Structural studies on Functional Abnormality of Factor XII and Fibrinogen.

(1)纤维蛋白原名古屋，-链上精氨酸取代谷氨酰胺-329。损害纤维蛋白单体聚合：对一种遗传性异常纤维蛋白原名古屋进行了结构研究，以确定导致纤维蛋白单体聚合障碍的异常。还原条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法。纤维蛋白原名古屋表明，除了正常的三条亚单位链外，还存在一条额外的蛋白质带，表观分子量为49,500。从纤维蛋白原名古屋cnbr片段的赖氨酰内切酶中分离出的多肽的氨基酸序列分析表明，链上的Gln-329已被Arg取代。这种替换可以用Gln-329(CAG-CGG)密码子上的一条非整合子链来解释。我们得出结论，链上的Gln-329对于纤维蛋白单体的正常聚合是必不可少的。(2)凝血因子IX Niigata：催化区域丙氨酸-390被Valine取代：因子IX Niigata是S突变的因子IX，导致中重度血友病B患者凝血因子IX抗原水平正常，凝血活性降低(正常的1-4%)。我们以前报道过，纯化的突变蛋白可以被Xia/Ca^&lt；2+&gt；因子以与正常因子IX相似的速度转化为因子IXa，但在因子XIII、Ca^&lt；2+&gt；和磷脂存在的情况下，所产生的突变因子IXa不能激活因子X。在本研究中，我们从结构水平上分析了因子IX Niigata，以阐明凝血活性丧失的分子异常。对赖氨酰内肽酶和SP-V8双酶切得到的多肽进行氨基酸序列分析表明，Niigata因子IX分子的催化域中第390位的丙氨酸被Valine取代。

(1) Fibrinogen Nagoya, a Replacement of Glutamine-329 by Arginine in the -chain. That Impairs the Polymerization of Fibrin Monomer: Structural studies on a hereditary abnormal fibrinogen, fibrinogen Nagoya were performed to identify the abnormality responsible for the impaired polymerization of fibrin nomomer. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. fibrinogen Nagoya showed the presence of an extra protein band with an apparent molecular weight of 49,500 in addition to the normal three subunit chains. Amino acid sequence analysis of a peptide isolated from a lysyl endopeptidase digest of one of the cnbr fragments derived from fibrinogen nagoya indicated that Gln-329 in the -chain had been replaced by Arg. This substitution can be explained by a single uncleotide chainge in the dodon for Gln-329 (CAG - CGG). We conclude that GLN-329 in the -chain is indispensable for the normal polymerization of fibrin monomer.(2) Blood Clontting Factor IX Niigata: Substitution of Alanine-390 by Valine in the Catalytic Domain: Factor IX Niigata is s mutant factor IX responsible for the moderately severe hemophilia B in a patient who has a normal level of factor IX antigen with reduced clotting activity (1-4% of normal). We reported previously that the purified mutant protein could be converted to the factor IXa form by factor XIa/Ca^<2+> at a rate similar to that in the case of normal factor IX, but the resulting mutant factor IXa could not activate factor X in the presence of factor XIII, Ca^<2+>, and phospholipids. In the present study, we analyzed factor IX Niigata at the structural level to elucidate the molecular abnormality responsible for the loss of clotting activity. Amino acid sequence analysis of a peptide obtained on lysyl endopeptidase digestion, coupled with subsequent SP-V8 digestion, demonstrated that the alanine at position 390 was substituted by valine in the catalytic domain of the factor IX Niigata molecule.