| Project/Area Number |
10670275
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | Tokushima Bunri University |
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Principal Investigator |
NAGAHAMA Masahiro Tokushima Bunri University, Faculty of Pharmaceutical Sciences, Assistant professor, 薬学部, 助教授 (40164462)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Keiko Tokushima Bunri University, Faculty of Pharmaceutical Sciences, Reserch associtate, 薬学部, 助手 (90170315)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Clostridium perfringens / Alpha-toxin / Phospholipase C / Site-directed mutagenesis / Ca ion / Hemolytic activity / Bacillus cereus / ビフェルメンタンス菌 / セレウス菌 / 溶血 / ハイブリッド酵素 / C-domain / キメラ酵素 |
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| Research Abstract |
N-domain(1 -250residues ; CP_<1-250>) of alpha-toxin and Bacillus cereus phospholipase C (BcPLC) possessed phospholipase C(PLC) activity of about 2% of wild-type alpha-toxin (WT) activity, but not hemolytic activity. PLC and hemolytic activities of C.bifermentans PLC(CbPLC) were about 10% and 1% of these activities of WT, respectively. These activities of the hybrid protein consisting of CP_<1-250> and the C-domain of CbPLC(CP_<1-250>-CB_<251-372>) were lower than those of WT and the activities of the hybrid protein consisting of the N-domain of CbPLC and the C-domain of alpha-toxin(CB_<1-250>-CP_<251-370>) were similar to those of WT.On the other hand, PLC activity of the hybrid protein consisting of BcPLC and the C-domain of alpha-toxin(BC-CP_<251-370>) or CbPLC(BC- CB_<251-372>) was not significantly different from that of BcPLC.However, BC-CP_<251-370> significantly hemolyzed rabbit erythrocytes, but BC-CB_<251-372> did not. WT, CB_<1-250>-CP_<251-370> and BC-CP_<251-370> strongly bound to the red cells, CbPLC and BC-CB_<251-372> bound faintly, and CP_<1-250> and BcPLC not at all. Incubation of CP_<1-250> with CP_<251-370> completely complemented hemolytic and PLC activities. Furthermore, CP_<251-370> significantly conferred hemolytic activity on BcPLC, but inhibited PLC activity of BcPLC.CP_<251-370> significantly stimulated PLC activity of CB_<1-250>, but induced no effect on hemolytic activity. CP_<251-370> significantly inhibited PLC activity of BcPLC.The C-domain mutants with acrylodan attached to residues at position 263 and 365 exhibited a marked blue shift, indicative of movement of the fluorophore to a hydrophobic environment. These observations suggest that interaction of the C-domain of alpha-toxin with fatty acyl residues of phosphatidylcholine plays an important role in biological activities of N-domain of alpha-toxin.
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