人红细胞膜带3蛋白重组人脂质体及其~(35)SO_4~(2-)运输活性

用Triton X-100选择性抽提法,部分提纯了人红细胞膜带3蛋白。在除去去污剂后,带3蛋白被插入由磷脂酰胆碱/胆固醇组成的脂质体中。研究表明,用0.05％Triton抽提膜能除去大部分唾液酸糖蛋白,不会损失太多的带3蛋白。再用0.5％Triton抽提,可增溶出大量带3蛋白和少量其它膜蛋白。把它重组入脂质体后,在最后超离心步骤中得到了小的单层囊泡和大的多层囊泡。本文研究了单层重组囊泡输入~(35)SO_4~(2-)的功能,并和那些由人红细胞“发芽”得到的天然囊泡输入~(35)SO_肋~(2-)的功能作了比较。     

红细胞膜的影细胞蛋白

一、发现哺乳动物(包括人)的红细胞膜蛋白在重量上约占膜总重量的50%。人红细胞膜经十二烷基硫酸钠处理、聚丙烯酰胺凝胶电泳和考马斯亮蓝染色后,至少可见14条谱带,多则可染21条,其中有12条与膜支架(skeleton)有关。最主要的一种支架蛋白叫影细胞蛋白(spec-trin),它既是红细胞膜支架的基本成份,又是红细胞膜的一种收缩性蛋白。影细胞蛋白首先是Marchesi与Steers利用豚鼠红细胞膜的血影(ghost)为材料,在5%聚丙烯酰胺(含8M     

带3蛋白反向转运Cl—HCO3^—的意义

带 3蛋白是红细胞膜上的 1种镶嵌蛋白 ,由于该蛋白在红细胞膜蛋白 SDS电泳时 ,处于凝胶上第 3条显带的位置 ,因此被称为带 3蛋白。带 3蛋白不仅在维持红细胞膜骨架方面起重要作用 ,而且还是阴离子反向交换器 (anion antiporter) ,它能够反向地转运 Cl-和HCO-3过膜。这种功能对于红细胞在肺部和组织中进行气体交换起着至关重要的作用。1　带 3蛋白的结构带 3蛋白分子量为 930 0 0 D,由 2个相同的肽链组成二聚体结构 ,每 1条肽链含有 92 9个氨基酸残基 ,其N端和 C端均朝向细胞质侧。带 3蛋白具有 2个结构域 ,其一是 N端在胞质面折叠成不…     

羟自由基对人红细胞氧化囊泡化的作用

红细胞经氧化处理后,发现红细胞膜区带１、２、２．１及３易形成高聚物,同时Ｈｂ亦有氧化变性。氧化过程中磷脂（ＰＳ、ＰＥ）逐渐减少,根据此结果提出氧化产生囊泡化的机制是：首先Ｈｂ氧化变性,膜骨架蛋白聚集,从而网架松散,膜磷脂脱离骨架的束缚,膜脂质与膜整合蛋白形成小囊泡从红细胞上脱落。     

红细胞内翻外囊泡带3蛋白活性测试方法的研究

从人血中提取红细胞膜,用注射器加压推打的方法首次获得了包含80mmol/L吡啶二羧酸(DPA)的封闭完好的内翻外囊泡(IOVs).离心除去囊泡外DPA,即可按Newton法测其阴离子转运活性.此法在红细胞膜内翻外囊泡体系上成功地建立了带3蛋白(Band 3)测活方法,具有简便迅速,重复性好等优点.     

氧化应激时Peroxiredoxin Ⅱ膜质转移对红细胞渗透脆性的影响

为了探讨氧化应激时peroxiredoxinⅡ(PrxⅡ)膜质转移对红细胞渗透脆性的影响,检测了H_2O_2处理后红细胞渗透脆性的变化,并利用蛋白质免疫印迹法(Western-blot)检测了红细胞内PrxⅡ膜质转移情况,以及红细胞膜蛋白——带3蛋白(band 3)和血影蛋白(spectrin)的变化情况。研究结果显示,氧化应激时红细胞渗透脆性增加,红细胞内PrxⅡ从细胞膜转移至细胞质中,同时维持红细胞膜稳定、细胞骨架结构功能完整的相关蛋白质——band 3和spectrin在红细胞膜上表达量减少。实验结果证明氧化应激时红细胞内PrxⅡ发生膜质转移,引起维持红细胞膜稳定、细胞骨架结构功能完整的相关蛋白质band 3和spectrin表达量降低,导致红细胞渗透脆性增加。     

