RESUMEN
Electron and hole Bloch states in bilayer graphene exhibit topological orbital magnetic moments with opposite signs, which allows for tunable valley-polarization in an out-of-plane magnetic field. This property makes electron and hole quantum dots (QDs) in bilayer graphene interesting for valley and spin-valley qubits. Here, we show measurements of the electron-hole crossover in a bilayer graphene QD, demonstrating opposite signs of the magnetic moments associated with the Berry curvature. Using three layers of top gates, we independently control the tunneling barriers while tuning the occupation from the few-hole regime to the few-electron regime, crossing the displacement-field-controlled band gap. The band gap is around 25 meV, while the charging energies of the electron and hole dots are between 3 and 5 meV. The extracted valley g-factor is around 17 and leads to opposite valley polarization for electrons and holes at moderate B-fields. Our measurements agree well with tight-binding calculations for our device.
RESUMEN
Measuring links among genotype, phenotype and survival in the wild has long been a focus of studies of adaptation. We conducted a 4-year capture-recapture study to measure survival by genotype and phenotype in the Southwestern Fence Lizard (Sceloporus cowlesi) at the White Sands ecotone (transition area between white sands and dark soil habitats). We report several unanticipated findings. First, in contrast with previous work showing that cryptic blanched coloration in S. cowlesi from the heart of the dunes is associated with mutations in the melanocortin-1 receptor gene (Mc1r), ecotonal S. cowlesi showed minimal association between colour phenotype and Mc1r genotype. Second, the frequency of the derived Mc1r allele in ecotonal S. cowlesi appeared to decrease over time. Third, our capture-recapture data revealed a lower survival rate for S. cowlesi individuals with the derived Mc1r allele. Thus, our results suggest that selection at the ecotone may have favoured the wild-type allele in recent years. Even in a system where a genotype-phenotype association appeared to be black and white, our study suggests that additional factors - including phenotypic plasticity, epistasis, pleiotropy and gene flow - may play important roles at the White Sands ecotone. Our study highlights the importance of linking molecular, genomic and organismal approaches for understanding adaptation in the wild. Furthermore, our findings indicate that dynamics of natural selection can be particularly complex in transitional habitats like ecotones and emphasize the need for future research that examines the patterns of ongoing selection in other ecological 'grey' zones.
Asunto(s)
Lagartos/genética , Pigmentación/genética , Receptor de Melanocortina Tipo 1/genética , Alelos , Animales , Ecosistema , Epistasis Genética , Flujo Génico , Pleiotropía Genética , Genética de Población , Genotipo , Fenotipo , Selección GenéticaRESUMEN
Fas ligand (FasL) has been shown to mediate both apoptotic and inflammatory reactions. To rigorously assess the physiological role of different forms of the FasL molecule with regard to these two distinct processes, we isolated stably transfected lymphoma cell lines that expressed either murine wild-type FasL, membrane-only FasL, or functionally distinct forms of soluble FasL. First, the ability of these lines to induce an inflammatory response was assessed in vivo by injecting the transfectants intraperitoneally and measuring subsequent neutrophil extravasation into the peritoneal cavity. Second, lines were assessed by injecting the transfectants subcutaneously and monitoring their growth as solid tumors. Our study clearly demonstrated that the extent of inflammation induced by the transfectants directly correlated with their relative cytotoxic activities. A neutrophil response could only be elicited in mice with intact Fas death domains although Fas expression by the neutrophils was not essential. Lymphoma cells expressing the soluble FasL form corresponding to the natural cleavage product could not trigger apoptosis and did not induce a neutrophil response. In contrast to the other FasL transfectants, these cells survived as tumor transplants. However, expression of soluble FasL was not benign, but actually suppressed the inflammatory response and protected other transfectants from the effector mechanisms elicted by membrane-bound FasL.
