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首页> 外文期刊>Journal of cell biology >Localization and Biosynthesis of NADH-Cytochrome b(5) reductase, an integral membrane protein, in rat liver cells. III. Evidence for the independent insertion and turnover of the enzyme in various subcellular compartments
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Localization and Biosynthesis of NADH-Cytochrome b(5) reductase, an integral membrane protein, in rat liver cells. III. Evidence for the independent insertion and turnover of the enzyme in various subcellular compartments

机译：在大鼠肝细胞中NADH细胞色素b（5）还原酶，一种完整的膜蛋白的定位和生物合成。三，各种亚细胞区隔中酶独立插入和转换的证据

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The biosynthesis and turnover of rat liver NADH-cytochrome b(5) reductase was studied in in vivo pulse-labeling and long-term, double-labeling experiments. Rats under thiopental anesthesia were injected into the portal vein with [(3)H]L-leucine and sacrificed at various times after the injection. NADH-cytochrome b(5) reductase was extracted from liver cell fractions by cathepsin D-catalyzed cleavage and was then immunoadsorbed onto antireductase-bearing affinity columns in the presence of excess unlabeled rat serum. After elution of the enzyme from the columns with a pH-2.2 buffer, the amount of the reductase protein in the samples was determined by radioimmunoassay, and the radioactivity in reductase was determined on SDS polyacrylamide gel reductase bands. The specific radioactivity of the reductase extracted from the homogenate as well as from rough and smooth microsomal, mitochondrial, and Golgi fractions, estimated at the end of the pulse (10 min after the injection) and at various time points thereafter, remained approximately constant over a 6-h period. These data suggest tha tth eenzyme is independently inserted into the various membranes where it is located. Moreover, the specific radioactivity of the mitochondrial reductase was lower than that of the other fractions, suggesting that it turns over at a slower rate. The lower turnover rate of the mitochondrial enzyme was confirmed by long-term, double-labeling experiments carried out according to the technique of Arias et al. (J. Biol. Chem. 244: 3303-3315.). The relevance of these findings in relation to the understanding of membrane biogenesis and turnover is discussed.
机译：大鼠体内NADH-细胞色素b（5）还原酶的生物合成和营业额在体内脉冲标记和长期，双标记实验中进行了研究。硫喷妥钠麻醉下的大鼠被注射[（3）H] L-亮氨酸进入门静脉，并在注射后的不同时间处死。通过组织蛋白酶D催化的裂解从肝脏细胞级分中提取NADH-细胞色素b（5）还原酶，然后在存在过量未标记大鼠血清的情况下将其免疫吸附到带有抗还原酶的亲和柱上。用pH-2.2缓冲液从柱中洗脱酶后，通过放射免疫测定法测定样品中还原酶蛋白的量，并在SDS聚丙烯酰胺凝胶还原酶谱带上测定还原酶的放射性。从匀浆以及粗糙和光滑的微粒体，线粒体和高尔基部分中提取的还原酶的比放射性在脉冲结束时（注射后10分钟）及其后的各个时间点估计，在整个过程中保持恒定6小时的时间。这些数据表明该酶被独立地插入其所处的各种膜中。此外，线粒体还原酶的比放射性低于其他部分，表明它以较慢的速度翻转。通过根据Arias等人的技术进行的长期双标记实验，证实了线粒体酶的较低转化率。 （J.Biol.Chem.244：3303-3315。）。讨论了这些发现与对膜生物发生和更新的理解的相关性。

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