Membrane disposition of the Escherichia coli mannitol permease: identification of membrane-bound and cytoplasmic domains.
Membrane disposition of the Escherichia coli mannitol permease: identification of membrane-bound and cytoplasmic domains.
复制标题
大肠杆菌甘露醇渗透酶的膜处置：膜结合域和细胞质域的鉴定。
DOI：
10.1021/bi00373a016
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Jacobson,GR
中科院分区：
文献类型：
--
作者：
Stephan,MM;Jacobson,GR
材料和方法化学品。[35S]甲醇（1000 Ci/mmol）、15种14 C标记氨基酸（55 mCi/毫碳原子）的混合物和[3 H]甘露醇（19 Ci/mmol）购自NewEngland Nuclear（Boston，MA）。125 I标记的金黄色葡萄球菌蛋白A（50 μ Ci/罐）购自ICN Biomedicals，Inc.。（Irvine，CA）。L-（对甲苯磺酰氨基）-2-苯乙基氯甲基酮（TPCK）处理的胰蛋白酶购自Sigma Chemical Co. (St. Louis，MO）。膜囊泡的制备。E.如前所述分离含有编码甘露醇操纵子的质粒pLC 15 -48的大肠杆菌菌株MV 1009（Lee等，1981年）。然后在合成过程中用[35 S]甲硫氨酸加其他19种未标记氨基酸中的每一种2 ng/mL或用以下物质的混合物选择性标记EIImt 1：
Materials and MethodsChemicals.[35S] Methionine (1000 Ci/mmol), a mixture of 15 14C-labeled amino acids (55 mCi/milliatom of carbon), and [3H] mannitol (19 Ci/mmol) were purchased from NewEngland Nuclear (Boston, MA). 125I-Labeled Staphylococcus aureus protein A (50/uCi/jug) was purchased from ICN Biomedicals, Inc.(Irvine, CA). Ll-(Tosylamido)-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin was pur-chased from Sigma Chemical Co.(St. Louis, MO). Preparation of Membrane Vesicles. Minicellsof E. coli strain MV1009 containing the plasmid pLC15-48, which en-codes the mannitol operon, were isolated as described previ-ously (Lee et al., 1981). EIImtl was then selectively labeled during itssynthesis with [35S] methionine plus 2 ng/mL each of the other 19 unlabeled amino acids or with a mixture of