ORGANIZATION OF THE PATHWAY OF UREA SYNTHESIS IN SITU

尿素原位合成途径的组织

基本信息

批准号：
3303854
负责人：
LUISA J RAIJMAN
金额：
$ 16.1万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-04-01 至 1994-03-31
项目状态：
已结题

项目摘要

Some soluble cellular enzymes in their natural environment have kinetic properties that are not evident under the conditions used in most enzymological studies. There is evidence that some soluble pathway-related enzymes are organized in cells, some associated with membranes, such that the pathway intermediates are channeled. These are important features of cells. The long term objective of this work is to obtain comprehensive information on the properties and organization of soluble enzymes in situ; this would extend our knowledge and understanding of normal cell structure and function and of regulatory mechanisms, as well as of the deviations from these which occur in disease. Several pathway-specific intermediates of urea synthesis are channeled, indicating that the enzymes of the pathway are organized in situ. The cytoplasmic urea cycle enzymes in permeabilized hepatocytes will be characterized with regard to their requirements for aspartate, ornithine, and ATP. Experiments involving inhibition of specific transporters and competition between endogenous and exogenous substrates will be done to obtain information on the channelling of intermediates. Immunohistochemical methods will be used to locate the cytoplasmic urea cycle enzymes in intact and permeabilized hepatocytes, and possibly in Bri j-treated and "nude" hepatocytes. Preparations of inner and outer mitochondrial membranes and of contact sites will be used to determine whether ornithine transcarbamylase, the ornithine transporter, and the arginase of the outer membrane form a complex; evidence will be obtained from sedimentation analysis, immunoblotting, and patterns of cross-linking.

一些可溶性细胞酶在自然环境中具有动力学在大多数情况下使用的条件下不明显的特性酶学研究。有证据表明一些可溶性途径相关酶在细胞中组织，其中一些与细胞膜相关，因此途径中间体被引导。这些是重要的特征细胞。这项工作的长期目标是获得全面的信息关于可溶性酶原位的性质和组织；这会扩展我们对正常细胞结构的知识和理解功能和监管机制，以及偏离这些是在疾病中发生的。尿素合成的几种途径特异性中间体被引导，表明该途径的酶是原位组织的。这透化肝细胞中的细胞质尿素循环酶将其特征在于对天冬氨酸、鸟氨酸的需求，和ATP。涉及抑制特定转运蛋白和内源底物和外源底物之间的竞争将进行获取有关中间体通道的信息。免疫组织化学方法将用于定位完整的细胞质尿素循环酶和透化肝细胞，并且可能是经过 Bri j 处理和“裸露”的肝细胞。线粒体内膜和外膜的制备接触位点将用于确定鸟氨酸是否转氨甲酰酶、鸟氨酸转运蛋白和精氨酸酶膜形成复合物；将从沉淀中获得证据分析、免疫印迹和交联模式。