VITAMIN K-DEPENDENT PROTEINS IN LIVER & ISOLATED CELLS

本提案的长期目标是了解维生素 K依赖的蛋白质生物合成以及这些过程是如何 监管. 我们将集中在肝脏和分离的细胞中的这种反应 从肺、上皮和人类癌中分离。 特别强调 血液凝固因子的生物合成。 一个主要 所提出的工作的一部分集中在内切核酸酶eta（endo eta）不敏感的蛋白质，其构成主要的γ- 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依赖性羧化酶的组分 酶复合物，我们认为该蛋白质是CO2载体 在γ-羧化反应中。 实验设计 来验证这个假设 该提案的另一部分侧重于 凝血因子蛋白前体的γ-羧化 在大鼠和人肝脏中。 我们想知道为什么 选择的前体在体外仅用作羧化酶底物。 实验方法是基于系统的比较 凝血因子II的两种前体形式， 125 I-碘化蛋白质的图谱和HPLC图谱。 单核细胞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，但似乎缺乏羧化酶活性。 另 另一方面，II型肺细胞表现出显著的羧化酶 活动 提出实验来测试是否 肺可以提供所有必要的凝血因子， 肺内的外源性凝血 众所周知， 刺激II型细胞增殖。 因此，II型细胞 将在条件培养基中培养， 巨噬细胞来测试 巨噬细胞和II型细胞，可以调节促凝血剂 II型细胞的输出。 抗大鼠凝血酶原抗体和 因子X将用于鉴定14 CO2标记的蛋白质 在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 人内皮细胞的K依赖蛋白。 识别 维生素K依赖性蛋白质的合成 也将尝试癌细胞系。 最后，净化 生理上重要的维生素K1 肝微粒体中的脱氢酶，介导 维生素K1的解毒作用将在蛋白水解后尝试 用蛋白酶切割微粒体膜。

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.