Erythrocytes Of Man Research Articles

Purification and Properties of 2,3-Bisphosphoglycerate Phosphatase-Mutase from Erythrocytes of Day-Old Chicks

1 Large quantities of 2,3-bisphosphoglycerate accumulate in the red blood cells of the chick embryo during the week prior to hatching; the 2,3-bisphosphoglycerate then abruptly decreases to very small amounts within a few days after hatching. 2 The enzyme 2,3-bisphosphoglycerate phosphatase was purified from the red blood cells of the day old chick. The elution profiles for bisphosphoglycerate phosphatase and 2,3-bisphosphoglycerate mutase were identical upon gradient elution from columns of hydroxylapatite and diethylaminoethylcellulose and by gel filtration on Sephadex G-100. At each stage of purification the ratio of phosphatase to mutase activity was the same. It was concluded that, just as in the human erythrocyte, both bisphosphoglycerate phosphatase and 2,3-bisphosphoglycerate mutase activities in chick erythrocytes reside on one protein. 3 Bisphosphoglycerate phosphatase has a pH of optimal activity of 7.0. Its activity is stimulated by 2-phosphoglycolate, phosphoenolpyruvate, bisulfite and dithionite and is inhibited by 2-phosphoglycerate, 3-phosphoglycerate, inorganic pyrophosphate and phytic acid. These properties are very similar to those described for bisphosphoglycerate phosphatase purified from human erythrocytes. 4 The purified bisphosphoglycerate phosphatase-mutase contained 3-phosphoglycerate mutase activity. Although the 2,3-bisphosphoglycerate mutase and 3-phosphoglycerate mutase activities were nearly equal in the purified enzyme, this protein accounts for a maximum of only 6% of the 3-phosphoglycerate mutase activity in these cells. 5 The total activity of the bisphosphoglycerate phosphatase in the erythrocytes was measured at intervals during development of the embryo, in the young chick, and in the mature chicken. Its activity increases during the time of 2,3-bisphosphoglycerate accumulation. The enzyme activity decreases gradually from its maximum in the two-day-old chick (1.67 μmol h−1 g hemoglobin−1) and by 27 days it has fallen nearly to the levels seen in the adult chicken (0.11 μmol h−1 g hemoglobin−1). 6 The factors which might be involved in inducing the 2,3-bisphosphoglycerate accumulation and degradation in the late chick embryo and young chick are discussed. It is concluded that this system is very similar to that found in the erythrocytes of man.

The role of complement in the clearance of cold agglutinin-sensitized erythrocytes in man.

To define the pathophysiologic mechanisms of cold agglutinin disease, we investigated a human model of this syndrome in normal volunteers and in patients with diminished levels of serum complement. Subjects received intravenous injections of autologous, chromated (51Cr) erythrocytes which had been exposed in vitro to purified cold agglutinin preparations and to fresh autologous serum (as a source of complement). In vitro tests confirmed that such cells were coated with activated complement components (C3b), but not with immunoglobulin. Studies of erythrocyte clearance and simultaneous organ scanning showed that erythrocytes sensitized with low levels of cold agglutinin primarily undergo reticuloendothelial sequestration by the liver rather than intravascular hemolysis. After the initial sequestration of C3b-coated erythrocytes, a fraction of the cells are released back into the circulation and survive normally thereafter. Both phenomena are dose dependent and closely follow the sequestration and release pattern observed with IgM isoagglutinin sensitization. Experiments that used heated autologous serum as a source of B3 inactivator demonstrated that functionally intact C3b is required for hepatic sequestration. Erythrocytes coated with C3d were not cleared from the circulation. In vitro assays that sued human macrophage monolayers suggested that the intrahepatic conversion of C3b to C3d is responsible for the release of sensitized erythrocytes back into the circulation. The clearance of cold agglutinin-sensititzed erythrocytes was compared to the clearance mediated by IgM isoagglutinin. We found that the rate of complement fixation by an IgM antibody proceeds rapidly in vivo that the time for complement activation is not a factor in limiting the rate of hepatic sequestration. The major limiting factor appears to be the rate of liver blood flow. Maximal in vitro coating of erythrocytes with C3d conferred protection from further cold agglutinin sensitization but not from IgM isoagglutinin-mediated clearance. This suggests a mechanism for the resistance to lysis observed in cells obtained from patients with the cold agglutinin syndrome and confirms the marked dependence of the site of C3 attachment on the site of membrane localization of the sensitizing antibody.