Asunto(s)
Glicoproteínas de Membrana/inmunología , Animales , Sitios de Unión , Membrana Celular/metabolismo , Supervivencia Celular , Proteína Ligando Fas , Femenino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Ratones Endogámicos MRL lpr , Neutrófilos/inmunología , Peritoneo/citología , Solubilidad , Células Tumorales CultivadasRESUMEN
Susceptibility to Fas-mediated apoptosis in nontolerant B cells is regulated in a receptor-specific fashion. To explore the regulation of Fas killing in tolerant, autoreactive B cells, mice doubly transgenic for hen egg lysozyme (HEL)-specific B cell receptors and soluble HEL were examined. Engagement of CD40 led to enhanced Fas expression and acquisition of sensitivity to Fas-mediated apoptosis in tolerant B cells, similar to that observed in nontolerant, receptor transgenic B cells. Engagement of surface immunoglobulin by specific (HEL) antigen failed to induce Fas resistance in tolerant B cells, in contrast to its effect on nontolerant B cells; however, cross-linking of biotinylated HEL with streptavidin induced similar levels of Fas resistance in tolerant and nontolerant B cells, which approximated the degree of Fas resistance produced by anti-Ig. Unlike surface Ig (sIg) engagement, physiological engagement of IL-4 receptors produced similar levels of Fas resistance in tolerant and nontolerant B cells. Thus, tolerant B cells differ from nontolerant B cells in the diminished capacity of surface immunoglobulin engagement to produce Fas resistance; however, tolerant B cells can be induced to become resistant to Fas-mediated apoptosis by IL-4 or by higher order cross-linking of sIg receptors.
Asunto(s)
Apoptosis , Linfocitos B/inmunología , Tolerancia Inmunológica , Interleucina-4/farmacología , Receptores de Antígenos de Linfocitos B/fisiología , Receptor fas/fisiología , Animales , Ligando de CD40 , Masculino , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Muramidasa/inmunologíaRESUMEN
Two hapten-inhibitable murine monoclonal antiidiotopic antibodies identified two idiotopes expressed by the heavy chain of hybridoma protein 36-65, whose amino acid sequence is encoded in the germ line of A/J mice. Among cross-reactive idiotype-positive hybridoma proteins and p-azophenylarsonate-immune antibodies, the two idiotopes were not always expressed together; some diversified antibodies expressed one idiotope without the other. Suppression that was induced by the two antiidiotopes was idiotope specific and corresponded to the fine specificities of these two reagents.
Asunto(s)
Compuestos Azo/inmunología , Epítopos , Idiotipos de Inmunoglobulinas/inmunología , Linfocitos T Reguladores/inmunología , p-Azobencenoarsonato/inmunología , Animales , Sitios de Unión de Anticuerpos , Unión Competitiva , Haptenos/inmunología , Idiotipos de Inmunoglobulinas/biosíntesis , Técnicas de Inmunoadsorción , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos BALB C , ConejosRESUMEN
The proximate cause of autoantibodies characteristic of systemic autoimmune diseases has been controversial. One hypothesis is that autoantibodies are the result of polyclonal nonspecific B cell activation. Alternatively, autoantibodies could be the result of antigen-driven B cell activation, as observed in secondary immune responses. We have approached this question by studying monoclonal anti-DNA autoantibodies derived from unmanipulated spleen cells of the autoimmune MRL/lpr mouse strain. This analysis shows that anti-DNAs, like rheumatoid factors (19), are the result of specific antigen-driven stimulation. In addition, correlation of sequences with fine specificity shows that: (a) somatic mutations can cause specificity for dsDNA and that such mutations are selected for; (b) arginine residues play an important role in determining specificity; and (c) anti-idiotypes that recognize the majority of anti-DNA are probably not specific for any one family of V regions.
Asunto(s)
Autoanticuerpos/genética , Enfermedades Autoinmunes/genética , ADN/inmunología , Mutación , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Enfermedades Autoinmunes/inmunología , Secuencia de Bases , Células Clonales , ADN de Cadena Simple/inmunología , Ensayo de Inmunoadsorción Enzimática , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Ratones , Ratones Endogámicos , Ratones Mutantes , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , ARN Mensajero/genética , Especificidad de la EspecieRESUMEN
The homologous, monoclonal antiidiotope, MB, induced idiotope suppression that was remarkably stable and could be transferred by B lymphocytes. Marked depletion of T cell function, confirmed by limiting diluting analysis, did not affect the ability of MB to suppress the corresponding idiotope. Suppression induced by MB appears to result from direct interaction with idiotope-positive B cells, without the intervention of idiotope-specific T suppressor cells.
