Development of Synthetic Fluorogenic Peptide Substrates for Blood Clotting Proteases
凝血蛋白酶合成荧光肽底物的开发
基本信息
- 批准号:63870017
- 负责人:
- 金额:$ 6.14万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Developmental Scientific Research
- 财政年份:1988
- 资助国家:日本
- 起止时间:1988 至 1989
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
The utility of the synthetic chromogenic and fluorogenic peptide substrates for specific assay of various proteases in body fluid has been established, because of their high sensitivities. The specific substrates for alpha-thrombin, factor Xa, plasma kallikrein, protein Ca, urokinase, factor XIIa, factor XIa, plasmin and limulus clotting enzyme developed by our group are now commercially available. The purpose of this project is to kind specific and sensitive substrates for factors VIIa and IXa and glandular kallikreins and to realize these synthetic substrates for determinations of various proteases in body fluids. The results are as follows:1. Boc-Met-Ala-Arg4-methylcoumaryl-7-amide was found to be the most sensitive substrate for mouse glandular (submaxillary) kallikrein. the substrate comprised the amino acid sequence adjacent to the NH_2- and COOH-terminal regions of the kinin moiety found in mouse low molecular weight kininogen. The mouse kallikrein cleaved this substrate (Km = 2 … More 30 muM, kcat = 1.4s^<-1>) approximately 80-fold faster than hog glandular kallikreins.2. We newly synthesized a chromogenic substrate of Boc-Leu-Thr-arg-p-nitrobenzylester for factor VIIa. This substrate was found to be most sensitive one for factor VIIa among the previously eynthesized 74 peptide substrates. This finding made it possible to investigate the molecular interaction between factro VII and tissue factor participated in the extrinsic coagulation system.3. A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized as the first step for specific assay of factor VII in body fluid. The antibody VII-M31 possessed a strong affinity only for factor VII in the presence of Ca^<2+> without any reactivities to prothrombin, factor X, factor IX, protein C, protein S and protein Z. Denaturation of factor VII by urea and SDS all its reduction with 2-mercaptoethanol did not destroy the antigenic site, measured by immunoblotting method. Moreover, the antibody bound specifically to the Gla-containing peptide corresponding to the NH_2-terminal 23-50 residues of factor VII. These results indicated that VII-M31 is available for specific enzyme-linked immunoassay of factor VII, in combination with the synthetic peptide substrate described above. Less
合成的显色和荧光肽底物用于体液中各种蛋白酶的特异性测定的效用已经建立,因为它们的高灵敏度。本课题组开发的α-凝血酶、Xa因子、血浆激肽释放酶、蛋白钙、尿激酶、XIIa因子、XIa因子、纤溶酶和鲎凝血酶的特异性底物现已上市。本项目的目的是为因子VIIa和因子IXa以及腺激肽释放酶筛选特异性和敏感性底物,并实现这些合成底物用于测定体液中的各种蛋白酶。研究结果如下:1.发现Boc-Met-Ala-Arg 4-甲基香豆酰-7-酰胺是小鼠腺(颌下)激肽释放酶的最敏感底物。该底物含有与小鼠低分子量激肽原中激肽部分的NH_2-和COOH-末端区域相邻的氨基酸序列。小鼠激肽释放酶切割该底物(Km = 2 ...更多信息 30 μ M,kcat = 1.4s^<-1>)比猪腺激肽释放酶快约80倍。我们新合成了一种凝血因子VIIa的显色底物Boc-Leu-Thr-Arg-p-nitrobenzylester。在以前合成的74种肽底物中,发现该底物对因子VIIa最敏感。这一发现使得研究凝血因子VII和组织因子参与外源性凝血系统的分子相互作用成为可能.制备了抗牛凝血因子VII的鼠单克隆抗体(VII-M31),并对其进行了初步鉴定。抗体VII-M31在Ca^2+存在下仅对因子VII具有强亲和力,而对凝血酶原、因子X、因子IX、蛋白C、蛋白S和蛋白Z没有任何反应性。用尿素和SDS变性VII因子,用2-巯基乙醇还原,并没有破坏抗原位点,用免疫印迹法测定。该抗体与VII因子NH_2端23-50位氨基酸残基所对应的含Gla肽段特异性结合。这些结果表明,VII-M31与上述合成肽底物组合可用于因子VII的特异性酶联免疫测定。少
项目成果
期刊论文数量(73)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Suehiro,K.: "Blood Clotting Factor IX BM Nagoya:Substitution of Arginene-180 by Tryptophan and Its activation by Chymotrypsin" J.Biol.Chem.264. 21257-21265 (1989)
Suehiro,K.:“凝血因子 IX BM Nagoya:色氨酸取代精烯-180 及其被胰凝乳蛋白酶激活”J.Biol.Chem.264。
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- 影响因子:0
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Sugimoto,M.: "Factor IX Kawachinagano:Impaired function of the Gladomain caused by attached propeptide region due to substitution of arginine by glutamine at position" Br.J.Haematol.72. 216-221 (1989)
Sugimoto,M.:“因子 IX Kawachinagano:由于在位置上用谷氨酰胺取代精氨酸,导致附着的前肽区域导致 Gladomain 功能受损”Br.J.Haematol.72。
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Kawabata, S. et al.: "Highly Sensitive Peptide-4-methylcoumary-7-amide Substrates for Blood Clotting Proteases and Tyrpsin." Eur. J. Biochem. 172(2), 17-25.1988.