Ontogenetic development of NADH-dependent methemoglobin reductase in erythrocytes of man and rat.

Ontogenetic development of NADH-dependent methemoglobin reductase was followed in humans and rats. The human kinetic profile differs from that in the rat. The low level of methemoglobin reductase in human infants at birth and for the first months of life may provide a partial explanation of the particular susceptibility to methemoglobinemic agents of this age group.

Monocyte involvement in PHA induced cytotoxicity of chicken erythrocytes in man

Phytohemagglutinin (PHA) induced cell cytotoxicity was studied in man using chromated chicken red blood cells (CRBC) as target cells. A phagocytic, adherent monocyte was found to be necessary for lysis of target cells. Results using E rosette depletion showed that this procedure markedly increased mitogen-induced cellular cytotoxicity (MICC). Carbonyl iron treatment of peripheral blood cell suspensions to remove phagocytic cells abolished MICC, as did removal of adherent cells by glass wool columns. Complement mediated lysis of B cells did not substantially reduce MICC. However, pretreatment of cells with silica or hydrocortisone did reduce MICC. The mechanism of mitogen-induced lysis appears to require direct cell contact between effector cells and target cells.

Evidence for three enzymatic activities in one electrophoretic band of 3-phosphoglycerate mutase from red cells

Electrophoresis of 3-phosphoglycerate mutase from erythrocytes of man and several animal species has been performed on cellulose acetate strips. In most cases the electrophoretic pattern of this enzymatic activity shows three bands. 2,3-diphosphoglycerate phosphatase and diphosphoglycerate mutase from erythrocytes of the same species have been revealed after migration during the same electrophoresis. We found that the band of 2,3-diphosphoglycerate phosphatase and the band of diphosphoglycerate mutase activities migrate at the same level as one of the bands corresponding to 3-phosphoglycerate mutase. Here, we discuss the possible existence of a single molecule carrying three enzymatic activities.

Distribution des diphenoloxydases (DPOx) dans divers tissus de diverses especes animales

An oxidative activity has been demonstrated in different tissues of various animals species (man, rabbit, hen, rat, mouse) measuring it by means of adrenaline, by starch gel electrophoresis and acrylamide gel electrofocusing, followed by incubation of these gels with DOPA. Kidney, liver and erythrocytes of rabbit, rat, mouse and hen, human erythrocytes and platelets display a high oxidative activity. This activity, which is not completely inhibited by KCN, is found in one band on starch gel electrophoresis and in two distinct bands on electrofocusing in the case of platelets and erythrocytes of man, rabbit and rat. The starch gel electrophoretic migration and the electrofocusing pattern are tissue and species dependent.

Adenosine uptake by erythrocytes of man, rat and guinea-pig and its inhibition by hexobendine and dipyridamole

Adenosine uptake by erythrocytes of man, rat and guinea-pig and its inhibition by hexobendine and dipyridamole

Hexokinase of human erythrocytes. Purification, kinetic model and its application to the conditions in the cell.

