Hósel W, Barz W
Eur J Biochem. 1975 Sep 15;57(2):607-16. doi: 10.1111/j.1432-1033.1975.tb02336.x.
Beta-Glucosidases specific for isoflavone 7-O-glucosides have been isolated from garbanzo plants, Cicer arietinum L. These aryl-beta-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 polyac rylamide gels the electrophoretically homogeneous glucohydrolases were found to consist of one or two major and several minor enzymically active molecular species. In roots the beta-glucohydrolase isoenzymes constitute a considerable portion of the extractable protein, so that purification to an electrophoretically homogeneous form is easily attainable. All beta-glucosidases analyzed possess molecular weights in the range of 125 000 (ultracentrifugation) to 135 000 (Sephadex G-200) and contain two subunits of molecular weight near 68 000. 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. alpha-Glucosides as well as disaccharides were not hydrolyzed at all. Isoflavone 7-O-glucosides are the most favoured substrates with a Km of 2 x 10(-5) M, while the Km with aromatic glucosides (i.e. salicin, 4-nitrophenyl glucoside) are 100 times larger. In addition the beta-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 beta-glucohydrolase activity is strongly inhibited by Hg2plus. This inhibition is partially reversible and preferentially influences the Km values of the enzymes compared to V. Agplus, glucono-1,5-lactone, ethyleneglycol monomethyl ether and glycerol are only weakly inhibitory, while glucose, p-chloromercuribenzoate and Cu2plus are without effect.
已从鹰嘴豆（Cicer arietinum L.）中分离出对异黄酮7 - O - 葡萄糖苷具有特异性的β - 葡萄糖苷酶。这些芳基 - β - 葡萄糖水解酶以多种分子形式存在于植物的不同器官中。根、叶和下胚轴的主要同工酶被纯化至电泳纯。当在聚丙烯酰胺凝胶中进行等电聚焦时，发现电泳纯的葡萄糖水解酶由一种或两种主要的以及几种次要的具有酶活性的分子种类组成。在根中，β - 葡萄糖水解酶同工酶占可提取蛋白质的相当一部分，因此很容易纯化至电泳纯形式。所有分析的β - 葡萄糖苷酶的分子量范围为125000（超速离心法）至135000（葡聚糖G - 200法），并且含有两个分子量接近68000的亚基。酶活性的最适pH为7 - 7.5，第二个最适pH为4.5 - 5。各种分子种类的等电点在pH 5.9至7.1之间。糖蛋白染色呈阳性。动力学分析表明，这些酶对以葡萄糖为糖基部分的芳香族底物具有明显的特异性。α - 葡萄糖苷以及二糖根本不被水解。异黄酮7 - O - 葡萄糖苷是最受青睐的底物，Km为2×10⁻⁵ M，而与芳香族葡萄糖苷（即水杨苷、4 - 硝基苯基葡萄糖苷）的Km值大100倍。此外，β - 葡萄糖苷酶对黄酮核7位上的葡萄糖具有明显的特异性。以异黄酮苷元为底物时，也可检测到葡萄糖转移酶活性。β - 葡萄糖水解酶活性受到Hg²⁺的强烈抑制。这种抑制部分可逆，与V相比，优先影响酶的Km值。Ag⁺、葡萄糖酸 - 1,5 - 内酯、乙二醇单甲醚和甘油的抑制作用较弱，而葡萄糖、对氯汞苯甲酸和Cu²⁺则无作用。
