Phospholipid dependence of the anion transport system of the human erythrocyte membrane. Studies on reconstituted band 3/lipid vesicles

Band 3 protein extracted from human erythrocyte membranes by Triton X-100 was recombined with the major classes of phospholipid occurring in the erythrocyte membrane. The resulting vesicle systems were characterized with respect to recoveries, phospholipid composition, protein content and vesicle size as well as capacity and activation energy of sulfate transport. Transport was classified into band-3-specific fluxes and unspecific permeability by inhibitors. Transport numbers (sulfate ions per band 3 per minute) served as a measure of functional recovery after reconstitution. The transport properties of band 3 proved to be insensitive to replacement of phosphatidylcholine by phosphatidylethanolamine, while sphingomyelin and phosphatidylserine gradually inactivated band-3-specific anion transport when present at mole fractions exceeding 30 mol%. The activation energy of transport remained unaltered in spite of the decrease in transport numbers. The results, which are discussed in terms of requirements of band 3 protein function with respect to the fluidity and surface charge of its lipid environment, provide a new piece of evidence that the transport function of band 3 protein depends on the properties of its lipid environment just as the catalytic properties of some other membrane enzymes. The well-established species differences in anion transport (Gruber, W. and Deuticke, B. (1973) J. Membrane Biol. 13, 19–36) may to some extent reflect this lipid dependence.     

Progressive oxidative membrane damage in erythrocytes after pulse treatment with t-butylhydroperoxide

Development of membrane damage in erythrocytes in the presence of the radical-forming oxidant t-butylhydroperoxide is a well established fact (see, for example, Deuticke et al. (1986) Biochim. Biophys. Acta 854, 169-183). We have now demonstrated that a mere pulse treatment of erythrocytes (5-15 min) with this agent leads to subsequent development of progressive oxidative membrane damage in spite of the absence of exogenous oxidant. Damage comprises the occurrence of ion leakiness and subsequent colloid-osmotic lysis, enhancement of the transbilayer mobility of phospholipid analogues, and lipid peroxidation. There is, however, only very little concomitant oxidation and precipitation of hemoglobin. Defect formation is not due to oxidation of SH-groups nor is it directly related to lipid peroxidation, since it can be suppressed by thiourea without concommitant inhibition of lipid peroxidation. This 'spontaneous' development of membrane damage can be antagonized by metabolic substrates and by desferrioxamine, indicating that lack of protective metabolic resources as well as the presence of catalytic metal (iron) complexes are required for the development of membrane damage. This progressive development of injury in cells only temporarily exposed to an exogenous oxidant may be regarded as a more appropriate model for oxidative membrane damage under pathophysiological conditions in vivo than cells exposed to continuous damage by exogenous oxidants.     

Relationship between gramicidin conformation dependent induction of phospholipid transbilayer movement and hexagonal HII phase formation in erythrocyte membranes

Addition of gramicidin in sufficient concentration from dimethylsulfoxide or trifluoroethanol to isolated erythrocyte membranes induces hexagonal HII phase formation for the phospholipids. In contrast, addition from ethanol does not change the overall bilayer organization despite a similar extent of peptide incorporation. The same solvent dependence is observed for the enhancement of transbilayer reorientation of lysophospholipids and unspecific leak formation in intact erythrocytes at lower gramicidin concentrations. These results indicate that the (beta 6.3) conformation of the peptide is essential for all three membrane perturbing effects.     

Possible relationship between membrane proteins and phospholipid asymmetry in the human erythrocyte membrane.

After incubation of human erythrocytes at 37 degrees C in the absence of glucose (A) for 24 h, (B) for 4 h with 8 mM hexanol or (C) for 3 h with SH reagents, phosphatidylethanolamine becomes partly susceptible to hydrolysis by phospholipase A2 from Naja naja. The presence of glucose during the pretreatments suppresses this effect, except in the case of SH reagents that inhibit glycolysis. After incubation with tetrathionate, up to 45% of the phosphatidylethanolamine is degraded by the enzyme, an amount considerably in excess of the 20% attacked in fresh erythrocytes. Pancreatic phospholipase A2, an enzyme unable to hydrolyse the phospholipids of intact erythrocytes, partially degrades phosphatidylcholine and phosphatidylethanolamine of erythrocytes pretreated with hexanol or SH reagents. Reagents capable of oxidizing SH groups to disulfides (tetrathionate, o-iodosobenzoate and hydroquinone) even render susceptible to pancreatic phospholipase A2 phosphatidylserine, a phospholipid supposed to be entirely located in the inner lipid layer of the membrane. Alkylating or acylating SH reagents have no such effect. It is postulated that disulfide bond formation between membrane protein SH groups leads to an alteration in protein-phospholipid interactions and consequently induces a reorientation of phospholipids between the inner and the outer membrane lipid layer.     

Experimental alteration of phospholipid-protein interactions within the human erythrocyte membrane. Dependence on glycolytic metabolism.

Phosphatidylethanolamine in freshly drawn human erythrocytes is trinitrophenylated by 2,4,6-trinitrobenzene sulfonic acid only slowly and to a maximum of 32%. After different preincubation procedures at 37 degrees C in saline media in the absence of glucose (24 h without additive, 1-5 h with 8 mM hexanol or 1-4 h with the SH reagent, 5 mM tetrathionate) the rate of subsequent trinitrophenylation of phosphatidylethanolamine, in the absence of the additives, is greatly enhanced and the amount of phospholipid reacting increased. Glucose or inosine prevent these effects, inhibitors of glycosis abolish this protection. The results indicate that in fresh as well as in glycolysing incubated erythrocytes phosphatidylethanolamine in the outer layer of the membrane lipid is shielded by a protein. Conformational changes of this protein induced by metabolic starvation and perturbing agents expose the phospholipid head group to 2, 4, 6-trinitrobenzene sulfonic acid. In addition, a "flip-flop" of phosphatidylethanolamine from the inner to the outer layer may also contribute to the effects observed.     

Dielectric breakdown of the erythrocyte membrane enhances transbilayer mobility of phospholipids

Dielectric breakdown of erythrocytes is shown to result in a loss of asymmetry of phosphatidylethanolamine and in a markedly enhanced transbilayer mobility of exogenous lysophosphatidylcholine. The effect is much more pronounced in non-resealed cells than in cells resealed after the breakdown. A casual relationship between the structural defects in the lipid phase, indicated by these results, and fusion by dielectric breakdown is discussed.     

Errata

Human erythrocytes were exposed to oxidative stress by iodate and periodate. Oxidation causes a time- and concentration-dependent increase in membrane permeability for hydrophilic molecules and ions. The induced leak discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy (E_a = 1–4 kcal · mol^?1). These results are reconcilable with the formation of aqeous pores. The pore size was approximated to be between 0.45 and 0.6 nm. This increase in permeability is reversible upon treatment with dithioerythritol. Blocking of membrane thiol groups with N-ethylmaleimide protects the membranes against leak formation. The oxidation causes dithioerythritol-reversible modification of membrane proteins as indicated by the gel electrophoretic behavior. These modifications can also be suppressed by blocking the membrane thiol groups with N-ethylmaleimide. About half of the membrane methionine is oxidized to acid hydrolysis-stable derivatives. A fast saturating increase in diene conjugation was observed in whole cells but not in isolated membranes, with only minor degradation of fatty acid chains. The oxidation of cell membrane lipids as well as oxidation of cell surface carbohydrates are not involved in leak formation. Taken together with earlier data (Deuticke, B., Poster, B., Lütkemeier, P., and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196–210), these findings indicate that formation of disulfide bonds by different oxidative mechanisms results in leaks with similar properties.