| Project/Area Number |
02557016
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Kyushu University |
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Principal Investigator |
IWANAGA Sadaaki Kyusyu Univ., Faculty of Sci., Professor, 理学部, 教授 (90029942)
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| Co-Investigator(Kenkyū-buntansha) |
KIMURA Terutoshi Peptide Institute., Co., Ltd., Derector, 研究部長 (30072771)
MIYATA Toshiyuki Kyusuy Univ., Faculty of Sci., Research Associate, 理学部, 助手 (90183970)
川畑 俊一郎 九州大学, 理学部, 助手 (90183037)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥9,400,000 (Direct Cost: ¥9,400,000)
Fiscal Year 1991: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1990: ¥6,600,000 (Direct Cost: ¥6,600,000)
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| Keywords | Factor VII / tissue factor / Synthetic Peptide substrate / Extrinsic coagulation / gamma-Gla domain / Blood protease / Monoclonal antibody / Esterase / VII因子 / 蛍光性ペプチド基質 / IX因子 / X因子 |
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| Research Abstract |
TISSUE FACTOR POTENTIATES THE HYDROLYSIS OF SYNTHETIC ESTER SUBSTRATE CATALYZED BY FACTOR VIIA.
We established a simple and sensitive assay method for the activity of factor VIIa-tissue factor complex using N^<alpha>-benzyloxycarbonyl-L-arginine P-nitrobenzyl ester (Z-Arg-ONb) as a substrate. The substrate has been synthesized previously (Zur, M., and Nemerson, Y. (1978) J. Biol. Chem. 253, 2203-2209). The new method was that the amount of P-nitrobenzyl alcohol released during the enzyme reaction is measured by reversed-phase HPLC, using a 3C_<18> column in 45% acetonitrile containing 0.1% trifluoroacetic acid with an isocratic elution. Z-Arg-ONb had a broad specificity for plasma serine proteases, and factor IXa could hydrolyze this substrate, indicating that the method is also useful for assay of the factor IXa activity. Using this assay method, we examined effect of tissue factor on the esterase activity of factor VIIa under various conditions, and found that tissue factor. . potenti
… More
ates greatly the hydrolysis of Z-Arg-ONb by both human and bovine factor VIIa's. These results did not agree with the previous report. Moreover, phospholipids were not required for the factor VIIa-mediated hydrolysis of Z-Arg-ONb even in the presence of tissue factor. We also determined the kinetic parameters (Km and kcat) of factor VIIa toward the substrate in the presence or absence of tissue factor. The Km value of factor VIIa alone was 6 times higher than that of factor VIIa-tissue factor complex (3.2 versus 0.54 mM), whereas the kcat value was 12 times lower than that of factor VIIa-tissue factor complex (14.3 ve rsus 173 s^<-1>). These results indicate that tissue factor affects the catalytic center of factor VIIa and enhances the factor VIIa-mediated hydrolysis of the ester substrate. The potentiating effect of tissue factor disappeared by removal of y-carboxyglutamic acid (Gla) -domain from factor VIIa, whereas the esterase activity in the absence of tissue factor was not affected by this modification, suggesting that the Gla-domain is required as the potent determinants on factor VIIa for the interaction with tissue factor, even if phospholipids are absent in the reaction mixture. Less
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