ISOLATION AND CHARACTERIZATION OF A MEMBRANE-PROTEIN FROM NORMAL HUMAN-ERYTHROCYTES THAT INHIBITS REACTIVE LYSIS OF THE ERYTHROCYTES OF PAROXYSMAL-NOCTURNAL HEMOGLOBINURIA
ISOLATION AND CHARACTERIZATION OF A MEMBRANE-PROTEIN FROM NORMAL HUMAN-ERYTHROCYTES THAT INHIBITS REACTIVE LYSIS OF THE ERYTHROCYTES OF PAROXYSMAL-NOCTURNAL HEMOGLOBINURIA
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DOI：
10.1172/jci114172
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发表时间：
1989-07-01
影响因子：
15.9
通讯作者：
PARKER, CJ
中科院分区：
文献类型：
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作者：
HOLGUIN, MH;FREDRICK, LR;PARKER, CJ
观察到，III型红细胞的阵发性睡眠性血红蛋白尿症（PNH）是敏感的激活眼镜蛇毒因子复合物（CoFBb），而正常的红细胞是抵抗启动溶血，这意味着PNH III细胞是缺乏在膜成分，调节这一过程。为了从正常红细胞中分离抑制因子，首先用丁醇提取膜蛋白，然后依次进行阴离子交换、羟基磷灰石和疏水层析。通过SDS-PAGE和银染对抑制级分的分析显示对应于具有表观Mr和18 kD的蛋白的单一条带。将PNH红细胞与递增浓度的放射性标记蛋白孵育，然后洗涤。以剂量依赖的方式，蛋白质纳入细胞膜和抑制CoFBb引发的裂解。这种蛋白质抑制剂通过在C7和C8掺入水平限制膜攻击复合物的组装而起作用。通过使用单特异性抗体来阻断抑制剂的功能，表明正常红细胞对CoFBb引发的溶血敏感。膜蛋白的Western印迹分析显示，PNH III红细胞缺乏18-kD蛋白。凭借其分子量和抑制活性，18-kD蛋白似乎是离散的其他先前描述的红细胞膜蛋白，调节补体。这些研究还表明，PNH III型红细胞对反应性裂解的敏感性与18-kD膜抑制剂的缺乏有因果关系。
The observation that type III erythrocytes of paraoxysmal nocturnal hemoglobinuria (PNH) are susceptible to hemolysis initiated by activated cobra venom factor complexes (CoFBb), whereas normal erythrocytes are resistant, implies that the PNH III cells are deficient in a membrane constituent that regulates this process. To isolated the inhibitory factor from normal erythrocytes, membrane proteins were first extracted with butanol and then subjected to sequential anion exchange, hydroxylapatite, and hydrophobic chromatography. Analysis by SDS-PAGE and silver stain of the inhibitory fractions showed a single band corresponding to a protein with an apparent Mr and 18 kD. PNH erythrocytes were incubated with incremental concentrations of the radiolabeled protein and then washed. In a dose-dependent fashion, the protein incorporated into the cell membrane and inhibited CoFBb-initiated lysis. This protein inhibitor function by restricting the assembly of the membrane attack complex at the level of C7 and C8 incorporation. By using a monospecific antibody to block the function of the inhibitor, it was shown that normal erythrocytes are rendered susceptible to CoFBb-initiated hemolysis. Analysis by Western blot of membrane proteins revealed that PNH III erythrocytes are deficient in the 18-kD protein. By virtue of its molecular weight and inhibitory activity, the 18-kD protein appears to be discrete from other previously described erythrocyte membrane proteins that regulated complement. These studies also indicate that the susceptibility of PNH III erythrocytes to reactive lysis is causally related to a deficiency of the 18-kD membrane inhibitor.