Chen X G, Nagai T, Yamada H
Oriental Medicine Research Center, Kitasato Institute, Tokyo, Japan.
Eur J Biochem. 1994 Apr 15;221(2):655-64. doi: 10.1111/j.1432-1033.1994.tb18778.x.
Sialidase activities of rabbit blood cells and serum were measured. The leucocyte particulate fraction showed the highest specific activity of sialidase towards mixed gangliosides and sialyllactose, and the cytosolic fraction showed for fetuin. Predominant sialidase activity in the blood was detected in erythrocyte particulate fraction when mixed gangliosides were used as substrate. The sialidase for ganglioside was solubilized from the erythrocyte ghosts by using Triton X-100. The solubilized sialidase was purified 1886-fold by sequential chromatographies on DEAE-cellulose, EAH-Sepharose 4B, Octyl-Sepharose CL-4B, Sephadex G-100, concanavalin-A--Sepharose, N-(p-aminophenyl)oxamic acid-agarose and Heparin-Sepharose CL-6B. The optimum pH of purified sialidase was 4.5 for ganglioside mixture, and this enzyme exhibited M(r) = 48,000 by gel filtration. When the purified sialidase was subjected to SDS/PAGE, a major sialidase-active protein band at M(r) = 54,000 and another fainter inactive protein band with M(r) = 115,000 were observed. The purified enzyme was active towards oligosaccharides, gangliosides, fetuin glycopeptide and 4-methylumbelliferyl alpha-D-N-acetylneuraminic acid except for glycoproteins tested. Fe2+, Fe3+ and dithiothreitol significantly inhibited the enzyme activity, while Triton X-100 activated the enzyme. Inside-out vesicles and unsealed ghosts of rabbit erythrocyte showed the sialidase activity for mixed gangliosides but not for resealed ghosts or intact erythrocytes. These results indicate that the active site of this sialidase is oriented mainly on the inside of the erythrocyte membrane and not on the outside. Treatment of rabbit erythrocyte unsealed ghosts with phosphatidylinositol-specific phospholipase C liberated no sialidase activity toward mixed gangliosides from the ghosts.
测定了兔血细胞和血清的唾液酸酶活性。白细胞颗粒部分对混合神经节苷脂和唾液乳糖显示出最高的唾液酸酶比活性，而细胞溶质部分对胎球蛋白显示出最高活性。当以混合神经节苷脂为底物时，血液中主要的唾液酸酶活性在红细胞颗粒部分被检测到。通过使用Triton X-100从红细胞血影中溶解出针对神经节苷脂的唾液酸酶。通过在DEAE-纤维素、EAH-琼脂糖4B、辛基-琼脂糖CL-4B、葡聚糖G-100、伴刀豆球蛋白A-琼脂糖、N-（对氨基苯基）草氨酸-琼脂糖和肝素-琼脂糖CL-6B上进行连续色谱法，将溶解的唾液酸酶纯化了1886倍。纯化的唾液酸酶对神经节苷脂混合物的最适pH为4.5，通过凝胶过滤该酶的分子量为48,000。当纯化的唾液酸酶进行SDS/PAGE时，观察到在分子量为54,000处有一条主要的唾液酸酶活性蛋白带，以及在分子量为115,000处有另一条较淡的无活性蛋白带。除了所测试的糖蛋白外，纯化的酶对寡糖、神经节苷脂、胎球蛋白糖肽和4-甲基伞形酮基α-D-N-乙酰神经氨酸有活性。Fe2+、Fe3+和二硫苏糖醇显著抑制酶活性，而Triton X-100激活该酶。兔红细胞的外翻小泡和未封闭血影对混合神经节苷脂显示出唾液酸酶活性，但对重新封闭的血影或完整红细胞则没有。这些结果表明，这种唾液酸酶的活性位点主要位于红细胞膜的内侧而非外侧。用磷脂酰肌醇特异性磷脂酶C处理兔红细胞未封闭血影不会从血影中释放出针对混合神经节苷脂的唾液酸酶活性。
