Characterization of Recombinant Human Coagulation Factor XFriuli
Characterization of Recombinant Human Coagulation Factor XFriuli
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重组人凝血因子 XFriuli 的表征
DOI：
10.1055/s-0038-1650267
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发表时间：
1996
影响因子：
6.7
通讯作者：
H. James
中科院分区：
文献类型：
--
作者：
D. J. Kim;A. Girolami;H. James
摘要天然存在的血浆因子XFriuli（pFXFr）通过外源性和内源性凝血途径轻微激活，并具有受损的催化潜力。启动这些研究以确认该分子由于催化结构域中位置343处的Ser取代Pro而具有多功能缺陷。采用Nelson-Long定点突变法，在pRc/CMV中构建了人胚肾细胞产生的重组因子XFriuli（rFXFr）的cDNA。纯化的rFXFr，并显示具有相同的分子大小的正常血浆因子X（pFX）的凝胶电泳，和氨基末端测序显示正常的加工裂解。以重组正常血浆因子X（rFXN）为参照，rFXFr的翻译后γ-羧基谷氨酸（Gla）和β-羟基天冬氨酸（β-OH-Asp）含量分别为预期水平的85%和100%。rFXFr的活化和催化活性与pFXFr相同。分子模拟表明，Ser-343和Thr-318的侧链之间存在一个新的氢键，因为它们出现在pFXFr的底物结合口袋附近的反平行β折叠片层中。这些结果支持这样的结论，即观察到的突变pFXFr是负责其功能失调的激活和催化潜力，它占中度出血倾向的纯合子个体谁拥有这种变体促凝血剂。
Summary Naturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.
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DOI：
10.1016/s0021-9258(18)54982-6
发表时间：
1991-10
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
J. Toomey;K. Smith;D. Stafford
通讯作者：
J. Toomey;K. Smith;D. Stafford
DOI：
10.1016/s0021-9258(18)61070-1
发表时间：
1987-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
P. Matsudaira
通讯作者：
P. Matsudaira
DOI：
10.1016/s0021-9258(18)60423-5
发表时间：
1989-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
A. Chattopadhyay;D. S. Fair
通讯作者：
A. Chattopadhyay;D. S. Fair
影响因子：
2.9
作者：
LEYTUS, SP;FOSTER, DC;DAVIE, EW
通讯作者：
DAVIE, EW
X 因子弗留利变体的分离和表征。
影响因子：
20.3
作者：
Fair,DS;Revak,DJ;Hubbard,JG;Girolami,A
通讯作者：
Girolami,A