LAMININ ALTERATIONS AFTER NON-ENZYMATIC GLUCOSYLATION

在糖尿病中，高血糖会扰乱许多代谢途径， 每一种都可能导致糖尿病并发症。 的一个主要 改变是蛋白质的非酶促葡糖基化;它 发生在两个步骤：第一，可逆的（形成阿马多里 第二种是不可逆的（形成 最终产品）。 由于其半衰期长，地下室 膜大分子是该过程的主要目标。 在 糖尿病肾病，其中最重要的改变是 肾小球的增厚和通透性增加 基底膜 我们的长期目标是了解 分子水平上这些改变的发病机制。 层粘连蛋白是最重要的基底膜之一 组件;它在其结构组织中起着关键作用， 与大多数其他基底膜大分子相互作用 和细胞表面。 因此，我们建议调查 非酶促葡萄糖基化对几种生物学功能的影响 层粘连蛋白和阿司匹林的作用（已知抑制可逆的 步骤）和氨基胍（已知可抑制不可逆步骤） 恢复受影响的功能。 在我们的研究中， 模型系统将被使用：首先，层粘连蛋白提取自 产生基底膜的小鼠肿瘤（EHS）将被 葡萄糖基化;第二，分离，完整肾小球基底 来自大鼠的膜将被葡萄糖基化，然后层粘连蛋白将被 提取。 糖基化方案将包括添加阿司匹林 或氨基胍。 在这些治疗后，将检测层粘连蛋白 其结构外观和功能分析， 非酶促葡糖基化对其性质的影响。 的 功能测定将包括：a）二聚体形成，B） c）结合IV型胶原，d）结合 肝素，e）与硫酸乙酰肝素蛋白聚糖结合。 生物物理 技术（浊度测定法）和生物化学技术（固相测定法） 和形态学（旋转阴影的电子显微镜） 样品）将用于这些测定。 拟议的研究将 a）扩大我们对结构和功能的了解 糖尿病条件下基底膜的改变， B）使我们能够测试功能影响和治疗 由于非酶促的修饰的每个阶段 葡萄糖基化，和c）允许我们确定可能的副作用， 这些化合物似乎有希望预防 糖尿病并发症

In diabetes, hyperglycemia disturbs many metabolic pathways and each may lead to diabetic complications. One of the major alterations is the non-enzymatic glucosylation of proteins; it occurs in two steps: a first, reversible one (formation of Amadori products) and a second, irreversible one (formation of endproducts). Because of their long half-life, basement membrane macromolecules are primary targets of this process. In diabetic kidney disease, among the most important alterations are the thickening and the increased permeability of the glomerular basement membrane. Our long term objective is to understand at the molecular level the pathogenesis of those alterations. Laminin is one of the most important basement membrane components; it plays a key role in their structural organization, by interacting with most other basement membrane macromolecules and cell surfaces. Therefore, we propose to investigate the effect of non-enzymatic glucosylation on several biological functions of laminin and the role of aspirin (known to inhibit the reversible step) and aminoguanidine (known to inhibit the irreversible step) in restoring affected functions. For our studies, two in vitro model systems will be used: first, laminin extracted from a basement membrane-producing murine tumor (EHS) will be glucosylated; second, isolated, intact glomerular basement membranes from rats will be glucosylated and then laminin will be extracted. Glucosylation protocols will include addition of aspirin or aminoguanidine. After these treatments laminin will be tested for its structural appearance and in functional assays to examine the effect of non-enzymatic glucosylation on its properties. The functional assays will include: a) dimer formation, b) polymerization, c) binding to type IV collagen, d) binding to heparin, e) binding to heparan sulfate proteoglycan. A biophysical technique (turbidometry) and biochemical one (solid-phase assays) and a morphological one (electron microscopy of rotary shadowed samples) will be used for these assays. The proposed studies will a) broaden our knowledge on structural and functional modifications of basement membranes under diabetic conditions, b) enable us to test for functional implications and treatment of each stage of the modifications due to non-enzymatic glucosylation, and c) allow us to determine possible side effects of compounds that appear promising for preventing progression of diabetic complications.