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1.
The hydrophobic probe phenylisothiocyanate is utilized for chemical modification of human erythrocyte band 3 protein. The binding of phenylisothiocyanate to this protein is characterized in whole erythrocytes, erythrocyte ghost membranes and in isolated band 3 protein. The label, reactive with nucleophiles in their deprotonated form, is found in all three preparations to be covalently bound to band 3 protein. Under saturation conditions, 4–5 mol phenylisothiocyanate are covalently bound per mol protein (molecular weight 95 000). The described modification effects inhibition of phosphate entry into erythrocytes. 50% inhibition of phosphate transport is obtained following a preincubation of erythrocytes with 0.45 mM phenylisothiocyanate. Both phenylisothiocyanate binding and transport inhibition are saturating processes. The relationship of the two parameters is non-linear. 相似文献
2.
Oxidation of erythrocyte membrane SH-groups by diamide and tetrathionate induces cross-linking of spectrin (Haest, C.W.M., Kamp, D., Plasa, G. and Deuticke, B. (1977) Biochim. Biophys. Acta 469, 226–230). This cross-linking was now shown to go along with a concentration- and time-dependent enhancement of membrane permeability for hydrophilic nonelectrolytes and ions. The enhancement is specific for oxidative SH-group modifications, is reversible by reduction of the induced disulfides, can be suppressed by a very brief pre-treatment of the cells with low concentrations of and is strongly temperature-dependent. The pathway of the induced permeability discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy (). These findings are reconcilable with the formation of a somewhat inhomogeneous population of aqueous pores with radii probably . Estimated pore numbers vary with the size of the probe molecule. Assuming a diffusion coefficient as in bulk water within the pore, at least 20 pores per cell have to be postulated; more realistic lower diffusion coefficients increase that number. Alterations of the lipid domain by changes of cholesterol contents and insertion of hexanol or nonionic detergents alter the number or size of the pores. Since aggregation of skeletal and intrinsic membrane proteins also occurs after the SH-oxidation, in parallel to the formation of membrane leaks, one may consider (a) defects in the disturbed bilayer interface, (b) a mismatch between lipid and intrinsic proteins or (c) channels inbetween aggregated intrinsic proteins as structures forming the pores induced by diamide treatment. 相似文献
3.
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. 相似文献
4.
The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate exchange in intact erythrocytes. 50% inhibition is obtained by binding 4.7 · 105 or 6.0 · 105 molecules/cell for eosin-NCS and IA-eosin, respectively. Both eosin probes are irreversibly bound and occupy common binding sites with 4,4′-diisothiocyano-1,2-diphenyl-ethane-2,2′-disulfonic acid (H2DIDS), although other sites are labeled as well. The inhibition of anion transport is light independent and can therefore not be attributed to a photosensitizing action of the eosin probes. Both eosin derivatives, however, inactivate acetylcholinesterase upon illumination of air-equilibrated samples of hemoglobin-free labeled ghosts. The inactivation of the enzyme is accompanied by the formation of protein aggregates as visualized by polyacrylamide gel electrophoresis. These effects are not observed when intact erythrocytes are illuminated in the presence of eosin probes suggesting a protective effect of hemoglobin during the labeling procedure. Protection of ghosts from photo-oxidation is achieved by displacing air with argon. These results are discussed in relation to the use of these and similar probes to measure protein diffusion in membranes. 相似文献
5.
The transmembrane movements of lactate and other monocarboxylate anions in mammalian erythrocytes have been claimed, by virtue of their sensitivity to SH-reagents, to involve a transfer system different from the classical anion system (Deuticke, B., Rickert, I. and Beyer, E. (1978) Biochim. Biophys. Acta 507, 137–155). Inhibition of monocarboxylate transfer by SH-reagents, however, was incomplete to an extent varying for different monocarboxylates. The transport component insensitive to SH-reagents has now been shown to involve (a) the classical anion-exchange system, as demonstrated by sensitivity to specific disulfonate inhibitors, and (b) nonionic diffusion, as indicated by the characteristic pH- and concentration dependency of this component and its stimulation by aliphatic alcohols. Under physiological conditions about 90% of total lactate movement proceed via the specific system, 5% via the classical anion-transfer system, 5% by nonionic diffusion. These three components of lactate exchange differ in their activation energies. The specific lactate system mediates net fluxes almost as fast as exchange fluxes, in marked contrast to the classical anion-exchange system which mediates halide exchange much faster than halide net movements. The underlying mechanism, for maintenance of electroneutrality, is an OH?-antiport or an H+-symport as indicated by the particular response of lactate net fluxes to changes of intra- or extracellular pH. 相似文献
6.
