ANALYTICALBIOCHEMISTRY 182,432-437 (1989) Bovine Testicular,&Galactosidase: Purification of Enzyme Fractions That Exhibit High Affinity for

An improved method is described for the preparation of bovine testicular B-galactosidase that allows the iso-lation of enzyme fractions that bind avidly to phospho-mannosyl receptors. The procedure permits removal of a contaminating @-hexosaminidase and yields nearly homogeneous &galactosidase. Enzyme eluted from DEAE-Sephacel was arbitrarily divided into pools that exhibited differing ability to bind phosphomannosyl re-ceptors. A high binding fraction was rapidly assimi-lated by cultured cells and bound to both low and high molecular weight phosphomannosyl receptors. Carbo-hydrate analysis of the high binding fraction indicates an average content of one complex and one high man-nose oligosaccharide chain per molecule and an average mannose 6-phosphate content of two residues per mole-cule. However, electrofocusing studies indicated that all the fractions were heterogeneous with respect to si-alit acid and phosphate content. The purification proce-dure also provides highly purified &galactosidase suit-able for removing b-galactosidase residues from a variety of complex carbohydrates. o 1999 Academic PAM, IN. Bovine testicular P-galactosidase, a lysosomal hydro-lase, was previously purified by affinity chromatography on phenylthio+-galactoside-substituted agarose an