Himeno M, Koutoku H, Tsuji H, Kato K
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka.
J Biochem. 1988 Nov;104(5):773-6. doi: 10.1093/oxfordjournals.jbchem.a122548.
Acid phosphatase in rat liver lysosomal contents, C-APase I, was purified about 5,700-fold over the homogenate with 8.0% recovery, to apparent homogeneity as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included; preparation of crude lysosomal contents, DEAE-Sephacel ion exchange chromatography, hydroxylapatite chromatography, and gel filtration with Sephacryl S-300. The enzyme is composed of three identical subunits with an apparent molecular weight of 48K. The enzyme contains about 11% carbohydrate and the carbohydrate moiety was composed of mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine in a molar ratio of 20:3:11:1. Sialic acid was not detected in the enzyme. Antisera against the purified C-APase I were raised in goat and the C-APase I was rapidly purified with high yield (10%) by using the specific antibodies coupled to Sepharose 6B.
大鼠肝脏溶酶体内容物中的酸性磷酸酶C-APase I，经纯化后比匀浆提高了约5700倍，回收率为8.0%，根据在有和没有十二烷基硫酸钠（SDS）的情况下聚丙烯酰胺凝胶电泳图谱判断，达到了表观均一性。纯化步骤包括：制备粗溶酶体内容物、二乙氨基乙基-葡聚糖凝胶离子交换色谱法、羟基磷灰石色谱法以及用Sephacryl S-300进行凝胶过滤。该酶由三个相同的亚基组成，表观分子量为48K。该酶含有约11%的碳水化合物，碳水化合物部分由甘露糖、岩藻糖、N-乙酰葡糖胺和N-乙酰半乳糖胺组成，摩尔比为20:3:11:1。在该酶中未检测到唾液酸。用山羊制备了针对纯化后的C-APase I的抗血清，并通过使用与琼脂糖6B偶联的特异性抗体以高产率（10%）快速纯化了C-APase I。
