VITAMIN K-DEPENDENT PROTEINS IN LIVER & ISOLATED CELLS

这项建议的长期目标是了解维生素 依赖钾的蛋白质的生物合成以及这些过程是如何 受监管的。我们将重点研究肝脏和分离细胞中的这种反应。 来自肺癌、上皮细胞和人类癌症。特别强调的是 放在血液凝血因子的生物合成上。一位少校 拟议工作的一部分集中在内切酶ETA(endo Eta)不敏感的蛋白质，构成主要的伽马- 维生素K依赖的羧化产物 体外反应。初步实验表明， 蛋白质是维生素K依赖的羧基酶的组成部分 酶复合体，我们认为该蛋白是一种二氧化碳载体 在伽马-羧化反应中。设计了实验 来检验这一假设。这项提案的另一部分集中在 凝血因子蛋白前体的伽玛羧化 II在大鼠和人的肝脏中。我们想知道为什么 在体外，选定的前体只能作为羧基酶底物。 实验方法是基于系统的比较。 凝血因子II的两种前体形式及其用途 ~(125)I-碘化蛋白质定位及高效液相色谱定位。 单核巨噬细胞是凝血的有效触发物 系统。肺巨噬细胞产生组织因子和因子 V，但似乎没有羧基酶活性。另一方面 手，II型肺泡细胞表现出显著的羧化酶 活动。建议进行实验，以测试是否 肺可以提供所有必要的凝血因子 肺内的外源性凝血。已知巨噬细胞 刺激II型细胞增殖。因此，II型细胞 将在受刺激的条件培养液中培养 以测试巨噬细胞之间通信的可能性 巨噬细胞和能调节促凝血剂的II型细胞 按类型II单元格输出。抗大鼠凝血酶原抗体和 因子X将用于鉴定14CO2标记的蛋白质 II型细胞体外羧化系统中的前体。 同样的方法也将用于研究维生素的生物合成。 人内皮细胞的钾依赖蛋白。鉴定 由不同人类产生的维生素K依赖蛋白 癌细胞株也将被尝试。最后，净化 具有重要生理意义的维生素K1还原 肝微粒体中的脱氢酶介导 蛋白分解后将尝试维生素K1的解毒作用 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.