Trepanier D J, Thibert R J, Draisey T F, Caines P S
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Clin Biochem. 1996 Aug;29(4):347-55. doi: 10.1016/0009-9120(96)00038-0.
To establish the degree of erythrocyte membrane protein carbamylation in uremic and nonuremic patients, and to characterize the in vitro binding of cyanate to the individual proteins of the cytoskeletal matrix.
For in vivo studies, erythrocyte ghosts were digested with proteinase K and the released peptides colorimetrically assayed for carbamylation, using the diacetyl monoxime reagent, and quantitated using homocitrulline. For in vitro studies, erythrocyte ghosts were incubated with [14C] cyanate, and the membrane proteins separated by SDS-PAGE. Cyanate incorporation was quantitated by liquid scintillation counting and imaging densitometry of the excised bands.
Erythrocytes from uremic patients were found to have a greater level of carbamylation than those from nonuremic patients (47.09 +/- 7.80 and 25.89 +/- 6.92 nmol homocitrulline/mg proteolyzed protein released, respectively). In vitro incorporation of [14C] cyanate into membrane protein followed the sequence: spectrin > ankyrin > Band 4.1 > Band 3 > actin > Band 7.
The increased level of erythrocyte membrane protein carbamylation in uremic compared to nonuremic patients may lead to membrane destabilization and contribute to the decreased erythrocyte survival time observed in uremia.
确定尿毒症患者和非尿毒症患者红细胞膜蛋白的氨甲酰化程度，并描述氰酸盐在体外与细胞骨架基质中单个蛋白质的结合情况。
对于体内研究，用蛋白酶K消化红细胞血影，使用二乙酰单肟试剂比色法测定释放肽的氨甲酰化，并使用高瓜氨酸进行定量。对于体外研究，将红细胞血影与[14C]氰酸盐孵育，然后通过SDS-PAGE分离膜蛋白。通过液体闪烁计数和对切下条带的图像密度测定法定量氰酸盐掺入情况。
发现尿毒症患者的红细胞氨甲酰化水平高于非尿毒症患者（分别为47.09±7.80和25.89±6.92 nmol高瓜氨酸/毫克释放的蛋白水解产物）。体外[14C]氰酸盐掺入膜蛋白的顺序为：血影蛋白＞锚蛋白＞4.1带＞3带＞肌动蛋白＞7带。
与非尿毒症患者相比，尿毒症患者红细胞膜蛋白氨甲酰化水平升高可能导致膜不稳定，并导致尿毒症患者红细胞存活时间缩短。