Asunto(s)
Anticuerpos Antiidiotipos/fisiología , Anticuerpos Monoclonales/fisiología , Idiotipos de Inmunoglobulinas/biosíntesis , Terapia de Inmunosupresión , Depleción Linfocítica , Linfocitos T/inmunología , Animales , Linfocitos B/inmunología , Linfocitos B/trasplante , Inmunización Pasiva , Idiotipos de Inmunoglobulinas/inmunología , Activación de Linfocitos , Cooperación Linfocítica , Ratones , Ratones Endogámicos A , p-Azobencenoarsonato/inmunologíaRESUMEN
The Na+/H+ antiport is stimulated by 12-O-tetradecanoylphorbol-13, acetate (TPA) and other phorbol esters in rat thymic lymphocytes. Mediation by protein kinase C is suggested by three findings: (a) 1-oleoyl-2-acetylglycerol also activated the antiport; (b) trifluoperazine, an inhibitor of protein kinase C, blocked the stimulation of Na+/H+ exchange; and (c) activation of countertransport was accompanied by increased phosphorylation of specific membrane proteins. The Na+/H+ antiport is also activated by osmotic cell shrinking. The time course, extent, and reversibility of the osmotically induced and phorbol ester-induced responses are similar. Moreover, the responses are not additive and they are equally susceptible to inhibition by trifluoperazine, N-ethylmaleimide, and ATP depletion. The extensive analogies between the TPA and osmotically induced effects suggested a common underlying mechanism, possibly activation of a protein kinase. It is conceivable that osmotic shrinkage initiates the following sequence of events: stimulation of protein kinase(s) followed by activation of the Na+/H+ antiport, resulting in cytoplasmic alkalinization. The Na+ taken up through the antiport, together with the HCO3- and Cl- accumulated in the cells as a result of the cytoplasmic alkalinization, would be followed by osmotically obliged water. This series of events could underlie the phenomenon of regulatory volume increase.
Asunto(s)
Proteínas Portadoras/metabolismo , Linfocitos/fisiología , Ésteres del Forbol/farmacología , Forboles/farmacología , Equilibrio Hidroelectrolítico/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Amilorida/farmacología , Animales , Calcio/metabolismo , Citoplasma/metabolismo , Diglicéridos/farmacología , Activación Enzimática/efectos de los fármacos , Masculino , Proteína Quinasa C , Proteínas Quinasas/metabolismo , Ratas , Ratas Endogámicas , Intercambiadores de Sodio-Hidrógeno , Trifluoperazina/farmacologíaRESUMEN
In mice functionally deficient for either Fas or Fas ligand expression, the failure of Fas-ligand-expressing cytotoxic T cells to eliminate autoreactive B cells can result in excessive autoantibody production. Recent in vitro studies have shown that B cells activated by CD40 ligand become extremely sensitive to Fas-mediated apoptosis while IL-4 and/or surface IgM receptor engagement protects B cells from Fas ligand cytolysis. Potential in vivo sites for Fas ligand regulation of self-reactive B cells have been suggested and implications for human disease have been investigated.
Asunto(s)
Enfermedades Autoinmunes/genética , Receptor fas/genética , Animales , Enfermedades Autoinmunes/inmunología , Proteína Ligando Fas , Humanos , Ligandos , Glicoproteínas de Membrana/genética , Ratones , Ratones MutantesRESUMEN
Proteins encoded by the fas and fas ligand (fasL) genes are involved in apoptotic cell death in lymphocytes. In this article we review the recent elucidation of the role of the Fas-FasL interactions in the maintenance of tolerance to self antigens and in the homeostatic regulation of lymphocyte clonal expansion, and discuss the mechanisms of autoimmunity in Fas- and FasL-deficient mutant mouse strains.