羟自由基对人红细胞氧化囊泡化的作用

红细胞经氧化处理后,发现红细胞膜区带１、２、２．１及３易形成高聚物,同时Ｈｂ亦有氧化变性。氧化过程中磷脂（ＰＳ、ＰＥ）逐渐减少,根据此结果提出氧化产生囊泡化的机制是：首先Ｈｂ氧化变性,膜骨架蛋白聚集,从而网架松散,膜磷脂脱离骨架的束缚,膜脂质与膜整合蛋白形成小囊泡从红细胞上脱落。     

慢性呼吸衰竭病人红细胞膜蛋白和红细胞变形能力的研究

目的：探讨红细胞膜蛋白在红细胞变形性改变中的作用。方法：参照Ｌｅａｍｍｌｉ和Ｐｅａｃｏｃｋ方法,测定了肺心病Ⅰ型呼吸衰竭（Ⅰ组）１８例、Ⅱ型呼吸衰竭（Ⅱ组）１８例和健康对照（ＣＧ）２０例的红细胞膜带３蛋白、膜收缩蛋白二聚体（ＳｐＤ）和四聚体（ＳｐＴ）的相对含量与红细胞变形能力。结果：Ⅰ、Ⅱ组带３蛋白、ＳｐＤ、ＳｐＴ相对含量和红细胞变形指数（ＤＩ）与对照组均有显著差异,且肺心病病人的ＤＩ与带３蛋白相对含量呈显著正相关,与ＳｐＤ／ＳｐＴ比值呈显著负相关。结论：带３蛋白和膜收缩蛋白的异常,可能是导致肺心病人红细胞变形能力降低的重要因素之一。     

红细胞膜带4.2蛋白的结构与功能

带4.2蛋白是一种重要的红细胞膜蛋白,与红细胞的形态、可变形性及携氧功能有至关重要的联系。它通过与带3蛋白(阴离子通道蛋白)、锚蛋白结合,稳定的连接在细胞膜的内表面,连接着膜骨架网架结构与细胞膜,是膜骨架与脂质双分子层连接的重要纽带。带4.2蛋白的缺失会引起球形或椭圆形红细胞增多症及不同程度的溶血性贫血,严重的情况需要摘除脾脏来进行治疗。近年来研究认为,带4.2蛋白在维持细胞膜骨架的完整性和稳定性方面扮演了重要角色。现对带4.2蛋白结构及功能的研究状况进行综述。     

大强度训练及恢复后大鼠红细胞变形能力和红细胞膜蛋白的变化

目的 :探讨运动对红细胞变形性和红细胞膜蛋白的影响及其相互关系。方法 :设计不同强度的训练方案 ,用激光衍射法测定红细胞变形能力 ,用SDS PAGE方法测定一定体积大鼠红细胞膜中的重要蛋白带 3蛋白 (band 3)和肌动蛋白 (actin)的含量 ,研究运动即刻和恢复后红细胞变形性及膜蛋白的变化。结果 :长期的运动训练会促进大鼠红细胞变形能力的改善和红细胞膜band 3蛋白和actin的良好发展 ,一次大强度训练会引起红细胞膜band 3蛋白和actin含量的减少 ,大鼠红细胞变形能力降低 ,一周和二周的大强度训练会提高恢复期大鼠红细胞的变形能力和红细胞膜band 3蛋白和actin含量。结论 :运动训练造成的红细胞膜蛋白含量的变化 ,导致了红细胞膜结构的改变 ,从而影响红细胞变形能力 ,可能是训练对红细胞变形能力的作用机制之一。     

Topology of the anion exchange protein AE1: the controversial sidedness of lysine 743

