Ankyrin is necessary for both drug-induced and ATP-induced shape change of human erythrocyte ghosts

Human erythrocyte membranes from ACD preseved blood were digested with trypsin (Protein: trypsin=100:1) and analyzed by SDS PAGE. After digestion for 20 sec at 0°C, only ankyrin disappeared but other bands including spectrin, band 4.1 and band 3 remained intact. In contrast to intact membranes, treatment with chlorpromazine or MgATP or HEPES did not induce shape change in these membranes. The number of transformable cells correlated closely with the amount of remaining ankyrin. We conclude that ankyrin is necessary for their shape change. This is the first direct evidence that ankyrin is involved in the maintenance of red cell shape. Three additional lines of indirect evidence are also presented.     

Dynamics of membrane structure of frog erythrocyte ghosts measured with a nanosecond fluorometer

The dynamics of membrane microstructure was studied as molecular motions of phospholipids for bullfrog erythrocyte ghosts by the DPH fluorescence depolarization technique with a nanosecond fluorometer. The bullfrog erythrocyte ghosts were obtained by hypotonic lysis and collagenase treatment. The constituents of membrane proteins were confirmed by the disk gel electrophoresis. The viscosity of erythrocyte membrane ghosts was estimated to be 3.3 +/- 1.0 at 10 degrees C, and 2.1 +/- 0.1 at 20 degrees C and 1.3 +/- 0.2 at 30 degrees C in the unit of poise and the wobbling angle of lipid molecule was 35 +/- 1, 41 +/- 1 and 43 +/- 1 degree at the respective temperatures on an average and +/- S.D. The viscosity is lower than that of human erythrocytes. The relatively low viscous phospholipid bilayer may be one of the factors for the deformability of bullfrog erythrocytes.     

The structural basis of ankyrin function. I. Identification of two structural domains

The structure of ankyrin, a major linking protein between spectrin and the erythrocyte membrane, was analyzed after restricted proteolytic digestion at 0 degree C. By the use of two-dimensional peptide mapping, we found that tryptic digestion of ankyrin (1 h, 0 degree C) resulted in the production of two nonoverlapping peptides of molecular weights 82,000 and 55,000. The 82,000-dalton peptide had a basic isoelectric point (7.9) and was remarkably sensitive to further proteolytic digestion; after 24 h at 0 degree C, trypsin completely digested this peptide into fragments too small to detect by gel electrophoresis. The 55,000-dalton peptide was neutral (isoelectric point = 6.9-7.2) and more resistant to further proteolytic cleavage. After a 24-h digestion with trypsin at 0 degrees C, the 55,000-dalton peptide was cleaved into two complementary fragments of molecular weight 32,000 and 15,000. Analysis of phosphorylated ankyrin indicated that the phosphates were exclusively found in these two complementary peptides. By comparison with larger fragments, we were able to align the constituent peptides of ankyrin and propose a low resolution model. Ankyrin appears to be a bipolar molecule containing a basic domain of 82,000 daltons and a neutral phosphorylated domain of 55,000 daltons.     

Alteration of rheological properties of human erythrocytes by crosslinking of membrane proteins

The crosslinking of membrane proteins of human erythrocytes by diamide (diazene dicarboxylic acid bis(N,N-dimethylamide) ) was quantified by 4% polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. The relation between the crosslinking of membrane proteins and erythrocyte functions (rheological and oxygen transporting) was quantitatively examined. (i) The crosslinking of membrane protein was induced by diamide, without changing the shape and the contents of intracellular organic phosphates (adenylates and 2,3-diphosphoglycerate). The intensity of spectrin 2 in SDS-polyacrylamide gel electrophoresis decreased proportionally to diamide concentration. The percentage decrease in spectrin 2 (using band 3 as an internal standard) was the most appropriate indicator for crosslinking ("% crosslinking'). (ii) The suspension viscosity of erythrocytes increased in proportion to the percentage of crosslinking, in the range of applied shear rates of 3.76-752 s-1. (iii) Erythrocyte deformability (measured by a high-shear rheoscope) was reduced by the crosslinking. The change was detectable even at 5% crosslinking. (iv) Rouleaux formation (measured by a television image analyzer combined with a low-shear rheoscope) was inhibited by the crosslinking. The inhibition was also sensitively detected at more than 5% crosslinking. (v) Hemoglobin in erythrocytes was chemically modified by higher dose of diamide (probably by the binding of diamide with sulfhydryl groups). Also the oxygen affinity of hemoglobin increased and the heme-heme interaction decreased. (vi) The reduction of the crosslinking of membrane proteins by dithiothreitol apparently reversed the intensity of spectrin bands in SDS-polyacrylamide gel electrophoresis and the erythrocyte functions (the suspension viscosity and the deformability), though not completely.     

