Localization of binding sites within human von Willebrand factor for monomeric type III collagen.
Localization of binding sites within human von Willebrand factor for monomeric type III collagen.
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人血管性血友病因子中单体 III 型胶原蛋白结合位点的定位。
DOI：
10.1021/bi00374a004
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Hickey,MJ
中科院分区：
文献类型：
--
作者：
Roth,GJ;Titani,K;Hoyer,LW;Hickey,MJ
摘要：纯化的人血浆血管性血友病因子(VWF)与胃酶消化的III型胶原以可饱和(Kd=1×10~8M)、特异和快速的方式结合，化学计量比约为1：15[VWF亚单位(Mr 270000)-胶原三聚体(Mr 300000)]。VWF的两个还原和烷化的CNBR多肽，称为Mil残基542-622和M20残基948-998[Titani，K.，Kumar，S.，Takio，K.，Ericsson，LH，Wade，RD，Ashida，K.，Walsh，K.A.，Chopek，M.W.，Sadler，J.E.，&Fujikawa，K.(1986)BioChemical 25,3171-3184]，抑制VWF与胶原的结合。以125I-VWF(2×10~(-7)M)为配基，Mil、M20、片段III(二聚体，V8蛋白水解酶，氨基末端片段，mt 320000)和未标记的VWF分别抑制与胶原的结合，EC50值分别为4.8X10~(-7)、9.4×10~(-7)、1.1×10~(-7)和0.2×10™~(-7)M。当以125I标记的多肽为配体时，mIL和M20直接与胶原结合。与MIL和M20相比，VWF的其他CNBR片段作为抑制剂的效果较差(5倍或更少)，与胶原的结合也较差(5倍或更少)。一种鼠抗人VWF单抗(MR5)，它阻断VWF与胶原的结合，在酶联免疫吸附分析中检测时，选择性地与MIL和M20结合。片段m1和m20包含氨基酸序列同源性区域597-621和969-992，由重复结构域A1和A3编码，通过分析人VWF的cDNA序列定义[Shelton-Inloes，BB，Titani，K.，&Sadler，JE(1986)BioChemical 25,3164-3171]，人VWF包含两个单体III型胶原的结合位点。这些位点位于残基542-622和948-998之间，并可能与VWF分子的这些部分共享的序列同源性区域有关。
Revised Manuscript Received November 3, 1986 abstract: Purified humanplasma von Willebrand factor (vWf) binds to pepsin-digested monomeric type III collagen in a saturable (KD= 1 X 10~ 8 M), specific, and rapid manner with a stoichiometry of ap-proximately 1: 15 [vWf subunit (Mr 270 000)¡ collagen trimer (Mr 300000)]. Two reduced and alkylated CNBr peptides of vWf, termed Mil residues 542-622 and M20 residues 948-998 [Titani, K., Kumar, S., Takio, K., Ericsson, LH, Wade, RD, Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., & Fujikawa, K.(1986) Biochemistry 25, 3171-3184], inhibited vWf binding to collagen. With 125I-vWf (2 X 10 “9 M) as ligand, Mil, M20, fragment III (a dimeric, V8 protease, NH2-terminal fragment, Mt 320000 referenced above), and unlabeled vWf inhibitedbinding to collagen with EC50 values of 4.8 X 10 “7, 9.4 X 10-7, 1.1 X 10" 7, and 0.2 X 10™ 7 M, respectively. Mil and M20 bindto collagen directly when 125I-labeled peptides are used as ligands. Other CNBr fragments of vWf were less effective as inhibitors (5-fold or less) and bound less avidly to collagen (5-fold or less) compared to Mil and M20. A murine anti-human vWf monoclonal antibody (MR5), which blocks the binding of vWf to collagen, bound selectively to both Mil and M20 when tested in an enzyme-linked immunoadsorbent assay. Fragments Ml 1 and M20 contain a region of amino acid sequence homology, residues 597-621 and 969-992, and are encoded by repeat domains Al and A3, defined byanalysis of cDNA sequences for human vWf [Shelton-Inloes, BB, Titani, K., & Sadler, JE (1986) Biochemistry 25, 3164-3171], Human vWf contains two binding sites for monomeric type III collagen. The sites are located between residues 542-622 and 948-998 and may relate to regions of sequence homology shared by these portions of the vWf molecule.