The diversity of sulfhydryl groups in the human erythrocyte membrane

Human bank blood erythrocytes were exposed to the mercurials p-chloromercuribenzoate (PCMB), chlormerodrin (CM), p-chloromercuribenzenesulfonate (PCMBS), and 1-bromomercuri-2-hydroxypropane (BMHP) for different time intervals, at different concentrations and in combination with n-ethylmaleimide (NEM) added before, and 2-mercaptoethylguanidine (MEG) and reduced glutathione (GSH) added after the mercurial. Binding patterns of the mercurials to the cells and effects on permeability of the cells were measured. The results indicate that the erythrocyte membrane contains multiple classes of sulfhydryl groups, alteration of which has a variety of effects on cell permeability. PCMB, chlormerodrin and PCMBS react with at least three classes of sulfhydryls, two of which are associated with the sodium-potassium barrier and, when altered, result in potassium loss, sodium accumulation and hemolysis. BMHP reacts with at least two classes of sulfhydryls, one of which is associated with permeability, and, when altered, results in hemolysis in isotonic solutions of choline chloride or lactose. The results provide additional insight into the structure and function of the erythrocyte membrane.     

Reactive sulfhydryl groups of the band 3 polypeptide from human erythroycte membranes. Location in the primary structure.

Human erythrocyte membranes contain a major transmembrane protein, known as Band 3, that is involved in anion transport. This protein contains a total of five reactive sulfhydryl groups, which can be assigned to either of two classes on the basis of their susceptibility to release from the membrane by trypsin. Two of the groups are located in the region COOH-terminal to the extracellular chymotrypsin-sensitive site of the protein and remain with a membrane-bound 55,000-dalton fragment generated by trypsin treatment. The three sulfhydryl groups NH2-terminal to the extracellular chymotrypsin site are released from the cytoplasmic surface of the membrane by trypsin. All three groups are present in a 20,000-dalton tryptic fragment of Band 3. Two of these groups are located very close to the sites of trypsin cleavage that generate the 20,000-dalton fragment. The third reactve group is probably located about 15,000-daltons from the most NH2-terminal sulfhydryl group. Two other well defined fragments of the protein do not contain reactive sulfhydryl groups. They are a 23,000-dalton fragment derived from the NH2-terminal end that is also released by trypsin from the cytoplasmic surface of the membrane and a 19,000-dalton membrane-bound region of the protein that is produced by treatment with chymotrypsin in ghosts. The 20,000-dalton tryptic fragment may, therefore, constitute a sulfhydryl-containing domain of the Band 3 protein.     

Isolation and characterization of band 3, the predominant polypeptide of the human erythrocyte membrane.

Band 3 is the predominant approximately 90,000-dalton polypeptide component of the human erythrocyte membrane. It was solubilized selectively, along with the other major glycoproteins, by extracting membrane ghosts with Triton X-100 under nondenaturing conditions. Two major polypeptides remained associated with Band 3 under these conditions; however one (Band 6) could be dissociated at an ionic strength of 0.15 and the other (Band 4.2) by treatment with p-chloromercuribenzoate. Band 3 was then purified (greater than or equal to 97%) by aminoethyl cellulose ion exchange chromatography. The isolated protein was free of phospholipid and was moderately enriched in apolar amino acid residues; it contained galactose and glucosamine but very little sialic acid and galactosamine. When Band 3 was labeled by treatment of ghosts with galactose oxidase plus KB3H4 and then purified, the electrophoretic mobility of its radioactivity lagged slightly behing that of its Coomassie blue staining profile. Variation in glycosylation could therefore cause the diffuse trailing zone characteristically observed for Band 3 on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The ultraviolet circular dichroism of Band 3 was stable in nonionic detergent and suggested an alpha helix content of 43%, a value close to that estimated for this polypeptide in the membrane.     

Proteolytic dissection of band 3, the predominant transmembrane polypeptide of the human erythrocyte membrane.

Band 3 is the major, membrane-spanning, approximately90 000 dalton polypeptide of the human erythrocyte membrane. To facilitate the analysis of its structural integration into the membrane, we have cleaved this protein in situ into large fragments and ascertained their disposition. Digestion of intact cells with chymotrypsin yielded band 3 fragments with apparent molecular weights of 38 000 and 55 000. Both fragments resisted elution by NaOH and acetic acid, suggesting that they are anchored in the apolar core of the membrane. Both pieces communicate with the extracellular space, and the 55 000 dalton species extends to the cytoplasmic surface as well. Digestion of unsealed ghosts with chymotrypsin produced a hydrophobic 17 000 dalton species, a segment of the 55 000 dalton fragment, which spans and is firmly anchored in the core of the membrane. Trypsin and papain at low concentration generated integral band 3 fragments of 52 000 daltons and released major band 3 fragments of less than or equal to 41 000 daltons from the cytoplasmic side of the membrane. The latter water-soluble polypeptides remained associated in discrete complexes which retained the capacity to bind glyceraldehyde-3-phosphate dehydrogenase. An interchain disulfide bond, which can be induced only at the cytoplasmic surface, cross-linked intact band 3, and certain of its water-soluble fragments. Finally, fragments of 23 000 daltons were generated from the innersurface domain by reacting disulfide-linked band 3 dimers with cyanide or reduced polypeptides with 2-nitro-5-thiocyanobenzoate. A provisional ordering of these fragments is proposed.     