The disulfonatostilbene spin label, NDS-TEMPO, was synthesized (purity over 96%) and the binding of the spin label to human red-cell ghosts was studied. NDS-TEMPO is readily adsorbed to the membrane surface. Both pretreatment of the ghosts with FDNB and DIDS and the presence of DNDS completely prevent the binding of NDS-TEMPO to red-cell ghosts. Chloride and sulfate competitively inhibit the binding of NDS-TEMPO. Conversely, NDS-TEMPO is a strong, competitive inhibitor of chloride and of sulfate transport. The dissociation constants of NDS-TEMPO from the ESR studies were in the range 1.0–2.0 μM (pH 7.6, 20°C). The inhibition constants of NDS-TEMPO as obtained from the flux experiments were in the range 0.5–2.5 μM (pH 7.3, 25°C). The close accordance of the NDS-TEMPO dissociation constants from the ESR studies with the NDS-TEMPO inhibition constants from the flux measurements indicate a specific labeling of the inorganic-anion-transport system. 相似文献
7.
The putative hexose transport component of Band 4.5 protein of the human erythrocyte membrane was covalently photolabelled with [3H]cytochalasin B. Its transmembrane topology was investigated by electrophoretically monitoring the effect of proteinases applied to intact erythrocytes, unsealed ghosts, and a reconstituted system. Band 4.5 was resistant to proteolytic digestion at the extracellular face of the membrane in intact cells at both high and low ionic strengths. Proteolysis at the cytoplasmic face of the membrane in ghosts or reconstituted vesicles resulted in cleave of the transporter into two membrane-bound fragments, a peptide of about 30 kDa that contained its carbohydrate moiety, and a 20 000 kDa nonglycosylated peptide that bore the cytochalasin B label. Because it is produced by a cleavage at the cytoplasmic face and because the carbohydrate moiety is known to be exposed to the outside, the larger fragment must cross the bilayer. It has been reported that the Band 4.5 sugar transporter may be derived from Band 3 peptides by endogenous proteolysis, but the cleavage pattern found in the present study differs markedly from that previously reported for Band 3. Minimization of endogenous proteolysis by use of fresh cells, proteinase inhibitors, immediate use of ghosts and omission of the alkaline wash resulted in no change in the incorporation of [3H]cytochalasin B into Band 4.5, and no labelling of Band 3 polypeptides. These results suggest that the cytochalasin B binding component of Band 4.5 is not the product of proteolytic degradation of a Band 3 component. 相似文献
8.
Uptake of the water soluble 1,2-dimercaptopropanol (BAL) derivative 2,3-dimercapto-1-sulfonate (DMPS) into human red blood cells was found in vitro and the mode of penetration studied in detail. The compound entered erythrocytes in a concentration dependent manner. In contrast to sealed ghosts where inside and outside concentrations reached the same value, DMPS accumulated in intact erythrocytes. Since no binding of DMPS could be detected, the reason for accumulation was assumed to be a conversion of DMPS into chelates or metabolites which penetrated the membrane in a slower rate. A facilitated transport of DMPS mediated by the anion carrier protein was concluded on the basis of the following similarities with the anion transport: inhibition of [14C]DMPS-uptake by N-ethylmaleimide (NEM), tetrathionate (90%), sulfate (50%), 5,5′-dithio bis(2-nitrobenzoic acid) (DTNB) (25%); inhibition of uptake and efflux by 4,4′-diisothiocyano-2,2′-stilbene disulfonate (DIDS) (80%), dipyridamole (55%); temperature dependency (activation energy 24 Kcal/mol); pH-dependency (pH optimum about 6.9); counter-transport; activation of uptake by preincubation with DMPS (transmembrane effect). 相似文献