Is there any connection between heat inactivation of spectrin-dependent ATPase and loss of smooth biconcave shape of red cells?

Spectrin-dependent ATPase activity was measured in membranes from native human erythrocytes and erythrocytes heated for 20 min at different temperatures. This activity was found to decline when the erythrocytes were heated at 48 degrees C and higher. The break in ATPase activity corresponds to morphological changes in erythrocytes found by Crome and Mollison [Brit. J. Haematol. (1964) 10, 137]. The role of spectrin-dependent ATPase in erythrocyte shape maintenance is discussed.     

The effect of membrane preparation and cellular maturation on human erythrocyte adenylate cyclase

We found that adenylate cyclase activity of human erythrocytes is potentially labile during isolation of their plasmalemma. Addition of 1 mM EGTA to solution used to remove hemoglobin from lysed cells protected activity. Human erythrocyte adenylate cyclase is minimally activated by catecholamines, in the absence or presence of exogenous guanyl nucleotide, but substantially by 5'-guanylyl imidodiphosphate or sodium fluoride and concentration-dependently by Mg2+ or Mn2+. Basal catalytic activity is an age-dependent component of the human erythrocyte; 5'-guanylyl imidodiphosphate- or fluoride-activated activities decline with cellular maturation proportionally to the decrease in basal activity.     

Decay of proteinase and peptidase activities of human and rabbit erythrocytes during cellular aging

Variations in activity of the membrane-bound and cytosolic proteinases and peptidases were analyzed in human and rabbit erythrocytes at various stages of their life-span. The patterns observed with human erythrocytes were the following. (a) The acidic endopeptidase activity associated with the membranes undergoes a substantial decline during cellular aging, with an estimated half-life of 65 days. Concomitantly it appears to become progressively more latent. (b) All cytosolic proteinase and peptidase activities described previously (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., Benatti, U., Morelli, A. and De Flora, A. (1980) Eur. J. Biochem. 110, 421–430) decline exponentially throughout the erythrocyte life-span, with the exception of dipeptidyl aminopeptidase III. The calculated half-lives were: 60 days for the neutral endopeptidase; 87 days for the total acidic endopeptidase activity which is accounted for by three distinct enzymes; 49 days for aminopeptidase B and 133 days for a second aminopeptidase with broad substrate specificity; 84 days for dipeptidyl aminopeptidase II. The results obtained with the rabbit erythrocytes were: (a) no significant decline of leucine aminopeptidase, dipeptidyl aminopeptidase II and III activities in the transition from reticulocytes to mature erythrocytes; (b) very limited decline of aminopeptidase B activity; (c) a pronounced age-dependent decay, in increasing order, of neutral endopeptidase, aminopeptidase A, carboxypeptidase and acidic endopeptidase activities.     

Is red blood cell survival limited by the activity of a critical enzyme?

Erythrocyte glutathione reductase is responsible for generating reduced glutathione, which has been implicated in maintaining the integrity of the red blood cell.Erythrocytes from peripheral blood were separated into fractions of increasing age and the activity of glutathione reductase and aspartate amino transferase determined in each fraction.The age-related decline in activity of both enzymes was confirmed, but with detailed resolution of the cells by age a significant secondary rise in only glutathione reductase activity was found in very old cells. As red blood cells from the same cohort survive in the circulation for varying periods they must vary in some way from one another. It is postulated that glutathione reductase is a critical enzyme which limits erythrocyte survival and that the rate of decline in activity varies from cell to cell. A simple mathematical model based on this postulate accounted quantitatively for both the pattern of glutathione reductase activity and the erythrocyte survival curve. In addition, a simplified model of the passage of erythrocytes through the circulation was designed and run. The predicted erythrocyte survival curve and pattern of glutathione reductase activity were very similar to observed patterns. This model may be useful in other situations where a finite resource is degraded at different rates by random passages through different pathways.     

Comparison of life span of erythrocytes in some inbred strains of mouse using 14C-labelled glycine.

