| Project/Area Number |
61570149
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Fukuoka University |
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Principal Investigator |
HAMASAKI Naotaka DEPART. OF CLINICAL CHEMISTRY AND LAB. MED., FUKUOKA UNIV. SCHOOL OF MED., 医学部, 教授 (00091265)
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| Co-Investigator(Kenkyū-buntansha) |
河野 洋一 福岡大学, 医学部・生化学教室, 助手 (50152989)
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| Project Period (FY) |
1986 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1988: ¥100,000 (Direct Cost: ¥100,000)
Fiscal Year 1987: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1986: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Anion transport system / Erythrocyte membrane / Transmembrane protein / Amino acid sequence / Active center / Band 3 / 膜タンパク質の一次構造 / 赤血球膜蛋白質 / Band3 / 膜貫通ペプチド / アミノ酸配列 / ピリドキサルリン酸アフィニティラベル |
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| Research Abstract |
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.
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