Kawabata, S. 等人:“用于凝血蛋白酶和酪氨酸的高度敏感肽 4-甲基香豆基-7-酰胺底物”。
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Miyata,T.: "Facter XII Washington D.C.:Inactive Factor XIIa Resulting from Replacement of Cysteine-571 by Serine." Proc.Natl.Acad.Sci.,U.S.A.86. 8319-8322 (1989)
Miyata,T.:“华盛顿特区的因子 XII:由丝氨酸取代半胱氨酸 571 导致的失活因子 XIIa。”
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Nishimura,H.: "Identification of a Disaccharide(xyl-Glc)and a Trisaccharide(xyl2-Glc)O-Glycosidically Linked to a Serine Residue in the First Epidermal Growth Factor-like" J.Biol.Chem.Domain of Human Factors VII and Protein Z and Bovine Protein Z.264. 203
Nishimura,H.:“鉴定与第一类表皮生长因子中丝氨酸残基相连的二糖 (xyl-Glc) 和三糖 (xyl2-Glc)O-糖苷” J.Biol.Chem.Domain of Human Factors
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IWANAGA Sadaaki其他文献
IWANAGA Sadaaki的其他文献
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{{ truncateString('IWANAGA Sadaaki', 18)}}的其他基金
Role of Limulus Hemocytes in the Biological Defense System.
鲎血细胞在生物防御系统中的作用。
- 批准号:
04404090 - 财政年份:1992
- 资助金额:
$ 6.14万 - 项目类别:
Grant-in-Aid for General Scientific Research (A)
Basic studies on Development of Anti-thrombotic Agents
抗血栓药物开发的基础研究
- 批准号:
04557015 - 财政年份:1992
- 资助金额:
$ 6.14万 - 项目类别:
Grant-in-Aid for Developmental Scientific Research (B)
Molecular Mechanism of Extrinsic Blood Coagulation Pathway
外源性凝血途径的分子机制
- 批准号:
03044113 - 财政年份:1991
- 资助金额:
$ 6.14万 - 项目类别:
Grant-in-Aid for international Scientific Research
Studies on the Activity Measurement for Blood Proteases using their Monoclonal Antibodies
用单克隆抗体测定血液蛋白酶活性的研究
- 批准号:
02557016 - 财政年份:1990
- 资助金额:
$ 6.14万 - 项目类别:
Grant-in-Aid for Developmental Scientific Research (B)
Mechanism of Hemolymph Coagulation System in Invertebrates
无脊椎动物血淋巴凝固系统的机制
- 批准号:
02454539 - 财政年份:1990
- 资助金额:
$ 6.14万 - 项目类别:
Grant-in-Aid for General Scientific Research (B)
Initiation Mechanism of Extrinsic Blood Coagulation System
外源性凝血系统的启动机制
- 批准号:
63044110 - 财政年份:1988
- 资助金额:
$ 6.14万 - 项目类别:
Grant-in-Aid for international Scientific Research
Hemolymph Coagulation and Defence Systems in Invertebrate Animals
无脊椎动物的血淋巴凝固和防御系统
- 批准号:
62480453 - 财政年份:1987
- 资助金额:
$ 6.14万 - 项目类别:
Grant-in-Aid for General Scientific Research (B)
Development and Application of Fluorogenic Peptide Substrates for Determination fo Blood Clotting Proteases
凝血蛋白酶测定荧光肽底物的研制及应用
- 批准号:
61880016 - 财政年份:1986
- 资助金额:
$ 6.14万 - 项目类别:
Grant-in-Aid for Developmental Scientific Research
Studies on Molecular Abnormality of Blood Coagulation and Fibrinolytic Factors
凝血及纤溶因子分子异常研究
- 批准号:
60480497 - 财政年份:1985
- 资助金额:
$ 6.14万 - 项目类别:
Grant-in-Aid for General Scientific Research (B)
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Effect of ectopically synthesized coagulation factor VII on venous thromboembolism and malignancy
异位合成凝血因子VII对静脉血栓栓塞和恶性肿瘤的影响
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该项目的目的是确定第七因子激活蛋白酶(FSAP)在血管疾病中的作用机制,并进一步开发其诊断和治疗潜力
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Coagulation factor VII producing tumors and their release of microparticles with coagulation initiating capacity
产生凝血因子VII的肿瘤及其释放具有凝血启动能力的微粒
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21590455 - 财政年份:2009
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ANALYSIS OF DYSFUNCTIOANAL FACTOR VII ASSOCIATED WITH HOMOZYGOUS MISSENSE MUTATION 331GLY TO SER
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Characterization of Factor VII From Transgenic Fish
转基因鱼中因子 VII 的表征
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6402973 - 财政年份:2001
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