THE ROLE OF THE VITAMIN K CYCLE IN COAGULATION

该项目的目的是了解维生素K依赖的一级结构如何 底物控制其与γ谷氨酰羧化酶的相互作用，并鉴定相关维生素K环氧化物还原酶的基因。这些酶负责参与止血、骨代谢和细胞生长的蛋白质的维生素K依赖性翻译后修饰。了解这些酶的结构和功能肯定会导致在治疗人类疾病的许多领域的临床应用。具体目标是：一。启动一种全新的方法来识别维生素K环氧化物还原酶。华法林抗性基因和环氧化物还原酶基因显然定位于同一位点，这使得将该基因定位于人类16号染色体的一个狭窄区域成为可能。因此，人们可以使用直接测定？击倒？特异性信使RNA分子与小的双链RNA一起鉴定细胞培养物中的基因。两个.测定因子IX的残留量？在羧化酶的前肽结合位点结合特定残基的前肽。将使用传统蛋白质化学和噬菌体展示方法的组合。三.使用毛细管电泳与质谱联用，以确定向因子IX的Gla结构域的12个Gla残基添加二氧化碳的顺序。这一假说是，对功能最关键的谷氨酸将首先被羧化。四使用各种底物继续研究羧化的机制。将通过利用具有相同前肽但不同Gla结构域的底物来检查Gla结构域的作用。这些实验将 使用预稳态条件在酶过量下进行。前肽和Gla结构域之间的相互作用将通过利用具有相同Gla结构域但不同前肽的底物来检查。

The purpose of this project is to understand how the primary structure of vitamin K-dependent substrates controls their interaction with the gamma glutamyl carboxylase and to identify the gene of the related vitamin K epoxide reductase. These enzymes are responsible for the vitamin K dependent post-translational modification of proteins involved in hemostasis, bone metabolism and cell growth. Understanding the structure and function of these enzymes will certainly lead to clinical applications in the treatment of many areas of human disease. The specific aims are: One. Initiate an entirely new approach to identify the vitamin K epoxide reductase. The genes for warfarin resistance and epoxide reductase apparently map to the same locus making it possible to localize the gene to a narrow region of human chromosome 16. Thus one can use a direct assay to ?knock out? specific messenger RNA molecules with small double stranded RNA to identify the gene in cell culture. Two. Determine the residues of factor IX?s propeptide that bind to specific residues at the propeptide binding site of the carboxylase. A combination of traditional protein chemistry and phage display methods will be used. Three. Use capillary electrophoresis coupled with mass-spectrometry to determine the order of the addition of carbon dioxide to the twelve Gla residues of the Gla domain of factor IX. The hypothesis is that the glutamic acids most critical for functions will be carboxylated first. Four. Continue to examine the mechanism of carboxylation using various substrates. The role of the Gla domain will be examined by utilizing substrates that have the same propeptide but different Gla domains. These experiments will be done in enzyme excess using pre steady state conditions. The interaction between the propeptide and Gla domain will be examined by utilizing substrates that have the same Gla domain but different propeptides.