Biosynthesis of Oligogalactosyl Side Chains of the Cell Envelope Glycoprotein of <em>Cryptococcus laurentii</em>

A particulate enzyme preparation from the fungus imperfectus var. flavescens (NNRL Y-1401) catalyzes the transfer of galactosyl residues from UDP-[14C]-galactose to an endogenous acceptor. The radioactive reaction product is particle-bound, but is solubilized by pronase treatment. The solubilized radioactive material migrates as a single peak on a column of Sepharose 4-B, indicating that the galactosyl residues are transferred to an endogenous polymeric acceptor of high molecular weight. After elution from Sepharose 4B the material was found to contain 70% carbohydrate and 30% protein. The monosaccharides released after acid hydrolysis are mannose, galactose, and xylose, galactose being the only radioactive monosaccharide constituent. The radioactive reaction product is retained on DEAE-cellulose, from which over 80% of it is eluted with 0.5mNaCl together with endogenous carbohydrate and protein. After treatment with NaOH-NaBH4, the radioactive material no longer binds to DEAE-cellulose. Subsequent gel filtration on Sephadex G-25 results in a symmetrical radioactive peak of approximately 2000 molecular weight, suggesting liberation of a dodecasaccharide chain. Treatment with NaOH-NaB3H4followed by complete acid hydrolysis allows the isolation of tritium-labeled alanine and α-aminobutyric acid, indicating that carbohydrate side chains are linked through seryl and threonyl residues to protein. The major radioactive disaccharides isolated after partial acid hydrolysis are 6--α-[14C]galactosyl--[14C]galactose and -β-[14C]-galactosyl--mannose. The data suggest addition of multiple galactosyl residues to an endogenous glycoprotein acceptor: the first galactosyl residue is linked to a mannosyl residue forming a β linkage; subsequently additional galactosyl residues are transferred sequentially forming linear chains containing 6--α-galactosyl--galactosyl linkages.

来自黄变不完全真菌变种（NNRL Y - 1401）的一种颗粒状酶制剂催化半乳糖基残基从UDP - [14C] - 半乳糖转移至一种内源性受体。放射性反应产物与颗粒结合，但经链霉蛋白酶处理可溶解。溶解后的放射性物质在琼脂糖4 - B柱上呈单峰迁移，表明半乳糖基残基转移至一种高分子量的内源性聚合物受体。从琼脂糖4B洗脱后，发现该物质含有70%的碳水化合物和30%的蛋白质。酸水解后释放的单糖为甘露糖、半乳糖和木糖，其中半乳糖是唯一具有放射性的单糖成分。放射性反应产物保留在DEAE - 纤维素上，其中80%以上可与内源性碳水化合物和蛋白质一起用0.5mol/L氯化钠洗脱。经氢氧化钠 - 硼氢化钠处理后，放射性物质不再与DEAE - 纤维素结合。随后在葡聚糖凝胶G - 25上进行凝胶过滤，得到分子量约为2000的对称放射性峰，表明释放出一条十二糖链。经氢氧化钠 - 硼氢化钠处理后再进行完全酸水解，可分离出氚标记的丙氨酸和α - 氨基丁酸，这表明碳水化合物侧链通过丝氨酸和苏氨酸残基与蛋白质相连。部分酸水解后分离出的主要放射性二糖为6 - α - [14C]半乳糖基 - [14C]半乳糖和 - β - [14C] - 半乳糖基 - 甘露糖。这些数据表明多个半乳糖基残基添加到一种内源性糖蛋白受体上：第一个半乳糖基残基与一个甘露糖基残基相连形成β键；随后其他半乳糖基残基依次转移，形成含有6 - α - 半乳糖基 - 半乳糖基键的直链。