RICIN-A CHAIN CAN BE CHEMICALLY CROSS-LINKED TO THE MAMMALIAN RIBOSOMAL-PROTEINS L9 AND L10E
RICIN-A CHAIN CAN BE CHEMICALLY CROSS-LINKED TO THE MAMMALIAN RIBOSOMAL-PROTEINS L9 AND L10E
复制标题
DOI：
10.1074/jbc.270.21.12933
复制
发表时间：
1995-05-26
影响因子：
4.8
通讯作者：
GOLDMACHER, VS
中科院分区：
文献类型：
--
作者：
VATER, CA;BARTLE, LM;GOLDMACHER, VS
间接免疫荧光研究表明，当固定的，透化培养的人细胞与蓖麻毒素A链孵育，毒素分子定位在染色模式指示结合到内质网和核仁。进行化学交联实验以鉴定介导蓖麻毒素A链结合的细胞组分。标记的蓖麻毒素A链和两种蛋白质之间形成的共轭物存在于总细胞膜的制剂和纯化的哺乳动物核糖体的样品中，蓖麻毒素A链-核糖体相互作用的特异性被证明由过量的未标记的蓖麻毒素A链，但不是由过量的未标记的gelonin，另一种核糖体失活蛋白的复合物的形成的抑制。复合物的蓖麻毒素A链交联的核糖体蛋白进行纯化，并进行蛋白水解消化与胰蛋白酶。内部胰蛋白酶肽的氨基酸测序能够鉴定蓖麻毒素A链结合蛋白作为哺乳动物核糖体大亚基的L9和L10 e。
Indirect immunofluorescence studies revealed that when fixed, permeabilized cultured human cells were incubated with ricin A chain, the toxin molecule localized in a staining pattern indicative of binding to the endoplasmic reticulum and to nucleoli. Chemical crosslinking experiments were performed to identify the cellular components that mediated the binding of ricin A chain. Conjugates were formed between I-125-labeled ricin A chain and two proteins present in preparations of total cell membranes and in samples of purified mammalian ribosomes, Specificity of the ricin A chain-ribosome interaction was demonstrated by inhibition of formation of the complexes by excess unlabeled ricin A chain, but not by excess unlabeled gelonin, another ribosome-inactivating protein. Complexes of ricin A chain cross-linked to the ribosomal proteins were purified and subjected to proteolytic digestion with trypsin. Amino acid sequencing of internal tryptic peptides enabled identification of the ricin A chain-binding proteins as L9 and L10e of the mammalian large ribosomal subunit.