用马来酰亚胺自旋标记研究库存血红细胞膜蛋白质构象

用两种马来酰亚胺自旋标记物—马来酰亚胺Ⅰ和马来酰亚胺Ⅴ研究了红细胞膜蛋白质构象及巯基结合位点性质在ACD-B方库存血保存期间的动态变化。结果发现,在35天的血液保存期间,马来酰亚胺Ⅰ所标记红细胞膜的S/w值很快下降到一低水平,而马来酰亚胺Ⅴ所标记红细胞膜的旋转相关时间则呈现迅速下降后缓慢升高的双相性变化。作者结合膜蛋白构象及其周围微观环境进行了讨论。     

金属硫蛋白与红细胞的相互作用

根据金属硫蛋白(MT)对标记在膜上的马来酰亚胺自旋标记物的ESR波谱的影响,研究了MT与红细胞膜的相互作用,发现不同种属的MT对膜构象的影响不同.体外实验表明,MT可以吸附在红细胞的表面,用CdCl₂诱导家兔,对血浆和红细胞溶血液中的MT组分进行色谱分离,发现血液中的MT主要存在于血细胞中.进而对血液MT的来源、分布及其重要的生物学意义进行了讨论.     

锌_7-与镉_7-金属硫蛋白对羟自由基损伤大鼠红细胞膜保护作用的比较

用电子自旋共振自旋标记物氮氧自由基硬脂酸和马来酰亚胺标记大鼠红细胞膜脂和膜蛋白,测定膜脂流动性和膜蛋白构象改变,以硫代巴比妥酸法测定脂质过氧化产物丙二醛含量．结果表明,锌7-与镉7-金属硫蛋白对羟自由基引起的膜脂流动性减低、脂质过氧化反应增强双膜蛋白构象改变有明显抑制作用,而且,前者的作用明显强于后者．     

肾性贫血红细胞膜蛋白巯基结合位置性质的ESR研究

用四种不同链长的马来酰亚胺氮氧自由基标记物研究了慢性肾衰竭(CRF)贫血病人红细胞膜蛋白巯基结合位置的性质。从所得的ESR波谱计算了弱、强固定化成分之比(W/S)。结果表明,由M(Ⅰ)、M(Ⅱ)和M(Ⅲ)标记病人红细胞膜所得的W/S值都比标记正常人红细胞膜得到的W/S位高(P<0.05),这说明慢性肾衰竭贫血病人红细胞膜蛋白的巯基结合位置的构象发生了变化;用M(Ⅳ)标记正常人红细胞和病人红细胞所得到的ESR波谱都只含有弱固定化波谱,没有强固定化波谱。得出的这个结果将为探讨CRF贫血的发病机理提供理论证据。     

锌7—与镉7—金属硫蛋白对羟自由基损伤大鼠红细胞膜保护作用的比较

用电子自旋共振自旋标记物氮氧自由基硬脂酸和马来酰亚胺标记大鼠红细胞膜脂和膜蛋白,测定膜脂流动性和膜蛋白构象改变,以硫代巴比妥酸法测定脂质过氧化产物丙二醛含量。结果表明,锌７－与镉７－金属硫蛋白对羟自由基引起的膜脂流劝性减低,脂质过氧化反应增强及膜蛋白构象及改变有明显的抑制作用,而且,前的作用明显强于后。     

用顺磁共振及自旋标记研究活性氧对铜锌超氧化物歧化酶结构的影响

用抗坏血酸－Ｆｅ（Ⅲ）或过氧化氢为活性氧体系分别作用于牛红细胞铜锌超氧化物歧化酶（ＣｕＺｎ－ＳＯＤ）,发现酶活性降低的同时,其活性中心中Ｃｕ（Ⅱ）被还原成Ｃｕ（Ⅰ）。进一步用抗坏血酸－Ｆｅ（Ⅲ）或过氧化氢作用于马来酰亚胺标记ＣｕＺｎ－ＳＯＤ,用ＥＳＲ测定的结果表明酶分子亚基的缔合受到影响。结果提示酶的失活与活性中心中Ｃｕ（Ⅱ）的还原及酶构象的变化有关。     

用激光衍射法研究红细胞在低粘切变流场中的取向和变形及其生物物理意义

采用一种新型激光衍射法 ,对不同浓度WGA处理的红细胞进行测量 ,进一步研究了红细胞在低粘切变流场中的流变特性 .通过用DPH探剂标记的荧光偏振法 ,用马来酰亚胺 (MSL)标记的电子顺磁共振法 ,揭示了新激光衍射法所描写的红细胞在低粘切变流场中取向指数 (DI_or)与等容小变形指数 (DI_d)的生物物理意义     

细胞内、外源活性氧致人多形核白细胞膜理化特性改变的研究

DPH和N-(3芘)马来酰亚胺标记光敏氧化反应及黄嘌呤/黄嘌呤氧化酶反应生成外源性单线态氧(O_2)和超氧阴离子自由基(O_2)作用人多形核白细胞膜脂及膜蛋白质、荧光激发发射光谱形状、峰位未发生改变,荧光强度减小,其中以N-(3芘)马来酰亚胺标记O_2作用的膜蛋白质更为明显.荧光偏振度增大,相应清除剂L-组氨酸、超氧化物歧化酶和过氧化氢酶有抑制效应.调理的酵母多糖刺激中性粒细胞呼吸爆发产生膜、胞内活性氧损伤膜脂、膜蛋白质,测定荧光参数变化与前者不尽相同,DPH荧先强度显著增加,L-组氨酸,超氧化物歧化酶和过氧化氢酶似无抑制效果.     