Hexokinase was purified 680‐fold from erythrocytes of man. The preparation was not contaminated by other glycolytic enzymes.Initial reaction rate measurements were performed at pH 7.2, ionic strength 0.15 and 37°C by use of coupled optical assays. The experimental data were used to construct a kinetic model of the enzyme, the kinetic parameters of which were estimated by means of a computer‐fit program.The hexokinase of erythrocytes operates by a rapid‐equilibrium random mechanism. The dissociation constant of the E · glucose complex is 0.038 to 0.054 mM, of the E · MgATP complex 1.0 to 2.1 mM. Uncomplexed Mg2+ up to 4 mM is capable of activating the enzyme velocity twofold (KMg2+= 1.0 mM). An inhibition was observed at higher Mg2+ concentrations. α‐d‐Glucose 6‐phosphate and α‐d‐glucose 1,6‐bisphosphate inhibit the hexokinase competitive to MgATP with dissociation constants of 0.069 mM. The enzyme is also inhibited by 2,3‐bisphosphoglycerate competitive with respect to MgATP with a KP2G= 2.7 mM. An inhibition by uncomplexed ATP was not detected.The parameters of the kinetic model of the enzyme in conjunction with the free intracellular Concentrations of substrates and effectors were used to calculate the actual intracellular activity of the hexokinase in dependence on variations of pH, temperature and the state of haemoglobin oxygenation. The calculated hexokinase activities correspond to the glycolytic flux in all conditions considered, except at pH 8.

Persistence of Colorado tick fever virus in red blood cells.

Previous studies have indicated that Colorado tick fever (CTF) virus persists in erythrocytes of man and experimental animals for long periods. In this study, blood samples were obtained from nine patients at various intervals during and after clinical illness with CTF. Virus was isolated from erythrocytes of some patients as early as 1 day and as late as 120 days after onset of symptoms. Similar studies performed on experimentally infected Swiss mice and laboratory rats demonstrated that CTF virus persisted in their red blood cells for 60 and 50 days after inoculation, respectively. A single treatment with trypsin proved more efficient and rapid than multiple saline washes for removing neutralizing antibody from erythrocytes to allow the detection of CTF virus and viral antigens during any stage of illness or convalescence.

Arylesterase and acetylcholinesterase in the erythrocytes of man, cow and pig

1. 1. The esterase activities of human, bovine and porcine erythrocytes have been determined gasometrically with phenyl acetate and acetylcholine iodide as substrates. The activity of human erythrocytes is twice that of bovine and porcine cells. Intact cells and hemolysates have about the same activity. The three species differ in regard to the readiness of enzyme release from the cells by simple hemolysis against a hypotonic solution 2. 2. The effect of ultrasonic waves, together with other results obtained, suggest that both arylesterase (aryl-ester hydrolase, EC 3.1.1.2) and acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) are probably bound to the erythrocyte membrane 3. 3. The inhibitory effects of physostigmine and mercuric ions suggest that 10r-20% of the esterase activity, measured with phenyl acetate, is due to arylesterase (or carboxylesterase). The three species studied differ slightly regarding the relative activity level of each esterase type 4. 4. Hemoglobin-free esterase fractions have been prepared by gel filtration on Sephadex G-100. The properties and composition of these fractions have been studied with thiophenyl acetate and acetlythiocholine iodide as substrates using a quantitative spectrophotometric technique as well as a histochemical one for disc electropherograms.

The influence of pH and methylene blue on the pathways of glucose utilization and lactate formation in erythrocytes of man.

The influence of pH on the contributions of the Embden‐Meyerhof and of the oxidative pentose‐phosphate pathways in the utilization of glucose by human erythrocytes in the presence and absence of methylene blue were determined from yields of 14CO2 and [14C]lactate.In the absence of methylene blue both the NADP+ concentration and the phosphofructokinase are controlling factors. At pH 7.0 the oxidative pentose‐phosphate pathway accounts for nearly one‐half of the metabolic flux. Its contribution nearly vanishes at pH 8.2.In the presence of methylene blue the limitation by the NADP+ concentration disappears. At pH values of 7.4 and below the phosphofructokinase is completely inhibited and is circumvented for the formation of lactate. The limitation of the glucose influx resides in the hexokinase. At pH 8.2 both the Embden‐Meyerhof and the oxidative pentose‐phosphate pathway participate in the metabolic flux, since the inhibition of the phosphofructokinase is released.With methylene blue metabolic pools are formed. They are derived exclusively from the oxidative pentose‐phosphate pathway up to pH 7.5–7.6 and entirely from the Embden‐Meyerhof pathway at pH 8.2.

