Brüss M, Hammermann R, Brimijoin S, Bönisch H
Institut für Pharmakologie und Toxikologie, Universität Bonn, Federal Republic of Germany.
J Biol Chem. 1995 Apr 21;270(16):9197-201. doi: 10.1074/jbc.270.16.9197.
We have raised polyclonal antibodies (N6-28, L211-226, L371-384, and C590-607) against peptides corresponding to hydrophilic sequences of the human norepinephrine transporter (hNET). The antisera immunoprecipitated the [35S]Met-labeled hNET. Antiserum L211-226, directed against a sequence of the putative second (large) extracellular loop of hNET, also immunoprecipitated the human dopamine transporter. Antisera N6-28 and C590-607, raised against a hNET peptide region of the N and the C termini, respectively, recognized a 58-kDa protein from transfected COS-7 cells expressing the hNET. This 58-kDa species represents a functional, glycosylated form of the hNET and not a degradation product. Tunicamycin treatment of transfected COS-7 cells as well as peptide-N-glycosidase F digestion of the transporter converted the 58-kDa species to a 50-kDa form, indicating that the latter represents the hNET core protein. In indirect immunofluorescence studies, our antisera confirmed the originally proposed topology of hNET. Antisera N6-28 and C590-607 detected hNET only in permeabilized cells. In contrast, antisera L211-226 and L371-384 directed against peptide sequences of the second and fourth putative extracellular loop displayed fluorescence signals with the intact cells.
我们针对与人去甲肾上腺素转运体（hNET）亲水区序列相对应的肽段制备了多克隆抗体（N6 - 28、L211 - 226、L371 - 384和C590 - 607）。这些抗血清免疫沉淀了[35S]甲硫氨酸标记的hNET。抗血清L211 - 226针对hNET假定的第二个（大的）细胞外环序列，也免疫沉淀了人多巴胺转运体。分别针对hNET N端和C端肽段区域制备的抗血清N6 - 28和C590 - 607，识别来自表达hNET的转染COS - 7细胞中的一种58 kDa蛋白。这种58 kDa的蛋白代表hNET的一种功能性糖基化形式，而非降解产物。用衣霉素处理转染的COS - 7细胞以及对转运体进行肽 - N - 糖苷酶F消化，可将58 kDa的蛋白转化为50 kDa的形式，表明后者代表hNET核心蛋白。在间接免疫荧光研究中，我们的抗血清证实了最初提出的hNET拓扑结构。抗血清N6 - 28和C590 - 607仅在通透细胞中检测到hNET。相反，针对假定的第二个和第四个细胞外环肽序列的抗血清L211 - 226和L371 - 384在完整细胞中显示出荧光信号。
