Kinetics of chloride-bicarbonate exchange across the human red blood cell membrane

We use a fluorescent probe of [Cl-], 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), to study Cl-/HCO-3 exchange in human erythrocyte ghosts in a stopped-flow apparatus at 4 degrees C. The quench constant of SPQ in our Cl-/HCO-3/HPO=4 system at pH 7.4 is 0.065 +/- 0.005 mM-1. The time course of Cl-/HCO-3 exchange does not follow a single exponential function at 4 degrees C and we propose an extended ping-pong model in which slippage is explicitly considered in order to account for this phenomenon. The solution of the system of equations generated by our model is a double exponential function which fits the time course of Cl-/HCO-3 exchange. Our results confirm the predictions of the model concerning the functional dependence of the two rate constants. One rate constant (k1) is independent of medium composition; it is determined by the sum of the two slippage rate constants and its value is 1.04 +/- 0.14 sec-1. The other rate constant (k2) varies inversely with [Cl-]; the regression line is 1/k2 = 18.8 sec - 0.095 mM-1sec [Cl-].     

Kinetics of bicarbonate-chloride exchange across the human red blood cell membrane

The kinetics of bicarbonate-chloride exchange across the human red cell membrane was studied by following the time course of extracellular pH in a stopped-flow rapid-reaction apparatus during transfer of H+ into the cell by the CO2 hydration-dehydration cycle, under conditions where the rate of the process was determined by HCO3--Cl- exchange flux across the membrane. The flux of bicarbonate increased linearly with [HCO3-] gradient from 0.6 to 20 mM across the red cell membrane at both 37 degrees C and 2 degrees C, and decreased as transmembrane potential was increased by decreasing extracellular [Cl-]. An Arrhenius plot of the rate constants for the exchange indicates that the Q10 is strongly dependent on temperature, being about 1.7 between 24 degrees C and 42 degrees C and about 7 between 2 degrees C and 12 degrees C. These data agree well with the published values for Q10 of 1.2 between 24 degrees C and 40 degrees C and of 8 between 0 degrees C and 10 degrees C. The results suggest that different processes may determine the rate of HCO3--Cl- exchange at low vs. physiological temperatures, and that the functional (and/or structural) properties of the red cell membrane vary markedly with temperature.     

Rabbit red blood cell hexokinase. Purification and properties

Rabbit red blood cell hexokinase (EC 2.7.1.1.) has been purified 300,000-fold by a combination of ion exchange chromatography, affinity chromatography, and preparative polyacrylamide gel electrophoresis. The hexokinase activity has been isolated in 35% yield as a protein that is homogeneous by polyacrylamide and sodium dodecyl sulfate gel electrophoresis. The highest specific activity obtained was 145 units/mg of proteins. The native protein has a molecular weight of 110,000 by gel filtration on Ultrogel AcA 44 and 112,000 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a molecular weight of 110,000 indicating that hexokinase is a monomer. The enzyme had a pI of 6.20 to 6.30 pH units by isoelectric focusing. The enzyme was specific for Mg . ATP and Mg . ITP as the nucleotide substrates. Several hexokinase with different affinities.     

X-ray and neutron scattering density profiles of the intact human red blood cell membrane

Hemoglobin-free human red blood cell membranes have been prepared with glutaraldehyde to maintain an intact structure on partial dehydration. Treatment of resealed ghosts with poly(L-lysine) produced an essentially constant structural unit and permitted correlation of electron microscopy results with x-ray and neutron diffraction profiles. These profiles provide detailed information, for the intact membrane, on the location and relative distribution of lipids and proteins.     

The effect of 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate on CO2 permeability of the red blood cell membrane

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. The disappearance of 12C18O16O from a red cell suspension as the 18O exchanges between labeled CO2 + HCO3- and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or A) inside the cell and of the membrane self-exchange permeability to HCO3- (Pm,HCO-3). To test this technique, we added sufficient 4, 4'-diisothiocyanato-stilbene-2,2'-disulfonate (DIDS) to inhibit all the HCO3-/Cl- transport protein (Band III or capnophorin) in a red cell suspension. We found that DIDS reduced Pm,HCO-3 as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in Pm,CO2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO2 and HCO3-, we found that DIDS inhibited both Pm,HCO-3 and Pm,CO2, whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO2 exchange.     

