COUPLED CELL-FREE SYNTHESIS, SEGREGATION, AND CORE GLYCOSYLATION OF A SECRETORY PROTEIN
COUPLED CELL-FREE SYNTHESIS, SEGREGATION, AND CORE GLYCOSYLATION OF A SECRETORY PROTEIN
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DOI：
10.1073/pnas.75.5.2338
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发表时间：
1978-01-01
影响因子：
11.1
通讯作者：
BLOBEL, G
中科院分区：
文献类型：
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作者：
LINGAPPA, VR;LINGAPPA, JR;BLOBEL, G
将产后13-15天大鼠乳腺的m[信使]RNA移植到小麦胚芽无细胞系统中，无论是否存在从狗胰腺分离的粗微粒体制备的去除核糖体的膜。新合成的α-用抗大鼠α-乳清蛋白的单特异性兔抗血清通过免疫沉淀鉴定乳清蛋白。乳清蛋白，其特征在于部分氨基末端序列测定和凝集素亲和层析。在不存在膜的情况下，α-葡萄糖基化的可能未糖基化形式存在于细胞中。合成了既不与伴刀豆球蛋白A-琼脂糖凝胶结合也不与蓖麻凝集素-琼脂糖结合并且含有包含19个氨基酸残基的氨基末端信号肽区域的乳清蛋白。在膜的存在下，合成了加工形式，其缺乏信号肽部分并且具有与成熟α-的氨基末端序列相同的氨基末端序列乳白蛋白。发现这种加工形式被分离，推测在微粒体囊泡内，因为它对翻译后蛋白水解具有抗性。还发现它是糖基化的，并且因为它与伴刀豆球蛋白A-Sepharose结合，它可以被α-葡聚糖特异性洗脱。甲基甘露糖苷，而不是R. communis lectin-琼脂糖，它可能是核心糖基化的。加工，分离和核心糖基化进行观察，只有当膜翻译过程中存在。
m[messenger]RNA from rat mammary glands 13-15 days post partum was transplated in a wheat germ cell-free system either in the absence or in the presence of ribosome-denuded membranes prepared from isolated rough microsomes of dog pancreas. Newly synthesized .alpha.-lactalbumin was identified by immunoprecipitation with a monospecific rabbit antiserum against rat .alpha.-lactalbumin and was characterized by partial amino-terminal sequence determination and by lectin affinity chromatography. In the absence of membranes a presumably unglycosylated form of .alpha.-lactalbumin was synthesized that bound neither to concanavalin A-Sepharose nor to Ricinus communis lectin-agarose and that contained an amino-terminal signal peptide region comprising 19 amino acid residues. In the presence of membranes a processed form was synthesized that lacked the signal peptide portion and that had an amino-terminal sequence identical to that of mature .alpha.-lactalbumin. This processed form was found to be segregated, presumably within the microsomal vesicles, because it was resistant to post-translational proteolysis. It was also found to be glycosylated, and because it bound to concanavalin A-Sepharose, from which it could be eluted specifically by .alpha.-methyl mannoside, but not R. communis lectin-agarose, it was presumably core-glycosylated. Processing, segregation and core glycosylation were observed to proceed only when membranes were present during translation.