PROTEIN CHEMISTRY AND BIOCHEMICAL STUDY ON THE ANION TRANSPORT SYSTEM IN HUMAN ERYTHROCYTE MEMBRANES.

采用凝胶渗透和反相高效液相色谱相结合的方法，纯化了一种用磷酸吡哆醛亲和标记的人红细胞带3肽。通过对赖氨酰内肽酶和葡萄球菌蛋白酶V8消化得到的多肽进行测序，确定了跨膜肽的氨基酸序列。当含有人红细胞带3的COOH末端的多肽也被纯化和测序时，亲和标记的多肽位于带3的COOH末端附近，在那里它可以与小鼠红细胞带3的氨基酸残基852-927比对，该氨基酸残基来自于一个克隆的核苷酸序列。COOH末端的氨基酸序列与小鼠带3的氨基酸序列高度同源，从而建立了带3的COOH末端肽序列。H^1LFTGIQIIX^&lt；10&gt；LAVLWVVKST^&lt；20&gt；PASLALPFVL^&lt；30&gt；ILTVPLRRVL^&lt；40&gt；LPLIF RNVEL^&lt；50&gt；QCLDADDAKA^&lt；60&gt；TFDEEEGRDE^&lt；70&gt；YDEVAMPV^&lt；78&gt；吡哆醛磷酸结合位点为Lys-18，对应于推导序列的Lys-869。似乎带3的COOH末端区域至少构成了人红细胞膜负离子转运的活性中心的一部分。焦碳酸二乙酯抑制了人红细胞膜上的磷酸盐交换。只有当膜被来自重新密封的幽灵的胞液表面的试剂修饰时，交换速率才被抑制。二乙基焦碳酸酯的胞内修饰抑制了[~3H@]-DIDS与带3蛋白的胞外结合。此外，胞外DND保护膜不受焦碳酸二乙酯的细胞内修饰。测序得到的COOH-端肽与DDS结合部位密切相关。因此，焦碳酸二乙酯的胞内修饰可能会跨膜改变COOH末端多肽的构象。

A human erythrocyte band 3 peptide, affinity labeled with pyridoxal phosphate, was purified by a combination of gel permeation and reverse-phase high performance liquid chromatography. The amino acid sequence of the transmembrane peptide was determined by sequencing subfragments of the peptide obtained from lysyl endopeptidase and staphlococcal proteinase V8 digestions.When a peptide containing the COOH-terminal of human erythrocyte Band 3 was also purified and sequenced, the affinity-labeled peptide was found to be located close to the COOH-terminal of Band 3, where it could be aligned with amino acid residues 852-927 of a murine erythrocyte Band 3, deduced from a nucleotide seguence of a cDNA clone. The amino acid sequence of the COOH-terminal region was highly homologous to that of murine Band 3.As a result, the sequence of the COOH-terminal peptide of band 3 was established as follows. H^^1LFTGIQIIX^^<10> LAVLWVVKST^^<20> PASLALPFVL^^<30> ILTVPLRRVL^^<40> LPLIF RNVEL^^<50> QCLDADDAKA^^<60> TFDEEEGRDE^^<70> YDEVAMPV^^<78>The pyridoxal phosphate binding site was identified as Lys-18 which corresponded to Lys-869 of the deduced sequence. It appears that the COOH-terminal region of band 3 constitutes at least a part of the active center for anion transport in human erythrocyte membrans.Diethylpyrocarbonate inhibited the phosphate exchange across the human erythrocyte membrane. The exchange rate was inhibited only when the membranes were modified with the reagent from the cytosolic surface of resealed ghosts. The intracellular modification by diethylpyrocarbonate inhibited the extracellular binding of [^3H@]-DIDS to Band 3 protein. Furthermore, the extracellular DNDS protected the membranes from the intracellular modification by diethylpyrocarbonate.The sequenced COOH-terminal peptide closely related to the DIDS binding site. Thus, the intracellular modification by diethylpyrocarbonate may transmembranously change the conformation of the COOH-terminal peptide.