Calculation of a Gap restoration in the membrane skeleton of the red blood cell: possible role for myosin II in local repair.

Human red blood cells contain all of the elements involved in the formation of nonmuscle actomyosin II complexes (V. M. Fowler. 1986. J. Cell. Biochem. 31:1-9; 1996. Curr. Opin. Cell Biol. 8:86-96). No clear function has yet been attributed to these complexes. Using a mathematical model for the structure of the red blood cell spectrin skeleton (M. J. Saxton. 1992. J. Theor. Biol. 155:517-536), we have explored a possible role for myosin II bipolar minifilaments in the restoration of the membrane skeleton, which may be locally damaged by major mechanical or chemical stress. We propose that the establishment of stable links between distant antiparallel actin protofilaments after a local myosin II activation may initiate the repair of the disrupted area. We show that it is possible to define conditions in which the calculated number of myosin II minifilaments bound to actin protofilaments is consistent with the estimated number of myosin II minifilaments present in the red blood cells. A clear restoration effect can be observed when more than 50% of the spectrin polymers of a defined area are disrupted. It corresponds to a significant increase in the spectrin density in the protein free region of the membrane. This may be involved in a more complex repair process of the red blood cell membrane, which includes the vesiculation of the bilayer and the compaction of the disassembled spectrin network.

Cl- -HCO3- exchange in developing neonatal rat cardiac cells. Biochemical identification and immunolocalization of band 3-like proteins.

The Cl- -HCO3- exchanger is the main anionic exchanger (AE) that alleviates alkaline loads in cardiac cells. We recently identified in adult ventricular cells two membrane proteins (80 and 120 kD) immunologically related to the erythroid band 3 and likely to mediate the anion exchange. In the present study, we further investigated the Cl- -HCO3- exchanger activity concomitantly with the expression and intracellular localization of the band 3-like proteins during the development of neonatal rat cardiac cells maintained in culture for 17 days. Microspectrofluorometric measurements of pHi in single cells show that neonatal rat cardiomyocytes display a fully functional DIDS-sensitive Cl- -HCO3- exchanger at early stages of development. Neither basal pHi nor the anion exchange activity changes with different stages of the culture. In Western blotting with an anti-whole erythroid band 3 antibody, we found both the 80- and the 120-kD band 3-like proteins in whole heart and cultured neonatal cardiac cells. The 80-kD protein was also recognized by an anti-AE1 antiserum, whereas the 120-kD protein was specifically detected by an anti-cardiac AE3 antibody. Thus, we propose that the proteins are encoded by two different genes, AE1 and AE3, respectively. Subcellular fractionation of isolated and cultured cardiomyocytes revealed the presence of both proteins in the membrane, nuclear, and myofibril fractions. The results obtained in biochemical experiments corroborate the confocal images of immunostained neonatal cells, which demonstrate perinuclear location of band 3-like proteins at an early stage of development and their appearance within myofilaments after cell maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Tubulin is not phosphorylated in resting and thrombin-activated platelets.

We have investigated tubulin phosphorylation in human platelets, in order to evaluate whether it might be involved in the microtubular marginal band reorganization during platelet activation. Tubulin was identified with the use of specific monoclonal antibodies directed against alpha and beta subunits of tubulin. After metabolic 32P-labeling of platelets and analysis of separated proteins from whole cells, no phosphorylation of tubulin could be detected on autoradiography of platelet proteins either in resting platelets or during thrombin-induced activation. We also analyzed tubulin-enriched cytoskeletal fractions of resting or thrombin-stimulated platelets prepared in the presence of taxol, in comparison with tubulin-deprived cytoskeletal fractions prepared in the absence of this microtubule-stabilizing drug. Neither polymeric tubulin, assembled in microtubules and belonging to the platelet cytoskeleton, nor dimeric soluble tubulin showed significant 32P labeling. Finally, no tubulin was recovered among tyrosine-phosphorylated platelet proteins immunoprecipitated with a specific anti-phosphotyrosine protein monoclonal antibody. Thus, human platelet tubulin is not phosphorylated either in unstimulated platelets or in thrombin-stimulated platelets. The fact that both alpha and beta subunits are involved appears to be a unique feature of platelets in comparison with other cells. Microtubule-associated proteins are more likely to be involved in the unbundling of the platelet marginal band.