The topology of the band 3 (AE1) polypeptide of the erythrocyte membrane is not fully established despite extensive study. Residues near lysine 743 (K743) have been reported to be extracellular in some studies and cytoplasmic in others. In the work presented here, we have attempted to establish the sidedness of K743 using in situ proteolysis. Trypsin, papain, and proteinase K do not cleave band 3 at or near K743 in intact red cells, even under conditions that cause cleavage on the C-terminal side of the glycosylation site (N642) in extracellular loop 4. In contrast, trypsin sealed inside red cell ghosts cleaves at K743, as does trypsin treatment of inside-out vesicles (IOVs). The transport inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H(2)DIDS), acting from the extracellular side, blocks trypsin cleavage at K743 in unsealed membranes by inducing a protease-resistant conformation. H(2)DIDS added to IOVs does not prevent cleavage at K743; therefore, trypsin cleavage at K743 in IOVs is not a consequence of cleavage of right-side-out or leaky vesicles. Finally, microsomes were prepared from HEK293 cells expressing the membrane domain of AE1 lacking the normal glycosylation site. This polypeptide does not traffic to the surface membrane; trypsin treatment of microsomes containing this polypeptide produces the 20 kDa fragment, providing further evidence that K743 is exposed at the cytoplasmic surface. Therefore, the actions of trypsin on intact cells, resealed ghosts, unsealed ghosts, inside-out vesicles, and microsomes from HEK293 cells all indicate that K743 is cytoplasmic and not extracellular.     

High-pressure-induced hemolysis in papain-digested human erythrocytes is suppressed by cross-linking of band 3 via anti-band 3 antibodies

Upon exposure of human erythrocytes to a high pressure of 200 mPa, both hemolysis and vesiculation occur. The hemolysis of erythrocytes at 200 mPa was enhanced by removal of sialic acids from the membrane surface with papain. However, such enhancement was suppressed by cross-linking of band 3 via an anti-band 3 antibody (AB3A), which recognizes the exofacial domain of band 3, or by clustering of band 3 via Zn2+. On the other hand, the size of high-pressure-induced vesicles increased from 423 to 525 nm in diameter upon exposure to papain of erythrocytes, but decreased to 444 nm with following treatment with AB3A. In these vesicles, the content of spectrin relative to band 3 was almost the same. Furthermore, the band 3-cytoskeleton interactions in erythrocyte membranes remained unaltered upon treatment with papain and AB3A. Flow cytometric analysis demonstrated that papain-pretreated erythrocytes mainly produce open ghosts at 200 mPa and that the production of such open ghosts is suppressed by AB3A. Thus, upon removal of negative charges from the membrane surface, open ghosts are readily produced due to the release of larger vesicles under pressure. Upon cross-linking of band 3 via AB3A, however, the release of smaller vesicles at 200 mPa is facilitated so that high-pressure-induced hemolysis is suppressed.     

Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

Characterization of the chicken erythrocyte anion exchange protein

The avian erythrocyte anion exchange protein (band 3), after labeling with [3H2]4,4'-diisothiocyanodihydrostilbene-2, 2'-disulfonic acid appears as a doublet of polypeptide chains with apparent Mr = 105,000 and 100,000 by sodium dodecyl sulfate gel electrophoresis. The structures of the two species are almost identical as determined by partial proteolysis. The copy number of band 3 molecules per chicken erythrocyte was determined to be 800,000 by quantitating the amount of [3H2]4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid covalently bound to the cell surface. A comparison of human and chicken band 3 has revealed differences in their structure. Chicken band 3 differs from the human polypeptide in isoelectric point and proteolytic patterns. Antisera raised against human and chicken band 3 do not cross-react, implying that the two sera do not recognize any common antigenic determinants. There is a 6.5-fold lower activity per cell in the rate of phosphate exchange in the chicken erythrocyte which can be entirely explained by the 1.5-fold decrease in copy number per cell and the increased size of the chicken erythrocyte. This would suggest that there is no difference in the enzyme turnover number between chicken and human band 3. A major functional difference resulting from the structural differences is the inability to bind glyceraldehyde-3-phosphate dehydrogenase, a function associated with the NH2 terminus of human band 3.     

