Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
|Section||University-to-University Cooperative Research|
|Research Institution||Fukuoka University|
OKUBO Kenshi(1992-1993) Fukuoka University School of Medicine, Fukuoka, Japan, 医学部, 助手 (40194097)
浜崎 直孝(1991-1992) 福岡大学, 医学部, 教授
濱崎 直孝(1991) 福岡大学, 医学部, 教授 (00091265)
森 朗 福岡大学, 医学部, 大学院生
JENNINGS Mic テキサス大学, 医学部, 教授
HAMASAKI Naotaka Kyushu University, Fukuoka, Japan, 医学部, 教授 (00091265)
JENNINGS Michael l. University of Texas, Medical Branch, Galveston, U.S.A.
MICHAEL L.Je テキサス大学, 医学部, 教授
|Project Period (FY)
1991 – 1993
Completed(Fiscal Year 1993)
|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1993 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1992 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1991 : ¥1,000,000 (Direct Cost : ¥1,000,000)
|Keywords||Red Cell Membrane / Band 3 Protein / Anion Transport Mechanism / Structure-Function Relationship / Protease Sensitivity / Structure of Membrane Domain / Conformational Change / Inhibitor Binding Site / 細胞膜の物質透過 / 陰イオン透過の分子機構 / 透過活性中心 / Hydrophilic connector loop / 膜蛋白質の高次構造 / Transmembrane spanning region / 陰イオン透過系 / DIDS / ピリドキサルリン酸 / NaBH_4 / 1次構造決定|
This project is concerned with the structure-function relationship of the erythrocyte anion transporter, band 3 protein. Results of this project are summarized as follows :
1. Identification of a glutamic acid involved in anion transport ;
Woodward's reagent K modification of intact red cells causes in inhibition of anion transport. Isolation and sequencing of a fragment labeled with the reagent showed that Glu-681 is heavily modified, suggesting that it lies within the transport pathway. This is a first amino acid residue in band 3 which has been idenified as a binding site for a transported substrate.
2. Topological study of the membrane domain of band 3 in situ ;
(1) To determine the three-dimensional structure of band 3 in membranes, alkali-stripped membranes were digested with trypsin. Five peptides were released into the supernatant and isolated by reversed phase HPLC followed by sequencing. These peptides are parts of hydrophilic connecting loops of band 3. The amount of these water
-soluble tryptic peptides increased as NaOH concentration was raised, suggesting that the conformation of band 3 in situ is apparently changed by alkali treatment into the more protease-sensitive configure.
(2) In addition to trypsin, the same structural studies as (1) with chymotrypsin and pepsin determined the protease-susceptible regions, which would not be buried in the phospholipid bilayer completely but exposed in extra-or intra-cellular media. Results are not coincided with the hydropathy prediction (e.g. some membrane spanning domains deduced by the hydropathy prediction of AE1 cDNA are released by protease treatment). These topological studies are in progress.
3. Identification of the two lysine labeled with H_2DIDS ;
For years it is known that the anion transport-specific inhibitor H_2DIDS can react with two lysine residues in the same band 3, causing in formation of the intramolecular cross-link. With the informations of protease digestion of band 3 in situ described above, we could release the cross-linked peptides with [^3H]H_2DIDS from the membranes by combination of pepsin and chymotrypsin digestion. The labeled peptides were isolated by reversed phase HPLC and sequenced, consequently it is revealed that Lys-539 and Lys-851 react with H_2DIDS. Lys 851 si also affinity-labeled with pyridoxal phosphate, a substrate for the anion transport, described previously [Kawano et al. (1988) J.Biol. Chem. 263, 8232-8238]. It is suggest that these two lysine residues in situ, which are separated in the primary structure, are located in the transport pathway and into sufficiently close proximity no more than 13 A, the distance between the two reactive isothiocyano groups of H_2DIDS. Less