Role of sulfhydryl groups in band 3 in the inhibition of phosphate transport across erythrocyte membrane in visceral leishmaniasis

Membrane destabilization in erythrocytes plays an important role in the premature hemolysis and development of anemia during visceral leishmaniasis (VL). Marked degradation of the anion channel protein band 3 is likely to allow modulation of anion flux across the red cell membrane in infected animals. The present study describes the effect of structural modification of band 3 on phosphate transport in VL using (31)P NMR. The result showed progressive decrease in the rate and extent of phosphate transport during the post-infection period. Interdependence between the intracellular ionic levels seems to be a determining factor in the regulation of anion transport across the erythrocyte membrane in control and infected conditions. Infection-induced alteration in band 3 made the active sites of transport more susceptible to binding with amino reactive agents. Inhibition of transport by oxidation of band 3 and subsequent reversal by reduction using dithiothreitol suggests the contribution of sulfhydryl group in the regulation of anion exchange across the membrane. Quantitation of sulfhydryl groups in the anion channel protein showed the inhibition to be closely related to the decrease of sulfhydryl groups in the infected hamsters. Downregulation of phosphate transport during leishmanial infection may be ascribed to the sulfhydryl modification of band 3 resulting in the impaired functioning of this protein under the diseased condition.     

Binding of rabbit muscle aldolase to band 3, the predominant polypeptide of the human erythrocyte membrane.

Aldolase is a trace protein in isolated human red cell membrane preparations. Following total elution of the endogenous enzyme by a saline wash, the interaction of this membrane with rabbit muscle aldolase was studied. At saturation, exogenous aldolase constituted over 40% of the repleted membrane protein. Scatchard analysis revealed two classes of sites, each numbering approximately 7 X 10(5) per ghost. Specificity was suggested by the exclusive binding of the enzyme to the membrane's inner (cytoplasmic) surface. Furthermore, milimolar levels of fructose 1,6-bisphosphate eluted the enzyme from ghosts, while fructose 6-phosphate and NADH (a metabolite which elutes human erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) from its binding site) were ineffectuve. Removing peripheral membrane proteins with EDTA and lithium 3,5-diiodosalicylate did not diminish the binding capacity of the membranes. An aldolase-band 3 complex, dissociable by high ionic strength or fructose 1,6-bisphosphate treatment, was demonstrated in Triton X-100 extracts of repleted membranes by rate zonal sedimentation analysis on sucrose gradients. We conclude that the association of rabbit muscle aldolase with isolated human erythrocyte membranes reflects its specific binding to band 3 at the cytoplasmic surface, as is also true of G3PD.     

Inhibition of phosphate transport across the human erythrocyte membrane by chemical modification of sulfhydryl groups.

Effects of sulfhydryl-reactive reagents on phosphate transport across human erythrocyte membranes were examined using 31P NMR. Phosphate transport was significantly inhibited in erythrocytes treated with sulfhydryl modifiers such as N-ethylmaleimide, diamide, and Cu2+/o-phenanthroline. Quantitation of sulfhydryl groups in band 3 showed that the inhibition is closely associated with the decrease of sulfhydryl groups. Data from erythrocytes treated with diamide or Cu2+/o-phenanthroline demonstrated that intermolecular cross-linking of band 3 by oxidation of a sulfhydryl group, perhaps Cys-201 or Cys-317, decreases the phosphate influx by about 10%. The inhibition was reversed by reduction using dithiothreitol. These results suggest that sulfhydryl groups in the cytoplasmic domain of band 3 may play an important role in the regulation of anion exchange across the membrane.     

A study of the dependence of the human erythrocyte glucose transport system on membrane sulfhydryl groups

Summary A brief review of the data relating the glucose transport system and other membrane functions of red cells to surface sulfhydryl groups is presented. The effect of a variety of sulfhydryl reagents on glucose efflux rates from loaded red cells was studied. Neither iodoacetate nor iodoacetamide at 5mm inhibited efflux. Several maleimide derivatives and disulfides inhibited efflux in 0.7 to 2.0mm concentrations. Organomercury compounds, on the other hand, were active in the 0.07 to 0.1mm range. These data suggest that, if sulfhydryl groups are important in the glucose efflux process, they are not equally accessible to the above reagents; and that the primary effect of these reagents may be on structural elements near membrane sulfhydryl groups.     

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.     

