Endocytosis in adenosine triphosphate-depleted erythrocytes.

The extent of membrane invagination or endocytosis in intact erythrocytes was quantified by measuring the loss of acetylcholinesterase activity. Primaquine-induced endocytosis was completely inhibited in ATP-depleted cells. However, chlorpromazine and vinblastine were capable of inducing membrane invagination in depleted cells. With both drugs, the loss of enzyme activity was less than that measured in fresh cells. We conclude that drug-induced endocytosis is not necessarily an energy-dependent process.     

Phosphoglycolate phosphatase in human erythrocytes.

Human erythrocytes were found to contain an enzyme capable of dephosphorylating phosphoglycolate. The rates of hydrolysis of 14 other metabolites by the enzyme preparation were less than 6% of the rate with phosphoglycolate. The pH optimum is in the range of 6 to 7 and the Km for phosphoglycolate is 0.76 mM. The molecular weight appears to be about 79,000. Such an activity has previously been reported only for plant cells.     

Iron-mediated oxidative stress in erythrocytes.

Erythrocytes subjected extracellularly to iron-mediated oxidant stress undergo haemoglobin oxidation and membrane damage, which can be modulated by maintaining the energy requirements of the cells. The results presented here suggest that a balance exists between the oxidation state of the haemoglobin and the oxidative deterioration of the membrane lipids, which is dependent on the metabolic state of the erythrocytes. These findings have important implications for thalassaemic erythrocytes that may be exposed to excess plasma iron levels, in which excessive membrane-bound iron in the form of haemichromes is a characteristic feature and in which cellular ATP levels are lowered.     

Membrane proteins in senescent erythrocytes.

The examination of erythrocyte senescence has been facilitated by recent advances in techniques for the isolation of aged red cells. One of these methods, which uses biotinylated rabbit erythrocytes, has been used to examine the state of membrane proteins in effete cells. These aged red cells were found to have normal ratios of alpha-spectrin and beta-spectrin as well as normal levels of ankyrin. The observation concerning ankyrin is particularly important due to the sensitivity of this protein to proteolysis and the postulated action of proteinases in the aging process. The senescent erythrocytes were also found to have an altered ratio of bands 4.1a and 4.1b without any apparent change in the total level of 4.1. In addition, the analysis of the aged cell membranes did not show any large-molecular-mass aggregated protein at the origin of the SDS/polyacrylamide gels, indicating a lack of transglutaminase activity in the senescence process for rabbit erythrocytes. These results indicate that aging of the rabbit erythrocyte is not accompanied by gross proteolytic degradation or transglutaminase-catalysed cross-linking of membrane components.     

Glucokinase and hexokinase in pig erythrocytes.

We have shown different enzymatic activities responsible for the phosphorylation of glucose in pig erythrocytes. These activities were observed after partial purification from hemolyzed red cells. One of the enzymes involved is the hexokinase which is present in all tissues; the other is similar to hepatic glucokinase. We have determined the kinetic properties of these activities in hemolysates and in partially purified preparations. Their electrophoretic-migration characteristics were studied too.     

Changes of nonelectrolyte permeability in cholesterol-loaded erythrocytes.

Membrane cholesterol in porcine and bovine erythrocytes was elevated up to 165% of its normal value by incubation of the cells in cholesterol/phosphatidylcholine dispersions with or without serum. This alteration of membrane lipid composition brought about only a minor (10-40%) decrease of the permeability to glycerol, erythritol and to organic acids penetrating by non-ionic diffusion, although additional cholesterol had actually been incorporated into the lipid bilayer, as indicated by determinations of cell surface area from the critical hemolytic volume, in combination with quantitative evaluation of freeze-etch electron micrographs. On the basis of this finding and of the previously demonstrated (Grunze, M. and Deuticke, B. (1974) Biochim. Biophys. Acta 356, 125-130) considerable increase of permeability in cholesterol-depleted cells, it is proposed that in the erythrocyte membrane a pronounced "specific" reduction of permeability by cholesterol occurs only up to a molar ratio cholesterol/polar lipid of 0.6. At higher ratios cholesterol affects permeability only slightly, owing to an "unspecific" rigidifying effect on the membrane lipid phase.     

Contact patterns in concanavalin A agglutinated erythrocytes.

Agglutination of human erythrocytes by the lectin concanavalin A is enhanced when the erythrocytes are pretreated with neuraminidase, which removes sialic acids, or with pronase, which degrades both the glycophorins and band 3 protein. In the present work transmission electron microscopy of the enzymatically pretreated erythrocytes shows a regular pattern of interruption of contact between interacting plasma membranes. The lengths characteristic of the pattern were 0.66 and 0.50 microns for pronase- and neuraminidase-pretreated cells, respectively. Agglutination of normal erythrocytes and of neuraminidase-pretreated erythrocytes can be fully reversed by exposure to the competitive inhibitor methyl alpha-D-mannopyranoside. Complete reversal of contact does not occur with pronase-pretreated cells. The comparatively greater tenacity of contact between cells that were treated with pronase before exposure to lectin argues for an involvement of nonspecific interactions in the agglutination process. The results are compared with previously published studies of spatially periodic contact patterns induced by a range of other polymers.     

