Subunit structure of thrombin-activated porcine factor VIII.
Subunit structure of thrombin-activated porcine factor VIII.
复制标题
凝血酶激活的猪因子 VIII 的亚基结构。
DOI：
10.1021/bi00428a038
复制
发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Parker,CG
中科院分区：
文献类型：
--
作者：
Lollar,P;Parker,CG
佛蒙特大学医学与生物化学系，佛蒙特州伯灵顿05405摘要：因子VIII （fVIII）为单链合成，结构域序列为A1-A2-B-A3-C1-C2。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析凝血酶对fVIII的蛋白水解裂解，鉴定出来自B结构域的三个片段，分别称为fVIIIA1， fVIIIA2和fVIIlA3-ci-C2片段，这些片段难以可视化。这些片段的出现与凝固剂活性的发展有关，但活性是不稳定的，没有进一步的明显的蛋白质水解。在本研究中，猪fVIII与凝血酶反应，直到获得凝血活性峰值，然后进行阳离子交换（Mono S）高压液相色谱。在0.65 M NaCl/0.01 M His-HCl/0.005 M CaCl2、pH 6.0、20℃条件下，混凝剂活性恢复为单峰。对活化的fVIII进行分析性超离心，以测试这三个片段是否相关。当负载浓度从0.06 mg/mL增加到0.16 mg/mL时，平衡沉淀活化fVIII的表观分子量从14.8万增加到16.1万。这与fVIIIA1、fVIIIA2和fVIIIA3的表观分子质量总和一致。cl。C2计算自SDS-PAGE分析（148 000）或人类fVIII的氨基酸序列（159000）。这就确定了该制剂中的主要物种为a [viii] y， A2//A3。Ci-c2异源三聚体，另外表明三聚体的弱自结合和/或单个亚基形成三聚体的不完全结合。活化fVIII的速度沉降呈现单一边界（j20 w= 7.2 S）。结合流速和平衡沉降数据，计算得到的摩擦系数比为1.39，表明活化fVIII具有中度不对称性。
Departments of Medicine and Biochemistry, University of Vermont, Burlington, Vermont05405 Received May 23, 1988; Revised Manuscript Received August 11, 1988 abstract: Factor VIII (fVIII) is synthesized as a single chain having a domainal sequence A1-A2-B-A3-C1-C2. Analysis of the proteolyic cleavage of fVIII by thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identifies three fragments designated fVIIIA1, fVIIIA2, and fVIIlA3-ci-C2 fragment (s) derived from the B domain being difficult to visualize. The appearance of these fragments is associated with the development of coagulant activity, but the activity is labile without further apparent proteolysis. In this study, porcine fVIII was reacted with thrombin until peak coagulant activity was obtained and then subjected to cation-exchange (Mono S) high-pressure liquid chromatography. Coagulant activity was recovered in a single peak that contained all three fragments and was stable for weeks at 20 C in 0.65 M NaCl/0.01 M His-HCl/0.005 M CaCl2 at pH 6.0. Analytical ultracentrifugation of activated fVIII was done to test whether all three fragments were associated. The apparent molecular weight of activated fVIII from equilibrium sedimentation increased from 148 000 to 161 000 as the loading con-centration was increased from 0.06 to 0.16 mg/mL. This agrees well with the summed apparent molecular weights of fVIIIA1, fVIIIA2, and fVIIIA3. cl. C2 calculated from SDS-PAGE analysis (148 000) or from the amino acid sequence of human fVIII (159000). This establishes the major species in the preparation as a fVIIIA] y, A2//A3. ci-c2 heterotrimer and additionally indicates eitherweak self-association of the trimer and/or incomplete association of the individual subunitsto form the trimer. Velocity sedimentation of activated fVIII revealed a single boundary (j20 w= 7.2 S). From the combined velocity and equilibrium sedimentation data, a frictional coefficient ratio of 1.39 was calculated, indicating that activated fVIII is moderately asymmetrical.