Incorporation of adenosine-8-14/C and inosine-8-14C into rabbit heart adenine nucleotides.

Incorporation of adenosine-8-14/C and inosine-8-14C into rabbit heart adenine nucleotides.

Regulatory factors in methylene blue catalysis in erythrocytes

In erythrocytes of man and rabbits methylene blue catalysis is limited by the following factors at the pH optimum 7.5; primarily by the cellular concentration of NADP+, next by that of glucose‐6‐P. Possible further limiting steps are glucose‐6‐phosphate dehydrogenase, phosphogluconate dehydrogenase and reduced‐NADP dehydrogenase. Calculation of the intracellular NADP+ concentration with the two‐substrate equation for the glucose‐6‐phosphate dehydrogenase‐reaction does not yield reasonable results. The existence of inhibiting factors of the methylene blue catalysis is postulated. Their amount is presumably lower in intact cells as compared with hemolysates. There is no pH dependence of the glucose‐6‐P concentration in presence of methylene blue. There exists a competition between the oxidative pentose‐phosphate‐pathway and the Embden‐Meyerhof pathway, in which the phosphofructokinase plays a key role. The proportions of the two pathways at different pH values are calculated from data on oxygen and glucose consumption. At pH values below 7.6 glucose is utilized practically exclusively via the oxidative pentose phosphate pathway, whereas the Embden‐Meyerhof pathway is inhibited. At pH 8.2 the participation of the oxidative pentose‐phosphate pathway amounts to 40% of the total glucose utilization, while the Embden‐Meyerhof pathway is little if any reduced as compared to cells without methylene blue. With increased degree of oxidation of the pyridine nucleotides there occur decreases in the levels of hexose‐ and triose‐phosphates. With these changes and the decreased flow to lactate an increased formation of 2,3‐diphosphoglycerate occurs.

Purification and properties of 3-phosphoglyceric acid mutase from caprine erythrocytes

Relatively simple procedures are described for preparing partially purified phosphoglycerate mutase from erythrocytes of the goat. The kinetic properties of this enzyme are described and compared to those of the same enzyme from human erythrocytes. The K m for 3-phosphoglyceric acid is 6 × 10 −4 m. The enzyme has an absolute requirement for 2,3-diphosphoglyceric acid with a K m of 5 × 10 −6 m. 2,3-Diphosphoglyceric acid phosphatase activity co-purified with the phosphoglyceric acid mutase. This activity, thought to be an activity of the same enzyme, is greatly stimulated by inorganic pyrophosphate. Its K m for pyrophosphate is 1 × 10 −2 m and for 2,3-diphosphoglyceric acid in the phosphatase assay is 1.2 × 10 −4 m. No kinetic differences were found between the enzymes of human and caprine origins. The concentration of 2,3-diphosphoglyceric acid in caprine erythrocytes was measured enzymically and found to be 0.03 μmoles per ml cells, approximately 1% of the amount present in the erythrocytes of man. That amount of 2,3-diphosphoglycerate is nevertheless 5-fold above the enzyme's K m value for this cofactor. A second 2,3-diphosphoglyceric acid phosphatase, stimulated by bisulfite, was detected in caprine red cells but its activity was far less than in human erythrocytes.

