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首页> 外文期刊>Biochemistry >Cytoplasmic disposition of aspartate 821 in anion exchanger from human erythrocytes
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Cytoplasmic disposition of aspartate 821 in anion exchanger from human erythrocytes

机译：天冬氨酸821在人类红细胞阴离子交换剂中的胞质分布

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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.
机译：通过用不渗透试剂标记由内而外的囊泡和完整的红血球，并通过定点免疫化学检测结果，可以确定红细胞型阴离子交换剂中天冬氨酸821相对于质膜的位置。用1-乙基-3- [3-（3-（三甲基氨））丙基]碳二亚胺和[35S]磺胺酸对同一容器中完整的红细胞和由内而外的囊泡进行矢量修饰。通过差速离心将由内而外的囊泡与红细胞分离，并用碱剥离由红细胞制成的囊泡和膜，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。使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。用酸洗脱特异性结合的肽，合并洗脱液并进行高压液相色谱分析。从里到外的囊泡和完整的红细胞的样品色谱图中均出现了对应于肽YHPDVPYVK的229 nm处的吸收峰。在由内而外的囊泡样品的色谱图中观察到另一个峰，该峰在229和250 nm处显示吸光度，对应于肽YHP（p- [35S]磺基-β-天冬酰胺）VPYVK。红细胞样本。该峰与每分钟[35S]硫的计数相关，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]硫的计数。当两种制剂分别标记在红细胞中时，[35S]磺胺酸在阴离子交换剂的天冬氨酸821中的掺入比在红细胞中[35S]磺胺酸在阴离子交换剂的天冬氨酸821中的掺入至少至少十倍。相同的解决方案。这些结果证明，在阴离子交换剂序列的两个疏水性区段之间发现的天冬氨酸821位于该跨膜蛋白的细胞质表面上。

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