ISOZYMES OF HEART ASPARTATE TRANSAMINASE

这是一个研究线粒体和胞浆天冬氨酸的建议 转氨酶同工酶 我们的目标是：a）了解分子力 负责每种蛋白质的稳定性， 与每个反应中特定作用相关的结构元件 B）研究其对细胞内 蛋白质加工事件（蛋白酶）和蛋白质通过的机制 进入细胞器，如线粒体（生物学）;和c）操纵 化学上和遗传上干扰特定的酶功能， 结构蛋白元件或识别传代所必需的那些 分子生物学（Molecular Biology） 为此，我们建议：a）利用差分扫描和批处理 量热法，31 P和13 CNMR，以及FTIR分析结构因素 影响修饰引起的各同工酶的稳定性 通过选择的蛋白酶的作用或化学操作B）表征 同工酶的载脂蛋白形式的焦磷酸酶样活性; c）研究 蛋白酶作用或同工酶的化学修饰对 线粒体识别和/或蛋白质转运机制; d）产生 半合成酶，其中14-19个残基的N-末端部分是 明智地改变，并遵循这种改变的后果; e） 寻找因子的作用机制，例如多磷酸盐， 控制同工酶的胞内蛋白酶加工;和f） 分离并在细胞中表达线粒体同工酶的cDNA， 这种同工酶的前体形式，研究的分子特性的 前体，其转运到线粒体的机制，以及 转化为成熟同工酶。

This is a proposal to study mitochondrial and cytosolic aspartate transaminase isozymes. We aim to: a) understand molecular forces responsible for each protein's stability, and the characterization of structural elements associated with specific roles in each of reaction mechanisms (chemistry); b) investigate their susceptibility to endocellular protein processing events (proteases) and the mechanisms of protein passage into organelles such as mitochondria (biology); and c) manipulate chemically and genetically to perturb specific enzymatic functions, structural protein elements or those necessary for recognition for passage through membranes (molecular biology). To this end we propose to: a) utilize differential scanning and batch calorimetry, 31P and 13CNMR, as well as FTIR to analyze structural factors affecting each isozyme's stability induced as a consequence of modification by action of selected proteases or chemical manipulation b) characterize the pyrophosphatase-like activity of the apo form of the isozymes; c) study the effects of protease action or chemical modification of the isozymes on mitochondrial recognition and/or protein transport mechanisms; d) produce semisynthetic enzymes in which the 14-19 residue N-terminal portion is judiciously altered and follow the consequences of such alterations; e) search for a mechanisms of action of factors, such as polyphosphates, controlling endocellular protease processing of the isozymes; and f) isolate and express the mitochondrial isozyme's cDNA in cells to produce precursor forms of this isozyme to study the molecular properties of the precursor, its mechanism of transport into mitochondria, and process of conversion into mature isozyme.