用电子自旋标记法研究γ辐照对红细胞膜的影响

本文用5-氮氧自由基硬脂酸、12-氮??氧自由基硬脂酸和16-氮氧自由基硬脂酸三种脂肪酸自旋标记物对人红细胞膜(血影)的不同深度膜脂的流动性作了进一步的研究,所得到的电子自旋共振波谱表明它们的序参数都随γ照射剂量的增大而增大。本文还用马来酰亚胺氮氧自由基标记在膜蛋白的SH基团上,所得到的波谱表明其旋转相关时间和强固定化对弱固定化组分的谱线高度的比值也都随γ辐照剂量增大而增大。结果说明γ辐照后,人红细胞膜的流动性降低。这与我们用荧光探针研究所得到的结果相似。     

大脑皮层神经元膜蛋白构象改变的ESR研究

采用马来酰亚胺标记完整的大脑皮层细胞,观察由低氧引起的ESR谱线的变化及脑细胞脂质过氧化程度,低氧引起细胞过氧化物生成增加,膜蛋白构象改变．金属硫蛋白(10^-5mol/L)能明显抑制过氧化反应,具有一定的抗氧化作用．并对实验的分子机理进行了简要的讨论．     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

Abstract

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (^*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H₂O₂; ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

Erythrocyte membranes - compression of lipid phases by increased cholesterol content

Lipid and protein interactions were studied in guinea pig erythrocytes containing a normal or a two-fold increased amount of cholesterol. The electron spin resonance (ESR) spectra of cholesterol-loaded cells labeled with fatty-acid probes showed an increase in the local viscosity of the membrane as compared with control cells. This increase reflects changes in the interior of the lipid matrix of the membrane because the probes resisted destruction by ascorbate, were unaffected by the action of pronase, and gave spectra similar to those of liposomes. No differences were observed between control and cholesterol-loaded cells in the conformation of the membrane proteins by either the infrared spectra or the ESR spectra of cells labeled with maleimide probes.     

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

山莨菪碱对人红细胞膜蛋白及膜脂运动的影响

本文采用自旋标记顺磁共振波谱技术,研究了山茛菪碱对人红细胞膜蛋白和膜脂运动的影响.结果表明:用马来酰亚胺标记的人红细胞膜,加入山茛菪碱后,其顺磁共振波谱中强、弱固定化作用谱的峰值比增大,膜蛋白的运动受到限制.山茛菪碱对红细胞膜脂的作用部位主要在极性头部,并影响膜脂的流动性.本文还对山茛菪碱与红细胞膜作用的可能机制进行了讨论.     

A spin-label study of membrane proteins and internal microviscosity of erythrocytes in hereditary spherocytosis

Electron spin resonance (ESR) spectra of erythrocyte membranes of patients with hereditary spherocytosis (HS) and of healthy controls labeled with a maleimide spin label did not differ significantly both before and after prolonged incubation at 37 degrees C. It suggests that the different behavior of spin-labeled HS erythrocyte membranes upon incubation at a higher temperature reported previously is due indeed to structural abnormalities of HS red cell membranes and not to alterations in their proteolytic activity. Measurements of the rotational correlation time of Tempamine spin probe demonstrated a significant elevation of internal microviscosity of erythrocytes in HS, more pronounced in non-splenectomized patients.     

Effect of proteolysis on the electron spin resonance spectra of maleimide spin labeled erythrocyte membrane

The ratio of low-field amplitudes of weakly and strongly immobilized signals of ESR spectra of a maleimide spin label bound to erythrocyte membranes (hw/hs) increases progressively during incubation at 37 degrees C. This increase is due to the 'self-digestion' of membrane proteins by endogenous proteinases and is attenuated by proteinase inhibitors. Digestion of membranes with chymotrypsin also increases the hw/hs ratio. These results suggest a need for a careful interpretation of data from spin-labeled membrane proteins, especially in experiments involving prolonged incubations of membrane preparations when the proteolytic effects may be significant.     

ESR studies of the erythrocyte membrane skeletal protein network: influence of the state of aggregation of spectrin on the physical state of membrane proteins, bilayer lipids, and cell surface carbohydrates

The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell-surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bisphosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P less than 0.002 and P less than 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding site is observed (P less than 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P less than 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network.