Identification of band 3-like proteins and Cl-/HCO3- exchange in isolated cardiomyocytes.

The identification of the protein that exerts the function of Cl-/HCO3- exchange is still unresolved in cardiac tissue. We have addressed this issue by using a multiple technical approach. Western blotting analysis with an antibody raised against human erythroid whole band 3 protein, the so-called protein that mediates the Cl-/HCO3- exchange in erythrocytes, showed that adult cardiomyocytes expressed two proteins immunologically related to the erythroid band 3. These proteins migrated in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 80 and 120 kDa. They were specifically found in the membrane but not in the cytosolic or the myofibril fractions of adult cardiomyocytes. Confocal microscopy further indicated that the immunostained proteins were mainly located at the sarcolemma and along T-tubules, typical membrane structures of adult cardiomyocytes. Using an antibody raised against a cardiac amino-terminal domain of rat AE3, we found that the 120-kDa protein is the translation product of the AE3 gene specifically expressed in heart and brain. Using an antiserum raised against a specific domain of mouse erythroid band 3 (AE1), which is not shared by AE3, we showed that the 80-kDa protein is likely to be a truncated translation product of the AE1 gene. Microinjection of the anti-human erythroid whole band 3 antibody into single isolated cardiac cells significantly inhibited the Cl-/HCO3- exchange activity. Furthermore, the anti-AE1 antibody strongly decreased the efficiency of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate to inhibit the ionic exchange. We thus suggest that the 80-kDa or both the 80- and the 120-kDa proteins immunologically related to the erythroid band 3 protein perform the anionic exchange in rat cardiomyocytes.

Purification and characterization of erythrocyte caldesmon. Hypothesis for an actin-linked regulation of a contractile activity in the red blood cell membrane.

We have previously shown that in human or pig whole erythrocytes, only a single 71-kDa polypeptide cross-reacts with the affinity-purified antibody to pig platelet caldesmon (der Terrossian et al., 1989). In the present paper, we demonstrate that this polypeptide represents a genuine caldesmon which remains attached to the membrane prepared in the presence of an excess of free Mg2+ but not in its absence. Immunoreactivity of this peptide is specific towards the antibody to pig platelet caldesmon since it is not labelled with antibodies to other components of the red cell membrane. Erythrocyte caldesmon was purified to 95% homogeneity and displays well known characteristics of caldesmons from other sources. Together with tropomyosin, it has the ability to regulate platelet actin-activated rabbit skeletal muscle myosin ATPase activity. The stoichiometry of 1 caldesmon/1 tropomyosin/7-9 actin molecules indicates that the amount of caldesmon, in the red cell membrane, corresponds precisely to the amount of tropomyosin. Immunofluorescent labelling of whole erythrocytes gave similar punctate patterns with purified antibodies to myosin, to caldesmon, to tropomyosin and to actin (but not to spectrin), suggesting colocalization of these proteins. Together, and for the first time, our results give strong evidence that caldesmon, bound on the actin protofilament, might represent the inhibitory component, so far uncharacterized, of a thin-filament-like system in erythrocyte.

Heme binding to calmodulin, troponin C, and parvalbumin, as a probe of calcium-dependent conformational changes.

Heme-CO binds to the active (calcium-bound) form of calmodulin (CaM), but not to the inactive form. Despite a similarity in structure of another calcium-binding protein, skeletal muscle troponin C, both the affinity and the spectral red-shift of the absorption of the heme group are greatly decreased for troponin C relative to calmodulin. Parvalbumin, another calcium-binding protein, shows a twofold greater affinity for heme-CO relative to CaM. Unlike calmodulin and troponin C, the affinity of parvalbumin for heme-CO is even greater in the absence of calcium. The affinity of the tryptic and thrombic fragments of CaM for heme-CO are decreased relative to the entire calmodulin. The binding of heme-CO is specific as demonstrated by the discrimination of the calmodulin, troponin C, and parvalbumin pockets. The interaction of heme-CO with active (calcium-bound) CaM is rapid (ms) as determined by stopped flow measurements. No difference in kinetics was observed for mixing inactive (calcium free) CaM with a solution of [heme-CO plus calcium], indicating that the calcium-binding step and subsequent change in protein conformation are rapid.

Identification of tubulin-binding proteins of the chicken erythrocyte plasma membrane skeleton which colocalize with the microtubular marginal band.

