Protoporphyrin-induced photodynamic effects on band 3 protein of human erythrocyte membranes

In previous studies it has been shown that protoporphyrin-induced photodynamic effects on red blood cells are caused by photooxidation of amino acid residues in membrane proteins and by the subsequent covalent cross-linking of these proteins. Band 3, the anion transport protein of the red blood cell membrane, has a relatively low sensitivity to photodynamic cross-linking. This cannot be attributed to sterical factors inherent in the specific localization of band 3 in the membrane structure. Solubilized band 3, for instance, showed a similar low sensitivity to cross-linking. By extracellular chymotrypsin cleavage of band 3 into fragments of 60 000 and 35 000 daltons it could be shown that both fragments were about equally sensitive to photodynamic cross-linking. The 17 000 dalton transmembrane segment, on the other hand, was completely insensitive. Inhibition of band 3-mediated sulfate transport proceeded much faster than band 3 interpeptide cross-linking, presumably indicating that the inhibition of transport is caused by photooxidation of essential amino acid residues or intrapeptide cross-linking. A close parallel was observed between photodynamic inhibition of anion transport and decreased binding of 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (H₂DIDS), suggesting that a photooxidation in the immediate vicinity of the H₂DIDS binding site may be responsible for transport inhibition.     

Effects of anesthetic alcohols on membrane transport processes in human erythrocytes

1. 1. Anesthetic alcohols (pentanol, hexanol and heptanol) were found to increase the fluidity of red cell membrane lipids as monitored by the fluorescence depolarization of diphenylhexatriene. The relative potency of the alcohols was found to be parallel to their relative membrane/water partition coefficients.

2. 2. Hexanol had biphasic effect on erythritol uptake by simple diffusion by red cells. At concentrations less than 9 mM, hexanol had no significant effect. At concentrations greater than 9 mM, there was an approximately linear increase in erythritol permeability with increasing alcohol concentration.

3. 3. The facilitated transport of uridine was markedly inhibited by hexanol. Hexanol at 6 mM produced a 65% inhibition of uridine (4 mM) uptake. Hexanol decreased both the apparent K_m and V values for the equilibrium exchange of uridine.

4. 4. The facilitated transport of galactose was only slightly inhibited by hexanol.

5. 5. Hexanol was without effect on the passive and active fluxes of Na⁺ and K⁺ in red cells with altered cation contents. Cells that were slightly depleted of K⁺ and cells that were highly K⁺-depleted were both insensitive to hexanol.

Dual effect of membrane cholesterol on simple and mediated transport processes in human erythrocytes

The influence of cholesterol on simple and facilitated transport processes across the membrane of intact human erythrocytes was studied after graded depletion or enrichment of membrane cholesterol by incubation of the cells in phospholipid or phospholipid/cholesterol suspensions.

1. 1. The carrier-mediated transfer of L-lactate and of L-arabinose proved to be enhanced by cholesterol. In the case of L-lactate, a decrease in K_m seems to be involved in this effect. In contrast, the self-exchange of SO₄²⁻, mediated by the inorganic anion-exchange system, and the simple diffusion of erythritol via the lipid phase of the membrane are inhibited by cholesterol.

2. 2. Reversibility of these two opposite effects of cholesterol was demonstrated by measurements on cells depleted again after cholesterol enrichment and enriched again after previous depletion.

3. 3. Certain phospholipids used for preparing the lipid dispersions that are required for cholesterol variation have effects on permeability of their own, due, for example, to traces of contaminants. A discrimination of such artifacts from the effects of cholesterol is only possible by demonstrating reversibility.

4. 4. The opposite effects of cholesterol on various facilitated transfer processes, which have a correlation in the opposite effects of other modifications of the membrane lipid phase (Deuticke, B., Grunze, M. and Haest, C.W.M. (1979) Alfred Benzon Symposium 14, Munksgaard, Copenhagen, in the press), are indicative of different types of lipid-protein interaction in the erythrocyte membrane.

Adrenergic stimulation of membrane protein phosphorylation in human erythrocytes

Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocytes. Micromolar norepinephrine increased ³²P incorporation into Band 2 by 70%, and into Band 3 by 40%. Phosphorylation levels observed with a series of specific agonists and antagonists suggest that an α-adrenergic receptor is involved in this effect. The mechanism of linkage between this receptor and protein phosphorylation does not appear to involve modulation of intracellular concentrations of ATP, cyclic AMP, or cyclic GMP.     