Human erythrocyte galactosyltransferase. Characterization, membrane association and sidedness of active site

Human erythrocyte UDPgalactose : 2-acetamido-2-deoxy-alpha-D-galactopyranosylpeptide galactose beta(1 lead to 3) transferase (Galactosyltransferase) has been characterized in terms of detergent and metal ion requirements. Michaelis constants for donor and acceptor substrates, inhibition constant for N-acetylgalactosamine, pH optimum and ionic strength effects. The assay thus optimized permits initial velocity measurements. Galactosyltransferase was shown to be membrane-bound by demonstrating its association with erythrocyte ghosts after high and low ionic strength treatments to remove weakly-associated proteins. In the absence of detergents, no activity was detectable in sealed ghosts and inside-out vesicles derived from erythrocyte membranes. Enzyme activation by detergents paralleled solubilization of membrane proteins. Both latency and solubilization studies indicated a substrate inaccessible active site for the enzyme in situ in the membrane. Galactosyltransferase activity in resealed ghosts, leaky ghosts and inside-out vesicles was resistant to the action of trypsin, chymotrypsin or pronase applied as single agents. A mixture of these proteases, however, strongly reduced the enzyme activity in inside-out vesicles and leaky ghosts, indicating a cytosolic orientation for the active site of the galactosyltransferase.     

Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. II. Formation of membrane leaks in ghost membranes after limited proteolysis of skeletal proteins by trypsin.

Limited proteolysis of human erythrocyte ghost membranes by low levels of trypsin (10-240 ng/ml) added bilaterally at 0 degrees C together with the proteinase inhibitor, phenylmethylsulfonyl fluoride (PMSF) before resealing at 37 degrees C leads to a graded digestion of spectrin and ankyrin and the disappearance of band 4.1 protein, while band 3 is cleaved only to a very low extent. These alterations are accompanied by an increase of membrane permeability of the resealed ghosts to hydrophilic nonelectrolytes (erythritol to sucrose), taken to reflect impaired resealing. Moreover, the membrane begins to vesiculate. Shedding of vesicles during the efflux measurements can not be responsible for the increased release of test solutes, since the ghosts do not loose hemoglobin and discriminate the nonelectrolytes according to their size. Moreover, the vesiculation site itself does not seem to act as the leak site, since ghosts prepared from erythrocytes pretreated with a carbodiimide which induces membrane rigidification still exhibit a pronounced protein degradation and vesiculation while the permeability enhancement induced by trypsination is markedly suppressed. The trypsin-induced leak has the properties of an aqueous pore as indicated, besides size selectivity, by its inhibition by phloretin and the very low activation energy. In analogy with concepts developed in the preceding paper (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)) the impaired resealing after limited proteolysis is assumed to be related to a perturbation of interactions of membrane skeletal elements with themselves and/or with the bilayer domain constituting the permeability barrier.     

Structure and thermotropic phase behaviour of detergent-resistant membrane raft fractions isolated from human and ruminant erythrocytes

Detergent-resistant membrane raft fractions have been prepared from human, goat, and sheep erythrocyte ghosts using Triton X-100. The structure and thermotropic phase behaviour of the fractions have been examined by freeze-fracture electron microscopy and synchrotron X-ray diffraction methods. The raft fractions are found to consist of vesicles and multilamellar structures indicating considerable rearrangement of the original ghost membrane. Few membrane-associated particles typical of freeze-fracture replicas of intact erythrocyte membranes are observed in the fracture planes. Synchrotron X-ray diffraction studies during heating and cooling scans showed that multilamellar structures formed by stacks of raft membranes from all three species have d-spacings of about 6.5 nm. These structures can be distinguished from peaks corresponding to d-spacings of about 5.5 nm, which were assigned to scattering from single bilayer vesicles on the basis of the temperature dependence of their d-spacings compared with the multilamellar arrangements. The spacings obtained from multilamellar stacks and vesicular suspensions of raft membranes were, on average, more than 0.5 nm greater than corresponding arrangements of erythrocyte ghost membranes from which they were derived. The trypsinization of human erythrocyte ghosts results in a small decrease in lamellar d-spacing, but rafts prepared from trypsinized ghosts exhibit an additional lamellar repeat 0.4 nm less than a lamellar repeat coinciding with rafts prepared from untreated ghosts. The trypsinization of sheep erythrocyte ghosts results in the phase separation of two lamellar repeat structures (d=6.00; 5.77 nm), but rafts from trypsinized ghosts produce a diffraction band almost identical to rafts from untreated ghosts. An examination of the structure and thermotropic phase behaviour of the dispersions of total polar lipid extracts of sheep detergent-resistant membrane preparations showed that a reversible phase separation of an inverted hexagonal structure from coexisting lamellar phase takes place upon heating above about 30 degrees C. Non-lamellar phases are not observed in erythrocytes or detergent-resistant membrane preparations heated up to 55 degrees C, suggesting that the lamellar arrangement is imposed on these membrane lipids by interaction with non-lipid components of rafts and/or that the topology of lipids in the erythrocyte membrane survives detergent treatment.     

