Coffe G, Raymond M N
Laboratoire de Structure et Réactivité des Protéines, CNRS URA 1131, Université Paris XI, Orsay, France.
Biol Cell. 1990;70(3):143-52. doi: 10.1016/0248-4900(90)90371-9.
We report an isolation procedure of trans-Golgi vesicles (GVs) from rat parotid glands. Various organelle markers were used, particularly galactosyl transferase as a trans-Golgi marker, to test the purity of the GV fraction. A quantitative in vitro binding assay between microtubules and GVs is described. The vesicles were incubated with taxol-induced microtubules, layered between 50% and 43% sucrose cushions and subjected to centrifugation. Unlike free microtubules which were sedimented, the GV-bound microtubules co-migrated upward with GVs. Quantification of these bound microtubules was carried out by densitometric scanning of Coomassie blue-stained gels. The association between microtubules and GVs followed a saturation curve, with a plateau value of 20 micrograms of microtubule protein bound to 500 micrograms of GV fraction. The half-saturation of the GV sites was obtained with a microtubule concentration of 20 micrograms/ml. Electron microscopy of negatively stained re-floated material showed numerous microtubule-vesicle complexes. Coating of microtubules with an excess of brain microtubule-associated proteins (MAPs) abolished binding. In the absence of exogenous microtubules, we showed that the GV fraction was already interacting with a class of endogenous rat parotid microtubules. This class of colcemid and cold-stable microtubules represents 10-20% of the total tubulin content of the parotid cell.
我们报道了一种从大鼠腮腺中分离反式高尔基体囊泡（GVs）的方法。使用了各种细胞器标志物，特别是半乳糖基转移酶作为反式高尔基体标志物，来检测GV组分的纯度。描述了微管与GVs之间的定量体外结合测定。将囊泡与紫杉醇诱导的微管一起孵育，铺在50%和43%的蔗糖垫层之间并进行离心。与沉降的游离微管不同，与GV结合的微管与GVs一起向上迁移。通过考马斯亮蓝染色凝胶的光密度扫描对这些结合的微管进行定量。微管与GVs之间的结合遵循饱和曲线，平台值为20微克微管蛋白与500微克GV组分结合。GV位点的半饱和状态在微管浓度为20微克/毫升时获得。对负染的再漂浮材料进行电子显微镜观察，显示出大量微管-囊泡复合物。用过量的脑微管相关蛋白（MAPs）包被微管可消除结合。在没有外源微管的情况下，我们表明GV组分已经在与一类内源性大鼠腮腺微管相互作用。这类秋水仙酰胺和冷稳定微管占腮腺细胞微管蛋白总量的10%-20%。
