Bovine testicular beta-galactosidase: purification of enzyme fractions that exhibit high affinity for phosphomannosyl receptors.
نویسندگان
چکیده
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.
منابع مشابه
Binding of phosphorylated oligosaccharides to immobilized phosphomannosyl receptors.
We have purified phosphomannosyl-enzyme receptors from bovine liver on an affinity column composed of glycoproteins isolated from Dictyostelium discoideum secretions. Binding of human fibroblast beta-hexosaminidase B to receptors reconstituted into phosphatidylcholine liposomes was 1) specifically inhibited by mannose 6-phosphate, but not mannose 1-phosphate or glucose 6-phosphate, and 2) had p...
متن کاملImmobilization and assay of low-molecular-weight phosphomannosyl receptor in multiwell plates.
A novel sensitive binding assay for quantitation of a low-molecular-weight phosphomannosyl receptor (41-46 K) was devised. The receptor was immobilized by immunochemical means in the wells of polystyrene multiwell plates. The lysosomal enzyme ligand, testicular beta-galactosidase, was added and receptor-bound beta-galactosidase was measured by conventional colorimetric analysis using p-nitrophe...
متن کاملThe 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 n...
متن کاملPhosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes.
beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Mo...
متن کاملPurified Human Liver Acid A - D - Galactosidases Possessing Activity towards GMl - Ganglioside and Lactosylceramide By ARNOLD
Our studies with purified human liver acid 6-D-galactosidases (EC 3.2.1.23) indicate that 4-methylumbelliferyl fi-D-galactosidase and GM1-ganglioside /-D-galactosidase activities are identical with lactosylceramidase II activity. Evidence for this includes co-purification of all enzyme activities by affinity chromatography to yield a single band on polyacrylamide-gel electrophoresis and coincid...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Analytical biochemistry
دوره 182 2 شماره
صفحات -
تاریخ انتشار 1989