红细胞膜生物物理特性的测定方法

本文介绍几种完整红细胞膜生物物理特性的系列测定方法,包括膜对阴离子的通透性、膜对葡萄糖的跨膜运输、渗透脆性、低渗溶血速率等,以期对红细胞膜结构和功能特性的研究在完善理论意义的同时更具临床等实用意义。     

脱氧血红蛋白与红细胞膜的相互作用

人红细胞中血红蛋白主要以游离态存在,也有小部分与膜结合。文献报道,脱氧状态下两者相互作用加强,我们研究了这种相互作用的生物学意义,主要测定了脱氧状态下:(1)红细胞的低渗溶血速率;(2)红细胞膜对阴离子的通透性;(3)红细胞的变形性。     

不同状态血红蛋白对红细胞膜功能的影响

用荧光猝灭法测定了不同状态游离血红蛋白和红细胞血影膜间的相互作用,观察到和氧合血红蛋白相比,脱氧血红蛋白和膜上高亲和位点的作用加强,而高铁血红蛋白和低亲和位点的作用加强。本文用低渗溶血速率、阴离子通透性、变形性等三个参量测定了这种相互作用的变化对红细胞膜功能的影响,并讨论了脱氧状态下这种改变的生理意义。     

麦胚凝集素对红细胞膜阴离子通透性的影响

本文研究了WGA通过红细胞骨架对Band 3主要功能——阴离子通透性的影响。采用NO_2~-测定法和NH_4Cl等渗膨胀法,发现WGA作用使膜阴离子通透性降低,并且这种作用随WGA浓度增加而愈加明显。在特定条件下,高浓度WGA会引起部分红细胞明显变形。并由此改变WGA对膜阴离子通透性效应。此外,还通过NGA对WGA作用的恢复效应及对红细胞渗透脆性的研究,进一步探讨了WGA的作用机制。     

pCMBS对完整红细胞膜阴离子通透性的影响

根据等渗ＮＨ４Ｃｌ溶血动力学,探讨了ｐＣＭＢＳ（对氯汞苯磺酸）对完整红细胞膜阴离子通透性的影响．０．０５ｍｍｏｌ／ＬｐＣＭＢＳ使阴离子通透系数Ｐｃｌ下降为对照的８６．５％;１．０ｍｍｏｌ／Ｌ以上浓度时Ｐｃｌ值反而变大。ｐＣＭＢＳ浓度高于０．３ｍｍｏｌ／Ｌ时,细胞悬浮液在１０ｓ之前光密度下降过快,偏离理论拟合方程且不受ＤＩＤＳ抑制．半胱氨酸对ｐＣＭＢＳ引起的效应有恢复作用。结果表明ｐＣＭＢＳ和股骨架蛋白上特殊－ＳＨ基相互作用,导致ｂａｎｄ３构象改变,致使改变膜时阴离子的通透性。     

精胺对红细胞膜阴离子通透性的影响

本文采用改进的NH_4Cl等渗膨胀法研究了精胺对红细胞膜阴离子通透性的影响.结果表明精胺对膜阴离子通透性有增大作用,并且这种作用随精胺浓度的增加及作用时间的延长而增大.本文还就精按的作用机制进行了探讨.     

麦胚凝集素对红细胞膜阴离子通透性的影响

本文研究了ＷＧＡ通过红细胞骨架对Ｂａｎｄ３主要功能－阴离子通透性的影响。采用ＮＯ３＾－测定法和ＮＨ４Ｃｌ等渗膨胀法,发现ＷＧＡ作用使膜阴离子通透性降低,并且这种作用随ＷＧＡ浓度增加而明显。在特定条件下,高浓度ＷＧＡ会引起部分红细胞明显变形。并由此改变ＷＧＡ对膜阴离子通透性效应,此外,还通过ＮＧＡ对ＷＧＡ作用的恢复效应及对红细胞渗和渗透脆性的研究,进一步探讨了ＷＧＡ的作用机制。     

人红细胞膜上阴离子交换蛋白和葡萄糖运输蛋白的相互…

用细胞松弛素Ｂ抑制红细胞膜上葡萄糖运输蛋白对葡萄糖的运输,观察到对阴离子运输有促进作用。当ＧｌｕＴ－１结合其底物分子－葡萄糖后同样加快了阴离子运输速率。另一方面,实际亦给出了葡萄糖跨膜运输特性和Ｃｌ＾－离子浓度的关系,表明随着Ｃｌ＾－离子浓度的加大葡萄糖运输过程中Ｋｍ减小,Ｖｍａｘ增大。     

