VITAMIN K, METABOLISM AND FUNCTION

维生素 K、新陈代谢和功能

• 批准号: 2596409
• 负责人: REIDAR WALLIN
• 金额: $ 24.09万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2596409
• 关键词: Sf9 cell line; carboxylation; chemical binding; chemical models; coagulation factor IX; coagulation factor VII; cofactor; decarboxylases; drug resistance; enzyme complex; enzyme reconstitution; glutathione transferase; laboratory rabbit; laboratory rat; molecular site; oxidoreductase; pharmacokinetics; polymerase chain reaction; posttranslational modifications; protein biosynthesis; protein sequence; protein structure function; vitamin K; vitamin metabolism; warfarin

DESCRIPTION: Understanding vitamin K-dependent proteins will require insight into their biosynthesis and regulation. Unique for these proteins is their vitamin K-dependent post-translational modification. The modification is carried out by the _-carboxylase, an integral protein of the ER membrane which uses the reduced form of vitamin K (vitamin KH2) as cofactor. The reduced cofactor is produced by the enzyme Vitamin K 3,4-Epoxide reductase (VKOR) which is the target for the anticoagulant drug warfarin. Despite numerous attempts to purify VKOR, the molecular components that constitute the enzyme have not been identified. The applicants have been able to show that VKOR is an enzyme complex in the ER membrane and they propose that assembly of the complex is similar to assembly of the lipoxygenase complex on the nuclear envelope membrane. Both enzymes incorporates a member of the glutathione-S-transferase gene family as part of the lipid-protein enzyme complex. Experiments are proposed to complete the model of the VKOR enzyme complex and unveil the basis for genetic resistance to warfarin. Recombinant VKOR will be used to increase the capacity of cell lines to produce functional clotting factors VII and IX. It is our hypothesis that vitamin KH2 cofactor production by VKOR is a limiting factor in cellular production of these recombinant proteins. Although 8-10 different vitamin K-dependent proteins have been shown to be made extrahepatically only some have been identified. They include the coagulation factors prothrombin and protein S, the growth arrest specific gene 6 product Gas6 and the bone and cartilage resident proteins, osteocalsin and matrix GLA protein (MGP). In the last specific aim of this application experiments are proposed to identify new vitamin K-dependent proteins by vitamin K-dependent radioactive labeling. The proposed experiments will provide a better understanding of vitamin K metabolism and vitamin K function and have an impact on prophylactic medicine concerned with recombinant vitamin K-dependent coagulation factors and warfarin anticoagulation. The experiments should also contribute to the exciting, rapidly expanding area of research on extra-hepatic vitamin K-dependent proteins.

描述：了解维生素 K 依赖性蛋白质需要 深入了解它们的生物合成和调控。 这些蛋白质是独一无二的 是它们的维生素 K 依赖性翻译后修饰。 这 修饰是由 _-羧化酶进行的，该酶是 ER 膜使用还原型维生素 K（维生素 KH2）作为 辅因子。 还原的辅因子由维生素 K 酶产生 3,4-环氧还原酶（VKOR）是抗凝药物的靶标 华法林。 尽管人们多次尝试纯化 VKOR，但分子 构成该酶的成分尚未被鉴定。 这 申请人已经能够证明 VKOR 是 ER 中的酶复合物 膜，他们提出复合物的组装类似于 脂氧合酶复合物在核膜上的组装。 两个都 酶包含谷胱甘肽-S-转移酶基因家族的成员 作为脂质-蛋白质酶复合物的一部分。 建议进行实验 完成 VKOR 酶复合物模型并揭示了基础 对华法林的遗传耐药性。 重组VKOR将用于增加 细胞系产生功能性凝血因子 VII 的能力和 九． 我们假设 VKOR 产生的维生素 KH2 辅因子是 这些重组蛋白的细胞生产的限制因素。 尽管 8-10 种不同的维生素 K 依赖性蛋白质已被证明 肝外制造的仅部分已被鉴定。 它们包括 凝血因子凝血酶原和蛋白S，生长停滞特异性 基因 6 产物 Gas6 以及骨和软骨驻留蛋白， 骨钙素和基质 GLA 蛋白 (MGP)。 在本次活动的最后一个具体目标中 提出应用实验来鉴定新的维生素 K 依赖性 蛋白质通过维生素 K 依赖性放射性标记。 拟议的 实验将有助于更好地了解维生素 K 代谢和 维生素K功能及对有关预防药物的影响 含有重组维生素 K 依赖性凝血因子和华法林 抗凝。 这些实验还应该有助于令人兴奋的、 肝外维生素 K 依赖性研究领域迅速扩大 蛋白质。