Localization of the carboxyl terminus of Band 3 to the cytoplasmic side of the erythrocyte membrane using antibodies raised against a synthetic peptide

Polyclonal antibodies were raised in rabbits against a synthetic peptide which corresponds to the 12-amino acid carboxyl-terminal sequence of murine erythrocyte Band 3. Immunoblots of ghost membrane proteins showed that the antibody specifically recognized murine or rat Band 3 but not human or canine Band 3. The antibody also bound to murine ghost membranes applied directly to nitrocellulose but not to human ghost membranes. This shows that the carboxyl terminus of Band 3 is available for antibody binding in ghost membranes and that the carboxyl-terminal sequences of human and mouse Band 3 are not identical. The specificity of the antibody for the carboxyl terminus of Band 3 was confirmed by the loss of antibody binding after digestion of detergent-solubilized ghost membrane proteins with carboxypeptidase Y. In addition, carboxyl-terminal fragments of Band 3 generated by protease treatment of cells or ghost membranes were positive on immunoblots while amino-terminal fragments were negative. In contrast, protease-treated stripped ghost membranes did not contain a carboxyl-terminal fragment of Band 3 that was detectable on immunoblots. The carboxyl terminus of Band 3 was localized to the cytoplasmic side of the erythrocyte membrane since antibody binding as determined by immunofluorescence occurred in ghosts and permeabilized cells but not in intact cells. In addition, competition studies using enzyme-linked immunosorbent assays and immunoblots showed that cells and resealed ghosts competed poorly for antibody compared to ghost membranes, inside-out vesicles, or albumin-conjugated peptide.     

Proteolytic degradation of human erythrocyte band 3 by membrane-associated protease activity

Summary Antisera directed against the cytoplasmic portion of human erythrocyte Band 3 were used to follow the degradation of the band 3 molecule. Small amounts of Band 3 were degraded when well-washed red cell membrane ghosts were incubated in the cold; this process was greatly accelerated by incubating ghosts at 37°C. Band 3 labeled with pyridoxal-phosphate was digested at comparable rates. Band 3 digestion also took place when alkali-extracted ghost membranes were incubated at 37° for prolonged periods. These results suggest that human erythrocytes contain tightly bound, membrane-associated proteolytic activity.     

Trypsin-induced changes in the orientation of latent ATPase in protoplast ghosts from Mycobacterium phlei.

Latent ATPase, located on the inner surface of protoplast ghosts of Mycobacterium phlei, was unmasked either by trypsin or an impermeable form of trypsin, ethylene maleic anhydride-trypsin. Density gradient experiments showed that the ghost preparations remained intact following trypsin treatment. Evidence was obtained that 125I-trypsin failed to penetrate the ghost membranes. Thus, attempts were made to determine whether the ATPase molecule in the ghost membranes is accessible from the outer surface. Treatment of protoplast ghosts and trypsin-treated ghosts with 125I by the lactoperoxidase method resulted in the labeling of ATPase only in the trypsin-treated ghost preparations. The antibody to latent ATPase inhibited ATPase activity in trypsin-treated ghosts. The changes in the fluorescence polarization of diphenyl hexatriene indicated that trypsin treatment of the ghost membranes resulted in an increase in membrane fluidity. These studies suggest that the latent ATPase moiety has undergone translocation to the outer surface or it became accessible to trypsin digestion from the outer surface of the membranes as a result of removal of some proteins covering ATPase molecule in the membranes.     

Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin.

