NONENZYMATIC GLYCATION: ENZYMATIC MECHANISM FOR CONTROL

描述：（改编自申请人摘要）糖尿病并发症 仍然是糖尿病管理中的一个严重和棘手的问题 糖尿病。非酶糖基化假说认为糖尿病患者 并发症至少部分是由于非酶修饰的结果， 蛋白质由葡萄糖该过程的初始产物是果糖赖氨酸（FL） 随后经历一系列进一步的重排和反应 糖基化终产物（Advanced Glycation Endproducts，AGEs）该提案旨在 测试FLPT是细胞内去糖化系统的一部分的假设。 研究计划包括三个具体目标：1）使用完整的细胞 孵育，通过比较非酶的量来测试FLPT的作用。 由葡萄糖产生的糖化与由不含葡萄糖的糖产生的糖化 2）从人红细胞中纯化FLPT并对其进行表征 充分考虑其功能和结构特性，以及3）启动 致力于纯化FLPT的一种明显的伴侣酶FL 3 P-水解酶。 在第一个目的下描述的实验将使用红细胞（稀释至100 μ g/ml）。 0.5%红细胞压积），血红蛋白已通过 苯硼酸盐和血管内皮细胞。之所以包括后者，是因为 研究人员认为，富含赖氨酸的组蛋白是一种重要的糖基化， 通过评估生长、蛋白质和DNA合成， 不被FLPT代谢的糖将具有比 葡萄糖对这些参数的影响。他将前两个系统称为体内系统， 但这些都是用词不当红细胞孵育，尤其是重度 稀释，不等同于体内情况。血红蛋白通过 PBA将构象扭曲和变性。关于下列问题的结论： 基于糖化产物生成量的FLPT系统的存在 在红细胞与处理的血红蛋白与不同糖孵育后 会充满错误附带说明的是， 在背景技术部分中示出了混淆， 体外与体内血红蛋白糖化位点的差异作为支持 FLPT-去糖化系统的存在，而不考虑 体外情况会导致可及性、局部pKa等发生变化， 控制赖氨酸残基对糖化的敏感性。酶 纯化时间表是直接的，纯化的FLPT样品具有 进行氨基酸分析。调查员已经建立了 用于监测纯化的FLPT和FL 3 P活性测定 过程

DESCRIPTION: (Adapted from the applicant's abstract) Diabetic complications remain a serious and intractable problem in the management of diabetes mellitus. The nonenzymatic glycation hypothesis postulates that diabetic complications are a result, at least in part, of nonenzymatic modifications of proteins by glucose. The initial product in this process is fructoselysine (FL) which subsequently undergoes a series of further rearrangements and reactions to form the Advanced Glycation Endproducts (AGEs). This proposal intends to test the hypothesis that FLPT is part of an intracellular deglycating system. The research plan consists of three Specific Aims: 1) Using intact cell incubations, test of the role of FLPT by comparing the amount of nonenzymatic glycation produced by glucose vs. that produced by sugars which are not substrates of FLPT; 2) Purify FLPT from human erythrocytes and characterize it fully in terms of its functional and structural properties, and 3) Initiate work to purify an apparent companion enzyme of FLPT, FL3P-hydrolase. The experiments described under the first aim will use erythrocytes (diluted to 0.5% hematocrit), hemoglobin that has been "deglycated" by passage through phenylboronate, and vascular endothelial cells. The latter are included because the investigator believes that lysine-rich histones are an important glycation target and that, by assessing growth, protein and DNA synthesis, he may show that sugars not metabolized by FLPT will have a greater inhibitory effect than glucose on these parameters. He refers to the first two systems as in vivo and in vitro, but these are misnomers. Incubation of red cells, especially heavily diluted, does not equate with the in vivo situation. Hemoglobin passed through PBA will be conformationally distorted and denatured. Conclusions regarding the existence of a FLPT system based on the amount of glycated product generated after incubation of erythrocytes vs. processed hemoglobin with different sugars would be fraught with error. It is parenthetically noted that the same confusion is shown in the background section wherein the investigator cites differences in sites of hemoglobin glycation in vitro vs. in vivo as supporting the existence of a FLPT-deglycation system, without regard to the fact that the in vitro situation imposes changes in accessibility, local pKa, etc. that govern the susceptibility of lysine residues to glycation. The enzyme purification schedules are straightforward and a sample of purified FLPT has been submitted for amino acid analysis. The investigator has set up functioning assays for FLPT and FL3P activity that are used to monitor the purification process.