The effects of lactose on human erythrocytes

Human erythrocytes suspended in isotonic lactose solution lost potassium and continued to lose potassium even when resuspended in isotonic sodium chloride. The same phenomenon was observed when the cells were suspended in an isotonic solution of the sodium salt of glutamate, a nonpenetrating anion. The presence of 5 mEq per liter of sodium chloride in the lactose or sodium glutamate suspensions greatly reduced the initial potassium loss and the potassium loss when the cells were resuspended in sodium chloride solution. Salts of nonpenetrating anions were less or not effective in blocking lactose damage. The results indicate that absence of penetrating anions in the suspending media is the initiating condition of lactose damage. Chloride and consequently potassium are lost from the erythrocyte. Changes in cellular ionic pattern and/or changes in the cell membrane result in a nontransient damage manifested by continued potassium loss by lactose-treated cells resuspended in isotonic NaCl.     

Changes of reduced glutathion, glutathion reductase, and glutathione peroxidase after radiation in guinea pigs

In this series of experiments the protective action of reduced glutathion due to ionizing radiation has been studied. In the experimental group 18 guinea pigs were exposed to successive radiations of 150 rad 3 or 4 days apart. Total dose given amounted to 750 rad which is the LD50 for guinea pigs. Blood samples were taken 30 min after each exposure. The control series were sham radiated but otherwise treated identically. The cells of the removed blood samples were separated by centrifugation and were subjected to the reduced glutathion stability test. GSSGR, GPer, and LDH enzyme activities were also measured of which the latter served as a marked enzyme. It was found that LDH did not show any alteration after radiation. The reduced glutathion stability test showed a consistent but minor reduction (P greater than 0.05), in the experimental group. GSSGR enzyme activity on the other hand was reduced significantly (from 176.48 +/- 11.32 to 41.34 +/- 1.17 IU/ml of packed erythrocytes, P less than 0.001) in the same group. GPer activity showed a consistent but minor elevation during the early phase of the experimental group. It was later increased significantly beginning after 600 rad total radiation on the fourth session (P less than 0.050).     

Glutathione reductase from human erythrocytes. Catalytic properties and aggregation.

The catalytic properties of glutathione reductase from human erythrocytes have been studied over a range of buffer conditions and substrate concentrations. This study provides optimal conditions for determining the basic kinetic parameters of the enzyme. The catalytic behaviour of glutathione reductase is consistent with spatially separated binding sites for its substrates. In certain assays anomalies were observed which are correlated with an inactivation of the enzyme by NADPH. Concurrent sedimentation experiments showed that NADPH promoted aggregation of the enzyme. Both inactivation and aggregation could be connected with oxidation of thiols at the active site. The relation of the properties of glutathione reductase to cellular conditions is discussed.     

Role of NADPH and the NADPH-dependent methemoglobin reductase in the hydroxylase activity of human erythrocytes

Human erythrocytes were shown previously to catalyze the oxyhemoglobin-requiring hydroxylation of aniline, and the reaction was stimulated apparently preferentially by NADPH in the presence of methylene blue (K. S. Blisard and J. J. Mieyal,J. Biol. Chem.254, 5104, 1979). The current study provides a further characterization of the involvement of the NADPH-dependent electron transport system in this reaction. In accordance with the role of NADPH, the hydroxylase activity of erythrocytes or hemolysates from individuals with glucose-6-phosphate dehydrogenase deficiency (i.e., with diminished capacity to form NADPH) displayed decreased responses to glucose or glucose 6-phosphate, respectively, in the presence of methylene blue in comparison to samples from normal adults; maximal activity could be restored by direct addition of NADPH to the deficient hemolysates. Kinetic studies of the methylene blue-stimulated aniline hydroxylase activity of normal hemolysates revealed a biphasic dependence on NADPH concentrations: a plateau was observed at relatively low concentrations (K_m^NADPH ~ 20 μm), whereas saturation was not achieved at the higher concentrations of NADPH. The latter low efficiency phase (i.e., at the higher concentrations of NADPH) could be ascribed to a direct transfer of electrons from NADPH to methylene blue to hemoglobin. The high efficiency phase suggested involvement of the NADPH-dependent methemoglobin reductase; accordingly 2′-AMP, an analog of NADP⁺, effectively inhibited this reaction, but the pattern was noncompetitive. This behavior is suggestive of a mechanism by which both NADPH and methylene blue are substrates for the reductase and interact with it in a sequential fashion. The kinetic patterns observed for variation in NADPH concentration at several fixed concentrations of methylene blue, and vice versa, are consistent with this interpretation.     

