| Project/Area Number |
04454173
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Kyushu University |
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Principal Investigator |
TAKESHIGE Koichiro (1993) Kyushu University, Biochemistry, Professor, 医学部, 教授 (10037450)
水上 茂樹 (1992) 九州大学, 医学部, 教授 (90037325)
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| Co-Investigator(Kenkyū-buntansha) |
SUMIMOTO Hideki Kyushu University, Biochemistry, Associate Professor, 医学部, 助教授 (30179303)
竹重 公一朗 九州大学, 医学部, 助教授 (10037450)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1993: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1992: ¥5,500,000 (Direct Cost: ¥5,500,000)
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| Keywords | Neutrophils / NADPH oxidase / Active oxygens / Superoxide / Signal transduction / 細胞障害 / スーパーオキシド |
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| Research Abstract |
(1)Studies in a cell-free system (i)Because the alignments of the amino acid sequence of the large subunit of the cytochrome b558 with those of previously characterized flavoproteins revealed that the middle and C-terminal portions of the cytochrome are likely to be FAD-and NADPH-binding domains, respectively, cytochrome b558 appears to be a flavoprotein with an NADPH- binding site of the NADPH oxidase. (ii)We showed that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore we found that sodium dodecyl sulfate and arachidonic acid, activators of the oxidase in vitro, caused exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appeared to play a crucial role in the assembly of the oxidase components, because p47-SH3 bound to b
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oth p22phox ana p67phox but failed to interact with a mutant p22phox carrying Pro156Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a novel signal-transducing mechanism that normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins. (iii)Phorbol 12-myristate 13-acetate (PMA) induced a decrease in the phosphotyrosine phosphatase (PTPase) activity in human neutrophils. The decrease in the activity induced by PMA was blocked by the treatment of the cells with staurosporine, indicating that protein kinase C is involved in the decrease. The findings obtained suggest that conformational changes of the enzyme induced by PMA result in the decrease in PTPase activity.
(2)Studies with intact or electropermeabilized neutrophils We showed by using electropermeabilized human neutrophils that cAMP inhibited the activation of the NADPH oxidase not only the at the site before the protein kinase C but also at another site after the kinase, that tyrosine phosphorylation is involved in the activation of the NADPH oxidase at a step before diacylglycerol formation by phospholipase C and phospholipase D may not be involived the signal pathway in permeabilized cells and that phosphatidic acid activates the oxidase at a site down stream of protein kinase C. Less
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