Iron and copper in plasma membranes

Nonheme iron has been found in pig erythrocyte and mouse liver plasma membranes. The amount found, 8.2 nmol/mg protein in erythrocyte membranes and 7.4 nmol/mg protein in liver plasma membrane, is slightly lower than values reported for endoplasmic reticulum and Golgi apparatus. Less than one-third of the erythrocyte membrane iron can be released by acid treatment, which indicates that most of it is not in the typical iron-sulfur structure. Copper has been found in pig erythrocyte plasma membrane at a concentration of 0.45 nmol/mg protein. These metals may be associated with the redox enzymes of plasma membranes.     

Lysosomal breakdown of erythrocytes in the sheep placenta

Summary The breakdown of erythrocytes within the lysosomal apparatus of trophoblastic epithelial cells of the sheep placenta was studied at the ultrastructural level. Acid phosphatase activity could be demonstrated in the interspace between the erythrocyte membrane and the lysosomal membrane, but not inside ingested erythrocytes. The erythrocyte plasma membrane remained observable until the final stage of the breakdown process. Together with a peripheral layer of indigestible hemoglobin it might form a barrier for further penetration of lysosomal enzymes into the ingested erythrocyte. The hemoglobin of the erythrocyte is suggested to diffuse through the erythrocyte plasma membrane into the interspace between this membrane and the lysosomal membrane. Subsequently, the hemoglobin is digested in the interspace or in fragments pinched off from erythrocyte-containing lysosomes (=erythrolysosomes). The fragmentation of erythrolysosomes is considered to be the most efficient mechanism for the breakdown of red blood cells in the trophoblastic epithelium of the sheep placenta. The method of entry of hydrolytic enzymes into erythrocyte-containing phagosomes is discussed.     

The effect of carnosine on the activity of Na,K,ATPase: prospective uses in clinical cardiology]

The effects of carnosine on erythrocyte membrane Na,K-ATPase and isolated enzyme in vitro as well as on membrane Na,K-ATPase activity and lipid peroxidation (LPO) in chronic heart failure (CHF) and acute myocardial infarction (AMI) have been studied. CHF and AMI have been shown to be associated with significant inhibition of the erythrocyte membrane Na,K-ATPase activity and LPO activation. Marked activation of erythrocyte membrane Na,K-ATPase by carnosine in comparison with the isolated enzyme has been established. The ability of carnosine to induce Na,K-ATPase activation and prevent membrane depolarization indicates that the dipeptide may be a useful tool in the pathogenetic therapy of CFH and AMI.     

Endogenous proteolysis of the human erythrocyte membrane as studied by two-dimensional gel electrophoresis and electron spin resonance

1. Endogenous proteolysis in human erythrocyte membranes was studied in human erythrocyte membranes incubated at 37 degrees C by monitoring changes in 2-D electrophoretic pattern of membrane polypeptides and in the spectra of maleimide-spin labeled membranes. 2. A strong effect of exogenous proteases derived from contaminating other blood elements was found, resulting in formation of specific spots on 2-D electropherograms, requiring very careful leukocyte removal in investigations of red cell membrane protein composition and proteolysis. 3. Studies of the effects of protease inhibitors and Ca2+ confirmed a complex pattern of endogenous red cell membrane proteolysis ("self-digestion") involving many substrates and enzymes. 4. A promoting effect of high concentrations (150 mM) of Ca2+ on endogenous red cell membrane proteolysis was found.     

Hypothetical structure of the glycolytic enzyme complex (glycolytic metabolon) formed on erythrocyte membranes

A hypothetical structure of the glycolytic enzyme complex (glycolytic metabolon) adsorbed on the inner surface of the erythrocyte membrane has been proposed. Oligomers of integral membrane protein, band 3 protein (anion-transport system), are the anchor site for the complex. The complex is supposed to have a three-fold symmetry axis, perpendicular to the membrane plane, and contains a triple set of the glycolytic enzymes. The complex is in equilibrium with free enzymes; the equilibrium state depends on the physiological state of the erythrocyte.     

