Beta-glycosylamidine as a ligand for affinity chromatography tailored to the glycon substrate specificity of beta-glycosidases.
An affinity adsorbent for beta-glycosidases has been prepared by using beta-glycosylamidine as a ligand. beta-Glucosylamidine and beta-galactosylamidine, highly potent and selective inhibitors of beta-glucosidases and beta-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a beta-glycosylamine and 2-iminothiolane.HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for beta-glucosidases and beta-galactosidases, respectively. This one-pot procedure enables various beta-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a beta-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a beta-glucosidase and a beta-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The beta-glucosidase and beta-galactosidase were inhibited competitively by a soluble form of the corresponding beta-glycosylamidine ligand with an inhibition constant (K(i)) of 2.1 and 0.80 microM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities.
Inoue
Kazuko
K
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Hiratake
Jun
J
Mizutani
Masaharu
M
Takada
Masayasu
M
Yamamoto
Mikio
M
Sakata
Kanzo
K
D000578:Amidines / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D001667:Binding, Competitive; D044602:Cellulases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D008024:Ligands; D008956:Models, Chemical; D010407:Penicillium / Q000201:enzymology; D013379:Substrate Specificity; D013662:Tea / Q000201:enzymology; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000578:Amidines; D004791:Enzyme Inhibitors; D008024:Ligands; D013662:Tea; D044602:Cellulases; D001616:beta-Galactosidase