THERMODYNAMICS OF LIGAND BINDING TO HUMAN ERYTHROCYTE

这项拟议的研究考察了底物结合到 人红细胞葡萄糖-6-磷酸脱氢酶(G6PD) 磷酸戊糖途径。该项目对健康的影响是由 从G6PD的重要性出发。G6PD维持着人类的还原环境 红细胞，以及在遗传缺陷的个体中，怀疑 会导致溶血。这项建议的具体目的是 定量测定烟酰胺腺嘌呤二核苷酸的数量 磷酸(NADP)和葡萄糖-6-磷酸(G6P)分子与G6PD和 这两个分子的缔合常数的大小。这个 项目将直接测量放射性物质的平衡量 标记的G6P或NADP与酶结合。这些测量结果将是 在潜在调节分子的pH和浓度下进行 (例如，ATP)已知的影响G6PD酶活性的条件。 绑定数据将通过直接适配到一系列绑定来分析 用非线性回归方法求解日益复杂的方程 算法。该算法将为数字和 配体结合的强度。这些测量之间的比较将 指出这些配体对G6PD的影响，并分析其结合情况 通过关联函数方法的配置文件将提供定量估计 调节分子对底物结合的影响。

The proposed research examines the thermodynamics of substrate binding to human erythrocyte glucose-6-phosphate dehydrogenase (G6PD), a key enzyme of the pentose phosphate pathway. The project's health implication is derived from the importance of G6PD. G6PD maintains the reducing environment of the erythrocyte, and in genetically deficient individuals, it is suspected of contributing to hemolysis. The specific aims of the proposal are to measure quantitatively 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. These measurements will be conducted under pH and concentration of potential regulatory molecules (e.g. ATP) conditions known to influence the enzymatic activity of G6PD. The binding data will be analyzed by direct fitting to a series of binding equations of increasing complexity using a non-linear regression algorithm. The algorithm will yield quantitative values for the number and strength of the ligand binding. Comparison between these measurements will indicate the effect of these ligands on G6PD, and analysis of the binding profiles by a linked-functions approach will provide quantitative estimates of the impact of regulatory molecules upon substrate binding.