Specific Binding of Glycosylated ?-Galactosidase to Bovine Brain Synaptic Membrane

Abstract

The binding of a series of glycosylated .beta.-galactosidases to a fraction rich in synaptic membrane of bovine brain was examined. .beta.-Galactosidase modified with p-aminophenyl .beta.-D-galactopyranoside (.beta.-D-Gal .beta.-gal) was found the most effective in binding to synaptic membrane, followed by the modified with .beta.-D-glucopyranoside, whereas the enzyme modified with p-amino-phenyl derivatives of .alpha.-D-galactopyranoside, .alpha.-D-glucopyranoside, and .alpha.- and .beta.-L-fucopyranoside were found not to bind to the membrane. The binding was dependent on time, temperature, and pH; the maximal binding was obtained within 15 min at 4.degree. C and the optimal pH was approximately 4.0. The binding of .beta.-D-gal was inhibited by free p-aminophenyl .beta.-D-galactopyranoside and by the treatment of synaptic membrane with trypsin or phospholipase A2 or C. The equilibrium dissociation constant and the maximal concentration of binding sites were determined by Scatchard analysis to be 470 .+-. 35 nM and 27.5 .+-. 3.1 pmol/mg protein (n = 1). The results suggest that a specific binding site for the specified carbohydrates exists in synaptic membrane and is involved in the internalization of glycoconjugates into nerve terminals.