Baldwin G S, Chandler R, Seet K L, Weinstock J, Grego B, Rubira M, Mortiz R L, Simpson R J
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Parkville, Victoria, Australia.
Protein Seq Data Anal. 1987;1(1):7-12.
A 75-kDa glycoprotein (P75) has been purified to homogeneity from washed membranes isolated from the corpus of porcine gastric mucosa. The purification procedure employed chromatography on concanavalin A-Sepharose and DEAE-Sepharose, and preparative polyacrylamide gel electrophoresis. Reversed-phase microbore high-performance liquid chromatography was employed to fractionate and purify a number of tryptic peptides generated from approximately 1100 pmol purified P75. The use of reversed-phase microbore (2.1 mm internal diameter) columns facilitated the purification of subnanomole amounts of polypeptides in small volumes (40-60 microliter) suitable for loading onto the gas-phase sequencer without further concentration. N-Terminal amino acid sequence analyses were performed on the intact polypeptide and on 13 tryptic peptides and one Staphylococcus protease V8 peptide, yielding 170 unique assignments; this corresponds to approximately 26% of the molecule. A comparison of this amino acid sequence information with the cDNA-deduced primary structure of a 70-kDa heat-shock-related protein (P72), which is expressed in normal rat liver reveals that these protein sequences are almost identical, differing in only 1 of the 170 positions positively assigned thus far. The probable correspondence of P72 with the 78-kDa glucose-regulated protein (GRP78) isolated from hamster fibroblasts has been reported.
一种75千道尔顿的糖蛋白（P75）已从猪胃黏膜体分离的洗涤膜中纯化至同质。纯化过程采用伴刀豆球蛋白A-琼脂糖和二乙氨基乙基-琼脂糖柱层析以及制备性聚丙烯酰胺凝胶电泳。采用反相微孔高效液相色谱对约1100皮摩尔纯化的P75产生的一些胰蛋白酶肽进行分级分离和纯化。使用反相微孔（内径2.1毫米）柱有助于在小体积（40 - 60微升）中纯化亚纳摩尔量的多肽，这些小体积适合直接加载到气相测序仪上而无需进一步浓缩。对完整多肽以及13个胰蛋白酶肽和1个葡萄球菌蛋白酶V8肽进行了N端氨基酸序列分析，得到170个独特的序列信息；这大约相当于该分子的26%。将此氨基酸序列信息与在正常大鼠肝脏中表达的70千道尔顿热休克相关蛋白（P72）的cDNA推导的一级结构进行比较，发现这些蛋白质序列几乎相同，在迄今确定的170个位置中仅有1个位置不同。已有报道称P72可能与从仓鼠成纤维细胞中分离的78千道尔顿葡萄糖调节蛋白（GRP78）相对应。
