Ligand Binding and Kinetic Studies on Human Erythrocyte Glucose-6-Phosphate Dehydrogenase Dimers

人红细胞葡萄糖-6-磷酸脱氢酶二聚体的配体结合和动力学研究

• 批准号: 9005512
• 负责人: Chris Craney
• 金额: $ 11.3万
• 依托单位: Occidental College
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9005512&HistoricalAwards=false
• 关键词: Ligand; Binding; Kinetic; Studies; Human

The project's goal is to use a new approach to simplify the kinetic and thermodynamic study of human erythrocyte glucose-6- phosphate dehydrogenase (G6PD) that involves the control of the enzyme's state of association. G6PD is a key enzyme in the hexosemonophosphate pathway and over 370 genetic variants of the human erythrocyte enzyme have been identified. Individuals with G6PD deficiencies can suffer hemolytic crisis on exposure to a variety of drugs or foods. The large number of genetic variants, many with reduced activity, mae G6PD an attractive target for cloning and sequencing. The interpretation of the changes in amino acid sequence are dependent on the measurement of G6PD's biochemical properties. Unfortunately, there is a lack of agreement on many of the basic enzymatic properties of erythrocyte G6PD, namely, the binding of substrates and the effect of intercellular molecules on the enzyme's kinetics. In analogy with prior work on yeast G6PD, this proposal's hypothesis is past kinetic and thermodynamic studies of human erythrocyte G6PD measured the properties of a mixture of G6PD dimers and tetramers, each of which may have different biochemical properties. This lack of agreement concerning human erythrocyte G6PD's biochemical properties would arise because the relative amounts of G6PD dimers and tetramers changed during the kinetic or thermodynamic measurements. Thus proposal will focus upon measuring the biochemical properties of the G6PD dimer, the form presumed to be present in the erythrocyte. The three specific aims of the RUI proposal are (1) to measure the number of nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate (G6P) molecules bound to G6PD and the magnitude of the association constant for these two molecules. The project will directly measure the equilibrium quantity of radioactively labeled G6P, or NADP, bound to the enzyme. The proposal will also (2) quantitatively study the inhibition of erythrocyte G6PD kinetics by ATP and 2,3 diphosphoglycerate, two proposed physiologically important inhibitors of G6PD. The third (3) specific aim will measure the dimertetramer association constant using large zone (plateau) HPLC experiments on a size exclusion column.

该项目的目标是使用一种新方法来简化人红细胞葡萄糖-6-磷酸脱氢酶（G6PD）的动力学和热力学研究，其中涉及酶关联状态的控制。 G6PD 是己糖单磷酸途径中的关键酶，已鉴定出超过 370 种人类红细胞酶的遗传变异体。 G6PD 缺乏的个体在接触各种药物或食物时可能会遭受溶血危机。 大量的遗传变异（其中许多活性降低）使 G6PD 成为克隆和测序的有吸引力的目标。 对氨基酸序列变化的解释取决于 G6PD 生化特性的测量。 不幸的是，对于红细胞 G6PD 的许多基本酶学特性，即底物的结合和细胞间分子对酶动力学的影响，缺乏共识。 与之前关于酵母 G6PD 的工作类似，该提案的假设是过去对人类红细胞 G6PD 的动力学和热力学研究测量了 G6PD 二聚体和四聚体混合物的特性，其中每种都可能具有不同的生化特性。 由于 G6PD 二聚体和四聚体的相对量在动力学或热力学测量过程中发生变化，因此人类红细胞 G6PD 的生化特性缺乏一致性。 因此，提案将侧重于测量 G6PD 二聚体的生化特性，这种形式被认为存在于红细胞中。 RUI提案的三个具体目标是（1）测量与G6PD结合的烟酰胺腺嘌呤二核苷酸磷酸（NADP）和葡萄糖-6-磷酸（G6P）分子的数量以及这两个分子的关联常数的大小。该项目将直接测量与酶结合的放射性标记 G6P（或 NADP）的平衡量。 该提案还将 (2) 定量研究 ATP 和 2,3 二磷酸甘油酸（两种拟议的生理上重要的 G6PD 抑制剂）对红细胞 G6PD 动力学的抑制作用。 第三 (3) 个具体目标将使用尺寸排阻柱上的大区（平台）HPLC 实验来测量二聚四聚体缔合常数。