Glaser J H, Conrad H E
J Biol Chem. 1979 Jul 25;254(14):6588-97.
The beta-glucuronidase in homogenates of 12-day chick embryo livers catalyzed the release of glucuronic acid from 4-methylumbelliferyl-beta-D-glucuronide and from the nonreducing terminals of the hexasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4 at rates of 143, 114, and 108 nmol of glucuronic acid/h/mg of protein, respectively, when assayed at pH 3.5 in 0.05 M sodium acetate buffer. During a 60-fold purification of the enzyme, the ratios of the activities on these substrates did not change. When 4-methylumbelliferyl-beta-D-glucuronide was used as substrate the enzyme was active at pH values from 3.0 to 5.5, with maximal activity between pH values 4.0 and 4.5. Concentrations of NaCl from 0.15 to 0.3 M inhibited the activity at low pH values but activated the enzyme between pH 4.0 and 5.5. The enzyme was active on the chondroitin-6-SO4 hexasaccharide from pH 3.0 to 5.5, with a broad optimum between 3.0 and 4.5. NaCl inhibited the activity on the oligosaccharide substrate at all pH values. Eadie-Scatchard plots of rates of 4-methylumbelliferyl-beta-D-glucuronide hydrolysis at substrate concentrations ranging from 2 to 1000 microM showed multiple kinetic forms of the enzyme, a form with a Km of approximately 11 microM, and a second form with a Km of approximately 225 microM. The pH optimum of the low Km form was 3.5 to 4.0; that of the high Km form was pH 4.5. NaCl inhibited the activity of the low Km form, but activated the high Km form of the enzyme. Chondroitin SO4 oligosaccharides competed with 4-methylumbelliferyl-beta-D-glucuronide for the low Km form of the enzyme but had little effect on the hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide by the high Km form of the enzyme. The activities of the beta-glucuronidase on tetra-, hexa-, octa-, and decasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4, measured using a new assay procedure which can detect the formation of 1 nmol of product, were similar, although rates were somewhat lower for the higher oligosaccharides. With the exception of the chondroitin-4-SO4 tetrasaccharide, all of the oligosaccharide substrates saturated the enzyme at concentrations of 20 to 30 microM, indicating Km values of less than 10 to 15 microM for the oligosaccharides. Highly purified beta-glcuronidases from human placenta and from rat preputial gland also showed multiple kinetic forms when assayed using 4-methylumbelliferyl-beta-D-glucuronide as substrate.
12日龄鸡胚肝脏匀浆中的β-葡萄糖醛酸酶，在0.05M醋酸钠缓冲液中于pH 3.5测定时，催化从4-甲基伞形酮-β-D-葡萄糖醛酸苷以及硫酸软骨素-6-SO4和硫酸软骨素-4-SO4六糖的非还原末端释放葡萄糖醛酸，速率分别为143、114和108 nmol葡萄糖醛酸/小时/毫克蛋白质。在对该酶进行60倍纯化的过程中，其对这些底物的活性比值没有变化。当以4-甲基伞形酮-β-D-葡萄糖醛酸苷作为底物时，该酶在pH值3.0至5.5之间具有活性，在pH值4.0至4.5之间活性最大。0.15至0.3M的NaCl浓度在低pH值时抑制活性，但在pH 4.0至5.5之间激活该酶。该酶对硫酸软骨素-6-SO4六糖在pH 3.0至5.5之间具有活性，在3.0至4.5之间有一个较宽的最适pH范围。NaCl在所有pH值下均抑制对寡糖底物的活性。在底物浓度范围为2至1000μM时，对4-甲基伞形酮-β-D-葡萄糖醛酸苷水解速率的伊迪-斯卡查德图显示该酶有多种动力学形式，一种形式的Km约为11μM，另一种形式的Km约为225μM。低Km形式的最适pH为3.5至4.0；高Km形式的最适pH为4.5。NaCl抑制低Km形式的活性，但激活该酶的高Km形式。硫酸软骨素SO4寡糖与4-甲基伞形酮-β-D-葡萄糖醛酸苷竞争该酶的低Km形式，但对高Km形式的酶水解4-甲基伞形酮-β-D-葡萄糖醛酸苷的影响很小。使用一种能检测1 nmol产物形成的新测定方法测量，β-葡萄糖醛酸酶对硫酸软骨素-6-SO4和硫酸软骨素-4-SO4的四糖、六糖、八糖和十糖的活性相似，尽管较高寡糖的速率略低。除了硫酸软骨素-4-SO4四糖外，所有寡糖底物在20至30μM浓度时使该酶饱和，表明寡糖的Km值小于10至15μM。当以4-甲基伞形酮-β-D-葡萄糖醛酸苷作为底物进行测定时，来自人胎盘和大鼠包皮腺的高度纯化的β-葡萄糖醛酸酶也显示出多种动力学形式。
