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Structure and Function of Placental Anticoagulant Protein (PAP)

胎盘抗凝蛋白（PAP）的结构和功能

基本信息

批准号：
01571219
负责人：
FUNAKOSHI Takayuki
金额：
$ 1.28万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

1. Placental Anticoagulant Protein (PAP) was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephdex G-75, and Mono S. The yield of the purified protein was approximately 20 mg from one placenta.2. PAP is a member of "annexin" family of Ca^<2+>-dependent phospholipid binding proteins, and it inhibits the extrinsic and intrinsic pathways of blood coagulation.3. PAP rapidly lost its anticoagulant effect due to photooxidation in the presence of methylene blue at pH7.9 and 8 ^ﾟC. Photooxidized PAP failed to bind the phospholipid vesicle.4. Photooxidized PAP had significantly decreased histidine contents, whereas the contents of other amino acids remained essentially unchanged.5. The peptides which contain a histidine residue, SHLRKV and DHTLIR, corresponding to PAP residues 204-209 and 266-271, respectively, are included in the functional site of PAP and exhibit anticoagulant activity, but the peptides in which alanine is substituted for histidine does not. However, the peptide KHALKG, corresponding from Lys-97 to Gly-102, did not exhibit an anticoagulant activity, showing that it is not included in the functional site. It was suggested that His-205 and His-267 should be involved in the Ca^<2+>- or phospholipid-binding site of PAP, but His-98 was not.6. Monoclonal antibody against PAP was prepared according to the method of Kohler & Milstein.7. PAP was detected in human liver, kidney, colon and endotherial cells, and was located in the cell membrane of the cultured carcinoma cells.

1.采用硫酸铵沉淀、DEAE-Sepharose、Sephdex G-75和Monos S柱层析，从人胎盘可溶性部分中分离纯化出胎盘抗凝蛋白(PAP)，每个胎盘的产量约为20 mg。PAP是钙依赖的磷脂结合蛋白家族中的一员，它抑制外在和内在的血液凝固途径。在pH 7.9和8ﾟC亚甲基蓝存在下，PAP由于光氧化而迅速失去抗凝血作用，光氧化后的PAP不能结合磷脂小泡。光氧化PAP显著降低了组氨酸的含量，而其他氨基酸的含量基本保持不变。含有组氨酸残基的多肽SHLRKV和DHTLIR分别对应于PAP 204-209和266-271残基，它们包含在PAP的功能位点上并具有抗凝血活性，而丙氨酸取代组氨酸的多肽则不具有抗凝血活性。然而，对应于Lys-97和Gly-102的KHALKG多肽没有表现出抗凝血活性，表明它不包括在功能位点上。His-205和His-267可能与PAP的磷脂结合部位有关，而His-98不参与。按Kohler&Milstein方法制备抗PAP的单抗。PAP在人肝、肾、结肠和内皮细胞中均有表达，定位于培养的癌细胞的细胞膜中。