Asunto(s)
Antígenos de Superficie/inmunología , Apoptosis , Trastornos Linfoproliferativos/inmunología , Glicoproteínas de Membrana/inmunología , Animales , Autoantígenos/inmunología , Autoinmunidad , Modelos Animales de Enfermedad , Proteína Ligando Fas , Humanos , Tolerancia Inmunológica , Trastornos Linfoproliferativos/genética , Ratones , Ratones Mutantes , Receptor fasRESUMEN
Under appropriate conditions (presence of cation ionophores) net KCl efflux measured with a K+ electrode can be used to estimate conductive Cl- fluxes, a sensitive procedure that allows continuous recording. The procedure was tested in human red cells by demonstrating effects of ionophores and of an anion transport inhibitor, and in dissociated MDCK cells by demonstration of cAMP and volume-activated Cl- fluxes.
Asunto(s)
Cloruros/metabolismo , Eritrocitos/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Transporte Biológico , Colforsina/farmacología , Electrodos , Eritrocitos/efectos de los fármacos , Gramicidina/farmacología , Humanos , Potasio , Valinomicina/farmacologíaRESUMEN
Cation fluxes were measured in right-side-out and inside-out vesicles obtained from human red cells. Rubidium, which is spontaneously released at very slow rates, can be rapidly released from both types of vesicle by addition of valinomycin. P-Chloromercuriphenyl sulfonic acid (PCMBS) also increases the cation permeability of the vesicles with reversal to normal after addition of dithiothreitol. The effect of PCMBS is considerably larger and appears faster in the inside-out vesicles as compared to the right-side-out vesicles, the difference being greater at low temperatures. These data indicate that the SH groups responsible for the changes in cation permeability are more accessible from the inside face of the membrane. The response to PCMBS was not diminished after selective removal of extrinsic proteins by alkaline extraction, and/or after the membranes were exposed to proteolytic enzymes. The major polypeptide component remaining in vesicles after both treatments was a 17 000-dalton transmembrane fragment derived from band 3 which might, therefore, be responsible for the permeability response. Addition of Ca2+ to either right-side-out or inside-out vesicles, in the presence or absence of ionophore A23187, was without effect on monovalent cation permeability, indicating that the mechanism of Ca2+-induced K+ permeation was lost or inactivated during the preparation of the vesicles.
Asunto(s)
Proteínas Sanguíneas/fisiología , Membrana Eritrocítica/fisiología , Eritrocitos/fisiología , Proteínas de la Membrana/fisiología , Rubidio/metabolismo , 4-Cloromercuribencenosulfonato/farmacología , Calcio/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Humanos , Potasio/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Valinomicina/farmacologíaRESUMEN
A Na+/H+ exchanger is involved in the regulation of cytoplasmic pH and cellular volume in a variety of cells. Little is known about the molecular nature of this exchanger. The purpose of this study was to survey a variety of group-specific covalent reagents as potential inhibitors of the exchanger. Na+/H+ countertransport activity was assayed as the amiloride-sensitive rate of Na+-induced alkalinization in acid-loaded lymphocytes, or as the rate of swelling in cells suspended in sodium propionate medium. Activity was not affected by proteinases or by carboxyl-group and amino-group specific reagents. A significant inhibition was produced by diethylpyrocarbonate, a histidine-specific reagent and by N-ethylmaleimide, a sulfhydryl group reagent. A similarly reactive but nonpermeating sulfhydryl agent, glutathione-maleimide, failed to inhibit Na+-H+ exchange. Moreover, the reaction with N-ethylmaleimide was sensitive to changes in the cytoplasmic pH. The data suggest that the chemically reactive groups of the Na+/H+ exchanger of lymphocytes have limited exposure to the extracellular medium but that an internally located sulfhydryl group is critical for the cation-exchange activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Etilmaleimida/farmacología , Linfocitos T/metabolismo , Animales , Dietil Pirocarbonato/farmacología , Concentración de Iones de Hidrógeno , Masculino , Ratas , Ratas Endogámicas , Intercambiadores de Sodio-Hidrógeno , Linfocitos T/efectos de los fármacosRESUMEN
Right-side-out vesicles derived from red blood cells treated with chymotrypsin retain specific anion transport function (defined as transport sensitive to the specific inhibitor, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), even though the transport protein, band 3, is cleaved into two segments of 60 and 35 kdaltons. In contrast, vesicles derived from alkali-stripped ghosts treated with relatively high concentrations of chymotrypsin retain almost no specific anion function. The loss of function appears to be related to additional cleavages of band 3 protein that occur in treated ghosts, the 60-kdalton segment being reduced first to a 17- and then to a 15-kdalton segment and the 35-kdalton segment being reduced to a 9-kdalton segment plus a carbohydrate containing fragment. The chymotryptic cleavages of band 3 protein of ghosts are preferentially inhibited by high ionic strength, the production of the 9-kdalton segment being somewhat slower than that of the 15-kdalton segment. Vesicles derived from ghosts treated with chymotrypsin at different ionic strengths show a graded reduction in specific anion transport activity, but it was not possible to determine, definitively, which of the additional cleavages was inhibitory. In the light of these data and other information, the functional role of the segments of band 3 is discussed.
