Denaturation of hemoglobin--the first stage of erythrocyte thermohemolysis

Measurements were carried out of the rate constants of erythrocyte thermohemolysis and hemoglobin thermoaggregation in various animal species and also for the man under the action of hemoglobin-modifying factors (CO2, O2, ethanol). It is concluded from the obtained correlation dependences that hemoglobin thermodenaturation initiates erythrocyte thermohemolysis. The identity of the early stages of erythrocyte thermohemolysis and aging is suggested.     

Possible role of spectrin in thermohemolysis of erythrocytes

The activation energy of thermodenaturation of erythrocyte spectrin was defined by two different methods as Esp = 218 +/- 4 kcal/mole. This value was more than two times greater than that of thermohemolysis activation energy (Eth = 99 +/- 5 kcal/mole). The conclusion is made that spectrin does not play a significant role in thermohemolysis of erythrocytes.     

Thermohemolysis of erythrocytes in the temperature range including physiologic (autohemolysis)]

The activation energy of thermohemolysis of erythrocytes changes from 36 +/- 5 kcal/mol (35-45 degrees C) to 97 +/- 5 kcal/mol (45-55 degrees C) at the temperature about 45 degrees C in isotonic buffer. The break on Arhenius' plot is preserved also when erythrocytes are placed into plasma. The character of Arhenius' plot is the same when erythrocyte hemoglobin is totally oxidated into methemoglobin by chemical way, though thermal stability of such erythrocytes is decreased. The scheme is presented in which thermohemolysis of erythrocytes occurs by two independent ways: thermodenaturation of hemoglobin (limiting stage of the process when t greater than 45 degrees C) and modification of membrane proteins by hemin, the last being a product of hemoglobin oxidation (limiting stage of the process when t less than 45 degrees C).     

Thermohemolysis of erythrocytes

Thermohemolysis kinetic curves of erythrocytes in the temperature interval of 37-75 degrees C in different media and in the course of blood storage were studied. Mechanism of thermohemolysis is proposed. The rate constants of hemolysis stages are introduced. It is shown that these parameters are sensitive to the changes of structural state of erythrocytes.     

Incorporation of sphingomyelin increases thermostability of human erythrocyte membrane and resistance of erythrocytes against thermal hemolysis

Erythrocytes of various mammal species differ markedly in their resistance against thermally induced hemolysis that was assumed related to the different sphingomyelin content of their membranes (J. Therm. Biol. 18 (1993) 177). In this work, two choline-containing lipids, sphingomyelin and phosphatidylcholine, were incorporated into the membrane of human erythrocytes and the resulting effect on thermal resistance and membrane thermostability of these cells was studied measuring thermohemolysis upon exposure to constant temperature and electrolyte leakage during transient heating, respectively. While sphingomyelin increased thermal resistance by 56% and increased the inducing temperature T_m of electrolyte leakage by 1±0.2°C, phosphatidylcholine produced practically no effect. The results show that sphingomyelin alone stabilized the structure of plasma membrane providing thermal stability to membrane proteins and in turn to whole cells as well.     

Mechanisms of cytoskeletal regulation: functional and antigenic diversity in human erythrocyte and brain beta spectrin

A study of human erythrocyte and brain spectrin with particular emphasis on the beta subunits revealed a structural homology but functional dissimilarity between these two molecules. Six monoclonal antibodies raised to human erythrocyte beta spectrin identify three of the four proteolytically defined domains of erythrocyte beta spectrin. Five of these monoclonal antibodies cross-react with human brain spectrin. None of a previously identified set of alpha erythrocyte spectrin monoclonal antibodies [Yurchenco et al: J Biol Chem 257:9102, 1982] reacted with brain spectrin. A domain map generated by limited tryptic digestion shows that brain spectrin is composed of proteolytically resistant domains analogous to erythrocyte spectrin, but the brain protein is more basic. The binding of brain spectrin to erythrocyte ankyrin, both in solution and on erythrocyte IOVs, yielded an association constant approximately 100 time weaker than for erythrocyte spectrin. The binding of azido-calmodulin under native conditions was specific for the erythrocyte beta subunit but was not calcium dependent. In contrast, azido-calmodulin bound only to the alpha subunit of brain spectrin in a calcium-dependent manner. The similarity of structure but modified functional characteristics of the brain and erythrocyte beta spectrins suggest that these proteins serve different cellular roles.     