A specific association between spectrin and the inner surface of the human erythrocyte membrane has been examined by measuring the binding of purified [32P]spectrin to inside out, spectrin-depleted vesicles and to right side out ghost vesicles. Spectrin was labeled by incubating erythrocytes with 32Pi, and eluted from the ghost membranes by extraction in 0.3 mM NaPO4, pH 7.6. [32P]Spectrin was separated from actin and other proteins and isolated in a nonaggregated state as a So20,w = 7 S (in 0.3 mM NaPO4) or So20,w = 8 S (in 20 mM KCl, 0.3 mM NaPO4) protein after sedimentation on linear sucrose gradients. Binding of [32P]spectrin to inverted vesicles devoid of spectrin and actin was at least 10-fold greater than to right side out membranes, and exhibited different properties. Association with inside out vesicles was slow, was decreased to the value for right side out vesicles at high pH, or after heating spectrin above 50 degrees prior to assay, and was saturable with increasing levels of spectrin. Binding to everted vesicles was rapid, unaffected by pH or by heating spectrin, and rose linearly with the concentration of spectrin. Scatchard plots of binding to inverted vesicles were linear at pH 7.6, with a KD of 45 microng/ml, while at pH 6.6, plots were curvilinear and consistent with two types of interactions with a KD of 4 and 19 microng/ml, respectively. The maximal binding capacity at both pH values was about 200 microng of spectrin/mg of membrane protein. Unlabeled spectrin competed for binding with 50% displacement at 27 microng/ml. [32P]Spectrin dissociated and associated with inverted vesicles with an identical dependence on ionic strength as observed for elution of native spectrin from ghosts. MgCl2, CaCl2 (1 to 4 mM) and EDTA (0.5 to 1 mM) had little effect on binding in the presence of 20 mM KCl, while at low ionic strength, MgCl2 (1 mM) increased binding and inhibited dissociation to the same extent as 10 to 20 mM KCl. Binding was abolished by pretreatment of vesicles with 0.1 M acetic acid, or with 0.1 microng/ml of trypsin. The periodic acid-Schiff-staining bands were unaffected by trypsin digestion which destroyed binding; mild digestion, which decreased binding only 50%, converted Band 3 almost completely to a membrane-bound 50,000-dalton fragment resistant to further proteolysis. These experiments suggest that attachment of spectrin to the cytoplasmic surface of the membrane results from a selective protein-protein interaction which is independent of erythrocyte actin. A direct role of the major sialoglycoprotein or Band 3 as a membrane binding site appears unlikely.     

Proteolytic cleavages of cytochalasin B binding components of Band 4.5 proteins of the human red blood cell membrane

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.     

The interaction of human erythrocyte band 3 with cytoskeletal components

Band 3 of the human erythrocyte is involved in anion transport and binding of the cytoskeleton to the membrane bilayer. Human erythrocytes were treated to incorporate varying concentrations of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) a non-penetrating, irreversible inhibitor of anion transport, and both functions of Band 3 were analyzed. The rate of efflux of ³⁵SO₄. was measured and the binding of cytoskeletal components to the membrane was evaluated by extracting the membranes with 0.1 n NaOH and analyzing for the peptides remaining with the membrane. It was found that 0.1 n NaOH extracts all the extrinsic proteins from membranes of untreated cells, while, in the case of the membranes from cells treated with DIDS, a portion of the cytoskeletal components, spectrin (Bands 1 and 2) and Band 2.1 (ankyrin, syndein) remain with the membrane. The amount of these cytoskeletal components remaining with the membrane depends on the concentrations of DIDS incorporated. The effect of DIDS on the extractability of the spectrin-Band 2.1 complex correlates well with DIDS inhibition of anion transport (r = 0.91). At DIDS concentrations which completely inhibit anion transport, about 10% of total spectrin-Band 2.1 complex remains unextracted. Another anion-transport inhibitor, pyridoxal phosphate, has no effect on binding of the cytoskeleton to the membrane. On the other hand, digestion of DIDS-pretreated intact erythrocytes with Pronase, chymotrypsin, or trypsin releases the tight binding of Band 3 to cytoskeleton on the inside of the membrane. Since trypsin does not hydrolyze Band 3 the data suggest that a second membrane protein which is trypsin sensitive may be involved with Band 3 in cytoskeletal binding.     

