VITAMIN K-DEPENDENT PROTEINS IN LIVER & ISOLATED CELLS

肝脏中维生素 K 依赖性蛋白质

• 批准号: 3343306
• 负责人: REIDAR WALLIN
• 金额: $ 12.13万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-01-01 至 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3343306
• 关键词: affinity chromatography; alveolar macrophages; anticoagulants; autoradiography; cell population study; cellular oncology; chromatography; clotting factor; coagulation factor VII; coagulation factor X; decarboxylases; endonuclease; enzyme complex; enzyme inhibitors; enzyme substrate; gel electrophoresis; high performance liquid chromatography; human subject; immunochemistry; laboratory rabbit; laboratory rat; liver cells; metastasis; microsomes; neoplastic cell culture for noncancer research; oncoproteins; protein biosynthesis; protein sequence; prothrombin; radiotracer; respiratory epithelium; spectrometry; tissue /cell culture; vascular endothelium; vitamin K; warfarin

The long-term objective of this proposal is to understand vitamin K-dependent biosynthesis of proteins and how these processes are regulated. We will focus on this reaction in liver and isolated cells from lung, epithelium and human carcinomas. Special emphasis is placed on the biosynthesis of blood coagulation factors. A major part of the proposed work focusses on an endonuclease eta (endo eta) insensitive protein that constitutes the major gamma- carboxylated product of the vitamin K-dependent carboxylation reaction in vitro. Preliminary experiments suggest that the protein is a component of the vitamin K-dependent carboxylase enzyme complex and we propose that the protein is a CO2-carrier in the gamma-carboxylation reaction. Experiments are designed to test this hypothesis. Another part of this proposal focusses on gamma-carboxylation of protein precursors of coagulation factor II in rat and human liver. We would like to understand why selected precursors only serve as carboxylase substrates in vitro. The experimental approach is based on a systematic comparison of two precursor forms of clotting factor II and uses peptide mapping of 125I-iodinated proteins and mapping by HPLC. Mononuclear phagocytes are potent triggers of the coagulation system. Pulmonary macrophages produce tissue factor and factor V but appear to be devoid of carboxylase activity. On the other hand, Type II pneumocytes exhibit significant carboxylase activity. Experiments are proposed to test whether or not the lung can provide all the necessary coagulation factors for extrinsic coagulation in the lung. Macrophages are known to stimulate proliferation of Type II cells. Therefore, Type II cells will be cultured in conditioned medium from stimulated macrophages to test the possibility of communication between macrophages and type II cells which can regulate procoagulant output by Type II cells. Antibodies against rat prothrombin and factor X will be used to identify 14CO2-Labeled protein precursors in an in vitro carboxylation system of Type II cells. The same approach will be used to study biosynthesis of vitamin K-dependent proteins by human endothelial cells. Identification of vitamin K-dependent proteins produced by various human carcinoma cell lines will also be attempted. Finally, purification of the physiologically important vitamin K1 reducing dehydrogenase enzyme in liver microsomes that mediates the antidotic effect of vitamin K1 will be attempted after proteolytic cleavage of the microsomal membrane with protease.

该提议的长期目标是了解维生素 蛋白质的K依赖性生物合成以及这些过程如何 受监管。 我们将专注于肝脏和分离细胞中的这种反应 来自肺，上皮和人类癌。 特别强调 放置在血液凝血因子的生物合成上。 专业 拟议工作的一部分重点是核酸内切酶ETA（Endo ETA）构成主要γ-的不敏感蛋白 维生素K依赖性羧化的羧化产物 体外反应。 初步实验表明 蛋白质是维生素K依赖性羧化酶的组成部分 酶复合物，我们提出蛋白质是二氧化碳载体 在γ-羧化反应中。 设计实验 检验这一假设。 该提案的另一部分集中在 凝结因子蛋白质前体的伽马羧化 II在大鼠和人类肝脏中。 我们想了解为什么 选定的前体仅用作体外的羧化酶底物。 实验方法基于系统比较 凝血因子II的两种前体形式，并使用肽 映射125i碘化的蛋白质，并通过HPLC映射。 单核吞噬细胞是凝血的有效触发器 系统。 肺巨噬细胞产生组织因子和因子 V但似乎没有羧化酶活性。 另一方面 手，II型肺细胞表现出明显的羧化酶 活动。 提出了实验来测试是否 肺可以为 肺部外凝结。 巨噬细胞已知 刺激II型细胞的增殖。 因此，II型细胞 将在有刺激的条件培养基中培养 巨噬细胞测试之间交流的可能性 巨噬细胞和II型细胞，可以调节procogulans 按II型细胞输出。 针对大鼠凝血酶原和 因子X将用于识别14CO2标记的蛋白 II型细胞的体外羧化系统中的前体。 同样的方法将用于研究维生素的生物合成 人体内皮细胞的K依赖性蛋白质。 鉴别 由各种人产生的维生素K依赖性蛋白 还将尝试癌细胞系。 最后，纯化 降低生理上重要的维生素K1 肝微粒体中脱氢酶酶介导 蛋白水解后将尝试使用维生素K1的抗毒素作用 用蛋白酶裂解微粒体膜。