Inhibition of phosphate transport in human erythrocytes by water-soluble carbodiimides

Phosphate entry into human erythrocytes is irreversibly inhibited by treatment of the cells with the water-soluble carbodiimides 1-ethy1-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMC) in the absence of added nucleophile. EDC is the more potent inhibitor (40% inhibition, 2 mM EDC, 5 min, 37°C, 50% hematocrit, pH 6.9), while more than 20 mM CMC is required to give the same inhibition under identical conditions. EDC inhibition is temperature-dependent, being complete in 5 min at 37°C, and sensitive to extracellular pH. At pH 6.9 only 50% of transport is rapidly inhibited by EDC, but at alkaline pH over 80% of transport is inhibited. Inhibition is not prevented by modification of membrane sulfhydryl groups but is decreased in the presence of 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), a reversible competitive inhibitor of anion transport. EDC treatment leads to crosslinking of erythrocyte membrane proteins, but differences between the time course of this action and inhibition of transport indicate that most transport inhibition is not due to crosslinking of membrane proteins.     

Magneto-electro-fusion of human erythrocytes

In inhomogeneous (static) magnetic fields close contact between ‘magnetic’ human erythrocytes was established. The cells were made magnetic by incubating them in a medium containing small Fe₃O₄-particles which adsorbed to the outer membrane surface. Fusion was induced by applying two electric field pulses (field strength: 8.5 kV · cm^?1; duration: 60 μs) to the magnetically collected cells. This procedure allowed the use of electrically conductive media (3 · 10^?1 Ω^?1 · cm^?1). Fusion of red blood cells occured very often. If cell suspensions of high density were used fusion resulted in the formation of giant red blood cells with osmotically intact membranes.     

Hematoporphyrin-induced photo-oxidation and photodynamic cross-linking of nucleic acids and their constituents

The pH-dependency of photo-oxidation of the physiological purine and pyrimidine bases and some of their derivatives was studied, with hematoporphyrin as sensitizer. At high pH these bases (adenine, guanine, uracil, thymine and cytosine) were photo-oxidizable. In the physiological pH range only guanine, and to a much less extent thymine, were sensitive to photo-oxidation. At physiological pH values a slow photo-oxidation of RNA and DNA took place. The photo-oxidation of nuclei acids was strongly augmented by perturbation of their structure in 8 M urea. In model experiments photodynamic cross-linking of tryptophan and cysteine to DNA was demonstrated. No covalent binding of purine or pyrimidine bases to DNA was observed. In similar model experiments covalent photodynamic coupling of guanosine and guanosine-monophosphate to proteins could be shown, whereas no coupling of the other bases occured. These studies confirm the preferential photo-oxidation of guanine in nucleic acids and demonstrate the possible photodynamic cross-linking of proteins to the guanine moiety in other molecules.     

The binding and translocation steps in transport as related to substrate structure. A study of the choline carrier of erythrocytes

The relationships between structure, affinity and transport activity in the choline transport system of erythrocytes have been investigated in order to (i) explore the nature of the carrier site and its surroundings, and (ii) determine the dependence of the carrier reorientation process on binding energies and steric restraints due to the substrate molecule. Affinity constants and maximum transport rates for a series of trialkyl derivatives of ethanolamine were obtained by a method that involves measuring the trans effect of unlabeled analogs upon the movement of radioactive choline. The main conclusions are as follows: (1) An analysis of transport kinetics shows that the affinity constants determined experimentally differ from the actual dissociation constants in a predictable way. The better the substrate, the higher the apparent affinity relative to the true value, whereas the affinity of non-transported inhibitors is underestimated by a constant factor. (2) The carrier-choline complex undergoes far more rapid reorientation (translocation) than the free carrier. (3) The carrier imposes a strict upper limit upon the size of a substrate molecule that can participate in the carrier reorientation process; this limit corresponds to the choline structure. A smaller substrate such as tetramethylammonium, despite relatively weak binding forces, is unhindered in its translocation, suggesting that a carrier conformational change, dependent upon substrate binding energy, is not required for transport. (4) Small increases in the size of the quaternary ammonium head, as in triethylcholine, sharply lower affinity, consistent with a high degree of specificity for the trimethylammonium group. (5) Lengthening the alkyl substituent in derivatives of dimethyl- and diethylaminoethanol causes a regular increase in affinity, suggestive of unspecific hydrophobic bonding in a region very near the substrate site.     