The formation of vesicles retaining sodium-dependent transport systems for amino acids from protein-depleted membranes of pigeon erythrocytes

The process of the formation of vesicles from pigeon erythrocyte membranes was studied. Mildly alkaline solutions of low ionic strength, which reduce human erythrocyte membranes to small vesicles depleted of spectrin and other proteins, have no such effect on pigeon erythrocyte ghosts. A distinct phase of removal of membrane proteins, including spectrin, began to occur only when pigeon erythrocyte membranes were exposed to 0.2 mM EDTA adjusted to pH values above 10.2. Vesicles which demonstrated Na⁺-dependent amino acid transport were generated between the pH values 10.8 and 11.4. The results show that peripheral proteins, notably spectrin, maintain the integrity of the pigeon erythrocyte ghost. The interaction of these proteins with the membrane is rather different from that well studied in the human erythrocyte ghost and the possible significance of this for the pigeon erythrocyte is discussed.     

Characterization of biomimetic surfaces formed from cell membranes

A method for fabricating biomimetic surfaces from intact cell membranes is described. A monolayer of alkanethiol on gold is covered by a second layer derived from the components of erythrocyte membranes either by self-assembly or by Langmuir-Blodgett methods. The resulting asymmetric hybrid layer was characterized by ellipsometry, surface plasmon resonance (SPR), contact angle, capacitance, voltammetry, and electron and atomic force microscopy. The erythrocyte membrane layer was measured to be approximately 30-40 A in thickness. Using SPR, the presence of erythrocyte components on the surface was demonstrated by their selective removal by enzymatic action. The uniform deposition of membranous material on the substrate was shown by electron and atomic force microscopy. Demonstration of acetylcholinesterase (AChase) activity, a membrane-anchored enzyme, on the surface for at least 8 days, suggests that the outer leaflet of the erythrocyte membrane is present in its native form. Cyclic voltammetry demonstrates that enhanced electron transport from a solution redox species accompanies formation of the erythrocyte layer at the surface. This enhanced electron transport is blocked by 4,4'-diisothiocyanate stilbene-2,2'-disulfonic acid, a well known blocker of anion transport, suggesting that an erythrocyte anion transporter protein is incorporated into the surface layer in an active conformation.     

阵发性睡眠性血红蛋白尿症红细胞乙酰胆碱酯酶的研究

用化学方法测定了乙酰胆碱脂酶（ＡｃｈＥ）活性,阵发性睡眠性血红蛋白尿症（ＰＮＨ）红细胞远低于正常红细胞。为了进一步研究ＰＮＨＡｃｈＥ（—）的红细胞,采用Ｐｒｏｔｅｉｎ　Ａ　Ｓｅｐｈａｒｏｓｅ　６ＭＢ结合ＡｃｈＥ单抗亲和层析法分离出ＰＮＨＡｃｈＥ（—）的红细胞。用间接免疫荧光流式细胞术检测,ＰＮＨ细胞ＡｃｈＥ低于正常,而ＰＮＨＡｃｈＥ（—）红细胞未能检出ＡｃｈＥ。３Ｈ－肌醇标记实验证明,正常红细胞膜区带４．１处有较高的放射活性,而ＰＮＨ红细胞极低,ＰＮＨＡｃｈＥ（—）红细胞完全无放射活性。用ＡｃｈＥ抗体做免疫印渍实验证明了ＡｃｈＥ存在区带４．１部位。ＤＭＰＣ诱导正常和ＰＮＨ红细胞,检测二者囊泡化的程度,发现ＰＮＨ病人红细胞远比正常人红细胞易于囊泡化。     

Equine peptidases: Correspondence with human peptidases and polymorphism for erythrocyte peptidase a

Equine erythrocyte peptidases were compared to the six human erythrocyte peptidases, A, B, C, D, E, and F, regarding substrate specificity, relative activity, and electrophoretic mobility. Five equine erythrocyte peptidases appeared homologous to human peptidases A, B, D, E, and F. In contrast to human, equine peptidase C was absent in red cells, although it was weakly active in white cells. On the other hand, an equine peptidase, probably homologous to human peptidase S, was weakly active in red cells as well as present in white cells. Polymorphism for equine erythrocyte peptidase A is reported.     

