Zaremba S, Hogue-Angeletti R A
J Biol Chem. 1981 Dec 10;256(23):12310-5.
The topographical arrangement of membrane-bound dopamine beta-monooxygenase in the chromaffin granule was investigated by proteolytic treatment and radiolabeling of intact granules or the isolated granule membranes. Topographical analysis was furthered by precipitating detergent extracts of treated membranes with specific antibodies to dopamine beta-monooxygenase, followed by electrophoresis of the immunoprecipitate in acrylamide-dodecyl sulfate. Trypsin or pronase treatment of intact granules cleaved small portions of membrane-bound dopamine beta-monooxygenase, resulting in proteins with slightly lower molecular weights. Membranes bearing these smaller forms of membrane-bound dopamine beta-monooxygenase were fully active enzymatically. Pronase treatment of isolated membranes cleaved all of the membrane-bound dopamine beta-monooxygenase antigenic sites from the membrane and also removed the enzymatic activity. Lactoperoxidase-catalyzed iodination revealed that membrane-bound dopamine beta-monooxygenase can be labeled in intact granules, but to a greater extent in isolated membranes. Quantitative peptide mapping showed that the distribution of label among tryptic peptides of membrane-bound dopamine beta-monooxygenase differed, depending on whether intact granules or isolated membranes had been labeled. The results of these experiments provide evidence that membrane-bound dopamine beta-monooxygenase is a transmembrane protein which has its active site exclusively on the intragranular face, plus a unique portion of its polypeptide chain exposed on the cytoplasmic face of the intact granule.
通过对完整颗粒或分离的颗粒膜进行蛋白水解处理和放射性标记，研究了嗜铬颗粒中膜结合多巴胺β-单加氧酶的拓扑排列。通过用针对多巴胺β-单加氧酶的特异性抗体沉淀处理过的膜的去污剂提取物，然后在丙烯酰胺-十二烷基硫酸盐中对免疫沉淀物进行电泳，进一步进行拓扑分析。用胰蛋白酶或链霉蛋白酶处理完整颗粒会切割膜结合多巴胺β-单加氧酶的小部分，产生分子量略低的蛋白质。带有这些较小形式的膜结合多巴胺β-单加氧酶的膜具有完全的酶活性。用链霉蛋白酶处理分离的膜会从膜上切割所有膜结合多巴胺β-单加氧酶的抗原位点，同时也会去除酶活性。乳过氧化物酶催化的碘化反应表明，膜结合多巴胺β-单加氧酶可以在完整颗粒中被标记，但在分离的膜中标记程度更高。定量肽图谱分析表明，膜结合多巴胺β-单加氧酶的胰蛋白酶肽段中标记的分布有所不同，这取决于标记的是完整颗粒还是分离的膜。这些实验结果提供了证据，表明膜结合多巴胺β-单加氧酶是一种跨膜蛋白，其活性位点仅位于颗粒内表面，并且其多肽链的独特部分暴露在完整颗粒的细胞质表面。
