Characterization of glycolytic enzyme interactions with murine erythrocyte membranes in wild-type and membrane protein knockout mice
Characterization of glycolytic enzyme interactions with murine erythrocyte membranes in wild-type and membrane protein knockout mice
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DOI：
10.1182/blood-2008-03-146159
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发表时间：
2008-11-01
期刊：
影响因子：
20.3
通讯作者：
Low, Philip S.
中科院分区：
文献类型：
--
作者：
Campanella, M. Estela;Chu, Haiyan;Low, Philip S.
以往的研究表明，糖酵解酶（GEs）以多酶复合物的形式存在于人红细胞膜内表面。由于GE结合位点已被定位到膜蛋白第3带上的序列，这在其他哺乳动物的同源物中并不保守，因此问题出现了，GE是否可以在其他哺乳动物的红细胞膜上组织成复合物。为了解决这个问题，小鼠红细胞被甘油醛-3-磷酸脱氢酶、醛缩酶、磷酸果糖激酶、乳酸脱氢酶和丙酮酸激酶抗体染色，并在共聚焦显微镜下进行分析。在氧化红细胞中发现GEs定位于细胞膜，但在脱氧后重新分布到细胞质中，如在人红细胞中所见。为了鉴定参与GE组装的膜蛋白，我们检测了缺乏每种主要红细胞膜蛋白的小鼠红细胞的GE定位。条带3敲除小鼠的GEs与细胞膜无关，而是分布在整个细胞质中，与红细胞氧合状态无关。相比之下，缺乏α -谱蛋白、锚蛋白、蛋白4.2、蛋白4.1、β -内收蛋白或降糖素头戴的小鼠红细胞显示出与膜结合的GEs。这些数据表明，细胞膜上依赖氧的GEs组装可能是哺乳动物红细胞的普遍现象，这些相互作用的稳定性主要取决于带3。（血液。2008;112:3900-3906）
Previous research has shown that glycolytic enzymes (GEs) exist as multienzyme complexes on the inner surface of human erythrocyte membranes. Because GE binding sites have been mapped to sequences on the membrane protein, band 3, that are not conserved in other mammalian homologs, the question arose whether GEs can organize into complexes on other mammalian erythrocyte membranes. To address this, murine erythrocytes were stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase and analyzed by confocal microscopy. GEs were found to localize to the membrane in oxygenated erythrocytes but redistributed to the cytoplasm upon deoxygenation, as seen in human erythrocytes. To identify membrane proteins involved in GE assembly, erythrocytes from mice lacking each of the major erythrocyte membrane proteins were examined for GE localization. GEs from band 3 knockout mice were not membrane associated but distributed throughout the cytoplasm, regardless of erythrocyte oxygenation state. In contrast, erythrocytes from mice lacking alpha-spectrin, ankyrin, protein 4.2, protein 4.1, beta-adducin, or dematin headpiece exhibited GEs bound to the membrane. These data suggest that oxygenation-dependent assembly of GEs on the membrane could be a general phenomenon of mammalian erythrocytes and that stability of these interactions depends primarily on band 3. (Blood. 2008; 112:3900-3906)