Kömhoff M, Hollinshead M, Tooze J, Kern H F
Department of Cell Biology and Cell Pathology, University of Marburg, Germany.
Eur J Cell Biol. 1994 Apr;63(2):192-207.
We have studied the effect of increasing concentrations of Brefeldin A (BfA) on the rate of secretion in vitro of pulse-labeled proteins and individual enzymes and on the fine structure of the Golgi apparatus in pancreatic acinar cells derived from control rats and from animals stimulated in vivo by feeding a synthetic proteinase inhibitor (FOY 305). A half-maximal inhibition of intracellular transport of newly synthesized proteins was observed at 0.125 microgram/ml BfA which in FOY-stimulated pancreatic lobules was more pronounced. The Golgi apparatus at this low dose of BfA was still preserved and consisted of stacks of narrow cisternae devoid of secretory material. Granule formation from the trans-Golgi network (TGN) was greatly reduced. A nearly complete inhibition of both total protein and individual enzyme transport was observed at 0.5 microgram/ml BfA. This inhibitory effect was more pronounced with enzyme proteins which are transported slowly through the cellular compartments (e.g. lipase) as compared to faster moving proteins (e.g. chymotrypsinogen). The Golgi apparatus at 0.5 microgram/ml BfA was fragmented into clouds of small uniform vesicles which were stained with an antibody directed against TGN 38 and which were surrounded by a network of tubular membranes reacting with an antibody against p58, a marker protein of the intermediate compartment between rough endoplasmic reticulum (RER) and Golgi. Incubation of pancreatic lobules in 10 micrograms/ml BfA led to a disappearance of the small vesicles and a relocation of TGN 38 into the RER, while the tubuloreticular membranes of the intermediate compartment remained unaffected. Aggregates of clathrin cages devoid of membranes accumulated in the area of the previous Golgi vesicles. The inhibitory effect of 0.5 microgram/ml BfA on both intracellular transport and Golgi fine structure was readily reversible within 15 to 30 min upon removal of the drug, but took 1 h or longer after incubation in 5 or 10 micrograms/ml BfA.
我们研究了增加布雷菲德菌素A（BfA）浓度对脉冲标记蛋白和单个酶的体外分泌速率以及对来自对照大鼠和通过喂食合成蛋白酶抑制剂（FOY 305）在体内受到刺激的动物的胰腺腺泡细胞中高尔基体精细结构的影响。在0.125微克/毫升BfA时观察到新合成蛋白细胞内运输的半数最大抑制，这在FOY刺激的胰腺小叶中更为明显。在此低剂量BfA下，高尔基体仍得以保留，由缺乏分泌物质的狭窄扁平囊堆叠组成。从反式高尔基体网络（TGN）形成颗粒的过程大大减少。在0.5微克/毫升BfA时观察到总蛋白和单个酶运输几乎完全被抑制。与移动较快的蛋白（如胰凝乳蛋白酶原）相比，这种抑制作用对通过细胞区室运输较慢的酶蛋白（如脂肪酶）更为明显。在0.5微克/毫升BfA时，高尔基体破碎成小的均匀囊泡云，这些囊泡用针对TGN 38的抗体染色，并被与针对p58（粗面内质网（RER）和高尔基体之间中间区室的标记蛋白）的抗体反应的管状膜网络包围。将胰腺小叶在10微克/毫升BfA中孵育导致小囊泡消失，TGN 38重新定位到RER中，而中间区室的管状网状膜未受影响。缺乏膜的网格蛋白笼聚集体在前高尔基体囊泡区域积累。在去除药物后，0.5微克/毫升BfA对细胞内运输和高尔基体精细结构的抑制作用在15至30分钟内很容易逆转，但在5或10微克/毫升BfA中孵育后则需要1小时或更长时间。