The role of ankyrin in shape and deformability change of human erythrocyte ghosts

Human erythrocyte membranes (ghosts) from acid/citrate/dextrose preserved blood were digested with trypsin (protein/trypsin = 100:1) under hypotonic conditions and then analyzed by SDS-polyacrylamide gel electrophoresis. After digestion for about 20-30 s at 0 degree C, only ankyrin had disappeared and other bands including spectrin, actin, band 4.1 and band 3 remained intact. This observation was supported by electron micrographs showing that the horizontally disposed, filamentous structure was a little apart from the lipid bilayer and its components were not destroyed. In contrast to intact ghosts, treatment with chlorpromazine, or Mg-ATP did not induce shape change in these trypsin-treated ghosts. The number of transformable cells correlated closely with the amount of remaining ankyrin in the SDS-polyacrylamide gel electrophoresis pattern. Furthermore, the chlorpromazine- and Mg-ATP-induced decreases in viscosity of suspensions of erythrocyte ghosts were also prevented by trypsin treatment for 20-30 s at 0 degree C. These findings suggest that ankyrin plays an important role in the change in shape and deformability of erythrocyte ghosts. The molecular mechanism of drug-induced shape change and the role of undermembrane structure in regulating erythrocyte shape and deformability are discussed.     

Human erythrocyte galactosyltransferase. Characterization, membrane association and sidedness of active site

Human erythrocyte UDPgalactose : 2-acetamido-2-deoxy-α-d-galactopyranosylpeptide galactose transferase (Galactosyltransferase) has been characterized in terms of detergent and metal ion requirements, Michaelis constants for donor and acceptor substrates, inhibition constant for N-acetylgalactosamine, pH optimum and ionic strength effects. The assay thus optimized permits initial velocity measurements. Galactosyltransferase was shown to be membrane-bound by demonstrating its association with erythrocyte ghosts after high and low ionic strength treatments to remove weakly-associated proteins. In the absence of detergents, no activity was detectable in sealed ghosts and inside-out vesicles derived from erythrocyte membranes. Enzyme activation by detergents paralleled solubilization of membrane proteins. Both latency and solubilization studies indicated a substrate-inaccessible active site for the enzyme in situ in the membrane. Galactosyltransferase activity in resealed ghosts, leaky ghosts and inside-out vesicles was resistant to the action of trypsin, chymotrypsin or pronase applied as single agents. A mixture of these proteases, however, strongly reduced the enzyme activity in inside-out vesicles and leaky ghosts, indicating a cytosolic orientation for the active site of the galactosyltransferase.     

Temperature-dependent modes for the binding of the polyene antibiotic amphotericin B to human erythrocyte membranes. A circular dichroism study

The interaction of amphotericin B with isolated human erythrocyte ghosts was monitored by circular dichroism at 37 degrees C and 15 degrees C. Although different, these spectra were not concentration dependent over a concentration range covering the inducement of K+ leakage and hemolysis, which suggests the existence of only one bound amphotericin B species. At 15 degrees C, the spectra indicate that amphotericin B is complexed with membrane cholesterol; the complex formation is saturable but not cooperative. At 37 degrees C new spectra are observed, and their existence is conditioned by the presence of membrane proteins. The binding is cooperative but not saturable. The amphotericin B right side-out vesicles complexation is temperature as well as ionic strength dependent: at high ionic strength it is the same as with ghosts, with the same temperature dependence. At low ionic strength it is characteristic of an interaction with cholesterol, regardless of temperature. In the large unilamellar vesicles reconstituted from the total lipid extracts of erythrocyte membranes, amphotericin B is complexed with cholesterol, regardless of temperature and ionic strength. These results indicate that there are two different modes of amphotericin B complexation with erythrocyte membranes, reversible one in the other, depending on the molecular organization of the membrane and the presence of membrane proteins.