An allosteric pore model for sugar transport in human erythrocytes

Glucose transport in human erythrocytes is characterized by a marked asymmetry in the $\text{V}$ and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for entry and for exit. In addition, they show a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and a high $\text{V}$ for equilibrium exchange but low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for infinite cis and for infinite trans exit and entry. An allosteric pore model has been proposed to account for these characteristics. In this model, substrate-induced conformational changes destabilize the interfaces between protein subunits (the pore gates).Pores doubly occupied from inside destabilize the transport gates and result in high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and high $\text{V}$ transport parameters. This effect is less marked when pores are doubly occupied from outside and therefore transport asymmetry results.     

Formation of disulfide bonds between glutathione and membrane sh groups in human erythrocytes

In erythrocytes treated with the SH-oxidizing agent, diamide, mixed disulfide bonds between membrane proteins and GSH are formed involving 20% of the membrane SH groups. To study the distribution of these mixed disulfides over the membrane protein fractions, intracellular GSH was labelled biosynthetically with [2-³H]glycine prior to diamide treatment of the cells and the radioactivity of defined membrane peptide fractions determined. Mixed disulfides preferentially occur in the extrinsic protein, spectrin (six SH groups), in addition to the formation of peptide disulfides. Intrinsic proteins are much less reactive: only one SH group of the major intrinsic protein (band 3) reacts with GSH, which accounts for previously observed impossibility to dimerize band 3 via disulfide bonds in intact cells. The labelling method described offers a promising strategy to label and map exposed endofacial SH groups of membrane proteins with a physiological, impermeable marker, GSH.In ghosts treated with diamide and GSH the number of mixed disulfides formed is greater than in erythrocytes. Polymerization of spectrin via intermolecular disulfide bridges is suppressed, while intramolecular disulfides are still formed, providing a means for the analysis of spectrin structure.The diamide-induced mixed membrane-GSH disulfides are readily reduced by GSH. This suggests, that GSH may also be able to reduce mixed disulfides formed in the erythrocyte membrane under oxidative stress in vivo. The reversible formation of mixed disulfides may serve to protect sensitive membrane structures against irreversible oxidative damage.     

Bicarbonate/chloride transport kinetics at 37°C and its relationship to membrane lipids in mammalian erythrocytes

The rate of exchange of HCO₃^? with Cl^? at 37°C in erythrocytes of ten mammalian species was studied. The rate constant increases from 7s^?1 (ox) to 16s^?1 (rat), and is inversely proportional to the body size (log₁₀) of the species. It is found that the membrane permeability in different species is positively correlated to the red cell membrane phosphatidylcholine or arachidonate content, and is negatively correlated to the sphingomyelin or linoleate content.     

Kinetic mechanism of chlorpromazine inhibition of erythrocyte 3-O-methylglucose transport

The kinetic mechanism of chlorpromazine inhibition of erythrocyte hexose transport was investigated using the non-metabolizable glucose analog $\text{3-O-}\text{methylglucose}$. It was found that chlorpromazine added to the external medium is a non-competitive inhibitor of both equilibrium exchange and net $\text{3-O-}\text{methylglucose}$ transport at pH 7.8, 15°C. The $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ for equilibrium exchange is $\text{76 ± 21 μM}$. When net efflux and equilibrium exchange were measured on the same population of cells the equilibrium exchange was 2.5-times the maximum net efflux. The percent reduction of $\text{3-O-}\text{methylglucose}$ flux by chlorpromazine is dependent upon chlorpromazine concentration and not $\text{3-O-}\text{methylglucose}$ concentration as expected for a non-competitive inhibitor. Equilibrium exchange and net efflux show the same extent of inhibition at each concentration of chlorpromazine evaluated. These results suggest that exchange and net efflux of $\text{3-O-}\text{methylglucose}$ in the human erythrocyte may share a common transport system.     