人红细胞膜上阴离子交换蛋白和葡萄糖运输蛋白的相互影响

用细胞松弛素Ｂ抑制红细胞膜上葡萄糖运输蛋白（ＧｌｕＴ－１）对葡萄糖的运输,观察到对阴离子运输有促进作用。当ＧｌｕＴ－１结合其底物分子－葡萄糖后同样加快了阴离子运输速率．另一方面,实验办给出了葡萄糖跨膜运输特性和Ｃｌ－离子浓度的关系,表明随着Ｃｌ－离子浓度的加大能使葡萄糖运输过程中Ｋｍ减小、Ｖ（ｍａｘ）增大。这些结果表明了在完整红细胞膜上阴离子交换蛋白Ｂａｎｄ３和ＧｌｕＴ－１之间存在着双向联系,即一种膜蛋白的构象改变能影响另一种膜蛋白的功能。     

红细胞膜对NO_2~-通透性的研究

本文采用NO_2~-快速测量红细胞膜对阴离子的通透特性.NO_2~-跨膜运输进红细胞内后,和血红蛋白相互作用,使后者变为高铁血红蛋白,改变了红细胞内血红蛋白的吸收光谱,进而影响细胞悬浮液的光学特性.据此,测量加入NO_2~-后红细胞悬浮液在特定波长处光密度的时间变化,即能追踪阴离子跨膜运输的动态过程.红细胞膜阴离子运输专一性抑制剂DIDS能抑制约70%的NO_2~-跨膜运输.在15-38℃温度范围检测了NO_2~-跨膜通透速率的温度特性,给出活化能为60KJ·mol~(-1).     

Erythrocyte bisulfite transport

The wide range of transport rates for anions of differing chemical structure by the human erythrocyte anion transport protein (Band 3 protein) suggests that this protein is highly selective for anions that chemically resemble its natural substrate bicarbonate. To test this hypothesis, the influx of bisulfite (HSO3-), a bicarbonate analog, was compared to influxes of chloride, sulfate, and bicarbonate, as measured by the technique of colloid osmotic lysis in isotonic ammonium salt solution. The lysis time induced in chloride solution (much greater than 10 min) was markedly accelerated to 0.6 min by the addition of small amounts (5 mM) of bicarbonate, an effect characteristic of colloid osmotic lysis induced by the anion transport pathway. Lysis in bicarbonate solution was extremely rapid (0.09 min), and was markedly inhibited by acetazolamide (2.9 min). Lysis in bisulfite solution occurred spontaneously (2.2 min) but was markedly accelerated to a time similar to that of chloride (0.56 min) by addition of 5 mM bicarbonate. In contrast, sulfate induced lysis was extremely slow (less than 10% lysis at 40 min in the presence of bicarbonate). Preincubation of erythrocytes with SITS, an inhibitor of anion exchange, prevented lysis by chloride, but had no effect on lysis by bicarbonate, indicating that lysis by bicarbonate was predominantly through diffusion and not anion transport. SITS treatment of erythrocytes eliminated the catalytic effect of bicarbonate during lysis by bisulfite, indicating that anion transport of bisulfite and diffusion of the conjugate acid in the form of SO2 both contribute to the total membrane flux. When the contribution of diffusion is taken into account, the rate of bisulfite influx through the anion exchange pathway is at least 100-fold faster than that for sulfate.     

Structural modification of erythrocyte membranes during oxidative stress and activity of membrane bound NADH-methemoglobin reductase

The activity of NADH-methemoglobin reductase (metHb-reductase) in membranes isolated from human erythrocytes treated with phenylhydrazine at its sublytic concentration was studied. A decrease in the activity of membrane-bound metHb-reductase was shown to depend on the concentration of phenylhydrazine. Simultaneously, an increase in the level of membrane-bound methemoglobin and a change in the fluorescence parameters of membrane-bound 4,4'-diisothiocy-anatostilbene-2,2'-disulfonic acid were registered. In the case when Hb-free erythrocyte ghosts were treated with 0.2-2.0 mM phenylhydrazine, the activity of metHb-reductase did not change. The obtained results indicate that the inhibition of the activity of membrane-bound metHb-reductase by phenylhydrazine-induced oxidative stress in human erythrocytes is not caused by the direct action of the oxidant on the enzyme. The reason for this is the interaction of the products of hemoglobin oxidation with erythrocyte membrane (protein band 3) and structural changes in membrane proteins.     

