Nagaoka M, Saitoh M, Shiraishi T, Nagaoka H, Iriyama N, Furuhata K, Uda Y
Department of Health Chemistry, Niigata College of Pharmacy, Japan.
Biol Pharm Bull. 1998 Jul;21(7):682-7. doi: 10.1248/bpb.21.682.
Sialidase [E.C.3.2.1.18] has previously been purified from porcine liver by procedures including extraction, ammonium sulfate precipitation, concanavalin A-Sepharose adsorption, activation, CM-Sepharose ion exchange chromatography, and HPLC on a Shim pack Diol 300 column. Two sialidase preparations, sialidase I and II, were obtained by CM-Sepharose column chromatography and were eluted with pH 4.5 and 5.0 buffers, respectively. The two enzyme preparations showed the same optimum pH, pH stability, and specificities for natural substrates. The two final preparations contained beta-galactosidase activity and showed three protein components of 64, 30, and 21 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which are derived from the beta-galactosidase multimer. The anti-beta-galactosidase multimer antiserum was able to precipitate sialidase activity. It is likely that porcine liver sialidase exists as a multienzyme complex with beta-galactosidase and carboxypeptidase (protective protein).
唾液酸酶[E.C.3.2.1.18]先前已通过包括提取、硫酸铵沉淀、伴刀豆球蛋白A-琼脂糖吸附、活化、CM-琼脂糖离子交换色谱以及在Shim pack Diol 300柱上进行高效液相色谱等步骤从猪肝中纯化出来。通过CM-琼脂糖柱色谱获得了两种唾液酸酶制剂，即唾液酸酶I和II，分别用pH 4.5和5.0的缓冲液洗脱。这两种酶制剂对天然底物显示出相同的最适pH、pH稳定性和特异性。这两种最终制剂含有β-半乳糖苷酶活性，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中显示出64、30和21 kDa的三种蛋白质组分，它们源自β-半乳糖苷酶多聚体。抗β-半乳糖苷酶多聚体抗血清能够沉淀唾液酸酶活性。猪肝唾液酸酶很可能以与β-半乳糖苷酶和羧肽酶（保护蛋白）形成的多酶复合物形式存在。