Redox modulation of reductase and phosphatase activities in human erythrocytes

Summary We have previously reported that ferricyanide reductase activity in human erythrocytes depended on glycolysis and could be modulated by several compounds including oxidants and hormones like insulin. Insulin could activate glycolysis, probably as a consequence of tyrosine phosphorylation of protein band 3, implicating phosphorylation reactions as an important signal for activation of the reductase by insulin. Reversible phosphorylation of cellular proteins is also believed to play a key role in the action of insulin. Cytosolic acid phosphatase activity has been found in human erythrocytes. To further extend initial reports, we studied the effect of modulators on the cytosolic erythrocyte acid phosphatase. Mild oxidants like ferricyanide (1 mM), vanadate (1 mM), Mn²⁺ (0.5 and 1 mM), and phenylarsine oxide (10 and 100 M) inhibited the phosphatase activity. Similarly, insulin at concentrations that stimulate ferricyanide reduction (500, 1000 IU/ml) inhibited the activity of the phosphatase enzyme. The overall results indicated that oxidants are able to inhibit the acid phosphatase and stimulate the redox enzyme. In addition, a significant negative correlation (r = –0.400; P = 0.006) was observed between phosphatase and reductase activities. The observations discussed here, together with previous ones, emphasize that a close association between reductase and phosphatase enzymes may exist and also suggest a role for redox reactions in tyrosine phosphorylation/dephosphorylation-mediated signal transduction pathways.     

Soluble and microsomal forms of NADH-cytochrome beta 5 reductase from human placenta. Similarity with NADH-methemoglobin reductase from human erythrocytes.

In congenital methemoglobinemia associated with mental retardation a generalized deficiency of NADH-cytochrome beta 5 reductase (NADH : ferricytochrome beta 5 oxidoreductase, EC 1.6.2.2) has been found in soluble extracts of red blood cells, as well as in deoxycholate-treated extracts of leukocytes, muscle, liver and fibroblasts (Leroux et al. (1975) Nature 258, 619-620). In the present study the relationship between the microsomal (I) and the soluble (II) NADH-cytochrome beta 5 reductase was investigated, using human placenta as a source of enzyme. Both forms were compared to the human red-cell soluble NADH-methemoglobin reductase (III) and NADH-cytochrome beta 5 reductase (IV). The four entities exhibited great immunological similarities. It is concluded that the three soluble enzymes (II, III and IV) are identical. The detergent-solubilized microsomal NADH-cytochrome beta 5 reductase (I) is immunologically very similar to the soluble enzymes, but presents distinct features possibly due to the presence of a hydrophobic part.     

Enfuvirtide effects on human erythrocytes and lymphocytes functional properties.

