( K m of the order 10 −5 M) than for sialyllactose ( K m 0.68·10 −3 M), ovine submaxillary mucin and sialoglycopeptides; (c) higher V for gangliosides (1.26–2.12 units/mg protein), lower for sialyllactose (1.18 units/mg protein) and sialoglycoprotein substrates (0.27–0.41 units/mg protein); (d) inhibition by excess substrate (over 0.15-0.2 mM) only in the case of gangliosides; (e) maximum rate of hydrolysis of gangliosides at 70 °C (24 units/mg protein in the case of ganglioside GD1a); (f) considerable stability. 3. 3. Na + and Li + did not influence the enzyme activity; K + activated below 0.1 M; NH 4 + started inhibiting at 0.01 M. All bivalent cations tested inhibited the enzyme: Hg 2+ from 10 −6 M, Cu 2+ from 10 −5 M, Ca 2+ from 10 −3 M. Anions had no appreciable influence on the enzyme activity, at concentrations up to 5·10 −2 M.