STRUCTURE OF DEHYDROGENASES AND OTHER PROTEINS

脱氢酶和其他蛋白质的结构

基本信息

批准号：
3152535
负责人：
RALPH A BRADSHAW
金额：
$ 16.52万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
1982
资助国家：
美国
起止时间：
1982-12-01 至 1985-11-30
项目状态：
已结题

项目摘要

The amino acid sequences of the cytoplasmic and mitochondrial forms of pig heart malate dehydrogenase (MDH) as well as beta-hydroxyacyl CoA dehydrogenase (beta HADH) will be used to interpret the electron density maps determined by x-ray crystallography. Analyses will focus on determinants of folding, particularly in regions of similar conformation but highly dissimilar sequences. Lactate dehydrogenase will be included comparatively. The solution conformation of all of these enzymes will be compared to the x-ray derived model by various topographical probes. The biosynthesis of both MDH isozymes and beta HADH will be studied in cell-free systems and the structure of any additional segments found as amino or carboxyl terminal extensions will be sequenced by the incorporation of radiolabelled amino acids. The processing of these molecules will be examined relative to the enzyme(s) responsible. E. coli MDH, as an example of a prokaryotic version of the enzyme, will be isolated, characterized and sequenced and the structure compared to the eukaryotic forms. The translocators for L-malate and alpha-ketoglutarate from pig heart mitochondria will be solubilized by non-ionic detergents and purified by affinity chromatography. Several binding assays will be developed to identify the molecule. Characterization and sequence analysis will be performed on in the isolated translocator. The sequence of 17 beta-estradiol dehydrogenase will be sequenced and compared to the other dehydrogenases under study. In other studies, 1) various defined mutants of E. coli alkaline phosphatase will be isolated and defined in terms of the replacement. Crystals will be prepared and x-ray analysis performed (as difference maps with native enzyme) to correlate changes in three-dimensional structure with changes in kinetic/catalytic behavior. 2) The three-dimensional structure of E. coli biotin carboxyl carrier protein (9100) will be determined. 3) The sequences of chicken intestinal and liver lectins and muscle phosphoglycerate mutase will be determined.

猪心的细胞质和线粒体形式的氨基酸序列苹果酸脱氢酶（MDH）以及β-羟基酰基COA脱氢酶（β hadh）将用于解释由X射线确定的电子密度图晶体学。分析将集中于折叠的决定因素，特别是在类似构象但高度不同序列的区域中。乳酸脱氢酶将相对包括在内。所有的解决方案构象这些酶将通过各种酶进行比较地形探针。 MDH同工酶和βhadh的生物合成将在无细胞系统和任何其他段的结构中进行研究作为氨基或羧基末端扩展的发现将由放射性标记的氨基酸的掺入。这些分子的加工将对相对于负责的酶进行检查。大肠杆菌MDH，作为一个将酶的原核生物版本的示例将被隔离，表征与真核形式相比，测序和结构。这猪心脏线粒体的L-甲酸盐和α-酮戊二酸的转运剂将被非离子洗涤剂溶解并通过亲和力纯化色谱法。将开发几种约束分析，以确定分子。表征和序列分析将在孤立的转运剂。 17个β-雌二醇脱氢酶的序列将是测序并与正在研究的其他脱氢酶进行了比较。在其他研究，1）各种大肠杆菌碱性磷酸酶的突变体将是根据替代品进行了隔离和定义。晶体将准备并进行的X射线分析（作为与天然酶的差异图）相关三维结构的变化随动能/催化的变化行为。 2）大肠杆菌生物素羧基载体的三维结构将确定蛋白质（9100）。 3）鸡肠和将确定肝凝集素和肌肉磷酸甘油酸突变酶。