Grünberg J, Sterchi E E
Institute of Biochemistry and Molecular Biology, University of Berne, Switzerland.
Arch Biochem Biophys. 1995 Nov 10;323(2):367-72. doi: 10.1006/abbi.1995.9952.
Human lactase-phlorizin hydrolase [EC 3.2.1.23-3.2.1.62] is a disaccharidase located in the microvillus membrane of small intestinal epithelial cells. The enzyme is synthesized as a precursor protein in the endoplasmic reticulum and in addition to being glycosylated is subsequently proteolytically processed to the mature microvillus membrane-bound form after passing the trans-Golgi compartment. We studied the oligomerization of human lactase-phlorizin hydrolase in transfected polarized Madin Darby canine kidney cells using metabolic labeling and sucrose-density centrifugation analysis. We detected high mannose dimers of the lactase-phlorizin hydrolase precursor molecule after metabolic labeling with [35S]methionine at 37 and 15 degrees C. In addition, both complex-glycosylated lactase-phlorizin hydrolase precursor molecule and the mature microvillus membrane-bound enzyme showed this oligomeric structure. Chemical crosslinking resulted in the detection of covalently crosslinked lactase-phlorizin hydrolase dimers after sodium dodecyl sulfate polyacrylamide gel electrophoresis. These results provide evidence that oligomerization of lactase-phlorizin hydrolase is an early event and begins in the endoplasmic reticulum.
人乳糖酶 - 根皮苷水解酶[EC 3.2.1.23 - 3.2.1.62]是一种位于小肠上皮细胞微绒毛膜上的双糖酶。该酶在内质网中以前体蛋白形式合成，除了进行糖基化外，在通过反式高尔基体区室后随后被蛋白水解加工成成熟的微绒毛膜结合形式。我们使用代谢标记和蔗糖密度离心分析，研究了转染的极化马 - 达二氏犬肾细胞中人乳糖酶 - 根皮苷水解酶的寡聚化。在用[35S]甲硫氨酸在37℃和15℃进行代谢标记后，我们检测到了乳糖酶 - 根皮苷水解酶前体分子的高甘露糖二聚体。此外，复杂糖基化的乳糖酶 - 根皮苷水解酶前体分子和成熟的微绒毛膜结合酶均显示出这种寡聚结构。化学交联导致在十二烷基硫酸钠聚丙烯酰胺凝胶电泳后检测到共价交联的乳糖酶 - 根皮苷水解酶二聚体。这些结果提供了证据，表明乳糖酶 - 根皮苷水解酶的寡聚化是一个早期事件，并且在内质网中就开始了。
