Purification and characterization of acid beta-D-galactosidase from rabbit spleen.
beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.
Rodríguez-Berrocal
F J
FJ
Department of Biochemistry, Faculty of Biology, University of Salamanca, Spain.
Páez de la Cadena
M
M
Cabezas
J A
JA
Pérez-González
N
N
D000818:Animals; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005696:Galactosidases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D011817:Rabbits; D013154:Spleen / Q000378:metabolism*; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism
D005696:Galactosidases; D001616:beta-Galactosidase