Erickson H K
Department of Chemistry, 0506, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093-0506, USA.
Biochemistry. 1997 Aug 19;36(33):9958-67. doi: 10.1021/bi970493t.
The location with respect to the plasma membrane of aspartate 821 in erythrocytic anion exchanger has been determined by labeling inside-out vesicles and intact erythrocytes with impermeant reagents and following the outcome by site-directed immunochemistry. Intact erythrocytes and inside-out vesicles in the same container were vectorially modified with 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide and [35S]sulfanilic acid. The inside-out vesicles were separated from the erythrocytes by differential centrifugation, and both the vesicles and membranes made from the erythrocytes were stripped with alkali and digested with trypsin to liberate from each sample the peptide YHPDVPYVK containing aspartate 821. The tryptic digests were passed over an immunoadsorbent specific for peptides with the amino-and carboxy-terminal sequences YHPD- and -PYVK. Specifically bound peptides were eluted with acid, and the eluates were pooled and submitted to high-pressure liquid chromatography. A peak of absorbance at 229 nm corresponding to the peptide YHPDVPYVK was present in chromatograms of samples from both the inside-out vesicles and the intact erythrocytes. Another peak that displayed absorbance at 229 and 250 nm, corresponding to the peptide YHP(p-[35S]sulfo-beta-aspartanilide)VPYVK, was observed in the chromatogram of the sample from the inside-out vesicles but not in the chromatogram of the sample from the erythrocytes. This peak had associated with it a large number of counts per minute of [35S]sulfur, whereas no counts per minute of [35S]sulfur above background were detected on the chromatogram of the sample from the erythrocytes. The incorporation of [35S]sulfanilic acid into aspartate 821 of anion exchanger in inside-out vesicles was at least 10-fold greater than the incorporation of [35S]sulfanilic acid into aspartate 821 of anion exchanger in erythrocytes when the two preparations were labeled in the same solution. These results demonstrate that aspartate 821, found between two hydrophobic segments in the sequence of anion exchanger, is located on the cytoplasmic surface of this membrane-spanning protein.
通过用非渗透性试剂标记外翻囊泡和完整红细胞，并采用定点免疫化学方法追踪结果，确定了红细胞阴离子交换蛋白中天冬氨酸821相对于质膜的位置。同一容器中的完整红细胞和外翻囊泡用1-乙基-3-[3-(三甲基铵)丙基]碳二亚胺和[35S]磺胺酸进行定向修饰。通过差速离心将外翻囊泡与红细胞分离，然后用碱处理囊泡和由红细胞制成的膜，并用胰蛋白酶消化，以从每个样品中释放出含有天冬氨酸821的肽YHPDVPYVK。将胰蛋白酶消化物通过对具有氨基和羧基末端序列YHPD-和-PYVK的肽具有特异性的免疫吸附剂。特异性结合的肽用酸洗脱，合并洗脱液并进行高压液相色谱分析。在来自外翻囊泡和完整红细胞的样品色谱图中均出现了对应于肽YHPDVPYVK的229nm吸光度峰。在来自外翻囊泡的样品色谱图中观察到另一个在229和250nm处显示吸光度的峰，对应于肽YHP(p-[35S]磺基-β-天冬氨酰)VPYVK，但在来自红细胞的样品色谱图中未观察到。该峰与大量的每分钟[35S]硫计数相关，而在来自红细胞的样品色谱图中未检测到高于背景的每分钟[35S]硫计数。当两种制剂在同一溶液中标记时，外翻囊泡中阴离子交换蛋白的天冬氨酸821中[35S]磺胺酸的掺入量比红细胞中阴离子交换蛋白的天冬氨酸821中[35S]磺胺酸的掺入量至少高10倍。这些结果表明，位于阴离子交换蛋白序列中两个疏水片段之间的天冬氨酸821位于这种跨膜蛋白的细胞质表面。