高原低氧习服大鼠红细胞变形性的变化规律及其分子机制

目的：探讨高原低氧习服大鼠红细胞变形性的变化规律及其分子机制。方法：将健康雄性大鼠随机分为3组（n=10）：常氧对照组、急性低氧组和低氧习服组。模拟高原低氧环境对大鼠分别进行急性低氧和间断低氧习服,麻醉后心脏采血,分别测定大鼠红细胞变形性、膜流动性、膜胆固醇和总磷脂含量、膜磷脂成分的含量、红细胞ATP酶活性、红细胞内Na＋和Ca2＋浓度及建立红细胞膜蛋白质双向电泳图谱,寻找差异蛋白质点,对其进行质谱鉴定。结果：①急性低氧大鼠红细胞变形性、膜流动性、膜胆固醇和总磷脂含量、红细胞ATP酶活性均降低;红细胞内Na＋和Ca2＋浓度均增高;红细胞膜磷脂酰丝氨酸（PS）、鞘磷脂（SM）含量增加,磷脂酰胆碱（PC）含量降低;建立了红细胞膜蛋白质双向电泳图谱,选取7个差异蛋白质点,其中4个在急性低氧后表达降低。②低氧习服大鼠红细胞变形性、膜流动性、膜胆固醇和总磷脂含量、红细胞ATP酶活性明显均增高;红细胞内Na＋和Ca2＋浓度均降低;红细胞膜PS、SM含量降低,PC含量增加;上述7个差异蛋白质点中4个在低氧习服后表达增高,3个表达降低,质谱技术鉴定结果为补体结合蛋白、水通道蛋白、膜攻击复合物抑制因子、葡萄糖运载体、脂质移行酶、氨基磷脂转移酶、依赖ATP的翻转酶,其中后三个酶与红细胞膜磷脂翻转有关。结论：急性低氧引起红细胞变形性、膜流动性、膜蛋白质表达、红细胞ATP酶活性及胞内Na＋和Ca2＋浓度方面相应的改变;经低氧习服后,上述指标有所改善,低氧习服对急性低氧引起红细胞的影响具有一定的保护作用;红细胞膜上的3种蛋白质,包括脂质移行酶、氨基磷脂转移酶和依赖ATP的翻转酶在低氧习服改善红细胞变形性的机制中可能发挥重要的作用。     

Protein kinases of rabbit and human erythrocyte membranes. Solubilization and characterization.

Two protein kinases (EC 2.7.1.37) from rabbit and one from human erythrocyte membranes have been solubilized with 0.5 M NaCl. These enzymes have been partially purified by (NH4)2SO4 fractionation and gel filtration. The rabbit membrane enzymes have apparent Mr values of 100 000 and 30 000, as determined in the presence of 0.4 M NaCl. In the absence of salt, these enzymes aggregate into high molecular weight species. The kinase from human erythrocyte membranes has an apparent Mr of 30 000 and appears to have properties similar to those of the 30 000-dalton rabbit kinase. All three enzymes catalyze the phosphorylation of casein and phosvitin in salt-stimulated reactions. None of these enzymes appears to be related to cyclic AMP-dependent protein kinases.     

Interactions of glycolytic enzymes with erythrocyte membranes

Partition equilibrium experiments have been used to characterize the interactions of erythrocyte ghosts with four glycolytic enzymes, namely aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase and lactate dehydrogenase, in 5 mM sodium phosphate buffer (pH 7.4). For each of these tetrameric enzymes a single intrinsic association constant sufficed to describe its interaction with erythrocyte matrix sites, the membrane capacity for the first three enzymes coinciding with the band 3 protein content. For lactate dehydrogenase the erythrocyte membrane capacity was twice as great. The membrane interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase were mutually inhibitory, as were those involving either of these enzymes and lactate dehydrogenase. Although the binding of phosphofructokinase to erythrocyte membranes was inhibited by aldolase, there was a transient concentration range of aldolase for which its interaction with matrix sites was enhanced by the presence of phosphofructokinase. In the presence of a moderate concentration of bovine serum albumin (15 mg/ml) the binding of aldolase to erythrocyte ghosts was enhanced in accordance with the prediction of thermodynamic nonideality based on excluded volume. At higher concentrations of albumin, however, the measured association constant decreased due to very weak binding of the space-filling protein to either the enzyme or the erythrocyte membrane. The implications of these findings are discussed in relation to the likely subcellular distribution of glycolytic enzymes in the red blood cell.     

