HORMAL CONTROL OF HEPATIC GLUCONEOGENESIS

拟议的研究将集中在黄连的作用机制上。 激素对肝脏糖异生和糖酵解的影响。这一假设 将检验如下：激素对肝脏糖异生的控制 糖酵解是由cAMP依赖和/或非依赖介导的 F6P/F1，6-P2和F6P/F1中几种细胞质酶的磷酸化 PEP/丙酮酸底物循环及其参与的酶(S) 新近发现的二磷酸糖的合成和降解， 果糖2，6-二磷酸。研究将继续集中在L式的 丙酮酸激酶、果糖1，6-二磷酸酶、6-磷酸果糖1-激酶、 以及最近发现的两种酶6-磷酸果糖-2-激酶和果糖 2，6-二磷酸酶。6-磷酸果糖2-激酶/果糖2，6-二磷酸酶 将被提纯到均一性，以及它们的物理和动力学性质 学习。在体外，这些酶的磷酸化是由 CAMP依赖的蛋白激酶以及可能由其他激酶组成的 特色化的。胰高血糖素、胰岛素、儿茶酚胺及多种激素的作用 ~(32)P掺入6-磷酸果糖-2-激酶/果糖的底物 分离的肝细胞中的2，6-二磷酸酶将用 用免疫学方法分离酶。蛋白的磷酸化状态 完整细胞中的酶将与酶的活性相关。 用肝细胞提取液和cAMP水平测量。监管 这些酶的活性将在不同的饮食和激素中进行研究。 在体内和在隔离肝脏系统中的状态。我们还将 研究体内2，6-二磷酸果糖水平的调节 在分离的肝细胞中，并评估这一调节相对于 对肝脏糖酵解和糖异生的影响。为了实现这一目标 后一个目标我们将采用一种有意识的、完好无损的狗模型，其中内分泌 胰腺功能可以通过“胰腺夹”来控制。 技术和底物水平可以通过补充来控制 技巧。2，6-二磷酸果糖与其他化合物的相互作用 含6-磷酸果糖1-激酶、果糖的变构效应器 1，6-二磷酸酶、丙酮酸激酶和6-磷酸果糖2-激酶也将 被调查。最后，磷酸化对动力学和蛋白质组分的影响 这些酶的物理性质将通过以下几项进行评估 技巧。

The proposed research will concentrate on the mechanism of action of hormones on hepatic gluconeogenesis and glycolysis. The hypothesis which will be tested is as follows: Hormonal control of hepatic gluconeogenesis and glycolysis is mediated by cAMP-dependent and/or independent phosphorylation of several cytoplasmic enzymes involved in F6P/F1,6-P2 and PEP/Pyruvate substrate cycles and of the enzyme(s) involved in the synthesis and degradation of the recently discovered sugar diphosphate, fructose 2, 6-bisphosphate. Research will continue to focus on L-type pyruvate kinase, fructose 1, 6-bisphosphatase, 6-phosphofructo 1-kinase, and two recently discovered enzymes 6-phosphofructo 2-kinase and fructose 2,6-bisphosphatase. 6-Phosphofructo 2-kinase/fructose 2,6-bisphosphatase will be purified to homogeneity and their physical and kinetic properties studied. In vitro phosphorylation of these enzymes catalyzed by the cAMP-dependent protein kinase and perhaps by other kinases will be characterized. The effect of glucagon, insulin, catecholamines and various substrates on 32P incorporation into 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase in isolated hepatocytes will be investigated using immunological methods to isolate the enzymes. The phosphorylation state of the enzymes in intact cells will be correlated with enzyme activity measured in hepatocyte extracts and with cAMP levels. The regulation of activity of these enzymes will be studied in various dietary and hormonal states both in vivo and in isolated liver systems. We will also investigate the modulation of fructose 2, 6-bisphosphate levels in vivo and in isolated hepatocytes and assess the importance of this regulation vis a vis hepatic glycolysis and gluconeogenesis. In order to achieve this latter goal we will employ a conscious, intact dog model where endocrine pancreatic function can be controlled by virtue of a "pancreatic clamp" technique and substrate levels can be controlled by supplementation techniques. The interaction of fructose 2, 6-bisphosphate and other allosteric effectors with 6-phosphofructo 1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase, and 6-phosphofructo 2-kinase will also be investigated. Finally, the effect of phosphorylation on the kinetic and physical properties of these enzymes will be assessed by a number of techniques.