Current red blood cell transfusion practices

The appropriate use of blood transfusions remains variable among health-care institutions and patient populations. Transfusion practices are discussed in this article in relation to medical practice guidelines and utilization review. Specific transfusion practices in the settings of intensive care, orthopedic surgery, and open heart surgery are reviewed. A new, promising approach to improving transfusion outcomes is the use of transfusion algorithms. Transfusion algorithms may prove especially useful if they incorporate point-of-care testing that is both physiologic and patient-specific for transfusion decisions. Transfusion algorithms are discussed and data presented for cardiac surgical adults.     

The I antigen of human red cell membrane

A high-active I antigen was isolated from human red cells after papainization. Investigations on its chemical composition and its serological properties are reported. 1. The I antigen activity was clearly demonstrated by hemagglutination inhibition studies and by the immuno-double-diffusion with all available anti-I sera. 2. The I antigen did not react with other antibodies directed against red cell antigens thus proving its specificity. Any relationships to antigen activities within the Pr-1/Pr-2, MN, and ABO systems could be excluded. 3. The substance was shown to be a glycoprotein and not a glycolipid. This was confirmed by different delipidation procedures promoting always an increase of I activity. The delipidized material contained only traces of fatty acids, and did not move on thin-layer chromatography in solvent systems normally used for glycolipid development. 4. The I determinant resides on alkali-stable oligosaccharide chains. The main sugars are galactose and N-acetylglucosamine which might be involved in the immunodeterminants.     

Interaction of glucose and metformin with isolated red cell membrane

Isolated human erythrocyte membranes (red blood cell (RBC) ghosts) were incubated with glucose at 5, 10, 20 and 100 mmol/l concentrations, with insulin (0.01 to 200 mU/l) and metformin (CAS 657-24-9) 0.5 up to 50.0 mumol/l). Binding studies with 14C-glucose and subsequent gel electrophoresis revealed 60% of the radioactivity around ban 4.2-4.5 at 5 mmol/l, whereas a random distribution of radioactivity over all protein bands of the RBC membrane was found at 20 mmol/l concentration after incubation for 30 min or 48 h. Metformin does not bind covalently to RBC membranes, however, after photochemical linkage of 14C-metformin via the aminoreactive linker azidophenylglyoxal the highest radioactivity (21%) was counted in the range of band 4.2-4.5. In parallel with an increase of order parameters of 5-doxyl-stearic acid the thiol status of the membranes decreases as determined by monobromobimane fluorescence. 20 and 100 mmol/l concentrations of glucose decrease the reactivity of membrane thiols towards bromobimane significantly to 73 and 62% of the controls. Concomitantly, membrane fluidity at polar sites is diminished as measured by order parameters of spin label 5-doxyl stearic acid. In RBC membranes pretreated with 20 mmol/l glucose the decreased fluorescence is significantly raised again by insulin and metformin. This effect is even more pronounced, if insulin and metformin are incubated together. Reaction of membrane thiols with a maleimido spin label detects modification in the ratio of mobile and immobilized spin label populations in the electron paramagnetic resonance signal under the above conditions, indicative of conformational changes of membrane proteins.     

Dietary menhaden and corn oils and the red blood cell membrane lipid composition and fluidity in hyper- and normocholesterolemic miniature swine

This paper describes the design and synthesis of a tricationic transition state analogue (TSA 1) for the Diels-Alder reaction. TSA 1 contains a bicyclo[2.2.1]heptene ring system that mimics the boat conformation of the Diels-Alder transition state and is designed to bind tightly to antibodies, nucleic acids, and imprinted polymers by means of hydrogen bonds and salt-bridges. This paper also describes the syntheses of the Diels-Alder reaction substrates (diene 2 and dienophile 3) and a sensitive HPLC assay to monitor the formation of Diels-Alder product 4. In contrast to previously reported TSAs and dienophiles for the Diels-Alder reaction that are based upon maleimides, TSA 1 and dienophile 3 are based upon fumaramide. The fumaramide system should destabilize the initially formed boat conformer of Diels-Alder product 4 and stabilize a half-chair conformer. The conversion of the initially formed boat conformer to the half-chair conformer is designed to help prevent Diels-Alder product 4 from binding strongly to catalysts selected to strongly bind TSA 1. This feature should minimize product inhibition, which can be a problem in the catalysis of the Diels-Alder reaction.     