The transport of chloroquine across human erythrocyte membranes is mediated by a simple symmetric carrier

The kinetic properties of the mediated transport of chloroquine in human erythrocytes are investigated. The high rates of translocation across the cell membrane and high adsorbance properties to glass surfaces have led to the development of new techniques for measuring initial rates of transport. Three different methodological procedures are used to accomplish a complete kinetic characterization of the system. All measurements were done at 25°C. Under zero-trans conditions the system displays complete symmetry, the Michaelis constants being 39.2±2.4 μM for influx and 36.6±5.6 μM for efflux. The respective maximal velocities are 206.4±36.0 μM·min^?1 and 190.0±7.8 μM·min^?1. Under equilibrium-exchange conditions the Michaelis constant is 108.6±15.6 μM and the maximal velocity is 630.3±50.4 μM·min^?1. This 3-fold increase in both K and V over the zero-trans values indicates that the rate-limiting step in the transport of chloroquine is the movement of the unloaded carrier. The kinetic data are consistent with the prediction of a simple carrier model.     

Thermodynamics of ouabain binding to human erythrocytes

A previous study reported that the uptake and release kinetics of ouabain by human erythrocytes in suspension could well be explained by a physical model which involves the slow Langmuir binding of the drug to the erythrocyte membrane. The purpose of the present investigation was to assess quantitatively the thermodynamics of this drug-membrane receptor interaction in order to evaluate the consistency of these parameters with the proposed kinetics model.Cellular drug uptake and release experiments were conducted at 20, 30 and 40°C, and the Langmuir adsorption and desorption rate constants as well as the Langmuir adsorption isotherms determined from the rate data. With the knowledge of these Langmuir parameters, it was possible to estimate the magnitude of all relevant thermodynamic properties by the use of established physicochemical theories.The activation energies and entropies for the ouabain adsorption and desorption processes were computed as 105 kJ/mol, 231 J/K per mol, 180 kJ/mol and 245 J/K per mol, respectively. The kinetic and isosteric heats of adsorption were found to be ?75.0 and ?72.4 kJ/mol, respectively. These findings suggest that the ouabain-erythrocyte membrane interaction represents a case of activated chemisorption which follows the Langmuir isotherm, thus, further underscoring the appropriateness of the Langmuir binding kinetics model.     

Asymmetric binding of steroids to internal and external sites in the glucose carrier of erythrocytes

Steroids inhibit glucose transport in erythrocytes by binding to sites in the carrier which are exposed on both the outer and inner surfaces of the cell membrane. Some steroids are bound almost exclusively at inner sites (androstendione and androstandione), while others are bound about as firmly on one side as the other (corticosterone). Still others exhibit a moderate preference for the internal site (deoxycorticosterone). The inhibition is in all cases competitive with respect to a substrate which is bound at the same surface of the membrane as the inhibitor. However, in experiments on substrate entry, internally bound inhibitors act in an apparently non-competitive fashion, as expected if the carrier model is valid. This behaviour explains the appearance of competitive, non-competitive and mixed inhibitions with different steroids (Lacko, L., Wittke, B. and Geck, P. (1975) J. Cell Physiol. 86, 673–680).     

Erythrocyte membrane cholesterol levels and their effects on membrane proteins

The susceptibility of the band 3 protein of the erythrocyte membrane to proteolytic digestion at either surface of the membrane was not altered when the membrane cholesterol level was increased by 65–103%. Cross-linking of the major membrane proteins by o-phenanthroline · Cu, glutaraldehyde, dimethylsuberimidate and dimethyladipimidate was also unaffected.     

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.     

Calcium and ionophore A23187 induce the sickle cell membrane phosphorylation pattern in normal erythrocytes

Pre-treatment of normal erythrocytes with micromolar Ca²⁺ and ionophore A23187 induces abnormal phosphorylation of membrane polypeptides, as determined by labeling with exogenous ³²P_i. The Ca²⁺-induced effects, which include increased incorporation of ³²P into acid-stable linkages and increased labeling in the Band 3 and 4.5–4.9 regions of SDS gels, are similar to those seen in untreated sickle erythrocytes. Part of the abnormal phosphorylation of sickle cells may be caused by their elevated intracellular Ca²⁺ levels.