The transbilayer movement of phosphatidylcholine in vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

Vesicles have been prepared from 18 : 1_c/18 : 1_c-phosphatidylcholine with or without purified glycophorin or partially purified band 3 (obtained by organomercurial gel chromatography). The vesicles have been characterized by freeze-fracture electron microscopy, binding studies to DEAE-cellulose, ³¹P-NMR and K⁺ trap measurements. Pools of phosphatidylcholine available for exchange have been investigated using phosphatidylcholine exchange protein from bovine liver. The protein-containing vesicles both exhibit exchangeable pools larger than the fraction of phosphatidylcholine in the outer monolayer, whereas in the protein-free vesicles the exchangeable pool is consistent with the outer monolayer. The results indicate that both glycophorin and the partially purified band 3 preparation enhance the transbilayer movement of phosphatidylcholine.     

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.     

Effects of physical states of phospholipids on the incorporation and cytochalasin B binding activity of human erythrocyte membrane proteins in reconstituted vesicles

Proteoliposomes were reconstituted from a Triton extract of human erythrocyte membrane proteins and a mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of varying ratios. With mixtures of egg PC and soybean PE, the protein/lipid ratio of the reconstituted vesicles was maximal at 25% PC and 75% PE, the composition which is known to have a maximum bilayer disruption (highest occurrence of lipidic particles seen by freeze-fracture electron microscopy). With mixtures of 1-palmitoyl-2-oleoyl-PC and dilinoleoyl-PE, which gave vesicles with few isolated lipidic particles at room temperature, the effect was less pronounced. The specific activity of the cytochalasin B (CB) binding protein in the reconstituted vesicles, on the other hand, was increased monotonically up to severalfold as the PC content was increased in the egg PC/soybean PE mixture. A similar increase was observed when soybean PE was partially substituted by dimyristoyl-PC, cholesterol, or transphosphatidylated PE from egg PC. These findings indicate that preexisting defects in the lipid bilayer promote protein incorporation into the bilayer during reconstitution whereas reduction of the bilayer fluidity facilitates the CB binding activity in the reconstituted vesicles.     

Structural basis of human erythrocyte glucose transporter function in reconstituted vesicles

The transmembrane orientation of the human erythrocyte glucose transporter was assessed based on polarized Fourier transform infrared and ultraviolet circular dichroism spectroscopic data obtained from oriented multilamellar films of the reconstituted transporter vesicles. Infrared spectra revealed that there are distinct vibrations for alpha-helical structure while the vibrational frequencies specific to beta-structure are characteristically absent. Analysis of linear dichroism of the infrared spectra further indicated that these alpha-helices in the transporter are preferentially oriented perpendicular to the lipid bilayer plane forming an effective tilt of less than 38 degrees from the membrane normal. Such a preferential orientation was further supported by ultraviolet circular dichroism spectra which reveal that the 208 nm Moffit band found in the detergent-solubilized preparation is absent in the film preparation. Linear dichroism data further indicated that D-glucose, a typical substrate, further reduces this effective tilt angle slightly.     

Interaction of molybdenum with human erythrocyte membrane proteins

This study describes the interaction of molybdenum with blood components. Molybdenum-99 was added to blood, and after four washings, 3% of the total radioactivity was found in red cells. More specifically, the radioactivity was determined to be associated with the cell membrane. Molybdenum-99 in the +VI form did not interact with the human erythrocyte membrane; however, Mo(V) forms did interact. Of five different compounds, the highes uptake was observed with a brown Mo(V)-ascorbate complex generated from Mo(VI) and ascorbic acid in the molar ratio 1∶20. A membrane suspension of Mo-ascorbate-treated human erythrocytes was prepared and the solubilized proteins were separated on a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS). Molybdenum-99 binding to spectrin was demonstrated, as well as some minor interactions with membrane hemoglobin and bands 6 and 8.     

Permeability of human erythrocyte membrane vesicles to alkali cations

The permeability of inside-out and right-side-out vesicles from erythrocyte membranes to inorganic cations was determined quantitatively. Using ⁸⁶Rb as a K analog, we have measured the rate constant of ⁸⁶Rb efflux from vesicles under equilibrium exchange conditions, using a dialysis procedure. The permeability coefficients of the vesicles to Rb are only about an order of magnitude greater than that of whole erythrocytes. Furthermore, we have measured many of the specialized transport systems known to exist in erythrocytes and have shown that glucose, sulfate, ATP-dependent Ca and ATP-dependent Na transport activities are retained by the vesicle membranes. These results suggest that inside-out and right-side-out vesicles can be used effectively to study transport properties of erythrocyte membranes.     

Analysis of human erythrocyte membrane vesicles produced by shearing

Shearing of ghosts in a French pressure cell produces three classes of microvesicles that differ from endocytic vacuoles, exocytic vacuoles, and inside-out vesicles. It was thought that an analysis of these vesicles might provide some clues about the assembly of proteins within the human erythrocyte membrane. The microvesicles were separated into three visible bands, labeled top, middle, and bottom, and assayed for activity of Mg⁺⁺-ATPase, Na⁺, K⁺-ATPase, acetylcholinesterase, glyceraldehyde-phosphate dehydrogense, and NADH oxidoreductase. Their proteins were also characterized by polyacrylamide gel electrophoresis with both Coomassie blue staining, to assess total protein content and distribution, and PAS-staining, to characterize sialoglycopeptides. In order to minimize problems inherent in ghost preparation, Dodge or hypotonic ghosts and glycol or isotonic ghosts were used in all studies. Middle membrane vesicles most resembled intact ghosts. Top vesicles had reduced levels of NADH oxidoreductase and more PAS-2 at the expense of PAS-1. The bottom vesicle class was very much enriched with PAS-1 at the expense of PAS-2, and PAS-3 was completely absent. In addition bottom vesicles had highest NADH oxidoreductase activity but lowest activity of all the other enzymes measured. These vesicle classes could not have been produced by tangential shearing through the membrane, nor could radial shearing through a membrane in which all proteins were free to move laterally have accounted for the three discrete vesicle classes or for their different patterns of enzymes and proteins. The analysis of the microvesicles produced by shearing is most consistent with radial shearing through membranes where there may be fixed domains superimposed on the basic fluid-mosaic structure.     