The plasma membrane of nucleated erythrocytes contains a microtubular marginal band which appears to be associated with the plasma membrane skeleton. In this report, we identify two families of cytoskeletal proteins which may be involved in such an association. These proteins, of molecular mass 78 kDa and 48 kDa on SDS-PAGE, are shown to bind tubulin based on a 125I-labeled tubulin binding assay. Solubilization of isolated chicken erythrocyte plasma membranes in Triton X-100 shows that these proteins centrifuge with the pellet, indicating that they are bound to the membrane skeleton. Finally, immunofluorescence studies using antisera raised against the 78 kDa and 48 kDa proteins show that they colocalize with the marginal band in intact cells. Colocalization of cytoskeletal tubulin-binding proteins with the marginal band favors a hypothesis suggesting that the 78 kDa and 48 kDa proteins are involved in the association of the two molecular superstructures.

A mixing chamber to enucleate avian and fish erythrocytes: preparation of their plasma membrane.

Biochemical studies of the plasma membrane and the cytoskeleton of nucleated erythrocytes are strongly limited by the difficulties encountered in enucleating large quantities of cells. We describe an easily built hydrodynamic system which allows rapid preparation of large amounts of avian and fish erythrocyte plasma membranes. The contents of two 25-ml syringes containing hemolyzed nucleated erythrocytes are forced through four capillaries to a specially designed mixing chamber which fills a collecting syringe. The 50-ml erythrocyte suspension can be processed in 2 s. The high speed flow is achieved with a hand-activated piston. The turbulences in the mixing chamber are carried to an optimal efficiency by the vis-à-vis disposition of the four mixing jets. The enucleated membranes are separated from the nuclei and residual nucleated cells by differential centrifugations. They do not show contamination with nuclear material. Erythrocytes from chicken and trout have been used. They present striking differences in their stability toward hydrodynamic disruption, erythrocytes from chicken being far more stable. Ninety-five percent of trout erythrocytes are enucleated after only one run through the mixing chamber. Two runs performed at the maximal flow rate are necessary to enucleate chicken erythrocytes with a yield of 80%. In the former case most of the purified enucleated plasma membranes are fragmented in small vesicles while they retain a large size in the case of chicken erythrocytes. The proteins of the membranes thus prepared are characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: we found that erythrocyte membranes from trout are remarkable for their small spectrin content compared to those from chicken.

Caldesmon is present in human and pig erythrocytes.

Caldesmon, a major actin- and calcium-dependent calmodulin-binding protein, is now considered as an essential inhibitory factor of the actomyosin machinery in smooth muscle cells as well as in non-muscle cells. Since its structure seems to vary with the cell in a same species and because platelet and erythrocyte have a common embryonic origin, we have used the affinity purified antibody raised against the platelet caldesmon to determine whether this protein is present in erythrocyte. Using the immunoblotting technique, we show here that, in whole erythrocytes, only a single polypeptide crossreacts with this polyclonal antibody. A 71-72 kDa Mr has been calculated from SDS-PAGE. It is therefore different from those of the gizzard (Mr 145-150 kDa) or the platelet (Mr 80 kDa) proteins. Furthermore, we also give evidence that it is not adducin since this newly discovered calcium-dependent calmodulin-binding protein of erythrocyte, does not crossreact with the polyclonal affinity purified antibody raised against platelet caldesmon.

Kinetics of p-mercuribenzoate binding to sulfhydryl groups on the isolated cytoplasmic fragment of band 3 protein. Effect of hemoglobin binding on the conformation.

Hemoglobin binds to the cytoplasmic domain of band 3 protein (CDB3) at physiologic pH and ionic strength in an oxygen-linked fashion, with deoxyhemoglobin having the higher affinity. The evidence in the literature suggests functional communication between the hemoglobin-binding site on CDB3 and the anion transport sites within the membrane-bound domain of band 3. Since the hemoglobin-binding site is estimated to be over 200 A from the transport domain, the functional communication hypothesis would require the existence of long-range, global changes in the CDB3 dimeric quaternary structure consequent to hemoglobin binding. In this report sulfhydryl reactivity toward p-mercuribenzoate is studied in an attempt to identify such long-range conformational changes. Formation of stoichiometric hemoglobin/CDB3 complexes is shown to produce major changes in sulfhydryl reactivity. Since the sulfhydryl pocket of CDB3 is known to lie at the dimeric interface over 100 A from the hemoglobin-binding site, the observed changes in reactivity suggest that hemoglobin complexation induces a global change in quaternary structure of the CDB3 dimer. This change offers a mechanism to explain functional connections between CDB3-binding sites and the anion transport sites on band 3. The existence of such long-range conformational changes would imply that the CDB3 dimer is poised to function as a cytosolic arm or lever in order to modulate the global structure of the porter.