Contribution of whole cell and cytoplasmic polypeptides to apparent red cell membrane alterations

We have compared densitometric tracings of whole cell, cytoplasmic and membrane polypeptide electrophoretic patterns in an attempt to distinguish atypical partitioning from intrinsic membrane polypeptide changes occurring as a result of reticulocyte enrichment, metabolic depletion, N-ethylmaleimide treatment and hereditary xerocytosis. We report that membrane alterations seen in a reticulocyte-enriched population of normal cells are present in the whole cells prior to membrane isolation. Some of the membrane alterations in metabolically depleted cells and all of those in N-ethylmaleimide-treated cells are traced to modifications in tary xerocytosis. We report that membrane alterations seen in a reticulocyte-enriched population of normal cells are present in the whole cells prior to membrane isolation. Some of the membrane alterations in metabolically depleted cells and all of those in N-ethylmaleimide-treated cells are traced to modifications in the partitioning of polypeptides between membranes and supernatant (cytoplasm) at hemolysis. The power of this approach in resolving the sources of apparent red cell membrane protein alterations is demonstrated in studies with hereditary xerocytes. Suggested altered partitioning of these cells described earlier (Sauberman, N., Fortier, N.L., Fairbanks, G., O'Connor, R.J. and Snyder, L.M. (1979) Biochim. Biophys. Acta 556, 292-313) is further documented and found to be unrelated to the younger cell population or slight metabolic depletion that occurs during the washing of xerocytes prior to hemolysis.     

The effect of cetiedil on red cell membrane permeability

We report the effects of cetiedil, a new antisickling agent, on red cell membrane permeability. With fresh red cells containing normal levels of intracellular ATP, cetiedil increases membrane permeability to both sodium and potassium. With drug concentrations from 100 to 500 microM, net sodium gain exceeds net potassium loss, and the cells quickly swell. Changes are identical with normal and sickle red cells. Membrane permeability returns to normal after washing the cells in buffer free of cetiedil. In the absence of phosphate, ouabain potentiates the cetiedil effect. With external phosphate present, the effect of cetiedil is also enhanced, but ouabain is without effect. Our findings support the idea that the antisickling effect of cetiedil observed in vitro is secondary to cell swelling.     

The mechanism of chlorpromazine-induced red blood cell swelling

1. Chlorpromazine (CPZ)-induced red blood cell (RBC) swelling was investigated by determination of electrolyte, mean cell volume (MCV) and water content changes in CPZ-treated cells. 2. CPZ-induced RBC swelling is dose-, hematocrit- and pH-dependent, and is associated with a net increase in RBC monovalent cation and Cl- contents. 3. The partition coefficient (Kp) for the CPZ-RBC membrane interaction is pH dependent (Kp = 2500 at pH 7.8; Kp = 840 at pH 6.8). 4. Despite the pH dependence of Kp values an equal number of CPZ molecules is required to induce a 12% increase in MCV at pH 7.8 and 6.8.     

Oleic acid binding and transport capacity of human red cell membrane

Resealed human red cell membranes, ghosts, bind oleate (OL) by a limited number of sites when equilibrated at 37 degrees C, pH 7.3 with OL bound to bovine serum albumin (BSA) in molar ratios below 1.5. The binding capacity is 34 +/- 2.2 nmol g-1 ghosts with a dissociation equilibrium constant (37 degrees C) Kdm 1.38 +/- 0.15 fold Kd of albumin binding Kdm is temperature independent and approximately 7-8 nM. Exchange efflux kinetics at 0 degrees C to buffers of various albumin concentrations ([BSAy]) is biexponential and is analysed in terms of a three-compartment model. Accordingly the ratio of inner to outer membrane leaflet binding sites is 0.450 +/- 0.018 and the rate constant of unidirectional flux from inside to outside is 0.067 +/- 0.01 s-1. The rate constant of flux from the extracellular side of the membrane to BSAy increases with the square root of [BSAy] as expected of an unstirred layer effect. This provides an estimate of the dissociation rate constant of OL-BSA complex at 0 degrees C of 0.0063 +/- 0.0003 s-1. Exchange efflux from ghosts containing four different [BSAi] obeys the expected kinetics of a three-compartment approximation of the theoretical model. Accounting for the effect of an unstirred fluid inside ghosts, the rate coefficients fit the values predicted by the parameters obtained by the studies of albumin-free ghosts. The results show that the OL transport across the membrane is mediated exclusively by the asymmetrically distributed binding sites. The differences between transport sites of three long-chain fatty acids suggest that they are protein determined microdomains of phospholipids.