Gibeaut D M, Carpita N C
Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907-1155.
Proc Natl Acad Sci U S A. 1993 May 1;90(9):3850-4. doi: 10.1073/pnas.90.9.3850.
Membranes of the Golgi apparatus from maize (Zea mays L.) were used to synthesize in vitro the (1-->3), (1-->4)-beta-D-glucan (MG) that is unique to the cell wall of the Poaceae. The MG was about 250 kDa and was separated from a much larger (1-->3)-beta-D-glucan (callose) by gel-permeation chromatography. Diagnostic oligosaccharides, released by a sequence-dependent endoglucanase from Bacillus subtilis, were separated by HPLC and GLC. The trisaccharide beta-D-Glcp-(1-->4)-beta-D-Glcp-(1-->3)-D-Glc, the tetrasaccharide [beta-D-Glcp-(1-->4)]2-beta-D-Glcp-(1-->3)-D-Glc, and longer cellodextrin-(1-->3)-D-Glc oligosaccharides were synthesized in proportions similar to those found in purified MG. Activated charcoal added during homogenization enhanced synthesis of MG, presumably by removing inhibitory compounds. The Golgi apparatus was determined as the site of synthesis by a combination of downward and flotation centrifugations on sucrose step gradients. The rate of synthesis did not reach saturation at up to 10 mM UDP-Glc. Chelators completely abolished synthesis, but synthase activity was restored by addition of either MgCl2 or, to a lesser extent, MnCl2. Synthesis continued for well over 1 h; addition of KOH to raise the pH from 7.2 to 8.0 during the reaction increased the rate of synthesis, which indicates that a transmembrane pH gradient may facilitate synthesis of MG.
利用玉米（Zea mays L.）高尔基体膜在体外合成了禾本科植物细胞壁特有的（1→3），（1→4）-β-D-葡聚糖（MG）。MG约为250 kDa，通过凝胶渗透色谱法与一种大得多的（1→3）-β-D-葡聚糖（胼胝质）分离。由枯草芽孢杆菌的序列依赖性内切葡聚糖酶释放的诊断性寡糖，通过高效液相色谱法和气相色谱法进行分离。三糖β-D-葡糖基-（1→4）-β-D-葡糖基-（1→3）-D-葡糖、四糖[β-D-葡糖基-（1→4）]2-β-D-葡糖基-（1→3）-D-葡糖以及更长的纤维糊精-（1→3）-D-葡糖寡糖的合成比例，与纯化的MG中的比例相似。匀浆过程中添加活性炭可增强MG的合成，可能是通过去除抑制性化合物实现的。通过在蔗糖阶梯梯度上进行向下和浮选离心相结合的方法，确定高尔基体为合成位点。在高达10 mM UDP-葡萄糖的情况下，合成速率未达到饱和。螯合剂完全抑制了合成，但通过添加MgCl2或在较小程度上添加MnCl2可恢复合酶活性。合成持续超过1小时；反应过程中添加KOH将pH从7.2提高到8.0，可提高合成速率，这表明跨膜pH梯度可能促进MG的合成。
