Lin C W, Orcutt M L, Fishman W H
J Biol Chem. 1975 Jun 25;250(12):4737-43.
Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.
β-葡萄糖醛酸酶已从先前用促性腺激素诱导的小鼠肾脏中纯化出来，其比未诱导的肾脏的特异性酶活性高15倍。纯化过程包括超声处理以溶解酶、酸沉淀和硫酸铵沉淀、在Sephadex G-200中进行凝胶过滤、DEAE离子交换色谱和等电聚焦。所得产物的比活性为284,000菲什曼单位/毫克蛋白质，代表1090倍的纯化，比未诱导的肾脏中的水平高17,000倍。根据凝胶过滤、十二烷基硫酸钠凝胶电泳和免疫扩散的标准，纯化的β-葡萄糖醛酸酶显然是均一的。对纯化酶的表征表明，它在电泳上与溶酶体同工酶相同，亚基分子量为74,000（通过十二烷基硫酸钠凝胶电泳估计），寡聚体分子量为300,000。纯化的酶在高温（高达55摄氏度）和广泛的pH范围（4至11）内稳定。其活性的最适pH为4.7，Km为1.18×10⁻⁴M。从小鼠肾脏中纯化和表征这种酶对于理解β-葡萄糖醛酸酶同工酶的分子性质具有重要意义，并且将有助于未来关于这种水解酶的细胞内运输和分布机制的研究。