Mechanism of the change in erythrocyte osmotic resistance in rats exposed to valinomycin: the features seen in spontaneous hypertension

Introduction of valinomycin into erythrocyte incubation medium increased the cell stability to water-induced hemolysis. In these conditions the erythrocytes of spontaneously hypertensive and normotensive (control) rats release 63.2 +/- 1.5% and 80.9 +/- 1.6%, respectively, of the total hemoglobin content. Valinomycin effect is completely abolished with K+ substitution for Na+ and is independent of extracellular Ca2+ concentration. Valinomycin had no effect on human erythrocyte osmotic stability. It has been shown that valinomycin-induced kinetics of Na+ and K+ redistribution was different in human and rat erythrocytes. The distinctions are thought to be related to specific anion transport mediated by the third band protein--the main component of membrane cytoskeleton.     

Transmembrane Ca2+ gradient is essential for high anion transport activity of human erythrocytes

The role of a transmembrane Ca²⁺ gradient in anion transport by Band 3 of human resealed erythrocyte ghosts has been studied. The results show that a transmembrane Ca²⁺ gradient is essential for the conformation of erythrocyte Band 3 with higher anion transport activity. The dissipation of the transmembrane Ca²⁺ gradient by the ionophore A23187 inhibits the anion transport activity. The extent of this inhibition approaches 90% as the Ca²⁺ concentration on both sides of the ghost membrane is increased to 1.0 mM and half-maximum inhibition is observed at 0.25 mM Ca²⁺. Addition of ATP (0.4 mM) to the resealing medium can partly reestablish the transmembrane Ca²⁺ gradient by activation of Ca²⁺-ATPase and alleviate the inhibition to some extent. N-ethylmaleimide, an inhibitor of erythrocyte Ca²⁺-ATPase, prevents such restoration. Electron micrographs reveal that numerous larger intramembranous particles can be observed on the P-faces of freeze-fractured resealed ghosts in the absence of a transmembrane Ca²⁺ gradient.Abbreviations DPA dipicolinic acid - EITC eosin 5-isothiocyanate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - TES N-Tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid - PMSF phenylmethyl-sulfonylfluoride - NEM N-ethylamaleimide - BSA bovine serum albumin - EGTA ethyleneglycol-bis (aminoethylether)-tetra-acetic acid - EITC-Band 3 Band 3 labeled with EITC - Ca_i Ca²⁺ inside resealed ghosts - Ca_o Ca²⁺ outside resealed ghosts     

Injections of recombinant human erythropoietin increases lactate influx into erythrocytes.

Previous studies showed that erythropoietin not only increases erythrocyte production but is also essential in both the synthesis and the good functioning of several erythrocyte membrane proteins, including band 3. It is still unknown whether anion and/or H(+) fluxes are modified by erythropoietin. This study aimed to evaluate the effect of recombinant human erythropoietin (rHuEPO) injections on lactate transport into erythrocytes via band 3 and H(+)-monocarboxylate transporter MCT-1, two proteins involved in lactate exchange. Nine athletes received subcutaneous rHuEPO (50 U/kg body mass 3 times a week for 4 wk), and seven athletes received a saline solution (placebo group). All subjects were also supplemented with oral iron and vitamins B(9) and B(12). Lactate transport into erythrocytes was studied before and after the rHuEPO treatment at different lactate concentrations (1.6, 8.1, 41, and 81.1 mM). After treatment, MCT-1 lactate uptake was increased at 1.6, 41 (P < 0.01), and 81.1 mM lactate concentration (P < 0.001) although lactate uptake via band 3 and nonionic diffusion were unchanged. MCT-1 maximal velocity increased in the rHuEPO group (P < 0.05), reaching higher values than in the placebo group (P < 0.05) after treatment. Our results show that rHuEPO injections increased MCT-1 lactate influx at low and high lactate concentrations. The increase in MCT-1 maximal velocity suggests that rHuEPO may stimulate MCT-1 synthesis during erythrocyte formation in bone marrow.     