The erythrocyte life span in four inbred strains of mice--C57BL/10ScSnPh, B10.LP, BALB/c and CBA/JPh--was determined by means of erythrocyte labelling with 14C-glycine. Experimental data [decrease of 14C-activity of washed erythrocytes], corrected for blood loss, reincorporation of the label and delay in label incorporation during the initial period, were treated by a novel mathematical procedure based on the death probability function of the form: mu [t] = a + btlambda. The mean erythrocyte life span calculated using this function was, in the given sequence of strains, 42.1 +/- 0.6, 41.3 +/- 1.2, 39.3 +/- 0.9 and 38.6 +/- 0.6 days respectively. The rate of "random" destruction of erythrocytes was 1.20, 1.31, 0.70 and 0.63% of the total number of erythrocytes per day and a "mean potential" erthrocyte life span was found to be 58.9, 59.9, 46.2 and 44.5 days respectively. All the given parameters have similar numerical values in related strains and are apparently genetically conditioned. The erythrocyte life span determined simultaneously using DF32P labelling in the C57BL/10ScSnPh strain was 47.4 +/- 1.0 days. The implications of the results yielded by these two techniques is discussed.     

The effect of membrane preparation and cellular maturation on human erythrocyte adenylate cyclase

We found that adenylate cyclase activity of human erythrocytes is potentially labile during isolation of their plasmalemma. Addition of 1 mM EGTA to solution used to remove hemoglobin from lysed cells protected activity. Human erythrocyte adenylate cyclase is minimally activated by catecholamines, in the absence or presence of exogenous guanyl nucleotide, but substantially by 5′-guanylyl imidodiphosphate or sodium fluoride and concentration-dependently by Mg²⁺ or Mn²⁺. Basal catalytic activity is an age-dependent component of the human erythrocyte; 5′-guanylyl imidodiphosphate- or fluoride-activated activities decline with cellular maturation proportionally to the decrease in basal activity.     

Congenital Non-Spherocytic Hemolytic Anemia

A family with congenital non-spherocytic hemolytic anemia associated with glucose-6-phosphate dehydrogenase (G6PD) deficiency was studied. Two females, heterozygous for the enzyme deficency, had evidence of a hemolytic anemia. The results of chromium-51 erythrocyte life span studies prior to, during, and after periods of primaquine administration suggested that the hemolytic anemia in these women was due to the presence of two populations of red blood cells in their circulation. One population had normal G6PD levels and a normal life span, whereas the other had diminished enzyme activity and a shortened life span.In vitro metabolic studies of the erythrocytes of a heterozygous female and a hemizygous male suggested that, in spite of G6PD deficiency, the synthesis and breakdown of adenosine triphosphate and 2,3-diphosphoglyceric acid was similar to that in normal erythrocytes.     

Characteristics of endogenous proteolysis in preparations of human erythrocyte membranes

Endoproteolytic activity in human erythrocyte membrane preparations has been examined at 37 degrees C by one- and two-dimensional electrophoresis. Two-dimensional mapping has shown that the presence of leukocyte enzymes in erythrocytes prepared in a regular manner (centrifugation) cannot be excluded. Sedimentation in the 1.5% dextran 500,000 with the following erythrocyte purification on HBS-cellulose has made it possible to prepare erythrocyte membranes characterized by low level endoproteolytic activity without leukocyte enzymes. The marker peptide has been found. It is likely to be a specific product of the enzyme activity of membrane localization.     

Curcumin induced suicidal erythrocyte death.

