Trimble R B, Atkinson P H, Tschopp J F, Townsend R R, Maley F
New York State Department of Health, Wadsworth Center for Laboratories & Research, Albany, New York 12201-0509.
J Biol Chem. 1991 Dec 5;266(34):22807-17.
Saccharomyces SUC2 invertase, secreted by the methylotrophic yeast Pichia pastoris and purified to homogeneity from the growth medium by DE-52 chromatography, appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a diffuse ladder of species at 85-90 kDa, while the secreted Saccharomyces form migrated as a broad band from 100 to 150 kDa. Endo-beta-N-acetylglucosaminidase H released the Pichia invertase carbohydrate generating a 60-kDa protein with residual Asn-linked GlcNAcs and oligosaccharides separated on Bio-Gel P-4 into Man8-11GlcNAc. Nearly 75% of the oligosaccharides were equally distributed between Man8,9GlcNAc, while 17% were Man10GlcNAc and 8% were Man11GlcNAc. Oligosaccharide pools were analyzed for homogeneity by high-pH anion-exchange chromatography, and structures were assigned using 500 MHz one- and two-dimensional 1H NMR spectroscopy. Pichia Man8GlcNAc was the same isomer as found in Saccharomyces, which arises by removing the alpha 1,2-linked terminal mannose from the middle arm of the lipid-oligosaccharide Man9GlcNAc (Byrd, J. C., Tarentino, A. L., Maley, F., Atkinson, P. H., and Trimble, R. B. (1982) J. Biol. Chem. 257, 14657-14666). The Man9GlcNAc pool was 5% lipid-oligosaccharide precursor and 95% Man8GlcNAc isomer with a terminal alpha 1,6-linked mannose on the lower-arm alpha 1,3-core-linked residue (Hernández, L. M., Ballou, L., Alvarado, E., Gillece-Castro, B. L., Burlingame, A. L., and Ballou, C. E. (1989) J. Biol. Chem. 264, 11849-11856). An alpha 1,2-linked mannose on the new alpha 1,6-linked branch in Man9GlcNAc provided 80% of the Man10GlcNAc, which is the structure on Saccharomyces invertase (Trimble, R. B., and Atkinson, P. H. (1986) J. Biol. Chem. 261, 9815-9824). A minor Man10GlcNAc (12%) and the principal Man11GlcNAc (82%) were the major Man9,10GlcNAc with novel alpha 1,2-linked mannoses on the preexisting alpha 1,2-linked termini. Although Pichia glycans did not have terminal alpha 1,3-linked mannoses as found on Saccharomyces core oligosaccharides, over 60% of the structures were isometric configurations unique to lower eukaryotes.
甲基营养型酵母毕赤酵母分泌的酿酒酵母SUC2转化酶，通过DE - 52柱色谱法从生长培养基中纯化至同质，在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上呈现为85 - 90 kDa的弥散条带梯状图谱，而分泌的酿酒酵母形式迁移为100至150 kDa的宽带。内切β - N - 乙酰葡糖胺酶H释放毕赤酵母转化酶的碳水化合物，产生一个60 kDa的蛋白质，其带有残余的Asn连接的GlcNAc和寡糖，在Bio - Gel P - 4上分离为Man8 - 11GlcNAc。近75%的寡糖平均分布在Man8,9GlcNAc之间，17%为Man10GlcNAc，8%为Man11GlcNAc。通过高pH阴离子交换色谱分析寡糖库的同质性，并使用500 MHz一维和二维1H NMR光谱确定结构。毕赤酵母Man8GlcNAc与酿酒酵母中发现的异构体相同，它是通过从脂质寡糖Man9GlcNAc的中间臂去除α1,2连接的末端甘露糖产生的（伯德，J.C.，塔伦蒂诺，A.L.，马利，F.，阿特金森，P.H.，和特里姆布尔，R.B.（1982年）《生物化学杂志》257，14657 - 14666）。Man9GlcNAc库中5%是脂质寡糖前体，95%是Man8GlcNAc异构体，在较低臂的α1,3 - 核心连接残基上带有末端α1,6 - 连接的甘露糖（埃尔南德斯，L.M.，巴卢，L.，阿尔瓦拉多，E.，吉列斯 - 卡斯特罗，B.L.，伯林盖姆，A.L.，和巴卢，C.E.（1989年）《生物化学杂志》264，11849 - 11856）。Man9GlcNAc中新的α1,6 - 连接分支上的α1,2 - 连接甘露糖提供了80%的Man10GlcNAc，这是酿酒酵母转化酶上的结构（特里姆布尔，R.B.，和阿特金森，P.H.（1986年）《生物化学杂志》261，9815 - 9824）。少量的Man10GlcNAc（12%）和主要的Man11GlcNAc（82%）是主要的Man9,10GlcNAc，在预先存在的α1,2 - 连接末端带有新的α1,2 - 连接甘露糖。尽管毕赤酵母聚糖没有酿酒酵母核心寡糖上发现的末端α1,3 - 连接甘露糖，但超过60%的结构是低等真核生物特有的等距构型。
