Miura S, Erickson R H, Song I S, Kim Y S
Gastrointestinal Research Laboratory, Veterans Administration Medical Center, San Francisco, CA 94121.
Biochem Pharmacol. 1988 Nov 1;37(21):4081-8. doi: 10.1016/0006-2952(88)90099-8.
Tunicamycin, a known inhibitor of the lipid-dependent glycosylation of proteins, was used in vivo to study the biosynthesis of rat intestinal brush border membrane aminopeptidase N and dipeptidyl aminopeptidase IV. The incorporation of [3H]glucosamine into newly synthesized total protein of mucosal cell homogenates was inhibited by 60%, whereas incorporation of [3H]leucine was decreased only 21% by tunicamycin. This effect was much more pronounced in the brush border membrane fraction isolated from intestinal mucosal cells where incorporation of radiolabled leucine and glucosamine was reduced to 50 and 82% of control values respectively. An examination of the brush border membrane protein profile by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that there was a marked selective decrease in the amount of glycoproteins of molecular weights greater than 130 kD. In addition, there were decreased levels of assayable aminopeptidase N, dipeptidyl aminopeptidase IV and disaccharidase activity in intestinal mucosal cell homogenates and brush border membranes of tunicamycin-treated rats. Though tunicamycin decreased incorporation of newly synthesized aminopeptidase N and dipeptidyl aminopeptidase IV protein into brush border membranes by 70-75%, the newly synthesized enzyme that was incorporated was indistinguishable from that of controls. Further, non-glycoslyated forms of both enzymes were not detected in any other subcellular fractions. These results show that tunicamycin, an inhibitor of glycosylation, significantly affected the expression of brush border membrane glycoproteins, suggesting that both polypeptide synthesis and degradation of these proteins may be altered in the presence of this drug.
衣霉素是一种已知的蛋白质脂质依赖性糖基化抑制剂，被用于体内研究大鼠肠刷状缘膜氨基肽酶N和二肽基肽酶IV的生物合成。[3H]葡萄糖胺掺入黏膜细胞匀浆新合成的总蛋白中的量被抑制了60%，而衣霉素使[3H]亮氨酸的掺入量仅降低了21%。在从肠黏膜细胞分离的刷状缘膜部分，这种效应更为明显，其中放射性标记的亮氨酸和葡萄糖胺的掺入量分别降至对照值的50%和82%。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳对刷状缘膜蛋白图谱进行检查发现，分子量大于130 kD的糖蛋白量有明显的选择性减少。此外，在衣霉素处理的大鼠的肠黏膜细胞匀浆和刷状缘膜中，可检测到的氨基肽酶N、二肽基肽酶IV和双糖酶活性水平降低。虽然衣霉素使新合成的氨基肽酶N和二肽基肽酶IV蛋白掺入刷状缘膜的量减少了70 - 75%，但掺入的新合成酶与对照酶没有区别。此外，在任何其他亚细胞部分均未检测到这两种酶的非糖基化形式。这些结果表明，糖基化抑制剂衣霉素显著影响了刷状缘膜糖蛋白的表达，提示在这种药物存在的情况下，这些蛋白质的多肽合成和降解可能都发生了改变。
