Purification and characterization of Phaseolus vulgaris alpha-D-galactosidase isozymes.
A highly purified preparation of alpha-D-galactosidase [E.C. 3.2.1.22] isozymes was obtained from Phaseolus vulgaris (pinto bean) seeds by extraction, salt precipitation, ion exchange, and affinity chromatography. The final preparation was homogeneous by SDS-PAGE but revealed isozymes of relative mass of 38.3 and 39.6 kDa. The N-terminal sequence for both isozymes was identical, LANGLAKT (one letter code for amino acids). Relative native molecular mass was estimated at 149.3 kDa by Sephacryl S-200 chromatography. Activity was unaffected by ionic strength at high enzyme concentrations, and was specific for alpha-D-galactoside conjugates. No protease or hemagglutinin activity was detected, and activity was stable at 4 degrees C. Studies with soluble oligosaccharides demonstrated high activity against the selected straight and branched-chain substrates. The enzyme was active against terminal alpha 1-3 galactosyl residues on human and rabbit erythrocyte membranes. Because of its activity against membrane glycoconjugates, these isozymes may have potential utility for modifying membrane epitopes on native erythrocytes.
Dhar
M
M
Department of Biology, University of Missouri, Columbia 65211.
Mitra
M
M
Hata
J
J
Butnariu
O
O
Smith
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D004912:Erythrocytes / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology*; D005697:Galactosides / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D011817:Rabbits; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism
D005697:Galactosides; D007527:Isoenzymes; D000519:alpha-Galactosidase