Capsaicin-induced activation of erythrocyte membrane sodium/potassium and calcium adenosine triphosphatases

Capsaicin is the pungent ingredient present in hot peppers of the genus Capsicum. Capsaicin's effect on sensory neurons has been well studied; however, its effect on non-neuronal cells is still not fully understood. This study was undertaken to evaluate the effect of capsaicin on erythrocyte membrane enzymes: Na+/K(+)-ATPase and Ca(2+)-ATPase. Treatment with capsaicin (0.01-100 microM) caused a transient increase in the activities of both enzymes; the effect declined at lower concentrations of capsaicin, and no significant effect was observed at 0.01 microM capsaicin. The effect of capsaicin was fast with a significant (p<0.01) activation of enzyme activity observed within minutes of incubation. The findings on the effect of capsaicin on human erythrocyte membrane enzymes Na+/K(+)-ATPase and Ca(2+)-ATPase signify the importance of the non-neuronal effects of capsaicin, and the need for evaluating the physiological impact of high capsaicin (capsicum) consumption in some regions of the world.     

Relationship between membrane ATPase and shape changes in the dog erythrocyte

The effects of heating blood to 57 degrees C on intraerythrocytic calcium, membrane ATPase activity and cell shape have been studied in canine blood. Intraerythrocytic calcium was determined by use of arsenazo III, membrane ATPase activity was determined by inorganic phosphorous formation and erythrocyte shape was determined by scanning electron microscopy. The results of this study showed that this degree of thermal trauma would cause a 27% increase in intraerythrocytic calcium and a 38% decrease in ATPase activity. During these changes in calcium and ATPase activity the erythrocyte changed form from biconcave to spherical. Addition of catalase (3,200 U/ml) to the blood prior to heating prevented the changes observed in intraerythrocytic calcium, membrane ATPase activity and shape. The addition of the free-radical generating combination of hypoxanthine-xanthine oxidase to blood produced a 20% decrease in membrane ATPase activity and a change in erythrocyte shape, but did not alter intraerythrocytic calcium. These results suggest that free-radicals are responsible for the changes in membrane ATPase activity. The observation that shape change occurs when ATPase activity has been decreased, but calcium has not been increased, suggests that membrane ATPase activity levels are more important in producing changes in erythrocyte shape than are intraerythrocytic calcium levels.     

The effect of hypoxic hypoxia on the activity of glycolysis enzymes in rat erythrocytes

The oxygen-binding properties of hemoglobin, concentration of 2,3-diphosphoglycerate, activity of carbohydrate metabolism enzymes and kinetics of rat erythrocyte hemolysis have been studied at high altitudes. The hemoglobin affinity to oxygen, glycolysis enzyme activity and erythrocyte membrane resistance are established to increase at the initial period of adaptation. The activation of the pentose-phosphate pathway of the glucose, transformation and inhibition of the glycolytic process in these cells are observed on the 10th day.     

Human platelet glucose-6-phosphate dehydrogenase. Total purification, kinetic studies and relationship with enzyme from other blood cells.

Human platelet G-6-PD has been highly purified, to homogeneity, and its kinetic, electrophoretic and immunological characteristics have been studied. Platelet G-6-PD differs from erythrocyte or leukocyte enzymes by an increased Michaelis constant for G-6-P and a slow activity at the acid pHs. By electrofocusing only a main active band (band a) of platelet G-6-PD was found. The incubation at 37 degrees C in the presence of NADP+ and dithiothreitol normalize Km-G-6-P of platelet G-6-PD; the incubation with boiled and ultrafiltered leukemic granulocyte extracts led to an anodisation of G-6-PD active forms, a decrease of the molecular specific activity and a further increase of Km-G-6-P; these last modifications are the same as those undergone by G-6-PD incubated in crude extracts of normal or leukemic granulocytes.     

Isolation of two myosin light-chain kinases from bovine carotid artery and their regulation by phosphorylation mediated by cyclic AMP-dependent protein kinase

Glyceraldehyde phosphate dehydrogenase is one of four glycolytic enzymes in the human erythrocyte that together can catalyse exchange of isotope between the C-2 position of lactate and solvent. Detailed measurements of the exchange can be made by using a non-invasive spin-echo p.m.r. method that has been described previously [Brindle, Brown, Campbell, Foxall & Simpson (1982) Biochem. J. 202, 589-602]. By studying the dependence of the exchange on the activity of an individual enzyme, the specific isotope-exchange equilibrium velocity of the enzyme can be measured. Suggestions that glyceraldehyde phosphate dehydrogenase is bound to the membrane in the intact cell, and that it may, under certain conditions, be rate-limiting for glycolytic flux, were examined in the present study by comparing the exchange properties expressed by the enzyme in situ and in vitro. It is concluded that glyceraldehyde phosphate dehydrogenase is not rate-limiting for glycolytic flux and that it is unlikely that a significant fraction of the enzyme is bound to the erythrocyte membrane in situ.     