The formation of vesicles retaining sodium-dependent transport systems for amino acids from protein-depleted membranes of pigeon erythrocytes

The process of the formation of vesicles from pigeon erythrocyte membranes was studied. Mildly alkaline solutions of low ionic strength, which reduce human erythrocyte membranes to small vesicles depleted of spectrin and other proteins, have no such effect on pigeon erythrocyte ghosts. A distinct phase of removal of membrane proteins, including spectrin, began to occur only when pigeon erythrocyte membranes were exposed to 0.2 mM EDTA adjusted to pH values above 10.2. Vesicles which demonstrated Na⁺-dependent amino acid transport were generated between the pH values 10.8 and 11.4. The results show that peripheral proteins, notably spectrin, maintain the integrity of the pigeon erythrocyte ghost. The interaction of these proteins with the membrane is rather different from that well studied in the human erythrocyte ghost and the possible significance of this for the pigeon erythrocyte is discussed.     

Strong interactions between a spin-labeled cholesterol analog and erythrocyte proteins in the human erythrocyte membrane

We have used a spin label analog of cholesterol bearing a nitroxide on the alkyl chain (26-nor-25-doxylcholestanol) to study cholesterol-protein interactions in the human erythrocyte membrane. As judged from the ESR spectrum, the spin label is readily incorporated into the membrane when added from a concentrated ethanolic solution to a cell or ghost suspension. With intact erythrocytes or white ghosts in isotonic buffer, the ESR spectrum is a superposition of a mobile component and a strongly immobilized component (outer hyperfine splitting 61–63 G). The latter corresponds to approx. 45% of the signal, a percentage which is barely affected by varying the temperature between 5 and 37°C. Removal of the cytoskeletal proteins spectrin and actin by low ionic strength treatment or of all extrinsic proteins by alkali treatment of ghosts reduces the immobilized fraction to approx. 25%. The effect of controlled proteolysis of intrinsic proteins was also tested. Pre-treatment of cells with chymotrypsin or pre-treatment of unsealed ghosts with trypsin has no effect on the ESR spectrum obtained with alkali-treated membranes. On the other hand, after chymotrypsin treatment of unsealed ghost, which reduces the band 3 protein to a 17.5 kDa membrane fragment, the strongly immobilized component is no longer observable. These data show that the cholesterol analog 26-nor-25-doxylcholestanol interacts strongly with one or several proteins of the erythrocyte membrane. That the intrinsic protein band 3 is involved is suggested by the disappearance of the immobilized fraction occurring upon chymotrypsin digestion of this protein. Our results are thus consistent with the proposal of a selective cholesterol-band 3 interaction in the erythrocyte membrane (Schubert, D. and Boss, K. (1982) FEBS Lett. 150, 4–8). Our data also suggest that this interaction is influenced by cytoskeletal proteins, an effect which can be explained considering the known linking of band 3 to the erythrocyte cytoskeleton via ankyrin. Experiments have also been carried out with 3-doxylandrostanol, a more commonly used cholesterol spin-label analog. With this spin label, at all temperatures investigated, we found it impossible to demonstrate unambiguously the existence of two separate spectral components. It is suggested that 26-nor-25-doxylcholestanol is a better reporter of cholesterol behavior in membranes.     

Enzymatic methylation of band 3 anion transporter in intact human erythrocytes

Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77) [Freitag, C., & Clarke, S. (1981) J. Biol. Chem. 256, 6102-6108]. The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl-3H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl-3H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[3H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [3H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[3H]methyl ester or glutamyl gamma-[3H]methyl ester was detected. The formation of D-aspartic acid beta-[3H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl-3H]methionine.     

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.     

Intramembrane particle aggregation in erythrocyte ghosts. II. The influence of spectrin aggregation.