Molecular species of lecithins from erythrocytes and plasma of man

The lecithins of the plasma and erythrocytes of man were isolated by thin-layer chromatography, and the major molecular species were identified and quantitatively estimated by combined thin-layer and gas-liquid chromatography and specific enzymic hydrolyses. Using these techniques we could identify over 60 molecular species, accounting for some 98% of the total lecithin, in both plasma and cells, but only about 30 of them occurred in concentrations over 1%. The molecular species of lecithins in the cells and plasma were qualitatively similar; quantitatively, large differences were noted among and within the various classes of unsaturation. In the same blood, the erythrocyte lecithins contained 8-20 times as high a percentage of saturated lecithins and nearly twice as high a percentage of monounsaturated lecithins as did plasma lecithins. The differences in the relative amounts of a particular molecular species within a class of unsaturation were, however, most pronounced among the polyunsaturated lecithins. These results suggest that plasma and red cells possess distinct lecithin populations and that complete equilibration of the intact molecules between the two media is unlikely.

Phospholipid exchange between plasma and erythrocytes in man and the dog.

The turnover of the four major erythrocyte phospholipids has been studied with (32)P, both in vivo and in vitro, in man and the dog. Phosphatidyl serine and phosphatidyl ethanolamine appeared to be stable erythrocyte lipids in both species. Turnover of the phosphate moiety of lecithin and sphingomyelin in the circulating erythrocytes of these two species seems entirely due to an exchange of the whole molecule with the corresponding plasma compound. Exchangeable and nonexchangeable pools of these two cellular lipids were found. In man about 60% of erythrocyte lecithin is exchangeable. The 12 hr fractional turnover of this pool is approximately 13%. Only 30% of the sphingomyelin in human cells appeared exchangeable; this portion had a 12 hr fractional turnover of about 14%. Similar results were obtained in the dog except that in this species about 75% of the erythrocyte sphingomyelin was exchangeable. Inorganic (32)P was not incorporated into any of the four major phospholipids in either species. The present findings aid in estimating quantitatively the effect of plasmaerythrocyte lipid exchange on red blood cell phospholipids.

Fluorescence Microscopic Studies on the Appearance of Basophilic Stippled Erythrocytes in Man

Fluorescence Microscopic Studies on the Appearance of Basophilic Stippled Erythrocytes in Man

The metal composition of erythrocytes in different species and its relationship to the lifespan on the cells in the circulation

1. 1. Normal values are given for potassium, sodium, magnesium, calcium, zinc and copper in the erythrocytes of man, monkey, rabbit, rat, dog and duck. 2. 2. Calcium was consistently found in the erythrocytes of all these species except the duck in which it was detected in only one of three birds. 3. 3. A direct relationship was found between the average amounts of nitrogen, hemoglobin, water, total cation, potassium (dog excepted) and magnesium in the cells and the mean cell volume suggesting that with respect to these constituents variations between these species can be accounted for by differences in the size of the erythrocytes. 4. 4. The amount of calcium and sodium per cell bore no relationship to mean cell volume. 5. 5. Compared to the concentration in the cells of the other species erythrocyte copper was low in the monkey, zinc was low in the duck, and sodium was high in the rabbit and low in the dog. 6. 6. With the exception of the dog there was an indirect correlation between the lifespan of the erythrocyte in the species that were studied and the concentration of total cation/μl of erythrocyte water and a direct correlation between lifespan and the amount of zinc/μmole of hemoglobin in the cell.

Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in lens and blood of different species

The activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenase were measured in the lens and erythrocytes of man and several other species. There is a wide variation between individuals of the same species, but there seemed no correlation between the activity of either enzyme in the lens and their activity in the erythrocytes of the same species. There is a wide range of activity of both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the lens and in the blood of different species. Several species had glucose 6-phosphate dehydrogenase activity in erythrocytes below the level considered “deficient” in man. The sorbitol content of the lens was not correlated with glucose 6-phosphate dehydrogenase activity.

MONTHLY VARIATIONS IN THERMAL FRAGILITY OF ERYTHROCYTES IN MAN: INFLUENCE OF AGE AND SEX.

IN previous papers it has been shown that chronological ageing was accompanied in both sexes by significant increases in the thermal fragility of erythrocytes1. The present growing interest in the exploration of seasonal variations in biological functions has induced me to examine my data for seasonal changes. The results of this analysis are presented in this communication.