The purification and properties of beta-galactosidase from bovine testes.

A /3-galactosidase was purified 600-fold from bovine testes by ammonium sulfate precipitation, acetone fractionation, and at5nity chromatography on agarose substituted with terminal thio-fl-galactopyranosyl residues. The preparation was devoid of protease, a-fucosidase, ar-mannosidase, pglucosidase, P-glucuronidase, and hyaluronidase activities. A wide variety of compounds containing terminal nonreducing galactose residues were hydrolyzed by the enzyme, including proteoglycans, glycoproteins, gangliosides, disaccharides, and nitrophenylgalactosides. Kinetic studies showed that galactose was released at approximately the same maximal rate from several substrates, but that the K, values differed widely. The enzyme exhibited a high affinity for nitrophenylgalactosides (Km less than 10e4 M). The purified /3-galactosidase had a pH optimum of 4.3, required sulfhydryl groups for activity, and was neither stimulated nor inhibited by alkalimetal ions or by EDTA. A single band of P-galactosidase activity was observed after disc gel electrophoresis at several pH values. At each pH value the enzyme activity corresponded in migration to the major protein band. The pgalactosidase eluted as a single peak from Sephadex G-ZOO (apparent molecular weight 68,000). The enzyme catalyzed the transfer of galactose from p-nitrophenyl-@-galactoside to glucose, N-acetylglucosamine, and N-acetylgalactosamine. Galactose, galactal, galactonolactone, and thio+-galactopyranosides inhibited the enzyme. Two distinct fl-galactosidase activities were shown in bovine liver extracts. One enzyme (or enzymes) (type I) was bound to the affinity column whereas the second enzyme (or enzymes) (type II) passed through the column. While type I /Lgalactosidase had properties similar to those ascribed to the testicular enzyme, type II /3-galactosidase did not hydrolyze lactose or transfer galactose from /3-galactosides to glucose.