ISOZYMES OF HEART ASPARTATE TRANSAMINASE

这是一项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)了解分子作用力 负责每种蛋白质的稳定性，以及对 与每个反应中的特定角色相关联的结构元素 机制(化学)；b)调查它们对细胞内的敏感性 蛋白质加工事件(蛋白水解酶)与蛋白质传递机制 变成线粒体等细胞器(生物学)；以及c)操纵 从化学和基因上扰乱特定的酶功能， 结构蛋白元件或识别传代所必需的元件 通过膜(分子生物学)。 为此，我们建议：a)利用差异扫描和批处理 量热、31P和13CNMR，以及FTIR分析结构因素 修饰对各同工酶稳定性的影响 通过选定的酶的作用或化学操作b)表征 载脂蛋白亚型同工酶的焦磷酸酶活性；c)研究 蛋白水解酶作用或同工酶的化学修饰对酶活性的影响 线粒体识别和/或蛋白质运输机制；d)产生 其中14-19个残基N-末端部分是 明智地更改，并遵循此类更改的后果；e) 寻找因子的作用机制，如多聚磷酸盐， 控制同工酶的细胞内切酶加工；和f) 线粒体同工酶基因在细胞中的分离和表达 这种同工酶的前体形式，以研究其分子性质 前体，其转运到线粒体的机制，以及 转化为成熟的同工酶。

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.