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.     

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.     

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.     

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.     

Uptake and binding of dissaccharides in human erythrocytes.

Disaccharides (sucrose, lactose, melibiose, cellobiose, trehalose, maltose, and isomaltose) are not transported across the human erythrocyte membrane. Maltose alone is bound in appreciable amounts to the intact cell as well as ghost membranes and competes mutually for uptake with D-glucose. In (NH4)2-SO4-precipitated membrane preparations, maltose binds more strongly than other disaccharides (KD = 1.3 X 10(-5) M; maximum binding capacity, 71 pmol/mg protein) and again competes mutually with D-glucose. Phloretin inhibits the binding of glucose much more than that of maltose.     

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.     

Electro-orientation of ellipsoidal erythrocytes. Theory and experiment.

The frequency-dependent orientation of human and llama erythrocytes suspended in isotonic solutions and subjected to linearly polarized electric fields is examined. Human erythrocytes may be represented as oblate spheroids (3.9:3.9:1.1 microns) with two distinguishable orientations, while the llama cells are approximated as ellipsoids with three distinct axes (4.0:2.0:1.1 microns). Under appropriate experimental conditions, both orientations of the human cells and all three orientations of the llama cells are observed. A theoretical cell model which accounts for the membrane as a thin confocal layer of ideal capacitance is used to predict the orientational spectra. The predicted spectra compare favorably in frequency range and orientational sequence with experimental data. Estimates for cell internal conductivity and permittivity are obtained by adjusting the values of these important parameters to achieve the closet fit of the theoretical curves to the data. By the use of this method, the internal conductivity of llama erythrocytes is estimated to be 0.26 S/m (+/- 20%), while the effective internal dielectric constant and conductivity of Euglena gracilis are estimated to be 120 (+/- 10%) and 0.43 S/m (+/- 20%), respectively.     

Flicker in erythrocytes. I. Theoretical models and registration techniques

The phenomenon of stochastic low-frequency oscillations of erythrocyte cell membrane, termed usually the flicker of erythrocytes, is reviewed. The first part describes the theoretical models of erythrocyte flickering and the registration techniques. The relations are given and analyzed which connect the shape of both the frequency and the spatial spectra of stochastic membrane oscillations with geometrical and mechanical parameters of the erythrocyte and with the ambient physical characteristics. The existing concepts of excitation mechanisms of the membrane flickering are presented.     

Hypoxanthine phosphoribosyltransferase and hypoxanthine uptake in human erythrocytes.

A system of hypoxanthine uptake and IMP retention was studied and characterized in human erythrocytes. It follows closely the system already described for rabbit erythrocytes[7]. IMP formation and retention are dependent on the activity of hypoxanthine phosphoribosyl-transferase and on intracellular availability of phosphoribosyl pyrophosphate (P-Rib-PP), which is one of the substrates. In the extrecellular medium, neither P-Rib-PP nor GMP -- a potent inhibitor of the enzyme in vitro -- has any influence on IMP retention. The amount of residual hypoxanthine phosphoribosyltransferase in erythrocyte ghost preparations is directly related to the residual hemoglobin content. Thus the enzyme is characterized as typically soluble and "loosely bound" to membranes. There is a slight difference in the kinetic properties of the ghost-bound and the free soluble enzyme. The possible importance of these results for purine uptake and utilization in human red cells is discussed.