STRUCTURE AND FUNCTION OF PHOSPHOFRUCTOKINASE

本项目的目的是研究生物多样性的调节机制 磷酸果糖激酶(PFK)。虽然这种酶活性的调节是通过 各种代谢物在体外都有很好的特性，许多在体内 观察到的情况仍然不清楚。我们发现了一种“激活 PFK的因子是β-D-果糖-2，6-P2，它似乎是 PFK最重要的效应因子。聚乙二醇胺的合成与降解 将对果糖-2，6-P2及其周转的控制进行调查。 大鼠离体肝细胞α、β肾上腺素能作用的实验研究 将研究果糖-2，6-P2水平的激动剂和拮抗剂。 我们发现了一种名为果糖-6-P，2-激酶的酶，它催化 果糖-2，6-P2的合成，该酶将被提纯并 特色化的。我们将寻找一种酶，它负责 这种因子的降解，如果成功，这种酶将被分离 并以此为特征。我们将试图阐明其控制机制 这些酶。我们还将测定果糖-2，6-P2的分布 在大鼠和糖尿病大鼠的各种组织中。为了 阐明果糖-2，6-P2对纯化肌肉的作用机制 肝PFK，与磷酸和脱磷PFK结合的配体，构象 变化及其对缔合-解离反应的影响 学习。为了确定心脏的生理学意义 肌肉PFK在体内的磷酸化，将尝试关联 使用灌流的大鼠肌肉进行磷酸化收缩。如果 如果成功，我们将研究该反应的生化机理。

The purpose of this project is to investigate the regulatory mechanisms of phosphofructokinase (PFK). Although regulation of this enzyme activity by various metabolites in vitro is well characterized, many in vivo observations are still not understood. We discovered an "activation factor" for PFK which is Beta-D-fructose-2,6-P2, and which appears to be the most important effector of PFK. The synthesis and degradaton of fructose-2,6-P2 and the control of its turnover will be investigated. Using isolated rat hepatocytes the effect of Alpha- and Beta-adrenergic agonists and antagonists on the level of fructose-2,6-P2 will be studied. We found an enzyme termed fructose-6-P,2-kinase which catalyzes the synthesis of fructose-2,6-P2, and this enzyme will be purified and characterized. We will search for an enzyme which is responsible for the degradation of this factor and if successful, this enzyme will be isolated and characterized. We will attempt to elucidate the control mechanisms of these enzymes. We will also determine the distribution of fructose-2,6-P2 in various tissues of rats as well as in diabetic rats. In order to elucidate the mechanism of action of fructose-2,6-P2 on purified muscle and liver PFKs, ligand binding to phospho and dephospho PFKs, conformational changes and its effect on association-dissociation reactions will be studied. In order to determine the physiological significance of the phosphorylation of muscle PFK in vivo, attempts will be made to correlate muscle contraction with phosphorylation using perfused rat muscle. If successful, we will study the biochemical mechanism of the reaction.