Effects of domain-specific erythrocyte membrane modulators on acetylcholinesterase and NADH:cytochrome b5 reductase activities

Previously, we showed using electron paramagnetic resonance that the physical state of one side of erythrocyte membranes could be modulated by agents which interact with the opposite side (reviewed in Butterfield, 1989, Biological and Synthetic Membranes, A. R. Liss, Inc., New York). The present study was undertaken to determine whether membrane-bound enzymes would exhibit a similar transmembrane modulation effect. The effects of known, domain-specific modulators of the physical state of erythrocyte membranes on the activity of two membrane-bound enzymes were investigated. Acetylcholinesterase, an enzyme having its active site situated on the extracellular side of the membrane, seemed to be unaffected by most of the modulators employed in this study, with the exception of reversible inhibition by benzyl alcohol. Conversely, the activity of NADH:cytochrome b5 reductase, an enzyme whose active site is located on the cytoplasmic side of the erythrocyte membrane, was increased by those agents that interact primarily with skeletal proteins to increase skeletal protein-protein interactions; however, those agents which interact primarily with the skeleton to decrease protein-protein interactions decreased the activity of NADH:cytochrome b5 reductase. This enzyme's activity was also significantly altered by lectins which bind specifically to the external face of glycophorin A on the opposite side of the membrane, but it's activity was unaffected by concanavalin A, a lectin which binds to the external face of band 3. The results of these biochemical studies suggested that NADH:cytochrome b5 reductase can interact with and its activity can be modulated by skeletal or transmembrane proteins. In addition, these results support the hypothesis that in transmembrane signaling processes, biophysical and biochemical changes are correlated.     

A subset of hY RNAs is associated with erythrocyte Ro ribonucleoproteins.

The Ro autoantigen is a mammalian cellular ribonucleoprotein (RNP) of unknown function. We have demonstrated that hY1 and hY4 Ro RNAs are associated with erythrocyte Ro RNPs and represent a subset of the four hY RNAs found in HeLa cell and leukocyte Ro RNPs. We have cloned and sequenced hY4 RNA, the only hY RNA not sequenced previously, from a polymerase chain reaction amplified erythrocyte hY cDNA library. Sequencing of the erythrocyte hY RNAs in conjunction with Northern blot analysis confirms that the erythrocyte hY RNAs contain the same sequences as the respective HeLa cell RNAs of similar mobility. Ribonuclease inhibition activity has been found in erythrocytes and this activity inhibits the degradation of hY3 and hY5 in leukocyte lysates thereby favoring the possibility that the presence of hY1 and hY4 in erythrocytes is the result of differential expression of the hY RNAs in erythrocyte precursors.     

Proteolytic activity associated with human erythrocyte membranes. Self-digestion of isolated human erythrocyte membranes.

At least two kinds of enzymes are active in the proteolytic self-digestion of erythrocyte membranes. The specific activities of these enzymes do not decrease with repeated washings of purified stroma. The effects of a variety of inhibitors on the membrane preparation's capacity to digest 125-I-labelled casein, covalently linked to latex beads, have been examined. Pepstatin-inhibitable enzyme, active at low pH, digests the membrane extensively to small polypeptide fragments. Spectrin, located at the internal part of the membrane, is readily degraded. Diisopropylfluorophosphate-inhibitable enzyme, active at pH 8-9, has only limited digestive capacity. Some of the membrane components, such as the small molecular weight glycoproteins, are resistant to digestion. The restricted capacity of digestion is due to the membrane molecular arrangement; increased disaggregation removes the restriction and increases the activity. Spectrin is not digested unless the membrane topography is disrupted by NP-40 neutral detergent. These observations suggest that the enzymes active at basic pH are located external to the cell. Intact cells do possess a limited capacity to degrade 125-I-labelled casein when their surfaces are brought into contact with substrate-coated beads.     