The natural nutrient component Curcumin with anti-inflammatory and antitumor activity has previously been shown to stimulate apoptosis of several nucleated cell types. The present study has been performed to explore whether Curcumin could similarly induce suicidal death of erythrocytes or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Phosphatidylserine exposing cells are phagocytosed and thus rapidly cleared from circulating blood. Erythrocyte membrane scrambling may be triggered by increase of cytosolic Ca(2+) activity or formation of ceramide. To test for eryptosis, erythrocyte phosphatidylserine exposure has been estimated from annexin V binding, and erythrocyte volume from forward scatter in FACS analysis. Exposure of erythrocytes to Curcumin (= 1 microM) increased annexin V binding and decreased forward scatter, pointing to phosphatidylserine exposure at the cell surface and cell shrinkage. According to Fluo3 fluorescence Curcumin increased cytosolic Ca(2+) activity and according to immunofluorescence Curcumin increased ceramide formation. As shown previously, hypertonic shock (addition of 550mM sucrose), chloride removal and glucose depletion decreased the forward scatter and increased annexin V binding. The effects on annexin binding were enhanced in the presence of Curcumin. Exposure to Curcumin did, however, not significantly enhance the shrinking effect of hypertonic shock or Cl(-) removal and reversed the shrinking effect of glucose withdrawal. The present observations disclose a proeryptotic effect of Curcumin which may affect the life span of circulating erythrocytes.     

Sex-linked differences in activity of enzymes in the blood of the urodele amphibian Pleurodeles waltl.

Few data are available on enzyme activity in amphibian plasma or erythrocytes. We measured the activity of several blood enzymes in the urodele amphibian Pleurodeles waltl reared under standard laboratory conditions. In subsequent experiments, we will estimate and compare the physiological and biochemical conditions of P. waltl when reared under extreme temperature or microgravity conditions. The enzymes selected were glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, superoxide dismutase, catalase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. In fresh plasma samples, enzyme activity in females was higher than in males, except for aspartate and alanine aminotransferases, which were equivalent in females and males. Glutamate dehydrogenase activity was higher in males than in females. In female erythrocytes, the activity of all enzymes was higher than in male erythrocytes. We have also studied the storage conditions of samples and observed that for most enzymes, the activity in freshly isolated plasma and erythrocyte preparations decreased after storage at -18 or +4 degrees C.     

Hypoxanthine-guanine phosphoribosyltransferase deficiency and erythrocyte synthesis of pyridine coenzymes.

Purine and pyridine metabolism were studied in ten Lesch-Nyhan patients, with virtually no hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity in erythrocytes. Increased NAD erythrocyte concentrations were found in all patients. Raised activities of two enzymes catalysing NAD synthesis from nicotinic acid (nicotinic acid phosphoribosyltransferase: NAPRT, and NAD synthetase: NADs) was found in erythrocyte lysates from all patients. The two enzymes had normal apparent Km for their substrates and increased Vmax. The rate of synthesis of pyridine nucleotides from nicotinic acid by intact erythrocytes in vitro was also increased in most patients. These findings suggest that raised NAD concentrations in HPRT- erythrocytes are due to enhanced synthesis as a result of increased enzyme activities.     

Total body haematocrit iron kinetics and erythrocyte life span in pigeons (Columba livia)

1. The mean pigeon erythrocyte life span was found to be 17-25 days by Cr51-labeled erythrocytes and 21 +/- 3.4 days by iron kinetics. 2. Total red blood cell volume has been calculated by Cr51-labeled erythrocytes while total plasma volume was determined both by a dye method and iron kinetic data. From these results total blood volume and total body haematocrit were found to be 0.090 +/- 0.002 ml/g body wt and 36 +/- 4.3%, respectively. 3. Venous haematocrit, haemoglobin concentration, erythrocyte count, mean corpuscular haemoglobin concentration, plasma iron and red blood cell iron have also been measured. 4. A significant difference between total body and venous haematocrit and a short mean red blood cell life span, due to ageing and to random destruction of erythrocytes were shown. 5. The above observations are compared with analogous available data for human beings and their physiological significance is discussed.     