Enfuvirtide (T-20) is the first inhibitor of human Immunodeficiency Virus type-1 (HIV-1) entrance on a target cell approved for clinical use. Recent studies indicated that its action mechanism involves the interaction with the membrane surface, increasing the concentration in the site of action. In the present study, the in vitro interaction between enfuvirtide and blood cells of healthy human donors, namely erythrocytes and lymphocytes, and the peptide effect on plasma and lymphocyte suspensions supernatant ions were evaluated, in order to better characterize the action of this peptide. Enfuvirtide causes a decrease in the concentration of hemoglobin and in the percentages of methemoglobin and carboxyhemoglobin, together with increased values of P50, pCO2, and [HCO3-], and significant decreases of pO2 and pH, in blood plasma. The supernatants of lymphocyte suspensions derived from blood incubated with enfuvirtide presented a decrease in pH and [HCO3-]. Fluorescence anisotropy measurements of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), used to assess erythrocyte and lymphocyte membrane fluidity, did not yield enfuvirtide-induced changes (an effect could be expected due to peptide partition to lipid bilayers). Erythrocytes incubated with high enfuvirtide concentrations showed a significant decrease in osmotic fragility. As for erythrocyte deformability, enfuvirtide leads to increased elongation indexes for low shear stress values, whereas for high shear stress values it has the opposite effect. Despite the observed statistically significant variations in several parameters, these enfuvirtide-induced changes are not expected to lead to any detectable biomedical outcome for enfuvirtide-treated patients.     

Effects of some antibiotics on glutathione reductase activities from human erythrocytes in vitro and from rat erythrocytes in vivo

The effects of streptomycin sulfate, gentamicin sulfate, thiamphenicol, penicillin G, teicoplanin, ampicillin, cefotaxime, and cefodizime on the enzyme activity of glutathione reductase (GR) were studied using human and rat erythrocyte GR enzymes in in vitro and in vivo studies, respectively. The enzyme was purified 5,342-fold from human erythrocytes in a yield of 29% with 50.75 U/mg. The purification procedure involved the preparation of hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies, and rat erythrocyte hemolysate was used in the in vivo studies. In the in vitro studies, I50 and K(i) values were 12.179 mM and 6.5123 +/- 4.1139 mM for cefotaxime, and 1.682 mM and 0.7446 +/- 0.2216 mM for cefodizime, respectively, showing the inhibition effects on the purified enzyme. Inhibition types were noncompetitive for cefotaxime and competitive for cefodizime. In the in vivo studies, 300 mg/kg cefotaxime and 1000 mg/kg cefodizime when administered to rats inhibited enzyme activity during the first 2h (p < 0.01). Cefotaxime led to increased enzyme activity at 4h (p < 0.05), but neither cefotaxime nor cefodizime had any significant inhibition or activation effects over 6 h (p > 0.05).     

Effects of some antibiotics on glutathione reductase activities from human erythrocytes in vitro and from rat erythrocytes in vivo

The effects of streptomycin sulfate, gentamicin sulfate, thiamphenicol, penicillin G, teicoplanin, ampicillin, cefotaxime, and cefodizime on the enzyme activity of glutathione reductase (GR) were studied using human and rat erythrocyte GR enzymes in in vitro and in vivo studies, respectively. The enzyme was purified 5,342-fold from human erythrocytes in a yield of 29% with 50.75?U/mg. The purification procedure involved the preparation of hemolysate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies, and rat erythrocyte hemolysate was used in the in vivo studies. In the in vitro studies, I₅₀ and K_i values were 12.179?mM and 6.5123±4.1139?mM for cefotaxime, and 1.682?mM and 0.7446±0.2216?mM for cefodizime, respectively, showing the inhibition effects on the purified enzyme. Inhibition types were noncompetitive for cefotaxime and competitive for cefodizime. In the in vivo studies, 300?mg/kg cefotaxime and 1000?mg/kg cefodizime when administered to rats inhibited enzyme activity during the first 2?h (p<0.01). Cefotaxime led to increased enzyme activity at 4?h (p<0.05), but neither cefotaxime nor cefodizime had any significant inhibition or activation effects over 6?h (p>0.05).     

Age-dependent decay of cytochrome b5 and cytochrome b5 reductase in human erythrocytes.

Age-dependent decrease in cytochrome b5 was observed in erythrocytes from both a normal person and a patient with hereditary methaemoglobinaemia without neurological symptoms. With aging, concentrations of cytochrome b5 in erythrocytes from the patient were almost the same as those in the control. Age-dependent decrease in cytochrome b5 reductase activity in the control erythrocytes was also shown; however, the reductase activity was very low in erythrocytes from the patient over the whole age range. Our studies show that methaemoglobin content of erythrocytes seems to be dependent on the content of cytochrome b5 in the cells, both in the control subject and in the patient.     