Physicochemical properties of mixtures of spectrin and actin extracted from human erythrocyte ghosts have been correlated with ultrastructural changes observed in freeze-fractured erythrocyte membranes. (1) Extracted mixtures of spectrin and actin have a very low solubility (less than 30 mug/ml) near their isoelectric point, pH 4.8. These mixtures are also precipitated by low concentrations of Ca2+, Mg2+, polylysine or basic proteins. (2) All conditions which precipitate extracts of spectrin and actin also induce aggregation of the intramembrane particles in spectrin-depleted erythrocyte ghosts. Precipitation of the residual spectrin molecules into small patches on the cytoplasmic surface of the ghost membrane is thought to be the cause of particle aggregations, implying an association between the spectrin molecules and the intramembrane particles. (3) When fresh ghosts are exposed to conditions which precipitate extracts of spectrin and actin, only limited particle aggregation occurs. Instead, the contraction of the intact spectrin meshwork induced by the precipitation conditions compresses the lipid bilayer of the membrane, causing it to bleb off particle-free, protein-free vesicles. (4) The absence of protein in these lipid vesicles implies that all the proteins of the erythrocyte membrane are immobilized by association with either the spectrin meshwork or the intramembrane particles.     

Temperature-dependent abnormalities of the erythrocyte membrane in porcine malignant hyperthermia

The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24 degrees C in both MH and normal erythrocyte ghosts. However, below 24 degrees C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33 degrees C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.     

ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : I. Effects of Trypsin, Phospholipase C, and pH on the Topography of Bound Positively Charged Colloidal Particles

The effects of pH, trypsin, and phospholipase C on the topographic distribution of acidic anionic residues on human erythrocytes was investigated using colloidal iron hydroxide labeling of mounted, fixed ghost membranes. After glutaraldehyde fixation at pH 6.5–7.5, the positively charged colloidal particles were bound to the membranes in small randomly distributed clusters. The clusters of anionic sites were reversibly aggregated by incubation at pH 5.5 before fixation at the same pH. These results correlate with the distribution of intramembranous particles found by Pinto da Silva (J. Cell Biol. 53:777), with the exception that the distribution of anionic sites on a majority of the fixed ghosts at pH 4.5 was aggregated instead of dispersed. The randomly distributed clusters could be nonreversibly aggregated by trypsin or phospholipase C treatment of intact ghosts before glutaraldehyde fixation. Previous glutaraldehyde fixation prevented trypsin and pH induced aggregation of the colloidal iron sites. Evidence that N-acetylneuraminic acid groups are the principal acidic residues binding colloidal iron was the elimination of greater than 85% of the colloidal iron labeling to neuraminidase-treated cell membranes. Colloidal iron binding N-acetylneuraminic acid residues may reside on membrane molecules such as glycophorin, a sialoglycoprotein which contains the majority of the N-acetylneuraminic acid found on the human erythrocyte membrane.     

Sendai Virus Fusion Activity as Modulated by Target Membrane Components

We have studied the differences between erythrocytes and erythrocyte ghosts as target membranes for the study of Sendai virus fusion activity. Fusion was monitored continuously by fluorescence dequenching of R18-labeled virus. Experiments were carried out either with or without virus/target membrane prebinding. When Sendai virus was added directly to a erythrocyte/erythrocyte ghost suspension, fusion was always lower than that obtained when experiments were carried out with virus already bound to the erythrocyte/erythrocyte ghost in the cold, since with virus prebinding fusion can be triggered more rapidly. Although virus binding to both erythrocytes and erythrocyte ghosts was similar, fusion activity was much more pronounced when erythrocyte ghosts were used as target membranes. These observations indicate that intact erythrocytes and erythrocyte ghosts are not equivalent as target membranes for the study of Sendai virus fusion activity. Fusion of Sendai virus with both target membranes was inhibited when erythrocytes or erythrocyte ghosts were pretreated with proteinase K, suggesting a role of target membrane proteins in this process. Treatment of both target membranes with neuraminidase, which removes sialic acid residues (the biological receptors for Sendai virus) greatly reduced viral binding. Interestingly, this treatment had no significant effect on the fusion reaction itself.     