The modulatory effect of membrane viscosity on structural and functional properties of the anion exchange protein of human erythrocytes

The sterol content of human erythrocyte membranes was modified by polyvinylpyrrolidone (PVP)-mediated enrichment or depletion of cholesterol (CHL) or incorporation of cholesteryl hemisuccinate (CHS). The effects of these modifications on osmotic fragility and anion exchange protein (AEP) disposition and function were evaluated. CHS enrichment was fast (1 hr, 37 degrees C) and led to a concentration-dependent crenation as well as a decrease in osmotic cell fragility, in parallel with increased membrane microviscosity. CHL caused similar but considerably less marked effects due to slower incorporation rates into membranes. CHS enrichment of cells induced susceptibility of AEP to trypsin, a protease which otherwise does not affect AEP in intact cells. Although transport rates of monosaccharides, nucleosides, and anions were markedly slowed down by CHS enrichment of cells in parallel with increased membrane viscosity, anion transport was the most affected. The temperature profile of anion transport in CHS-enriched cells revealed a 10-kcal/mol increase in the enthalpy of activation relative to normal cells. Anion transport measured in heteroexchange conditions (Cl in--pyruvate out) and (Cl in-sulfate out) was relatively more susceptible to CHS modification than when it was measured in homoexchange conditions (Cl in-Cl out). The results of these measurements indicate that CHS-mediated increase in membrane viscosity affects AEP translocation capacity and transmembrane disposition via changes in lipid compressibility. Specific effects of CHS on AEP function, however, could not be ruled out.     

The osmoreactive betaine carrier BetP from Corynebacterium glutamicum is a sensor for cytoplasmic K+

The isolated glycine betaine uptake carrier BetP from Corynebacterium glutamicum was reconstituted in Escherichia coli phospholipid liposomes and its response to osmotic stress studied. The transport activity of BetP, which was previously shown to comprise both osmosensory and osmoregulatory functions, was used to identify the nature of the physicochemical stimulus related to hyperosmotic stress. Putative factors modulating transport activity in response to osmotic stress were dissected. These include type, osmolality and concentration of solutes in the internal and/or external compartment (cationic, anionic, zwitterionic, neutral), as well as membrane strain as a response to increased osmolality. Osmoresponsive activation of BetP was independent of any external factor and of physical alterations of the membrane, but was triggered by a change in the internal K+ concentration. Activation did not depend on the type of anion present and was K+ (or Cs+ and Rb+) specific, as choline and NH(4)+ did not trigger BetP activity. The half-maximal activation of BetP in E.coli phospholipid liposomes was correlated to an internal concentration of 221 +/- 23 mM K+.     

Molecular characterization of the human erythrocyte anion transport protein in octyl glucoside

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was purified in the presence of the nonionic detergent octyl glucoside. A molecular characterization was carried out to investigate whether the native structure of the protein was retained in the presence of this detergent. Band 3 bound octyl glucoside below the critical micelle concentration (cmc) of the detergent, approaching saturation above the cmc. At 40 mM octyl glucoside, close to saturating concentrations, 0.64 g of octyl glucoside is bound per gram of band 3 protein, corresponding to 208 molecules of detergent bound per monomer of band 3. Sedimentation velocity and gel filtration studies, performed at 40 mM octyl glucoside, indicated that the band 3-octyl glucoside complex had an average molecular weight of 1.98 X 10(6), which corresponds to a dodecamer. Sedimentation equilibrium experiments confirmed that band 3 in octyl glucoside exists in a heterogeneous and high oligomeric state. This high oligomeric state did not change dramatically over octyl glucoside concentrations ranging from 6 to 60 mM. The circular dichroism spectrum of band 3 changed only slightly over this range of octyl glucoside concentrations. The alpha-helical and beta-sheet contents of band 3 in 2 mM octyl glucoside were calculated to be 40% and 27%, respectively, indicating that no gross alteration in the secondary structure of the protein had occurred in octyl glucoside. The ability of band 3 to bind 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), a potent inhibitor (Ki = 1 microM) of anion transport, was measured to assess the integrity of the inhibitor binding site of the protein in octyl glucoside.(ABSTRACT TRUNCATED AT 250 WORDS)