Purification and properties of NADH-specific dihydropteridine reductase from human erythrocytes

NADH-specific dihydropteridine reductase (EC 1.6.99.7) has been purified from human erythrocytes in essentially homogeneous form. The molecular weight of the enzyme was estimated to be 46,000 by Sephadex G-100 gel filtration. The enzyme reacted with antiserum against NADH-specific dihydropteridine reductase from bovine liver and formed a single immunoprecipitin line in the Ouchterlony double-diffusion system. This precipitin line completely fused with that formed between the human liver enzyme and the antiserum. With use of 5,6,7,8-tetrahydro-6-methylpterin, Km values of the erythrocyte enzyme for NADH and NADPH were determined to be 0.94 and 47 mumol/l, respectively. Vmax values were 58.7 mumol/min/mg with NADH and 6.41 mumol/min/mg with NADPH. The average activity of NADH-specific dihydropteridine reductase of 9 human blood samples from healthy males (20-25 years old) was calculated to be approximately 600 mU/g of hemoglobin, 1.8 mU per 20 microliters of blood, or 1.9 mU per 10(8) erythrocytes.     

Prooxidative effects of TEMPO on human erythrocytes

The prooxidative effects of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were observed in human erythrocytes. Incubation of red blood cells with the membrane-permeable TEMPO leads to a decrease in the concentration of intracellular reduced glutathione, accompanied by the reduction of TEMPO. Extracellular ferricyanide inhibited the loss of glutathione and reduction of TEMPO. TEMPO induced glutathione release from the cells and oxidation of hemoglobin to methemoglobin; ferricyanide prevented these effects. These results indicate that TEMPO may act as an oxidant to erythrocytes, whilst extracellular ferricyanide protects against its effects.     

Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.     

The decreased membrane fluidity of in vivo aged, human erythrocytes. A spin label study.

The decreased membrane fluidity of the in vivo aged, human erythrocytes is found, by monitoring the electron paramagnetic resonance (EPR) spectra of fatty acid spin labels incorporated into the membrane. In addition, the decreased cell sizes and the decreased cholesterol and phospholipids contents, without significant changes of the quantity of the membrane proteins, also the decrease of ATP and 2,3-diphosphoglycerate and the increase of ADP and AMP, in the aged cells, were observed. Further the functional impairments of the aged cells, i.e. the increased oxygen affinity and the decreased deformability, were shown. On the basis of these quantitative data, the alteration of the protein-lipid organization, due to decreased lipid/protein ratio, the modified protein-lipid interaction and/or the influences of the diminished ATP content, is suggested to contribute towards the decreased membrane fluidity of the in vivo aged erythrocytes.     

A study on concanavalin a binding to human erythrocytes

Interactions of concanavalin A with human erythrocytes were studied using ¹²⁵I-labelled concanavalin A and a centrifugal technique with dibutyl phthalate which permitted complete separation of bound and free concanavalin A. Binding of ¹²⁵I-labelled concanavalin A to human erythrocytes was dependent on cell concentration, pH and temperature. Specificity of binding was confirmed by inhibition and dissociation studies with sugars and native concanavalin A. Positive cooperative binding of concanavalin A to human erythrocytes was observed at low concanavalin A concentrations (less than 1 μ/ml) in both buffers studied. Positive cooperativity at higher concanavalin A concentrations (more than 100 μ/ml) was seen in Tris-Hepes buffer but not in phosphate-buffered saline. Consistent with this cooperative effect was the observation that although dissociation of ¹²⁵I-labelled concanavalin A from the erythrocytes was complete in the presence of 1 mg/ml of the native lectin, release was inhibited by low concentrations (1 μ/ml). A comparison of concanavalin A binding with hemagglutination studies suggest that the amount of concanavalin A bound determines the rate of erythrocyte agglutination and the size of the aggregates formed.