Topology of membrane sulfhydryl groups in the human erythrocyte Demonstration of a non-reactive population in intrinsic proteins

A major fraction of the protein sulfhydryl groups of human erythrocyte membranes can be oxidized to disulfide bonds by the lipid soluble reagent, diamide, and the hydrophilic reagent, tetrathionate. Furthermore, the same fraction also reacts with the monofunctional reagent, $\text{N-}\text{ethylmaleimide}$. About 20% of the SH groups, however, do not react with any of these agents even upon prolonged treatment and increased concentrations.These ‘non-reacting’ SH groups were now localized by a procedure involving blockage of the accessible SH groups by non-labelled $\text{N-}\text{ethylmaleimide}$ or by diamide, subsequent isolation and solubilization of the membranes in SDS and labelling of the now accessible, residual SH groups with $\text{N-[ethyl-2-}{}^3\text{H]}\text{ethylmaleimide}$.The distribution of the radioactivity over the peptide fractions shows that the non-reacting SH groups are mainly localized in the intrinsic proteins, while essentially all of the SH groups of the extrinsic protein, spectrin, are reactive.After solubilization of the membranes with Triton X-100 the non-reacting SH groups became reactive towards $\text{N-}\text{ethylmaleimide}$. It is proposed that lack of reaction of SH groups in the native membranes is due to their localization within the hydrophobic core of the membrane.     

Specific cation modulation of anion transport across the human erythrocyte membrane

The specific modulation by three cations, Ca²⁺, Mg²⁺, and tetracaine of the equilibrium exchange of SO₄²⁻ across the erythrocyte membrane was investigated. While external calcium had no effect on SO₄²⁻ exchange, internal calcium, and external calcium in the presence of 10 μM A23187 were found to be potent inhibitors of the exchange reaction. The apparent inhibition constants (K₁) for Ca²⁺ were calculated to be 6.1 μM and 5 μM for the above two conditions, respectively.Unlike Ca²⁺, Mg²⁺ was shown to be a weak activator of SO₄²⁻ exchange with an apparent dissociation constant of 3.6 μM. Competition experiments demonstrated that the Ca²⁺ and Mg²⁺ sites associated with anion transport are distinct and noninteracting.Tetracaine, a cation at neutral pH, was also found to be an inhibitor of SO₄²⁻ exchange with an apparent K₁ of 0.8 mM. Although tetracaine was observed to displace calcium from non-specific sites on the erythrocyte membrane, it showed no effect on the apparent inhibition constant of Ca²⁺ for SO₄²⁻ exchange. Thus, the Ca²⁺ and tetracaine sites also appear to be independent. The difficulty of situating three mutually independent sites on a single subunit protein, i.e., band 3, is considered.Using the experimental data obtained from five individuals, the concentration of free calcium in the red cell cytoplasm was calculated to range from 0.2 to 0.7 μM. This concentration was sufficient to reduce SO₄²⁻ exchange only 3–8%. It was concluded that calcium inhibition of anion exchange, and, hence, impairment of CO₂ transport, may be physiologically significant only in senescent cells and in certain types of anemia where calcium concentrations are significantly increased.     

The phosphorylation of the major proteins of the human erythrocyte membrane.

Both the major sialoglycoprotein (PAS-1) and the component designated by Fairbanks et al. (G. Fairbanks, T. L. Steck, and D. F. H. Wallach, 1971, Biochemistry10, 2606–2617) as Band 3 are shown to be bonafide phosphoproteins by virtue of the presence of covalently bound serine and threonine phosphate residues. In agreement with the findings of others, PAS-1 does not seem to be phosphorylated when ghosts are incubated with [γ-³²P]ATP, but the phosphorylation is significant (about 0.15 mol/mol) when the cells are incubated in the presence of ³²P_i. Band 3 is phosphorylated to the extent of 0.90 mol/mol, and these sites are apparently distributed in several places along the polypeptide chain. Spectrin is also a phosphoprotein containing approximately four molecules of phosphate per 450,000 daltons of protein. The phosphorylation of these three polypeptides is not stimulated by the presence of cAMP.     

Calcium effects on human erythrocyte membrane proteins

The effects of Ca²⁺ on human erythrocyte membrane proteins were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Ca²⁺ had several effects on normal human erythrocyte membrane proteins. It affected the binding of cytoplasmic proteins to the membrane, produced a non-reversible aggregation of several membrane proteins and activated apparent proteolysis of membrane proteins. The Ca²⁺ effect could be obtained with isolated, washed membranes when the erythrocyte cytoplasm was added. These studies indicate that the Ca²⁺-induced membrane proteolysis and aggregation effects are not due simply to its presence at the time of hemolysis as previously suggested (Carraway, K.L., Triplett, R.B. and Anderson, D.R. (1975) Biochim. Biophys. Acta 379, 571–581), but are the result of more complex interactions between the erythrocyte membrane and cytoplasmic factors.