Asunto(s)
Aniones/sangre , Proteínas Sanguíneas , Quimotripsina/sangre , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Proteínas de la Membrana/sangre , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Proteína 1 de Intercambio de Anión de Eritrocito , Transporte Biológico/efectos de los fármacos , Membrana Eritrocítica/efectos de los fármacos , Humanos , Peso Molecular , Concentración Osmolar , Sulfatos/metabolismoRESUMEN
The specific anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and its reduced analog (H2DIDS), when irreversibly bound to band 3 protein of the red blood cell membrane, form amino acid conjugates through interaction with the epsilon-amino group of a particular lysine residue. The specific residue is located in a transmembrane segment of band 3 protein and appears to be a close neighbor of the transport site.
Asunto(s)
Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/sangre , Proteínas Sanguíneas/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Lisina/metabolismo , Proteínas de la Membrana/metabolismo , Estilbenos/sangre , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Proteína 1 de Intercambio de Anión de Eritrocito , Arginina/metabolismo , Transporte Biológico , Fenómenos Químicos , Química , Cromatografía en Capa Delgada , Humanos , HidrólisisRESUMEN
The binding site for 4,4'-diisothiocyano-2,2'-stilbenedi sulfonic acid, a specific, potent, irreversible inhibitor of anion transport in red blood cells is located in a 15 000 dalton transmembrane segment of band 3, produced by chymotrypsin treatment of ghosts stripped of extrinsic proteins. The segment was cleaved into three fragments of 7000 daltons by CNBr. The C-terminus of the segment is located in the 7000 daltons by the N-terminus in one of the 4000 dalton fragment; the N-terminus in one of the 4000 dalton fragments; and the binding site for 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid in the middle 4000 dalton fragment. The latter was cleaved by N-bromosuccinimide into two fragments of 2000 daltons. The binding site for 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid was located on the fragment containing the newly formed N-terminus. It is concluded that the binding site is located about 9000 daltons from the C-terminus (at the outside face of the membrane) and 6000 daltons from the N-terminus (at the cytoplasmic face). In view of the existing evidence that the binding site may be located near the outside face of the membrane, it is suggested that the 15 000 dalton segment is folded, so that it crosses the bilayer three times.
Asunto(s)
Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/metabolismo , Sitios de Unión , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Estilbenos/metabolismo , Proteínas Portadoras/metabolismo , Cromatografía en Gel , Quimotripsina/farmacología , Electroforesis en Gel de Poliacrilamida , Humanos , Peso MolecularRESUMEN
Sulfate efflux was measured in inside-out vesicles obtained from human red cells. Inhibition was observed in vesicles derived from cells pretreated with DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate) or after addition of dipyridamole to the vesicles, both agents being specific and potent inhibitors of anion transport in cells. Trypsinization of the cytoplasmic side of the membrane in order to release a 40 000 dalton fragment from band 3 (the purported anion transport protein) had no effect on sulfate efflux. Further degradation of band 3 to a 17 000 dalton segment, by trypsinization of inside-out vesicles derived from cells that had been pretreated with chymotrypsin, also showed little reduction in transport activity. Furthermore, such vesicles derived from DIDS pretreated cells were inhibited by over 90%. In DIDS-treated cells, the agent is highly localized in band 3. In trypsinized inside-out vesicles, it is largely found in a 55000 fragment and in trypsinized vesicles derived from cells pretreated with chymotrypsin it is largely located in the 17 000 fragment. The data suggest that both the anion transport and inhibitor binding sites are located in a 17 000 transmembrane segment of band 3.