Agonist-stimulated inositol phosphate metabolism in avian erythrocytes.

1. A screen for agonists capable of stimulating the formation of inositol phosphates in erythrocytes from 5-day-old chickens revealed the presence of a population of phosphoinositidase C-linked purinergic receptors. 2. If chicken erythrocytes prelabelled with [3H]Ins were exposed to a maximal effective dose of adenosine 5'-[beta-thio]diphosphate for 30 s, the agonist-stimulated increment in total [3H]inositol phosphates was confined to [3H]Ins(1,4,5)P3, Ins(1,3,4,5)P4 and InsP2. After 40 min stimulation, the radiolabelling of nearly all of the [3H]inositol phosphates that have been detected in these extracts [Stephens, Hawkins & Downes (1989) Biochem. J. 262, 727-737] had risen. However, some of these increases [especially those in Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5] were accountable for almost entirely by increases in specific radioactivity rather than in mass. 3. The effect of purinergic stimulation on the rate of incorporation of [32P]Pi in the medium into the gamma-phosphate group of ATP and InsP4 and InsP5 was also measured. After 40 min stimulation, the incorporation of 32P into Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5 was significantly elevated, whereas the mass of the last two and the specific radioactivity of the gamma-phosphate of ATP were unchanged compared with control erythrocyte suspensions. 4. In control suspensions of avian erythrocytes, the specific radioactivity of the individual phosphate moieties of Ins(1,3,4,6)P4 increased through the series 1, 6, 4 and 3 [Stephens & Downes (1990) Biochem. J. 265, 435-452]. This pattern of 32P incorporation is not the anticipated outcome of 6-hydroxy phosphorylation of Ins(1,3,4)P3 [the assumed route of synthesis of Ins(1,3,4,6)P4]. Although adenosine [beta-thio]diphosphate significantly stimulated the accumulation of [3H]Ins(1,3,4)P3, and despite the fact that avian erythrocyte lysates were shown to possess a chromatographically distinct, soluble, ATP-dependent, Ins(1,3,4)P3 6-hydroxykinase activity, purinergic stimulation of intact cells did not significantly alter the pattern of incorporation of [32P]Pi into the individual phosphate moieties of Ins(1,3,4,6)P4. These results suggest that the route of synthesis of this inositol phosphate species is not changed during the presence of an agonist.     

Phosphate compounds in rat erythrocytes and reticulocytes.

The concentrations of glycolytic intermediates, including 2,3-diphosphoglycerate, were similar in rat reticulocytes and erythrocytes. There were striking differences, however, in the content and kind of water-soluble nucleotides. Reticulocytes contained much higher concentrations of ATP, GTP, UTP and CTP and had nucleotides not detected in the mature cell including UDP-acetylhexosamine, guanosine diphosphomannose and an unidentified cytidine compound. A large fraction of the total GTP found in the reticulocyte was in the form of a 1:1 complex of ferric iron with GTP.     

cGMP and glutathione-conjugate transport in human erythrocytes.

The nature of cGMP transport in human erythrocytes, its relationship to glutathione conjugate transport, and possible mediation by multidrug resistance-associated proteins (MRPs) have been investigated. MRP1, MRP4 and MRP5 are detected in immunoblotting studies with erythrocytes. MRP1 and MRP5 are also detected in multidrug resistant COR-L23/R and MOR/R cells but at greatly reduced levels in the parent, drug sensitive COR-L23/P cells. MRP4 is detected in MOR/R but not COR-L23/R cells. Uptake of cGMP into inside-out membrane vesicles prepared by a spontaneous, one-step vesiculation process is shown to be by a low affinity system that accounts for more than 80% of the transport at all concentrations above 3 micro m. This transport is reduced by MRP inhibitors and substrates including MK-571, methotrexate, estradiol 17-beta-d-glucuronide, and S(2,4-dinitrophenyl)glutathione (DNP-SG) and also by glibenclamide and frusemide but not by the monoclonal Ig QCRL-3 that inhibits high-affinity transport of DNP-SG by MRP1. It is concluded that the cGMP exporter is distinct from MRP1 and has properties similar to those reported for MRP4. Furthermore the evidence suggests that the protein responsible for cGMP transport is the same as that mediating low-affinity DNP-SG transport in human erythrocytes.     

Sugar transport in reversibly hemolyzed avian erythrocytes.

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37 degrees C, 88% of the ghosts regained their permeability barrier to L-glucose. In these ghosts, the carrier-mediated rate of entry of 3-O-methylglucose was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.     