Blood group A activities of glycoprotein and glycolipid from human erythrocyte membranes.

It is known that ABO blood group substances in human erythrocyte membranes are sphingoglycolipids, but recently several authors have reported that the glycoproteins of the erythrocyte membranes also have ABO blood group activities in addition to MN blood group activities and virus hemagglutination inhibitor activity. We solubilized blood group A erythrocyte membranes with lithium diiodosalicylate and separated the glycoprotein fraction by phenol extraction and ethanol precipitation. This fraction was apparently not contaminated with glycolipid, but it showed weak blood group A activity. The activity of the glycoprotein of the erythrocyte membranes was one-sixth of that of the lgycolipid fraction from the same amount of membranes. The glycoprotein components were purified by Sephadex G-200 gel filtration in SDS. The main component isolated, PAS 1, still showed blood A activity.     

Rosette formation of concanavalin A-treated erythrocytes around polymorphonuclear leucocytes and lymphocytes.

Concanavalin A (Con A) induces rosette formation of erythrocytes around polymorphonuclear leucocytes and lymphocytes in cell suspensions of autologous human blood cells. The effect which is most characteristic in a concentration between 25 and 50 microgram/ml is due to Con A bound on the erythrocyte membrane. A similar effect, although less pronounced, was observed with phytohaemagglutinin at concentrations of 10 and 25 microgram/ml. The treated erythrocytes showed a higher affinity to polymorphonuclears when compared with lymphocytes. At the contact area, the membrane of the erythrocyte became highly folded while its free surface was smooth and spherical. The effect of the local concentration and immunobilization of the lectin on the erythrocyte membrane and the similarity of the contact pattern to that of erythrophagocytosis are discussed.     

Carbohydrate content of human erythrocyte membrane. Variations with ABO-blood group.

The study of the carbohydrates of human erythrocyte membranes has been mainly focused on their glycopeptidic and glycolipidic complexes. Modifications of these carbohydrates have been described in subjects with various pathological states. In order to characterize possible changes of the glycopeptides, or glycolipids obtained from erythrocyte membrane in various pathological situations, the determination of the carbohydrate content of the whole membrane appeared a necessary preliminary. This study concerns the determination of the normal values of the main carbohydrates of whole human erythrocyte membranes, with respect to their blood group. Erythrocyte membranes were prepared from donors of the four ABO blood groups. After acidic hydrolysis, the contents of fucose, mannose, galactose, glucose, glucosamine, galactosamine and N-acetylneuraminic acid in each blood group were determined and compared with one another. The galactosamine content of A, B and AB erythrocyte membranes is significantly higher than that of the O-erythrocyte. For galactose, the differences are significant for the following pairs: A/O; B/O; AB/O; A/B; A/AB. Significant differences in the mannose contents of O-erythrocytes and A, B and AB erythrocytes have also been found. This result suggests that a basic difference, in the core of the oligosaccharide chains, may exist between O and A, B, AB erythrocyte membranes.     

Similarities between the Mr 245,000 calmodulin-binding protein of the dogfish erythrocyte cytoskeleton and alpha-fodrin

The Mr 245,000 calmodulin-binding protein of the dogfish erythrocyte cytoskeleton (D245) has been compared with human erythrocyte spectrin and mammalian brain fodrin [J. Levine and M. Willard (1981) J. Cell Biol. 90, 631-643]. Mammalian erythrocyte alpha-spectrin, brain alpha-fodrin, and D245 are all localized in the cell surface-associated cytoskeleton, and have similar molecular weights. Like mammalian erythrocyte spectrin, D245 was extracted from erythrocyte ghosts under low-ionic-strength conditions. However, D245 failed to bind an antibody which reacted strongly with both subunits of human erythrocyte spectrin. Unlike mammalian erythrocyte alpha- and beta-spectrin, D245 bound calmodulin in the absence of urea both in a "gel-binding" assay and in situ using azidocalmodulin [D.C. Bartelt, R.K. Carlin, G.A. Scheele, and W.D. Cohen (1982) J. Cell Biol. 95, 278-284]. Striking similarities were noted between D245 and alpha-fodrin in that both exhibited (a) comparable calcium-dependent calmodulin binding properties, (b) strong reactivity with two different anti-fodrin antibody preparations, (c) similar reactivity with antibody to brain CBP-I, now believed to be fodrin, (d) proteolytic degradation yielding an Mr 150,000 calmodulin-binding fragment, and (e) lack of reactivity with an anti-spectrin antibody. A protein with calmodulin-binding and anti-fodrin-binding properties similar to D245 was detected in cytoskeletal preparations of chicken erythrocytes. Moderate and consistent cross-reactivity of anti-fodrin with human erythrocyte alpha-spectrin was also observed. The data indicate that D245 is functionally and immunologically more closely related to alpha-fodrin than to alpha-spectrin of the mammalian erythrocyte.     

