A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity
A new selenoprotein from human lung adenocarcinoma cells: Purification, properties, and thioredoxin reductase activity
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DOI：
10.1073/pnas.93.3.1006
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发表时间：
1996-02-06
影响因子：
11.1
通讯作者：
Stadtman, TC
中科院分区：
文献类型：
--
作者：
Tamura, T;Stadtman, TC
我们报告了一个新的硒蛋白的分离和鉴定，从人肺腺癌细胞系，NCI-H441。细胞在含有10%（体积/体积）胎牛血清和0.1 μ M [Se-75]亚硒酸盐的RPMI 1640培养基中生长。经DE-23、苯基琼脂糖、肝素琼脂糖和丁基琼脂糖层析，从细胞的声波提取物中分离到一种Se-75标记的蛋白质。该蛋白质是一种57 kDa亚基的同源二聚体，含有硒代半胱氨酸形式的硒;用碘乙酸酯或3-溴丙酸酯烷基化的蛋白质的水解分别产生Se-羧甲基-硒代半胱氨酸或Se-羧乙基-硒代半胱氨酸。硒蛋白在pH5.2和pH5.3有两个等电点。通过N-糖苷酶测定和高碘酸-丹酰肼试验表明，硒蛋白P上没有可检测到的糖基。硒蛋白含有FAD作为辅基，催化NADPH依赖的5，5 ′-二硫代双（2-硝基苯甲酸）（DTNB），以及在硫氧还蛋白（Trx）存在下的胰岛素还原，通过DTNB测定确定比活性为31单位/mg，DTNB、大肠杆菌Trx和大鼠Trx的表观Km值分别为116、34和3.7 μ M。DTNB还原被0.2mM亚砷酸盐抑制。虽然亚基组成和催化性质与哺乳动物硫氧还蛋白还原酶（TR）相似，但人肺硒蛋白在免疫印迹试验中不能与抗大鼠肝TR多克隆抗体反应。
We report the isolation and characterization of a new selenoprotein from a human lung adenocarcinoma cell line, NCI-H441. Cells were grown in RPMI 1640 medium containing 10% (vol/vol) fetal bovine serum and 0.1 mu M [Se-75]selenite. A Se-75-labeled protein was isolated from sonic extracts of the cells by chromatography on DE-23, phenyl-Sepharose, heparin-agarose, and butyl-Sepharose, The protein, a homodimer of 57-kDa subunits, was shown to contain selenium in the form of selenocysteine; hydrolysis of the protein alkylated with either iodoacetate or 3-bromopropionate yielded Se-carboxy methyl - selenocysteine or Se-carboxyethyl-selenocysteine, respectively. The selenoprotein showed two isoelectric points at pH 5.2 and pH 5.3. It was distinguished from selenoprotein P by N-glycosidase assay and by the periodate-dansylhydrazine test, which indicated no detectable amounts of glycosyl groups on the protein, The selenoprotein contains FAD as a prosthetic group and catalyzes NADPH-dependent reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and reduction of insulin in the presence of thioredoxin (Trx), The specific activity was determined to be 31 units/mg by DTNB assay, Apparent K-m, values for DTNB, Escherichia coli Trx, and rat Trx were 116, 34, and 3.7 mu M, respectively. DTNB reduction was inhibited by 0.2 mM arsenite, Although the subunit composition and catalytic properties are similar to those of mammalian thioredoxin reductase (TR), the human lung selenoprotein failed to react with anti-rat liver TR polyclonal antibody in immunoblot assays, The selenocysteine-containing TR from the adenocarcinoma cells may be a variant form distinct from rat liver TR.