Exofacial photoaffinity labelling of the human erythrocyte sugar transporter

The 4-azidosalicylate derivative of 1,3-bis(D-mannos-4'-yloxy)-2-[2-3H]propylamine (ASA-[2-3H]BMPA) has been tested as a photoaffinity label for the sugar transporter in human erythrocytes. When photolysed in the presence of intact erythrocytes, ASA-[2-3H]BMPA covalently binds to the exofacial surface of the transporter. This labelled protein appears as a broad band in the 4.5 region in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The peak of radiolabel incorporation gives an apparent Mr of approx. 50 000 on 5-20% acrylamide gels. The binding is 80% inhibitable by 320 mM 4,6-O-ethylidene-D-glucose, by 320 mM D-glucose and by 50 microM cytochalasin B. Photoirradiation of a saturating concentration of ASA-BMPA in the presence of erythrocytes results in a 25-30% loss of D-galactose transport activity. From transport inactivation data and estimations of the amount of ASA-[2-3H]BMPA binding to the transporter it is calculated that there are approx. 220 000 exofacial hexose-transport binding sites per erythrocyte. The labelling of the transporter has been carried out using freshly drawn blood and 4-weeks-old transfusion blood. No change in the binding profile on SDS-polyacrylamide gel electrophoresis was observed. Proteolytic digestion of the ASA-[2-3H]BMPA-labelled transporter with either trypsin or alpha-chymotrypsin results in the appearance of a labelled 19 kDa fragment on SDS-polyacrylamide gel electrophoresis.     

Cytoskeletal restraints of band 3 rotational mobility in human erythrocyte membranes

The interaction of band 3 with cytoskeletal proteins was investigated in erythrocyte membranes by measuring the rotational mobility of band 3 using the method of transient dichroism. It was found that selective proteolysis of ankyrin, a protein known to link band 3 to the spectrin-actin network, had no significant effect on band 3 rotation. Incubating ghosts to 70 degrees C, at which temperature ankyrin is expected to be denatured, also had no effect. It thus appears probable that linkage of band 3 to the cytoskeleton via ankyrin does not act as a restraint on band 3 rotational motion. It is suggested that this is a consequence of flexibility in the cytoskeletal structure. In further investigations of the effect of heat treatment, a large enhancement of band 3 rotational mobility was found to result from incubation of intact cells for 1 h at 50 degrees C. This effect was not observed if ghosts were subjected to the same treatment, nor did it occur if the incubation of cells was performed at 47 degrees C. These findings, in combination with previous studies of band 3 rotational mobility, indicate that the interactions which restrain band 3 are likely to be more complex than commonly envisaged.     

Binding of ras p21 to bands 4.2 and 6 of human erythrocyte membranes

The direct binding protein(s) of ras p21 was (were) investigated in inside-out vesicles of human erythrocyte ghosts using the pure v-Kirsten (Ki)-ras p21 synthesized in E. coli. The bound ras p21 was detected immunochemically using an anti-v-Ki-ras p21 monoclonal antibody, ras p21 bound to vesicles. Prior digestion of the vesicles with trypsin reduced this binding significantly. When ras p21 was laid over vesicle proteins immobilized on a nitrocellulose sheet by transfer from the gel of SDS-polyacrylamide gel electrophoresis, ras p21 bound to bands 4.2 and 6. ras p21 binding to these proteins was reduced by prior incubation of ras p21 with the purified band 4.2 or 6 protein. These results indicate that v-Ki-ras p21 can bind directly to bands 4.2 and 6 of human erythrocyte membranes as far as tested in an in vitro cell-free system.     

Cell-free synthesis of myosin by cardiac myofibrillar ribosomes

Human erythrocyte ghosts were treated with a bifunctional cross-linking reagent, dimethyl adipimidate dihydrochloride. On SDS-polyacrylamide electrophoresis of the cross-linked membrane proteins after solubilization, sialoglycoproteins and the proteins disappeared from the original band positions and appeared in a new band of aggregates.     

