Campanella M Estela, Chu Haiyan, Low Philip S
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2402-7. doi: 10.1073/pnas.0409741102. Epub 2005 Feb 8.
To characterize the location of glycolytic enzymes (GEs) in intact human erythrocytes, freshly drawn blood was fixed and stained with Abs to GAPDH, aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), carbonic anhydrase II, Hb, and band 3 (AE1). Confocal microscopy revealed that in cells where band 3 displays its expected membrane staining and Hb is evenly distributed across the cytoplasm, GEs are largely limited to the membrane. Biochemical studies confirmed that the membrane binding sites for GAPDH, aldolase, and PFK reside on band 3, but related analyses demonstrate that sites for PK and LDH do not. Four lines of evidence demonstrate that the GEs are at least partially assembled into multimeric complexes near the NH2 terminus of band 3. First, a mAb to residues 1-12 of band 3 displaces all of the above GEs from the membrane, including LDH and PK, which do not bind band 3. Second, tyrosine phosphorylation of the NH2 terminus of band 3 (Y8 and Y21) reversibly releases all of the GEs from the membrane, including LDH and PK. Third, deoxygenation of RBCs dislodges all GEs from the membrane, consistent with the established ability of deoxyHb but not oxyHb to bind the NH2 terminus of band 3. Fourth, a large increase in the accessibility of enzyme epitopes is observed upon dissociation of GEs from the membrane. We conclude, therefore, that GEs are organized into complexes on the membrane whose assembly is regulated by oxygenation and phosphorylation.
为了表征糖酵解酶（GEs）在完整人类红细胞中的定位，采集新鲜血液，固定并用抗甘油醛-3-磷酸脱氢酶（GAPDH）、醛缩酶、磷酸果糖激酶（PFK）、丙酮酸激酶（PK）、乳酸脱氢酶（LDH）、碳酸酐酶II、血红蛋白（Hb）和带3（AE1）的抗体进行染色。共聚焦显微镜显示，在带3呈现预期膜染色且Hb均匀分布于细胞质的细胞中，GEs主要局限于膜上。生化研究证实，GAPDH、醛缩酶和PFK的膜结合位点位于带3上，但相关分析表明PK和LDH的位点并非如此。四条证据表明，GEs至少部分组装成靠近带3 NH2末端的多聚体复合物。首先，针对带3第1 - 12位残基的单克隆抗体（mAb）可将上述所有GEs从膜上置换下来，包括不结合带3的LDH和PK。其次，带3 NH2末端（Y8和Y21）的酪氨酸磷酸化可使所有GEs从膜上可逆释放，包括LDH和PK。第三，红细胞的脱氧作用使所有GEs从膜上脱离，这与已确定的脱氧血红蛋白而非氧合血红蛋白结合带3 NH2末端的能力一致。第四，在GEs从膜上解离后，观察到酶表位的可及性大幅增加。因此，我们得出结论，GEs在膜上组织成复合物，其组装受氧合作用和磷酸化调节。