Asunto(s)
Proteínas Sanguíneas , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Proteínas de la Membrana , Sulfatos/sangre , Transporte Biológico , Proteínas Sanguíneas/metabolismo , Membrana Eritrocítica/ultraestructura , Humanos , Cinética , Proteínas de la Membrana/sangre , Peso Molecular , Péptido HidrolasasRESUMEN
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 cleavage 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.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Citocalasina B/metabolismo , Membrana Eritrocítica/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Portadoras/metabolismo , Quimotripsina/metabolismo , Citoplasma/metabolismo , Espacio Extracelular/metabolismo , Humanos , Peso Molecular , Proteínas de Transporte de Monosacáridos , Proteínas de Transporte de Nucleósidos , Pronasa/metabolismo , Tripsina/metabolismoRESUMEN
After prolonged treatment of red-cell ghosts with pepsin followed by SDS-urea-acrylamide gel electrophoresis of the membrane peptide fraction, a heavily stained band representing peptides of about 4 kDa (with traces of higher molecular weights) was found. If the cells were first labelled with the disulfonic stilbene, DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) or with N-ethylmaleimide, probes that react with specific sites in Band 3 the anion transport protein, both agents were largely located in the 4 kDA band. With less intensive pepsin treatment, Stained bands of about 17, 12 and 8 kDa were also visible, and DIDS labelling was associated with these higher molecular weight peptides. The 4 kDa band apparently contains at least five or six different peptides. A single peptide containing the DIDS-binding site was separated from others in the band by ion-exchange chromatography. The location of the DIDS-peptide in the primary structure of Band 3 was determined by matching the known location of DIDS and of a methionine residue cleavable by cyanogen bromide. It is concluded that two additional 4 kDA peptides are labelled with N-ethylmaleimide. Because the location of the N-ethylmaleimide-binding sites are known, these two peptides could also be mapped in the primary structure of Band 3. The findings are consistent with the suggestion that pepsin can digest those portions of Band 3 (and probably of other intrinsic peptides) that are exposed on either side of the membrane, leaving only those domains that cross the bilayer. For Band 3, the data are consistent with a structure containing five crossing strands per monomer, each crossing strand being about 4 kDa.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Pepsina A/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/metabolismo , Cromatografía por Intercambio Iónico , Bromuro de Cianógeno , Membrana Eritrocítica/metabolismo , Etilmaleimida/metabolismo , Humanos , Microscopía Electrónica , Peso MolecularRESUMEN
Isolated human blood platelets, loaded with the pH-sensitive fluorescence dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein show cytoplasmic alkalinization upon stimulation with thrombin but acidification with ADP stimulation. In both cases a Na+/H+ exchange is activated. This can be revealed by the sensitivity of the induced pH changes to amiloride and to 5-N-(3-aminophenyl)amiloride (APA), known inhibitors of the Na+/H+ exchanger, and by a dependence on sodium in the external medium. ADP-induced platelet aggregation is not affected by omission of sodium from the external medium. Furthermore, aggregation is barely inhibited (less than 10%) by amiloride or APA at concentrations up to 50 microM while the Ki values in affecting the Na+/H+ exchange are 5.9 and 1.6 microM for amiloride and APA, respectively. Platelet aggregation is inhibited by amiloride or APA at concentrations higher than 50 microM, but this inhibition is apparently due to a secondary effect of the agents. It is concluded that platelet aggregation induced by ADP is not dependent on activation of Na+/H+ exchange.