β‐Glucosidases from Cicer arietinum L.

Abstract

β-Glucosidases specific for isoflavone 7-O-glucosides have been isolated from garbanzo plants, Cicer arietinum L. These aryl-β-glucohydrolases occur in the different organs of the plant as multiple molecular forms. The major isoenzymes of the roots, the leaves and the hypocotyl were purified to electrophoretic homogeneity. When subjected to isoelectric focussing in polyacrylamide gels the electrophoretically homogeneous glucohydrolases were found to consist of one or two major and several minor enzymically active molecular species. In roots the β-glucohydrolase isoenzymes constitute a considerable portion of the extractable protein, so that purification to an electrophoretically homogeneous form is easily attainable. All β-glucosidases analyzed possess molecular weights in the range of 125000 (ultracentrifugation) to 135000 (Sephadex G-200) and contain two subunits of molecular weight near 68000. The pH optimum for enzymic activity is 7–7.5 with a second optimum of 4.5–5. The isoelectric points of the various species range between pH 5.9 and 7.1. Staining for glycoprotein was positive. Kinetic analysis demonstrated a pronounced specificity of the enzymes for aromatic substrates with glucose as the sugar moiety. α-Glucosides as well as disaccharides were not hydrolyzed at all. Isoflavone 7-O-glucosides are the most favoured substrates with a K_m of 2 × 10⁻⁵ M, while the K_m with aromatic glucosides (i.e. salicin, 4-nitrophenyl glucoside) are 100 times larger. In addition the β-glucosidases show a pronounced specificity for glucose in the 7-position of the flavonoid nucleus. Using isoflavone aglycones as substrates glucose transferase activity was also demonstrable. The β-glucohydrolase activity is strongly inhibited by Hg²⁺. This inhibition is partially reversible and preferentially influences the K_m values of the enzymes compared to to V. Ag⁺ glucono-1,5-lactone, ethyleneglycol monomethyl ether and glycerol are only weakly inhibitory, while glucose, p-chloromercuribenzoate and Cu²⁺ are without effect.