[Permeability of the erythrocyte membrane to pentoxifylline and propentofylline. Research on their effects on some structural and functional properties of the membrane]. / Etude de la perméabilité de la membrane érythrocytaire à la pentoxifylline et la propentofylline. Recherche de leurs effets sur quelques propriétés structurales et fonctionnelles de la membrane.

Contrarily to previous studies, we show that the red blood cell membrane is permeable to pentoxifylline and propentofylline. The kinetics of inward and outward flux are rapid. Both molecules do not transit via the pores for the water transport but instead seems to freely diffuse through the lipidic bilayer. It has not been possible to detect a significant compartimentation of the two drugs on the erythrocyte membrane. We have been searching for a possible modification of some of the structural and functional properties of the erythrocyte membrane in the presence of pentoxifylline and propentofylline. The experiments included measurement of the membrane fluidity, kinetics of water, glycerol and ethylene glycol transport, and studies on the stability of the membrane-cytoskeleton complex. No detectable differences were measured suggesting that the therapeutic action of pentoxifylline and propentofylline may result more from protective effect in the cases of pathological stresses occurring on the erythrocyte membrane than from their ability to repair the damaged membrane.

Interactions of actin and tubulin with human deoxyhemoglobin. Their possible occurrence within erythrocytes.

Short actin filaments are an essential component of the red-cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion-binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high-affinity complex with Hb. This is shown by measuring, by stopped-flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an alpha, beta-tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3-bisphosphoglycerate-binding site of Hb and a negatively charged domain, most likely the highly acidic N and C-terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.

Pig thyroid spectrin. A membrane-associated protein related in structure and function to brain spectrin.

Thyroid spectrin has been obtained pure from pig thyroid glands. This protein, composed of two non-identical polypeptide chains of 240 kDa and 235 kDa, appears to possess the same structural and immunological properties as well as the same calmodulin and actin-binding properties as brain spectrin. Through cross-linking of actin filaments it is a potent gelation factor for F-actin solutions. It represents one of the major protein of the cytoskeleton underlying the thyroid plasma membrane together with myosin, alpha-actinin and actin.

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate.

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.

Kinetics of water transport in sickle cells.

This paper reports the results of stopped-flow studies on differences in the kinetics of osmotic water transport of sickle and normal erythrocytes. The kinetics of inward osmotic water permeability are similar in sickle and normal red blood cells. In contrast, the kinetics of outward water flux are significantly (approx. 38%) decreased in sickle cells. Deoxygenation does not modify the water influx kinetics in either type of cells, but accelerates considerably the rate of water efflux in sickle cells. No significant variation of water transport kinetics was observed in density-separated cell fractions of either type. The results suggest that membrane-associated hemoglobin may decrease the outward water permeability and that in deoxygenated sickle cells the fraction of hemoglobin S near the lipid bilayer is lower than in oxygenated conditions.

High molecular weight microtubule-associated proteins from pig brain are immunologically related to human erythrocyte membrane proteins spectrin, ankyrin, proteins 4.1 and 4.2.

The microtubule-associated proteins MAPs 1 and 2 from pig brain have been found to react with antibodies directed against human ankyrin and spectrin, respectively (Bennett and Davis, 1981; Davis and Bennett, 1982). In a complementary approach we have prepared antibodies against MAP1 alpha. MAP1 gamma and MAP2 purified from pig brain and tested their reactivity with human erythrocyte membrane proteins. Anti-MAP1 alpha was shown to react with alpha and beta-spectrin and with protein 4.1; anti-MAP1 gamma reacted with alpha-spectrin and ankyrin and with a 60 K peptide which copurified with human spectrin. Finally anti-MAP2 was specific for beta-spectrin and protein 4.2. The biological function of protein 4.2 is still unknown but details on the interactions between ankyrin, spectrin and protein 4.1 and their role in mediating the linkage of oligomeric actin on the erythrocyte membrane are well documented. The present results, which demonstrate extended immunological analogies between pig brain high molecular weight MAPs and human erythrocyte membrane proteins, may reflect the presence, in the two families of proteins, of similar functionally important epitopes.