Localization of the C termini of the Rh (rhesus) polypeptides to the cytoplasmic face of the human erythrocyte membrane.

We have raised a rabbit antiserum to a synthetic peptide corresponding to the C terminus (residues 400-416) of the Rh30A polypeptide. The rabbit antiserum reacted with the Rh30B (D30) polypeptide in addition to the Rh30A (C/c and/or E/e) polypeptide(s), indicating that these proteins share homology at their C termini. The antiserum did not react with erythrocyte membranes from an individual with Rh(null) syndrome. The rabbit antiserum immunoprecipitated Rh polypeptides from erythrocyte membranes and alkali-stripped membranes, but not from intact erythrocytes. Treatment of intact red cells with carboxypeptidase Y did not affect the reactivity of the antiserum, whereas treatment of alkali-stripped and unsealed erythrocyte ghost membranes resulted in the loss of antibody binding. Carboxypeptidase A treatment of intact erythrocytes and alkali-stripped membranes had no effect on antibody binding, indicating that the C-terminal domains of the Rh polypeptides contain lysine, arginine, proline, or histidine residues. These results show that the C termini of the Rh polypeptides are located toward the cytoplasmic face of the erythrocyte membrane. Treatment of intact radioiodinated erythrocytes with bromelain followed by immunoprecipitation with monoclonal anti-D gave a band of M(r) 24,000-25,000, indicating that the Rh30B (D30) polypeptide is cleaved at an extracellular domain close to the N or C terminus, with loss of the major radioiodinated domain. Immunoblotting of bromelain treated D-positive erythrocyte membranes with the rabbit antiserum to the C-terminal peptide revealed a new band of M(r) 6000-6500, indicating that the extracellular bromelain cleavage site is located near the C terminus of the molecule. The band of M(r) 6000-6500 was not obtained in erythrocyte membranes derived from bromelain treated D-negative erythrocytes. Erythrocytes of the rare -D- phenotype appear to either totally lack, or have gross alterations in, the Cc/Ee polypeptide(s), since the bromelain treatment of these cells resulted in the total loss of staining in the M(r) 35,000-37,000 region and the concomitant appearance of the new band of M(r) 6000-6500.     

Freeze-etching study on membrane ultrastructural changes caused by freezing: cooling rate-dependent intramembrane particle aggregation in erythrocyte stripped ghosts

The changes of membrane ultrastructures by freezing stresses were examined on stripped ghosts which were made by removing almost all peripheral membrane proteins from human erythrocyte membranes. By freezing these stripped ghost membranes showed cooling rate-dependent intramembrane particle (IMP) aggregation. With the cooling rates at and faster than 30,000 degrees C/min, their IMPs were evenly distributed on the fracture faces. However, cooling rates at and slower than 8000 degrees C/min resulted in IMP aggregation. The degree of IMP aggregation increased in parallel with decreasing cooling rates. Without freezing, the IMP aggregation in stripped ghosts could be induced by exposing these ghosts to hypertonic salt solutions, but lowering the temperature did not affect IMP aggregation. The cooling rate-dependent IMP aggregation during freezing was suppressed by adding cryoprotective agents which were known to reduce the salt concentration of the medium during freezing. It is suggested that the IMP aggregation in stripped ghosts by freezing occurs by exposure to concentrated salt solutions during freezing. This result indicates the possibility that IMP aggregation may arise during slow freezing of some biomembranes as a result of an increase in salt concentration rather than as a result of reduction in temperature.     

