Bovine testicular β-galactosidase: Purification of enzyme fractions that exhibit high affinity for phosphomannosyl receptors

Abstract

An improved method is described for the preparation of bovine testicular beta-galactosidase that allows the isolation of enzyme fractions that bind avidly to phosphomannosyl receptors. The procedure permits removal of a contaminating beta-hexosaminidase and yields nearly homogeneous beta-galactosidase. Enzyme eluted from DEAE-Sephacel was arbitrarily divided into pools that exhibited differing ability to bind phosphomannosyl receptors. A high binding fraction was rapidly assimilated by cultured cells and bound to both low and high molecular weight phosphomannosyl receptors. Carbohydrate analysis of the high binding fraction indicates an average content of one complex and one high mannose oligosaccharide chain per molecule and an average mannose 6-phosphate content of two residues per molecule. However, electrofocusing studies indicated that all the fractions were heterogeneous with respect to sialic acid and phosphate content. The purification procedure also provides highly purified beta-galactosidase suitable for removing beta-galactosidase residues from a variety of complex carbohydrates.

Rat epididymal luminal fluid acid β-d-galactosidase optimally hydrolyses glycoprotein substrate at neutral pH

“…Concanavalin A-Sepharose 4B column chromatography. Since acid f-D-galactosidase in other tissues has been shown to be a glycoprotein containing high-mannose/hybrid-oligosaccharide units [12,[21][22][23][24][25], we first attempted to separate the PNP galactosidase from [3H]Gal galactosidase activities by affinity chromatography on a column of immobilized concanavalin A. Rat luminal fluid was applied to the column which was then extensively washed with the column buffer as described in the legend to Fig. 3.…”