The influence of melittin on the rotation of band 3 protein in the human erythrocyte membrane

The rotational mobility of band 3, a protein constituent of the human erythrocyte membrane, was measured by observing the flash-induced transient dichroism of the triplet probe eosin maleimide. In the presence of melittin, a pharmacologically active polypeptide from honey bee (Apis mellifera) venom, a dose-dependent loss of rotational mobility was detected. With acetylated melittin, the ability to immobilise is reduced. Succinylated melittin, however, is devoid of immobilising activity.The possible relevance of these findings to the normal mode of action of melittin was examined by comparing the relative abilities of the native, acetylated and succinylated melittins to lyse erythrocytes and synergise with phospholipase A₂, another constituent of bee venom. For both these properties, the order of effectiveness is native melittin > acetyl melittin > succinyl melittin = 0, the same as their order of effectiveness in immobilising band 3.A mechanism is proposed in which melittin is anchored in the membrane by its hydrophobic N-terminus, while its cationic C-terminal moiety binds to negatively charged residues on membrane proteins. This leads either directly or indirectly to protein aggregation and hence loss of mobility. From a detailed comparison of the different effects of the melittin derivatives, it is concluded that melittin may function in vivo by aggregating membrane proteins in order to allow phospholipase A₂ to gain access to the membrane bilayer and commence catalysis.     

The plasma membrane H+-ATPase of Neurospora crassa. Properties of two reactive sulfhydryl groups

Previous work with N-ethylmaleimide (NEM) has defined two sites on the Neurospora plasma membrane H+-ATPase. Modification of one (the "fast" site) by NEM is rapid but does not affect ATPase activity, while modification of the other (the "slow" site) inactivates the enzyme and is protectable by MgATP or MgADP. In the present study, a wider array of sulfhydryl reagents have been used to examine the properties of both sites. The results show the following. (a) Both fast and slow sites react preferentially with hydrophobic compounds (N-pyrenemaleimide, dithiobisnitropyridine greater than N-naphthylmaleimide, dithiobisnitrobenzoate greater than N-phenylmaleimide greater than N-ethylmaleimide) and are virtually insensitive to hydrophilic sulfhydryl reagents such as iodoacetamide and iodoacetic acid. (b) The reaction rate of the slow site with NEM is approximately 2000-fold less rapid than that of the fast site. The slow site also has an unusually high pKa (greater than 9.5). (c) Whether or not cysteine modification leads to inactivation of the ATPase depends upon the site and the reagent. For example, when the fast site reacts with NEM, enzymatic activity is retained; when it reacts with N-pyrenemaleimide, activity is lost. Likewise, when the slow site is modified by any of the maleimides or by dithiobisnitropyridine or dithiobisnitrobenzoate, the ATPase is inactivated; when it is modified by methylmethanethiosulfonate, activity remains intact. Thus, neither cysteine can be considered to play an essential role in the reaction cycle of the ATPase, but the introduction of a sufficiently bulky substituent at either site can disrupt activity. (d) Upon reaction of methylmethanethiosulfonate at the slow site, the K1/2 for MgATP hydrolysis is reduced from 0.65 to 0.25 mM. This result strengthens the evidence for a conformational relationship between the slow site cysteine and the nucleotide binding site of the ATPase.     

The interaction of hemoglobin with the cytoplasmic domain of band 3 of the human erythrocyte membrane

Previous studies point to the acidic amino-terminal segment of band 3, the anion transport protein of the red cell, as the common binding site for hemoglobin and several of the glycolytic enzymes to the erythrocyte membrane. We now report on the interaction of hemoglobin with the synthetic peptide AcM-E-E-L-Q-D-D-Y-E-D-E, corresponding to the first 11 residues of band 3, and with the entire 43,000-Da cytoplasmic domain of the protein. In the presence of increasing concentrations of the peptide, the oxygen binding curve for hemoglobin is shifted progressively to the right, indicating that the peptide binds preferentially to deoxyhemoglobin. The dissociation constant for the deoxyhemoglobin-peptide complex at pH 7.2 in the presence of 100 mM NaCl is 0.31 mM. X-ray crystallographic studies were carried out to determine the exact mode of binding of the peptide to deoxyhemoglobin. The difference electron density map of the deoxyhemoglobin-peptide complex at 5 A resolution showed that the binding site extends deep (approximately 18 A) into the central cavity between the beta chains, along the dyad symmetry axis, and includes Arg 104 beta 1 and Arg 104 beta 2 as well as most of the basic residues within the 2,3-diphosphoglycerate binding site. The peptide appears to have an extended conformation with only 5 to 7 of the 11 residues in contact with hemoglobin. In agreement with the crystallographic studies, binding of the peptide to deoxyhemoglobin was blocked by cross-linking the beta chains at the entrance to the central cavity. Oxygen equilibrium studies showed that the isolated cytoplasmic fragment of band 3 also binds preferentially to deoxyhemoglobin. The binding of the 43,000-Da fragment to hemoglobin was inhibited in the cross-linked derivative indicating that the acidic amino-terminal residues in the intact cytoplasmic domain also bind within the central cavity of the hemoglobin tetramer.