Allometric dependence of the life span of mammal erythrocytes on thermal stability and sphingomyelin content of plasma membranes

Thermal stability of erythrocyte membrane is a measure for its ability to maintain permeability barrier at deleterious conditions. Hence, it could impact the resistance of erythrocytes against detrimental factors in circulation. In this study the thermostability of erythrocyte membranes was expressed by the temperature, T(go), at which the transmembrane gradient of ion concentration rapidly dissipated during transient heating. T(go) is the inducing temperature of the membrane transition that activated passive ion permeability at hyperthermia causing thermal hemolysis. A good allometric correlation of T(go) to the resistance against thermal hemolysis and the life span of erythrocytes were found for 13 mammals; sheep, cow, goat, dog, horse, man, rabbit, pig, cat, hamster, guinea pig, rat, and mouse. For the same group, the values of T(go) were strictly related to the sphingomyelin content of erythrocyte membranes. The residual ion permeability, P, was temperature activated from 38 to 57 degrees C with activation energy of 250+/-15 kJ/mol that strongly differed from that below 37 degrees C. The projected value of P at 37 degrees C was about half that of residual physiological permeability for Na+ and K+ that build ground for possible explanation of the life span vs membrane thermostability allometric correlation.     

Deficiencies of glycolytic enzymes as a possible cause of hemolytic anemia

The critical minimum values of Na,K-ATPase and glycolytic enzyme activities at which the erythrocyte viability is lost were calculated using the mathematical model of the erythrocyte, which included all reactions of glycolysis, adenylate metabolism, ionic balance, and osmotic regulation of erythrocyte volume. The criterion for cell death was an increase in its volume to the level at which it is sequestrated from the circulation or is lysed. In hemolytic anemia associated with hexokinase or pyruvate kinase deficiency, activities of these enzymes measured in patient erythrocytes appeared to be close to the calculated critical values. By contrast, in hemolytic anemia associated with phosphofructokinase, glucosephosphate isomerase, triosephosphate isomerase, or phosphoglycerate kinase deficiency, activities of these enzymes measured in patient erythrocytes were significantly greater than the calculated critical values. In this case, if the deficient enzyme were stable, i.e. its activity in the cell were low, but constant in time, the deficiency observed would not account for the erythrocyte destruction observed and the development of hemolytic anemia. It was shown, however, that in phosphofructokinase, glucosephosphate isomerase, triosephosphate isomerase, or phosphoglycerate kinase deficiency, hemolytic anemia can arise because of the instability of these enzymes in time.     

Useful agents for the study of glutathione metabolism in erythrocytes. Organic hydroperoxides

t-Butyl hydroperoxide and cumene hydroperoxide, both known to be substrates for glutathione peroxidase, were used to oxidize erythrocyte GSH. Addition of concentrations of hydroperoxides equimolar with respect to GSH in the erythrocytes or whole blood quantitatively oxidizes GSH in the erythrocytes with a half-time of 4.5s at 37 degrees C and about three times as long at 4 degrees C. In the presence of glucose, normal erythrocytes regenerate all the GSH in about 25min. However, glucose 6-phosphate dehydrogenase-deficient erythrocytes failed to regenerate GSH. Treatment of erythrocytes with hydroperoxides does not affect erythrocyte survival in rabbits. Oxidation of erythrocyte GSH with equimolar concentrations of hydroperoxides does not lead to formation of mixed disulphides of haemoglobin and GSH. The hydroperoxides do not affect erythrocyte glycolytic and hexose monophosphate-shunt-pathway enzymes. Previous studies on transport of GSSG from erythrocytes were confirmed by using t-butyl hydroperoxide to oxidize erythrocyte GSH.     

