Valentijn J A, Sengupta D, Gumkowski F D, Tang L H, Konieczko E M, Jamieson J D
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510/USA.
Eur J Cell Biol. 1996 May;70(1):33-41.
This study reports of presence of rab3D, a low M(r) GTP-binding protein, in rat pancreatic acinar cells and islets using a combination of Western blot analysis, two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis/isoelectric focusing, and light and electron microscopic immunocytochemistry. For these purposes, we used an affinity-purified rabbit polyclonal antibody generated against the exclusive amino terminus of rab3D. Failure to detect rab3A, B or C in pancreatic acinar cells with their respective antisera indicated that the rab3D immunoreactivity was not due to cross-reaction with rab3A, B or C. A monoclonal antiserum which recognized rab3A, B, C and D (clone 42.1) detected a second lower M, band in gradient gels. This protein may be an additional member of the rab family. Double label electron microscopic immunogold localizations for rab3D, and the monoclonal antibody that recognizes all members of the rab3 family, showed a preferential localization of rab3D to zymogen granules. In contrast, clone 42.1 detected both zymogen granules and elements of the Golgi complex. Rab3D also localized to the secretory granule field in pancreatic islet cells which additionally expressed rab3A. The majority of rab3D in acinar cells was tightly associated with membrane fractions as indicated by its resistance to alkaline pH extraction. It is likely associated with membranes via isoprenyl groups as suggested by its partitioning into the detergent phase in Triton X-114 extractions. In contrast, bacterially expressed rab3D partitioned solely into the aqueous phase in Triton X-114 extractions. Because of its exclusive location on zymogen granules, rab3D may play a role in regulated exocytosis from pancreatic acinar cells.
本研究通过蛋白质免疫印迹分析、二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳/等电聚焦以及光镜和电镜免疫细胞化学相结合的方法，报道了低分子量GTP结合蛋白rab3D在大鼠胰腺腺泡细胞和胰岛中的存在情况。为此，我们使用了针对rab3D唯一氨基末端产生的亲和纯化兔多克隆抗体。用各自的抗血清未能在胰腺腺泡细胞中检测到rab3A、B或C，这表明rab3D免疫反应性不是由于与rab3A、B或C的交叉反应所致。一种识别rab3A、B、C和D的单克隆抗血清（克隆42.1）在梯度凝胶中检测到第二条较低分子量的条带。这种蛋白质可能是rab家族的另一个成员。rab3D与识别rab3家族所有成员的单克隆抗体的双标记电子显微镜免疫金定位显示，rab3D优先定位于酶原颗粒。相比之下，克隆42.1检测到酶原颗粒和高尔基体复合体的成分。Rab3D也定位于胰腺胰岛细胞的分泌颗粒区域，这些细胞还表达rab3A。腺泡细胞中大部分rab3D与膜组分紧密相关，这一点从其对碱性pH提取的抗性可以看出。正如其在Triton X-114提取中分配到去污剂相所表明的那样，它可能通过异戊二烯基与膜相关。相比之下，细菌表达的rab3D在Triton X-114提取中仅分配到水相。由于其仅位于酶原颗粒上，rab3D可能在胰腺腺泡细胞的调节性胞吐作用中发挥作用。