Study of erythrocyte sedimentation behavior by piezoelectric crystal impedance sensor

Based on the impedance characteristic of erythrocytes at high frequency, the response of piezoelectric crystal impedance (PCI) sensor in the erythrocyte suspension was derived and verified experimentally. A method of using PCI sensor to investigate erythrocyte aggregation-sedimentation phenomenon was proposed. From the frequency response of the PCI sensor, the erythrocyte aggregation time and sedimentation rate could be obtained during erythrocyte aggregation and sedimentation. With the present method, the effects of the erythrocyte deformability, the osmotic pressure and the coexisting macromolecules on the erythrocyte sedimentation rate were studied. The results show that the PCI sensor possesses some advantages, such as good sensitivity, simplicity of use and no thermal effect for the impedance study of erythrocyte aggregation and sedimentation.     

Determination of free choline in plasma and erythrocyte samples and choline derived from membrane phosphatidylcholine by a chemiluminescence method

A sensitive chemiluminescence method for assay of choline which has been developed for analysis of erythrocyte and plasma levels of choline is reported here. This method includes a charcoal purification step which yields consistent results with plasma and erythrocyte extracts. Further, choline derived from membrane phosphatidylcholine may also be measured by an extension of this method following digestion with phospholipase D. This method has been used to study abnormal levels of erythrocyte choline that occur in cluster headache patients compared to control subjects and migraine patients. In addition, the time course of changes in plasma and erythrocyte choline following a fatty meal have been monitored. Plasma choline levels rise to a maximum between 1 and 3 h after the meal and this is followed by a rise in erythrocyte choline levels which are maximal 3 h after the meal.     

A monomeric form of human erythrocyte membrane acetylcholinesterase

Purified detergent solubilized dimeric human erythrocyte acetylcholinesterase (6.3 S form) was converted to a stable monomeric 3.9 S species when treated with 2-mercaptoethanol and iodoacetic acid. More than 60% of the enzymatic activity were recovered after this treatment. A decreased susceptibility to reduction and alkylation was observed with purified, detergent depleted acetylcholinesterase aggregates. When erythrocyte membranes (ghosts) were subjected to the same treatment, acetylcholinesterase could subsequently be solubilized as monomeric 3.9 S form and and more than 90% of the activity were recovered. Monomeric acetylcholinesterase was less reactive towards antibodies raised against (dimeric) human erythrocyte membrane acetylcholinesterase and towards antibodies against human erythrocyte membranes. The results suggest that acetylcholinesterase is present as dimeric species in human erythrocyte membranes despite the fact that fully active monomers can be obtained.     

The isolation and functional identification of a protein from the human erythrocyte `ghost''

A protein, initially identified as a band on polyacrylamide-gel electrophoresis of erythrocyte ;ghosts', was isolated by selective extraction of ;ghosts' with EDTA solutions. The molecular weight of the polypeptide chain was estimated as 33000 and it represents approx. 5% of the membrane protein. The N-terminal sequence of the protein was established. Comparison with known protein sequences suggested that the protein might be the erythrocyte d-glyceraldehyde 3-phosphate dehydrogenase. This identification was confirmed by direct enzyme assay. It is suggested that this enzyme, which is strongly retained by erythrocyte ;ghosts' on haemolysis of erythrocytes, is unlikely to be an integral part of the structure of the erythrocyte membrane.     

Optimizing the diagnosis of anemia

The specific value of the erythrocyte parameters MCH and MCV for the differential-diagnostic arrangement of anaemias to the use of the haematological automations of analysis PHA 1/2 is represented. A prerequisite is the precise determination of haemoglobin concentration and erythrocyte number. MCHC has no sufficient discriminatory capacity.