Spiro M J, Spiro R G
J Biol Chem. 1985 May 10;260(9):5808-15.
The effect of anion-specific inhibitors on the utilization of the sugar nucleotides (UDP-glucose, GDP-mannose, and UDP-N-acetylglucosamine) required for the formation of the oligosaccharide-lipid involved in N-glycosylation has been studied in intact endoplasmic reticulum (ER) vesicles from thyroid. Of the reagents tested, the nonpenetrating probe DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) and its dihydro derivative (H2DIDS) were the most effective, causing a pronounced impairment in the synthesis from UDP-Glc of dolichyl phosphate (Dol-P) glucose (50% reduction at 60 microM DIDS) and in the incorporation of glucose into oligosaccharide-lipid and N-glycosylated protein; in contrast, no inhibition was observed in the formation from UDP-Glc of a glycogen-like proteoglucan. The specificity of the DIDS effect was indicated by the finding that methyl isothiocyanate, a nonanionic amino-reactive agent, demonstrated negligible inhibition. While DIDS also effected a block in the formation of Dol-P-P-GlcNAc from UDP-GlcNAc, no impairment in the utilization of GDP-Man for Dol-P-Man synthesis was observed. Since the DIDS inhibition of UDP-Glc and UDP-GlcNAc utilization was maintained after disruption of the ER vesicles with Triton, even when the incubations were supplemented with Dol-P, it appears that this reagent does not interact with sugar nucleotide translocator proteins but rather with the cytoplasmically oriented anion binding sites of glycosyltransferases (UDP-Glc- and UDP-GlcNAc:Dol-P glucosyl- and GlcNAc-1-P transferases). This is consistent with the protease sensitivity of these enzymes in the intact ER vesicles. Incubation of the vesicles with tritiated H2DIDS (8 microM) introduced radioactivity into membrane polypeptides with molecular weights of about 52,000 and 31,000 as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that this inhibitor may prove useful as an affinity label in further studies of some of the glycosyltransferases involved in the synthesis of lipid-monosaccharide intermediates.
阴离子特异性抑制剂对参与N-糖基化的寡糖脂形成所需的糖核苷酸（UDP-葡萄糖、GDP-甘露糖和UDP-N-乙酰葡糖胺）利用的影响，已在来自甲状腺的完整内质网（ER）囊泡中进行了研究。在所测试的试剂中，非穿透性探针DIDS（4,4'-二异硫氰酸-2,2'-二苯乙烯二磺酸）及其二氢衍生物（H2DIDS）最为有效，导致从UDP-葡萄糖合成磷酸多萜醇（Dol-P）葡萄糖受到显著损害（在60μM DIDS时减少50%），并使葡萄糖掺入寡糖脂和N-糖基化蛋白中；相反，在从UDP-葡萄糖形成类糖原蛋白聚糖时未观察到抑制作用。甲基异硫氰酸酯（一种非阴离子氨基反应剂）抑制作用可忽略不计，这一发现表明了DIDS作用的特异性。虽然DIDS也会阻断从UDP-N-乙酰葡糖胺形成Dol-P-P-GlcNAc，但未观察到对利用GDP-甘露糖合成Dol-P-甘露糖有损害。由于在用Triton破坏ER囊泡后，即使在孵育中添加了Dol-P，DIDS对UDP-葡萄糖和UDP-N-乙酰葡糖胺利用的抑制作用仍然存在，因此看来该试剂不与糖核苷酸转运蛋白相互作用，而是与糖基转移酶（UDP-葡萄糖和UDP-N-乙酰葡糖胺：Dol-P葡糖基和GlcNAc-1-P转移酶）面向细胞质的阴离子结合位点相互作用。这与这些酶在完整ER囊泡中的蛋白酶敏感性一致。如通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳所观察到的，用氚标记的H2DIDS（8μM）孵育囊泡将放射性引入分子量约为52,000和31,000的膜多肽中，这表明该抑制剂可能作为一种亲和标记物，在进一步研究参与脂质-单糖中间体合成的一些糖基转移酶中有用。