Topology of the anion exchange protein AE1: the controversial sidedness of lysine 743

The topology of the band 3 (AE1) polypeptide of the erythrocyte membrane is not fully established despite extensive study. Residues near lysine 743 (K743) have been reported to be extracellular in some studies and cytoplasmic in others. In the work presented here, we have attempted to establish the sidedness of K743 using in situ proteolysis. Trypsin, papain, and proteinase K do not cleave band 3 at or near K743 in intact red cells, even under conditions that cause cleavage on the C-terminal side of the glycosylation site (N642) in extracellular loop 4. In contrast, trypsin sealed inside red cell ghosts cleaves at K743, as does trypsin treatment of inside-out vesicles (IOVs). The transport inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H(2)DIDS), acting from the extracellular side, blocks trypsin cleavage at K743 in unsealed membranes by inducing a protease-resistant conformation. H(2)DIDS added to IOVs does not prevent cleavage at K743; therefore, trypsin cleavage at K743 in IOVs is not a consequence of cleavage of right-side-out or leaky vesicles. Finally, microsomes were prepared from HEK293 cells expressing the membrane domain of AE1 lacking the normal glycosylation site. This polypeptide does not traffic to the surface membrane; trypsin treatment of microsomes containing this polypeptide produces the 20 kDa fragment, providing further evidence that K743 is exposed at the cytoplasmic surface. Therefore, the actions of trypsin on intact cells, resealed ghosts, unsealed ghosts, inside-out vesicles, and microsomes from HEK293 cells all indicate that K743 is cytoplasmic and not extracellular.     

The role of ankyrin in shape and deformability change of human erythrocyte ghosts

Human erythrocyte membranes (ghosts) from acid/citrate/dextrose preserved blood were digested with trypsin (protein/trypsin = 100:1) under hypotonic conditions and then analyzed by SDS-polyacrylamide gel electrophoresis. After digestion for about 20-30 s at 0 degree C, only ankyrin had disappeared and other bands including spectrin, actin, band 4.1 and band 3 remained intact. This observation was supported by electron micrographs showing that the horizontally disposed, filamentous structure was a little apart from the lipid bilayer and its components were not destroyed. In contrast to intact ghosts, treatment with chlorpromazine, or Mg-ATP did not induce shape change in these trypsin-treated ghosts. The number of transformable cells correlated closely with the amount of remaining ankyrin in the SDS-polyacrylamide gel electrophoresis pattern. Furthermore, the chlorpromazine- and Mg-ATP-induced decreases in viscosity of suspensions of erythrocyte ghosts were also prevented by trypsin treatment for 20-30 s at 0 degree C. These findings suggest that ankyrin plays an important role in the change in shape and deformability of erythrocyte ghosts. The molecular mechanism of drug-induced shape change and the role of undermembrane structure in regulating erythrocyte shape and deformability are discussed.     

Photoaffinity labeling of glyceraldehyde-3-phosphate dehydrogenase by an aryl azide derivative of glucosamine in human erythrocytes

An aryl azide derivative of glucosamine, N-(4-iodoazidosalicyl)-2-amido-2-deoxy-D-glucopyranose (GlcNAs), was synthesized as a potential photoaffinity label for the facilitative hexose carrier. The derivative inhibited hexose uptake into intact human erythrocytes half-maximally at 3.5 mM and was itself slowly transported into cells. However, photolysis of iodinated GlcNAs with leaky erythrocyte ghosts produced appreciable labeling on gel electrophoresis only of Band 6, which is glyceraldehyde-3-phosphate dehydrogenase. Band 6 photolabeling in leaky ghosts by GlcNAs was: saturable, due mostly to the aryl azide moiety, inhibited by agents with known affinity for the enzyme including sulfhydryl reagents and the enzyme substrate glyceraldehyde-3-phosphate, and not inhibited by the free-radical scavenger p-aminobenzoic acid. Moreover, GlcNAs also inhibited erythrocyte glyceraldehyde-3-phosphate dehydrogenase activity in a dose-dependent fashion in the dark and more potently following irradiation. In resealed ghosts, Band 6 labeling was decreased by D-glucose, reflecting inhibition of carrier-mediated uptake of the agent. GlcNAs appears to be a specific photoaffinity label for erythrocyte glyceraldehyde-3-phosphate dehydrogenase, and therefore potentially useful for studies of enzyme activity, compartmentation, or membrane association.     

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.