Purines and pyrimidines in malarial parasites

In order for the plasmodium malarial parasite to replicate in the human erythrocyte it requires metabolic pathways which are not operative in the host erythrocyte. Thus, the malarial parasite not only synthesizes enzymes for purine salvage and interconversion, for the pyrimidine biosynthetic pathway de novo, and for the folate cycle, but it also alters the host erythrocyte membrane in respect to the transport of purines. Several of the plasmodium enzymes from these pathways have been cloned and these appear to be highly homologous to the corresponding human enzymes. However, enzymes which have been purified from Plasmodium, have demonstrated physicochemical and kinetic differences and may be potential targets for chemotherapy. Inhibition of individual enzymes, such as the dihydroorotate dehydrogenase (DHO-DHase), and inhibition of the inserted pathway from IMP to AMP and IMP to GMP hold considerable promise as chemotherapeutic targets. An entirely new approach in inhibiting malarial growth involves the altered nucleoside transporter in the infected cell membrane through which cytotoxic compounds may be selectively targeted into only the infected cell.     

Antioxidative activity of some quaternary ammonium salts incorporated into erythrocyte membranes

The antioxidative activity of two series of amphiphilic compounds from a group of quaternary ammonium salts has been investigated. They were so-called bifunctional surfactants synthesized to be used as common pesticides or as antioxidants. The latter application was to be ensured by providing the compounds studied with an antioxidant group. Studies on antioxidative possibilities of those compounds were performed on pig erythrocytes. Due to their hydrophobic parts, they anchor in the erythrocyte membrane and influence the degree of lipid oxidation in the erythrocyte membrane subjected to UV radiation. It was found that compounds of both series decreased the oxidation of the membrane lipids. The inhibition of this oxidation increased with the length of their hydrophobic chains up to fourteen carbon atoms. The compounds of the longest hydrophobic chains showed a somewhat weaker antioxidative activity. Of the two series studied compounds were more effective having bromide ions as counterions. The corresponding compounds of a second series (chlorides) protected erythrocyte significantly weaker against oxidation. The effect of the compounds on fluidity of the erythrocyte membrane has been studied in order to explain the oxidation results. Change in fluidity of the erythrocyte ghost membranes was found also dependent on length of the hydrophobic part of the compounds and was more pronounced in the case of bromide surfactants. The final conclusion is that the compounds studied can be succesfully used as antioxidant agents of good efficacy.     

The major fibrinolytic proteases of human leukocytes

The major fibrinolytic enzymes present in leukocyte granules and active at physiological pH have been identified. The fibrinolytic activity in extracts of leukocyte granules was bound to fibrinogen-Sepharose and eluted with 8.0 M urea. Two distinct zones of fibrinolytic activity were detected upon electrophoresis of leukocyte extracts on fibrinogen polyacrylamide gels, and both were qualitatively recovered in the 8.0 M urea eluate. Quantitatively, greater than 95% of the fibrinolytic activity was recovered in the urea eluate. Two major leukocyte proteases, elastase (EC 3.4.21.11) and cathepsin G (EC 3.4.21.-), were quantitatively recovered in the urea eluate. Both enzymes, when purified separately by affinity chromatography, were shown to: (a) possess fibrinolytic activity; (b) coincide in mobility and generate the two zones of fibrinolytic activity on fibrinogen polyacrylamide gels; and (c) quantitatively reconstitute the fibrinolytic activity of the leukocyte granules when combined at activity levels present in granular extracts. A highly significant correlation (r = 0.98) was found between the fibrinolytic activity and the sum of elastase and cathepsin G activity in leukocytes from five donors. Thus, elastase and cathepsin G are the major enzymes of the leukocyte fibrinolytic pathway, and fibrinogen-Sepharose chromatography may be used to obtain these enzymes.     

Cholesterol inhibits the lytic activity of melittin in erythrocytes

Although cell lysis by the hemolytic peptide, melittin, has been extensively studied, the role of specific lipids of the erythrocyte membrane on melittin-induced hemolysis remains unexplored. In this report, we have explored the modulatory role of cholesterol on the hemolytic activity of melittin by specifically depleting cholesterol from rat erythrocytes using methyl-beta-cyclodextrin (MbetaCD). Our results show that the hemolytic activity of melittin is increased by approximately 3-fold upon depletion of erythrocyte membrane cholesterol by approximately 55% without any appreciable loss of phospholipids. This result constitutes the first report demonstrating that the presence of cholesterol inhibits the lytic activity of melittin in its natural target membrane, i.e., the erythrocyte membrane. These results are relevant in understanding the role of cholesterol in the mechanism of action of melittin in the erythrocyte membrane.