Ionophore-induced oscillations in erythrocytes. A kinetic model

The kinetic model for K+, H+, Ca2+ concentrational self-oscillations in erythrocytes induced by A23187 and FCCP ionophores are considered. The model is based on the experimental data available and contains the minimal number of essential variables. The model was analysed by the method based on the graph representation of kinetic equations. The critical graph fragments provoking self-oscillatory trajectories in the system were revealed. It is shown that self-oscillatory behaviour is basically induced by conjugated processes produced by A23187. The parameter domain for self-oscillations is estimated including parameters of Ca2+-induced (through K+-channels) proton transport with FCCP participation. Numerical integration of kinetic equations was performed. The results obtained are in a good qualitative agreement with experimental data.     

Patterns of purine nucleotides in fish erythrocytes.

1. The purine nucleotides were determined in the whole blood of 9 fresh water teleosts and 2 marine selachians. 2. GTP and ATP accounted for 88-99% of the total erythrocytes purines. 3. The ATP/ADP ratio ranged from 11 to 60 in the erythrocytes of the fish examined. 4. GTP is widely distributed in fish erythrocytes but its level ranged from 1 to 33 nmol/mg Hb (0.4 to 9 mumol/ml erythrocyte). 5. Lepomis and Esox exhibited a GTP/ATP ratio as elevated as in Anguilla; moreover the concentration of GTP per mol of Hb (physiologically most indicative) is higher in Lepomis, Esox, Ictalurus and Silurus than in Anguilla.     

Actin-activated ATPase from human erythrocytes.

A fibrillar protein complex, possessing ouabain-insensitive Ca2+-ATPase activity was isolated from human erythrocyte membranes by using a low ionic strength extraction procedure. Mg2+-ATPase activity was revealed upon addition of rabbit skeletal muscle actin, thus demonstrating the presence of a myosin-like protein in the crude extract of the erythrocyte membrane. Upon sodium dodecylsulfate gel electrophoresis, the extract showed mainly the doublet of subunit molecular weight bands of 230 000 and 210 000, and more than 10 faster moving bands. Gel filtration of the erythrocyte membrane extract on Sepharose 4B furnished 4 fractions. Fraction I, containing the doublet and 80 000, 60 000 and 46 000 subunit molecular weight bands was 5-fold purified with respect to Ca2+-ATPase activity, but was devoid of actin-activated Mg2+-ATPase activity. Fraction II, containing only the doublet, was devoid of Ca2+ and actin-activated Mg2+-ATPase activity. The 210 000 subunit molecular weight protein could be phosphorylated in the presence of Mg2+ in the crude extract and Fraction I but not in Fraction II.     

Taurine uptake in chicken leukocytes and erythrocytes.

1. The intracellular taurine concentration and rate of taurine uptake of chicken erythrocytes and two leukocyte populations were determined from one to six weeks of age. 2. Plasma taurine concentrations increased significantly from the time of hatching to week 2 and remained constant thereafter. 3. Intracellular taurine concentrations in both leukocyte populations increased significantly with age without any significant change in the erythrocytes. 4. Taurine uptake rate for erythrocytes was significantly higher at weeks 1-3 while both leukocyte populations showed no significant change during the six week period studied.     

Lipid peroxidation in Plasmodium falciparum-parasitized human erythrocytes.

cis-Parinaric acid (PnA) was used as a fluorescent probe to study lipid peroxidation in nonparasitized and Plasmodium falciparum-parasitized erythrocytes, upon challenge by cumene hydroperoxide and tert-butyl hydroperoxide. Parasitized erythrocytes were less susceptible toward lipid peroxidation than nonparasitized erythrocytes with which they had been cultured. Furthermore, nonparasitized erythrocytes cultured together with parasitized cells, and thereafter isolated on a Percoll gradient, were less susceptible toward lipid peroxidation than erythrocytes kept under the same experimental conditions but in the absence of parasitized cells. We concluded, therefore, that the intracellular development of the parasite leads to an increase in the resistance against oxidative stress, not only of the host cell membrane of the parasitized erythrocyte, but also in the plasma membrane of the neighboring cells. The erythrocyte cytosol of parasitized cells and/or the intraerythrocytic parasite was required for the increased protection of the host cell membrane, since ghosts prepared from parasitized erythrocytes were more susceptible to lipid peroxidation than those prepared from nonparasitized ones. Vitamin E content of parasitized erythrocytes was lower than that of nonparasitized cells. However, parasitized erythrocytes promoted extracellular reduction of ferricyanide at higher rates, which might be indicative of a larger cytosolic reductive capacity. It is suggested that the improved response of intact erythrocytes is due to an increased reduction potential of the host-erythrocyte cytosol. The role of vitamin C as a mediator of this process is discussed.