Alhadeff J A, Miller A L, Wenaas H, Vedvick T, O'Brien J S
J Biol Chem. 1975 Sep 25;250(18):7106-13.
Human liver alpha-L-fucosidase has been purified 6300-fold to apparent homogeneity with 66% yield by a two-step affinity chromatographic procedure utilizing agarose epsilon-aminocaproyl-fucosamine. Isoelectric focusing revealed that all six isoelectric forms of the enzyme were purified. Polyacrylamide gel electrophoresis of the purified alpha-L-fucosidase demonstrated the presence of six bands of protein which all contained fucosidase activity. The purified enzyme preparation was found to contain only trace amounts of seven glycosidases. Quantitative amino acid analysis was performed on the purified fucosidase. Preliminary carbohydrate analysis indicated that only about 1% of the molecule is carbohydrate. Gel filtration on Sepharose 4B indicated an approximate molecular weight for alpha-L-fucosidase of 175,000 +/- 18,000. High speed sedimentation equilibrium yielded a molecular weight of 230,000 +/- 10,000. Sodium dodecyl sulfate polyacrylamide gels indicated the presence of a single subunit of molecular weight, 50,100 +/- 2,500. The enzyme had a pH optimum of 4.6 with a suggested second optimum of 6.5. Apparent Michaelis constants and maximal velocities were determined on the purified enzyme with respect to the 4-methylumbelliferyl and the p-nitrophenyl substrates and were found to be 0.22 mM and 14.1 mumol/mg of protein/min and 0.43 mM and 19.6 mumol/mg of protein/min, respectively. Several salts had little or no effect on fucosidase activity although Ag+ and Hg2+ completely inactivated the enzyme. Antibodies made against the purified fucosidase were dound to be monospecific against crude human liver supernatant fluids and the pure antigen. No cross-reacting material was detected in the crude liver supernatant fluid from a patient who died with fucosidosis.
利用琼脂糖ε-氨基己酰岩藻糖胺，通过两步亲和层析法，已将人肝脏α-L-岩藻糖苷酶纯化了6300倍，收率达66%，达到了表观均一性。等电聚焦显示该酶的所有六种等电形式均已纯化。纯化后的α-L-岩藻糖苷酶的聚丙烯酰胺凝胶电泳表明存在六条蛋白带，所有这些蛋白带均含有岩藻糖苷酶活性。发现纯化的酶制剂仅含有痕量的七种糖苷酶。对纯化的岩藻糖苷酶进行了定量氨基酸分析。初步碳水化合物分析表明，该分子中仅约1%是碳水化合物。在Sepharose 4B上进行凝胶过滤表明，α-L-岩藻糖苷酶的近似分子量为175,000±18,000。高速沉降平衡得出分子量为230,000±10,000。十二烷基硫酸钠聚丙烯酰胺凝胶表明存在分子量为50,100±2,500的单个亚基。该酶的最适pH为4.6，推测的第二个最适pH为6.5。针对纯化的酶，测定了相对于4-甲基伞形酮基和对硝基苯基底物的表观米氏常数和最大速度，发现分别为0.22 mM和14.1 μmol/mg蛋白质/分钟以及0.43 mM和19.6 μmol/mg蛋白质/分钟。几种盐对岩藻糖苷酶活性几乎没有影响，尽管Ag+和Hg2+可使该酶完全失活。发现针对纯化的岩藻糖苷酶制备的抗体对粗制人肝脏上清液和纯抗原有单特异性。在死于岩藻糖苷贮积症的患者的粗制肝脏上清液中未检测到交叉反应物质。
