Purification and immunological characterization of acid beta-galactosidase from dog liver.
1. Dog liver acid beta-galactosidase was isolated in high yield and purified to homogeneity using a series of chromatographies on Con A-Sepharose, decyl-agarose, anion-exchange HPLC and gel-filtration HPLC. 2. Non-denaturing gel filtration by HPLC gave a single homogeneous peak corresponding to molecular mass of 180-190 kDa. During SDS-PAGE analysis, the single peak dissociated into a major band corresponding to molecular mass of 32 kDa with minor bands at 18 and 13 kDa. 3. Polyclonal antibodies raised against the purified enzyme immunoprecipitated beta-galactosidase activity specifically from dog liver extracts and recognized a single 32 kDa band in Western blot analysis of dog tissue homogenates. This antibody did not crossreact with any protein band in tissue homogenates from other species examined except cat. 4. Western blot analysis of tissue extracts from dogs affected with GM1-gangliosidosis showed the presence of a 32 kDa band similar to that of controls.
Hotamisligil
S
S
Department of Biochemistry, E.K. Shriver Center for Mental Retardation, Waltham, MA 02254.
Hale
S
S
Alroy
J
J
Fischer
I
I
Raghavan
S
S
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D004285:Dogs / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009154:Mutation; D013045:Species Specificity; D001616:beta-Galactosidase / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
D001616:beta-Galactosidase