Purification and characterization of isoforms of beta-galactosidases in mung bean seedlings.
Five isoforms of beta-galactosidase (EC 3.2.1.23), designated as beta-galactosidases I-V, were isolated from five-day-old mung bean (Vigna radiata) seedlings. Beta-galactosidases II and III were purified to electrophoretic homogeneity by a procedure involving acid precipitation, ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose (DEAE-Cellulose) and con A-Sepharose. and chromatofocusing. Beta-galactosidases I, II and III have the same molecular mass of 87 kDa. comprising two nonidentical subunits with molecular masses of 38 and 48 kDa, while beta-galactosidases IV and V have molecular masses of 45 and 73 kDa, respectively. All the enzymes were active against p-nitrophenyl-beta-D-galactoside, and to a lesser extent, p-nitrophenyl-alpha-L-arabinoside and p-nitrophenyl-beta-D-fucoside. The enzymes were inhibited by D-galactono-1,4-lactone, D-galactose, Hg2+, Ag+ and sodium dodecyl sulfate (SDS). Beta-galactosidases I, II and III were shown to be competitively inhibited by either D-galactono-1, 4-lactone or D-galactose. Isoforms I, II and III have a common optimal pH of 3.6, while isoforms IV and V have pH optima at 3.8 and 4.0, respectively. Isoelectric points of isoforms I, II and III were 7.7, 7.5 and 7.3, respectively. Double immunodiffusion analysis indicated that beta-galactosidases I, II, III and V are immunologically similar to each other, while beta-galactosidase IV shares partially identical antigenic determinants with the other four isoforms. The purified beta-galactosidases II and III were capable of releasing D-galactose residue from the hemicellulose fraction isolated from mung bean seeds.
Li
S C
SC
Graduate Institute of Agricultural Chemistry, National Taiwan University, Taipei.
Han
J W
JW
Chen
K C
KC
Chen
C S
CS
D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007887:Fabaceae / Q000201:enzymology*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D007785:Lactose / Q000494:pharmacology; D008970:Molecular Weight; D009005:Monosaccharides / Q000494:pharmacology; D010946:Plants, Medicinal; D021122:Protein Subunits; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009005:Monosaccharides; D021122:Protein Subunits; D001616:beta-Galactosidase; D007785:Lactose