Kanno C, Hwangbo S, Azuma N
Department of Applied Biochemistry, Utsunomiya University, Japan.
Biosci Biotechnol Biochem. 1995 May;59(5):848-52. doi: 10.1271/bbb.59.848.
The isolation and partial characterization of PAS-4 glycoprotein (78 kDa) from bovine milk fat globule membrane (MFGM) is described. PAS-4 was selectively extracted with Triton X-114 nonionic detergent and then fractionated on DEAE-Sepharose at pH 7.5. The PAS-4 fraction that was not bound on DEAE-Sepharose gave a single band by SDS-PAGE. The recovery of PAS-4 was 57.4% from MFGM. An amino acid analysis found a high percentage of nonpolar residues. Approximately 7.2% of carbohydrate from PAS-4 was composed of mannose, galactose (Gal), N-acetylglucosamine, N-acetylgalactosamine (GalNAc), and sialic acid, most of the Gal and GalNAc in PAS-4 being released after mild alkaline hydrolysis. This indicated that PAS-4 contained both N- and O-linked sugar chains in concordance with the results of lectin affinity. PAS-4 had apparent isoelectric points of 7.45, 7.41, and 7.32, but these were shifted to pI 7.47 by a neuraminidase treatment. The apparent molecular weight of PAS-4 after deglycosylation with N-glycanase was approximately 57,000 by SDS-PAGE.
本文描述了从牛乳脂肪球膜（MFGM）中分离并部分鉴定PAS - 4糖蛋白（78 kDa）的过程。PAS - 4用Triton X - 114非离子去污剂进行选择性提取，然后在pH 7.5条件下于DEAE - 琼脂糖上进行分级分离。未结合在DEAE - 琼脂糖上的PAS - 4级分经SDS - PAGE显示为单一条带。PAS - 4从MFGM中的回收率为57.4%。氨基酸分析发现非极性残基的比例很高。PAS - 4中约7.2%的碳水化合物由甘露糖、半乳糖（Gal）、N - 乙酰葡糖胺、N - 乙酰半乳糖胺（GalNAc）和唾液酸组成，PAS - 4中的大部分Gal和GalNAc在温和碱性水解后被释放。这表明PAS - 4同时含有N - 连接和O - 连接的糖链，与凝集素亲和力结果一致。PAS - 4的表观等电点为7.45、7.41和7.32，但经神经氨酸酶处理后，这些等电点移至pI 7.47。用N - 聚糖酶进行去糖基化后，PAS - 4经SDS - PAGE显示的表观分子量约为57,000。
