Verbeke P, Perichon M, Schaeverbeke J, Bakala H
Laboratoire de Biologie Cellulaire, Université Paris VII, France.
Biochim Biophys Acta. 1996 Jun 13;1282(1):93-100. doi: 10.1016/0005-2736(96)00043-0.
Aging is associated with the loss of preferential urinary excretion of Amadori-product glycated albumin. We have measured the binding of 125I-labeled glycated albumin to the renal brush-border membrane vesicles from young and old rats to determine whether a specific receptor-mediated endocytosis system may be involved. 125I-Glycated albumin was specifically bound by renal brush-border membrane vesicles in a time- and temperature-dependent manner; the binding was concentration-dependent, saturable and reversible. Scatchard plots gave an apparent dissociation constant Km of 488 +/- 17 nM, and a number of binding sites N of 33.5 +/- 3.4 pmol/mg protein/min in membrane vesicles from young (3 months old) rats; the binding of native [125I]albumin, gave a Km of 1194 +/- 200 nM (P < 2%) and N of 82.4 +/- 16.3 pmol/mg protein/min (P < 3%). Vesicles from 10-month-old rats had a similar Km (619.6 +/- 135.3 nM) and N (21.91 +/- 2.98 pmol/mg protein/min), while those from older (30 months old) rats had significantly increased Km (1344 +/- 237 nM, P < 3%) and N (81.3 +/- 10.9 pmol/mg protein/min, P < 1%) for 125I-glycated albumin binding. 125I-Glycated HSA was not displaced by unlabeled native HSA in less than 100-fold excess and native [125I]HSA was only displaced by a 10-fold excess of unlabeled glycated HSA. The binding of native [125I]HSA was partly inhibited (85%) by unlabeled glycated HSA. Thus, there appear to be two different binding sites, one for glycated and the other for native albumin, lying close together; and the glycation site on albumin is the discriminatory recognition factor.
衰老与尿中阿马多里产物糖化白蛋白优先排泄的丧失有关。我们测定了125I标记的糖化白蛋白与年轻和老年大鼠肾刷状缘膜囊泡的结合，以确定是否可能涉及特定的受体介导的内吞系统。125I-糖化白蛋白以时间和温度依赖的方式被肾刷状缘膜囊泡特异性结合；结合是浓度依赖的、可饱和的和可逆的。Scatchard图显示，来自年轻（3个月大）大鼠的膜囊泡中，125I-糖化白蛋白结合的表观解离常数Km为488±17 nM，结合位点数N为33.5±3.4 pmol/mg蛋白/分钟；天然[125I]白蛋白的结合，Km为1194±200 nM（P<2%），N为82.4±16.3 pmol/mg蛋白/分钟（P<3%）。来自10个月大大鼠的囊泡具有相似的Km（619.6±135.3 nM）和N（21.91±2.98 pmol/mg蛋白/分钟），而来自老年（30个月大）大鼠的囊泡，125I-糖化白蛋白结合的Km（1344±237 nM，P<3%）和N（81.3±10.9 pmol/mg蛋白/分钟，P<1%）显著增加。125I-糖化人血清白蛋白在未标记的天然人血清白蛋白过量不到100倍时不会被置换，而天然[125I]人血清白蛋白仅在未标记的糖化人血清白蛋白过量10倍时才会被置换。未标记的糖化人血清白蛋白部分抑制（85%）天然[125I]人血清白蛋白的结合。因此，似乎存在两个不同的结合位点，一个用于糖化白蛋白，另一个用于天然白蛋白，它们靠得很近；白蛋白上的糖基化位点是鉴别识别因子。
