Radominska A, Berg C, Treat S, Little J M, Lester R, Gollan J L, Drake R R
Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock 72205.
Biochim Biophys Acta. 1994 Oct 12;1195(1):63-70. doi: 10.1016/0005-2736(94)90010-8.
The endoplasmic reticulum (ER) of rat liver contains several well characterized UDP-glucuronosyltransferases (UGTs), membrane-bound proteins of 50-54 kDa, and also less well identified UDP-glucosyltransferases, with nucleotide binding sites located on the lumenal surface. There is evidence that the substrates for these enzymes, UDP-glucuronic acid (UDP-GlcUA) and UDP-glucose (UDP-Glc), biosynthesized in the cytosol, are transported into the lumen of the ER via unknown mechanisms, the characteristics of which are poorly defined. A new approach for the study of the transport process has been devised using two active-site directed photoaffinity analogs, [beta-32P]5-azido-UDP-GlcUA and [beta-32P]5-azido-UDP-Glc. Photoincorporation of these probes into the lumenally oriented UGTs of intact rat liver microsomal vesicles was used as an indicator of transport. In intact vesicles, [32P]5N3UDP-GlcUA was efficiently incorporated into UGTs in a time, temperature and concentration dependent manner. In contrast, [32P]5N3UDP-Glc apparently was not transported effectively; maximal photolabeling of the 50-54 kDa proteins by this probe was dependent on detergent disruption of the vesicles. Vesicular uptake of and subsequent photolabeling of the 50-54 kDa proteins by [32P]5N3UDP-GlcUA were inhibited by UDP-GlcUA and 5N3UDP-GlcUA while UDP-Glc, 5N3UDP-Glc, UDP-xylose and UDP-N-acetylglucosamine were less inhibitory, suggesting a high degree of specificity for the uptake/photolabeling process. The anionic transport inhibitors DIDS and SITS inhibited [32P]5N3UDP-GlcUA photoincorporation into UGTs in intact vesicles, but also inhibited photolabeling of these and other enzymes in detergent disrupted vesicles. These data suggest the presence in rat liver microsomal vesicles of a specific, carrier-mediated transport process for UDP-GlcUA which is distinct from the mechanism of UDP-Glc transport.
大鼠肝脏的内质网（ER）含有几种特性明确的尿苷二磷酸葡萄糖醛酸基转移酶（UGTs），即50 - 54 kDa的膜结合蛋白，还有一些特性不太明确的尿苷二磷酸葡萄糖基转移酶，其核苷酸结合位点位于内质网腔表面。有证据表明，这些酶的底物——在胞质溶胶中生物合成的尿苷二磷酸葡萄糖醛酸（UDP-GlcUA）和尿苷二磷酸葡萄糖（UDP-Glc），通过未知机制被转运到内质网腔中，而这些机制的特性尚不清楚。利用两种活性位点定向光亲和类似物[β-32P]5-叠氮基-UDP-GlcUA和[β-32P]5-叠氮基-UDP-Glc，设计了一种研究转运过程的新方法。这些探针光掺入完整大鼠肝脏微粒体囊泡的面向内质网腔的UGTs中，被用作转运的指标。在完整囊泡中，[32P]5N3UDP-GlcUA以时间、温度和浓度依赖性方式有效地掺入UGTs中。相比之下，[32P]5N3UDP-Glc显然没有被有效地转运；该探针对50 - 54 kDa蛋白质的最大光标记依赖于囊泡的去污剂破坏。[32P]5N3UDP-GlcUA对50 - 54 kDa蛋白质的囊泡摄取及随后的光标记受到UDP-GlcUA和5N3UDP-GlcUA的抑制，而UDP-Glc、5N3UDP-Glc、尿苷二磷酸木糖和尿苷二磷酸-N-乙酰葡糖胺的抑制作用较小，这表明摄取/光标记过程具有高度特异性。阴离子转运抑制剂DIDS和SITS抑制完整囊泡中[32P]5N3UDP-GlcUA光掺入UGTs，但也抑制去污剂破坏的囊泡中这些酶和其他酶的光标记。这些数据表明，大鼠肝脏微粒体囊泡中存在一种特异性的、载体介导的UDP-GlcUA转运过程，该过程与UDP-Glc的转运机制不同。
