Trombetta S E, Gañan S A, Parodi A J
Instituto de Investigaciones Bioqímicas Fundación Campomar, Buenos Aires, Argentina.
Glycobiology. 1991 Mar;1(2):155-61. doi: 10.1093/glycob/1.2.155.
N-linked oligosaccharides devoid of glucose residues are transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum. The reaction products have been identified, depending on the organisms, as protein-linked Glc1Man5-9GlcNAc2. Incubation of right side-sealed vesicles from rat liver with UDP-[14C]Glc, Ca2+ ions and denatured thyroglobulin led to the glucosylation of the macromolecule only when the vesicles had been disrupted previously by sonication or by the addition of detergents to the glucosylation mixture. Similarly, maximal glucosylation of denatured thyroglobulin required disruption of microsomal vesicles isolated from the protozoan Crithidia fasciculata. Treatment of the rat liver vesicles with trypsin led to the inactivation of the UDP-Glc:glycoprotein glucosyltransferase only when proteolysis was performed in the presence of detergents. The glycoprotein glucosylating activity could be solubilized upon sonication of right side-sealed vesicles in an isotonic medium, upon passage of them through a French press or by suspending the vesicles in an hypotonic medium. Moreover, the enzyme appeared in the aqueous phase when the vesicles were submitted to a Triton X-114/water partition. Solubilization was not due to proteolysis of a membrane-bound enzyme. The enzyme could also be solubilized from C. fasciculata microsomal vesicles by procedures not involving membrane disassembly. About 30% of endogenous glycoproteins glucosylated upon incubation of intact rat liver microsomal vesicles with UDP-[14C]GLc could be solubilized by sonication or by suspending the vesicles in 0.1 M Na2CO3. These and previous results show that the UDP-Glc:glycoprotein glucosyltransferase is a soluble protein present in the lumen of the endoplasmic reticulum. In addition, both soluble and membrane-bound glycoproteins may be glucosylated by the glycoprotein glucosylating activity.
不含葡萄糖残基的N-连接寡糖在内质网中直接从UDP-葡萄糖进行短暂的糖基化。根据生物体的不同，反应产物已被鉴定为与蛋白质相连的Glc1Man5-9GlcNAc2。用UDP-[14C]葡萄糖、钙离子和变性甲状腺球蛋白孵育大鼠肝脏右侧封闭的囊泡，只有在囊泡先前通过超声处理或在糖基化混合物中添加去污剂而被破坏时，才会导致大分子的糖基化。同样，变性甲状腺球蛋白的最大糖基化需要破坏从原生动物克氏锥虫分离的微粒体囊泡。仅当在去污剂存在下进行蛋白水解时，用胰蛋白酶处理大鼠肝脏囊泡才会导致UDP-葡萄糖：糖蛋白葡萄糖基转移酶失活。糖蛋白糖基化活性可通过在等渗介质中超声处理右侧封闭的囊泡、使其通过法国压榨机或通过将囊泡悬浮在低渗介质中而溶解。此外，当囊泡进行Triton X-114/水分配时，酶出现在水相中。溶解不是由于膜结合酶的蛋白水解。该酶也可以通过不涉及膜拆卸的程序从克氏锥虫微粒体囊泡中溶解。完整的大鼠肝脏微粒体囊泡与UDP-[14C]葡萄糖孵育后，约30%的内源性糖蛋白糖基化可通过超声处理或通过将囊泡悬浮在0.1 M Na2CO3中而溶解。这些结果和先前的结果表明，UDP-葡萄糖：糖蛋白葡萄糖基转移酶是一种存在于内质网腔中的可溶性蛋白质。此外，可溶性和膜结合糖蛋白都可能被糖蛋白糖基化活性糖基化。