Decrease of transport of some polyols in sickle cells.

This paper reports the results of kinetic studies on the inward net-flux of small non-electrolytes (ethylene glycol, glycerol and erythritol) in sickle cells as compared to normal erythrocytes. Net transport rates were evaluated by turbidimetric measurements for ethylene glycol and glycerol and by hematocrit monitoring for erythritol. A 2-fold and 4-fold reduction in the permeability coefficient for ethylene glycol and glycerol, respectively, were found in sickle cells as compared to normal erythrocytes. In contrast, no significant changes in erythritol transport kinetics were observed. The dependence of glycerol permeability on temperature, pH and oxygenation is the same in both types of cells. A significant correlation was observed between glycerol permeability and cell density only for sickle cells. The results indicate that irreversible modifications of membrane proteins, responsible for the glycerol and ethylene glycol transport, do occur in sickle cells.

Interaction between microtubule-associated protein tau and spectrin.

Tau factor, one of the microtubule-associated proteins (MAPs), is shown here to bind to spectrin. Evidence for an interaction between these two proteins is provided by spectrin affinity chromatography of brain MAPs, gel overlay of electrophoresed MAPs with 125I-labelled spectrin, incorporation of tau factor in human erythrocyte ghosts, and demonstration that tau inhibits the F-actin cross-linking activity of tetrameric spectrin. The wide distribution of both tau and spectrin-like proteins in eukaryotic cells is in favor of the possible biological significance of this interaction. The results suggest that tau could be one of the proteins involved in the concerted regulation of microtubule and actin networks in the membrane vicinity.

A dynamical study on the interactions between the cytoskeleton components in the human erythrocyte as detected by saturation transfer electron paramagnetic resonance of spin-labeled spectrin, ankyrin, and protein 4.1.

Isolated human erythrocyte spectrin, ankyrin, and protein 4.1 have been labeled with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl, and studied by saturation transfer electron paramagnetic resonance spectroscopy. The presence of the labels does not affect the reassociation of these proteins with erythrocyte membranes selectively depleted of either spectrin-actin or of all the extrinsic proteins. When maleimide spin-labeled spectrin is reassociated with the erythrocyte membrane in presence of all the cytoskeleton components, including endogeneous or purified muscle actin, spectrin still preserves its flexible character. The rotational mobilities of maleimide spin-labeled ankyrin and maleimide spin-labeled protein 4.1 are of the same order of magnitude (tau c (L"/L) approximately 5 X 10(-5) and 8 X 10(-5) s, respectively, at 2 degrees C), while protein 4.1 is almost three times smaller in size than ankyrin. This result indicates that the movements of membrane-bound maleimide spin-labeled protein 4.1 are more restricted than those of ankyrin. This suggests that their respective binding sites have different structural properties. The rotational movements of both proteins are slowed down on the addition of spectrin indicating that protein 4.1 as well as ankyrin also represents one of the links of the cytoskeleton to the membrane.

Spectral and oxygen-release kinetic properties of human hemoglobin bound to the cytoplasmic fragment of band 3 protein in solution.

Binding of the cytoplasmic fragment of band 3 protein to oxyhemoglobin in solution caused a spectral change in the absorbance of the hemoglobin beta chain at a ratio of one monomer of band 3 protein per alpha beta dimer of hemoglobin. This spectral change was reversed at higher ratios of cytoplasmic fragment to hemoglobin. The unusual dependence on protein concentration was interpreted as indicating the formation of higher aggregates of the complex between hemoglobin and the cytoplasmic fragment of band 3 protein. Oxygen-release kinetic measurements also showed marked changes as a function of the concentration of the cytoplasmic fragment of band 3 protein. The higher ratio mixture had significantly different kinetic properties as compared with the lower ratio one, which in turn was different from oxyhemoglobin in solution. The significance of the formation of aggregates of band 3 protein containing oxyhemoglobin dimers is discussed in context with evidence suggesting that band 3 protein may exist as an equilibrium mixture of tetramers and dimers in the membrane.