1991 Fiscal Year Final Research Report Summary
Mechanism of the ATP-dependent membrane transporter
| Project/Area Number |
02660091
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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 |
応用生物化学・栄養化学
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| Research Institution | Kyoto University |
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Principal Investigator |
UEDA Kazumitsu Department of Agricultural Chemistry Kyoto University, Assistant Professor, 農学部, 助手 (10151789)
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| Co-Investigator(Kenkyū-buntansha) |
KOMANO Tohru Department of Agricultural Chemistry Kyoto University, Professor, 農学部, 教授 (30026413)
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| Project Period (FY) |
1990 – 1991
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| Keywords | Transmembrane protein / P-glycoprotein / Cystic fibrosis / Multidrug resistance / ATPase / Fusion protein / Cultured cell / Transport protein |
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| Research Abstract |
The ATP-dependent membrane transport proteins of eukaryotes includes P-glycoprotein and cystic fibrosis transmembrane conductance regulator(CFIR). P-glycoprotein functions as an ATP-dependent efflux pump of anticancer drugs and is involved in multidrug resistance of cancer cells which is a major obstacle of cancer chemotherapy. CFTR is involved in cystic fibrosis, the most common lethal, recessively inherited disease among Caucasians. This family of membrane transport proteins has a high degree of symmetry between its two halves, each composed of six membrane-spanning alpha-helices and a hydrophilic region containing an ATP-binding sites. To investigate the involvement of the two ATP-binding domains in the ATPase activity of P-glycoprotein, fulllength and the 5'-half of human MDR1 cDNA, which encodes P-glycoprotein, were fused with the E. coli lacZ gene and expressed in NIH3T3 cells. Immunoprecipitated full-length P-glycoprrotein-beta-galactosidase showed ATPase activity in the presenc
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e of phospholipids suggesting that stabilization of the transmembrane domains is necessary for ATP hydrolysis. N-terminal half P-glycoprotein-beta-galactosidase also showed ability to hydrolyze ATP but with slightly lower specific activity. Both ATPase activities showed similar characteristics when the effect of several inhibitors was analyzed, indicating that the N-terminal ATP-binding domain contains all residues necessary to hydrolyze ATP without interacting with the C-terminal ATP-binding domain. Then, MDRI cDNA fragment encoding the NH_2- and COOH-cytoplasmic domains of P-glycoprotein(N-half and C-half, respectively)were isolated and fused with E. coli lacZ gene. The fusion genes were expressed in E. coli strain BL21 using the T7 RNA polymerase expression system. The fusion proteins were purified by affinity chromatography against the beta-galactosidase moiety to near homogeneity. Both ATP-binding doipains of P-glycoprotein showed ATPase activity with similar Km values but the Vmax for the activity of C-half was about half that of N-half. This difference in turnover rates suggests different roles in the transport process. Less
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