Isolation of two high-molecular-mass proteinases from human erythrocytes

Two forms of a neutral--alkaline high-molecular-mass proteinase (termed A1 and A2) have been purified from human erythrocytes by a procedure including a DEAE-cellulose batchwise treatment of erythrocyte cytosol, gel filtration and DEAE-cellulose chromatography. Both enzymes show distinctive properties of multicatalytic proteinases. They have an apparent molecular mass of 700 kDa and are composed by eight major subunits (23-32 kDa). Both enzymes show a proteinase activity towards casein and hydrolyze synthetic peptides with tyrosine, arginine or lysine at the P1 position. Among the synthetic peptides tested, the tetrapeptide succinyl-leucyl-leucyl-valyl-tyrosyl-7-amido-4-methylcoumarin and tripeptides with arginine in the P1 position (benzyloxycarbonyl-valyl-leucyl-arginyl-4-methoxy-2-naphthylamide and benzyloxycarbonyl-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamide) are the most effective substrates. The proteinases are devoid of amino and diaminopeptidase activity. Both enzymes are completely inhibited by hemin, chymostatin and thiol-group reagents. However, the enzymes can be distinguished by the isoelectric point, the different effect of nucleotides, glutathione disulphide, sodium dodecyl sulfate and cations on the catalytic activity.     

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.     

Sex-linked differences in activity of enzymes in the blood of the urodele amphibian Pleurodeles waltl

Few data are available on enzyme activity in amphibian plasma or erythrocytes. We measured the activity of several blood enzymes in the urodele amphibian Pleurodeles waltl reared under standard laboratory conditions. In subsequent experiments, we will estimate and compare the physiological and biochemical conditions of P. waltl when reared under extreme temperature or microgravity conditions. The enzymes selected were glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, superoxide dismutase, catalase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. In fresh plasma samples, enzyme activity in females was higher than in males, except for aspartate and alanine aminotransferases, which were equivalent in females and males. Glutamate dehydrogenase activity was higher in males than in females. In female erythrocytes, the activity of all enzymes was higher than in male erythrocytes. We have also studied the storage conditions of samples and observed that for most enzymes, the activity in freshly isolated plasma and erythrocyte preparations decreased after storage at −18 or +4°C.     

Isoenzyme patterns of glutathione transferases from mammalian erythrocytes

The occurrence of glutathione transferase isoenzymes in mammalian erythrocytes was investigated. The enzymes present in the hemolysates of human, horse, beef, pig, and sheep erythrocytes were purified by a column of GSH-linked epoxy-activated Sepharose 6B and subjected to an isoelectric focusing run in the pH range 3.5-10. Human and horse preparations were resolved in a single peak of activity centered at pH 4.6 and 5.9, respectively. Two forms with a maximum of activity at pH 4.9 and 7.0 and four with a maximum at pH 5.9, 6.5, 7.1, and 8.1 were separated from bovine and porcine erythrocytes. At least six forms ranging from pH 4.3 to pH 7.1 were present in the ovine preparation, the neutral contributing more than 90% of total activity. The subunit composition of affinity-bound fractions was studied by sodium dodecyl sulfate-gel electrophoresis. The analysis revealed that erythrocyte glutathione transferases are composed of subunits of identical molecular weights. This result suggests that the polymorphism existing in beef, pig, and sheep may be due to charge isomers. The erythrocyte glutathione transferases did not express selenium-independent GSH peroxidase activity.     

A futile cycle in erythrocyte glycolysis

The storage lesion which limits the shelf life of human blood in blood banking is associated with a metabolic loss of 2,3-diphosphoglycerate and ATP. This metabolic loss is driven by intracellular ATPases which are usually considered to include the ion pumps and the reactions which maintain the discoid shape of the human erythrocyte. Under the acidic conditions of blood storage, the energy-yielding reactions of the glycolytic pathway are restricted at the hexokinase and phosphofructokinase steps. We show here that under such circumstances the enzyme of the diphosphoglycerate shunt, diphosphoglycerate mutase/phosphatase and the glycolytic enzyme phosphoglycerate kinase can form a futile cycle with ATPase activity. This ATPase activity responds to 2-phosphoglycolate which is known to activate both diphosphoglycerate mutase and diphosphoglycerate phosphatase reactions. When the enzymes of the futile cycle are combined with the enzymes of the lower glycolytic pathway in a reconstitution experiment designed to represent conditions within the stored erythrocyte, the futile cycle does provide an ATPase activity which results in the metabolic loss of 2,3-diphosphoglycerate. An isotope incorporation experiment demonstrates that the futile cycle is active in glucose-depleted erythrocytes.