INTRAMEMBRANE PARTICLE AGGREGATION IN ERYTHROCYTE GHOSTS : I. The Effects of Protein Removal

We have used freeze-etching and SDS-polyacrylamide gel electrophoresis to study the conditions under which the intramembrane particles of the human erythrocyte ghost may be aggregated. The fibrous membrane protein, spectrin, can be almost entirely removed from erythrocyte ghosts with little or no change in the distribution of the particles. However, after spectrin depletion, particle aggregation in the plane of the membrane may be induced by conditions which cause little aggregation in freshly prepared ghosts. This suggests that the spectrin molecules form a molecular meshwork which limits the translational mobility of the erythrocyte membrane particles.     

The role of spectrin in erythrocyte ghost endocytosis.

Endocytosis in white ghosts prepared from human erythrocytes was induced by three methods: incubation with Mg-ATP, incubation with 0.1 mM EDTA, and digestion with 20 nanograms (ng.) per ml. of trypsin. In each case the endocytic vacuoles that were produced when separated and analyzed on SDS-polyacrylamide gel electrophoresis were found to be depleted of spectrin. This observation suggested that a requirement for endocytosis is the establishment of spectrin-free domains in the membrane. This hypothesis was tested by pre-incubating ghosts with anti-spectrin antibodies. Pre-incubation with anti-spectrin antibody blocked white ghost endocytosis produced either by Mg-ATP, EDTA, or trypsin. Therefore, it is proposed that spectrin has a key role in the endocytosis process.     

Structural organisation of band 3 in Melanesian ovalocytes

The diffusional freedom of human erythrocyte band 3 (anion exchanger 1) has been measured in membranes from normacytic and ovalocytic erythrocytes. A dramatic reorganisation of band 3 in the ovalocyte membranes is indicated by a markedly restricted rotational mobility. Extraction of spectrin from erythrocyte membranes had no effect on normocyte band 3 mobility, but partially relieved the restrictions on ovalocyte band 3 mobility. Further removal of ankyrin and band 4.2 resulted in an increase in the rotational mobility of both ovalocyte and normocyte band 3 to similar levels. The results suggest that the molecular basis of the unusual shape and decreased deformability of ovalocytes resides in an altered interaction of band 3 with one or more of the peripheral proteins. We present a model which illustrates a possible role for band 3 aggregation in controlling erythrocyte deformability.     

Chemical Characterization and Pronase Susceptibility of the Na:K Pump-Associated Phosphoprotein of Human Red Blood Cells

The phosphoproteins formed by incubation of red cell ghosts with [γ-³²P]ATP in the presence of Mg and Na + Mg have been characterized by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The ³²P-labeled phosphoprotein was seen as a single peak confined to the region of the diffuse 90,000 dalton polypeptide band; labeling with Na + Mg considerably increased the quantity of ³²P-phosphoprotein contained in this band relative to labeling with Mg alone. Treatment of intact cells with Pronase known to partially hydrolyze the glycoproteins and the 90,000 daltons polypeptide did not change either the amount or the position of the ³²P-phosphoprotein present in the gels. The molecular weight of the ³²P-phosphoprotein is estimated to be 103,000. Pronase treatment of intact cells also did not significantly alter any of the transport parameters of the membrane such as the K pump flux, ouabain binding, or Na,K-ATPase. In contrast, treatment of ghosts with Pronase not only resulted in drastic alteration of the transport parameters but also inhibited the formation of the phosphoprotein under all conditions. Thus, while the Na:K pump is not intrinsically resistant to Pronase, those elements of the pump which are susceptible are not accessible from the outside of the cell. Further, SDS-polyacrylamide gel electrophoresis after Pronase treatment of intact cells results in a substantial increase in the purification of the phosphoprotein relative to that which was previously possible in ghosts.     

A new major transmembrane glycoprotein, gp155, in goat erythrocytes. Isolation and characterization of its association to cytoskeleton through binding with band 3-ankyrin complex

Erythrocyte membranes from goat contain a considerable amount, more than 10% of the total amount, of a glycoprotein with Mr = 155,000 (gp155) on sodium dodecyl sulfate-polyacrylamide electrophoresis gel. This report describes the first isolation and characterization of gp155. This gp155 has major trypsin-sensitive sites at each side of the plasma membrane to generate membrane-bound fragments, indicating that the gp155 spans the lipid bilayer several times. This protein consists of a single polypeptide containing about 1,200 amino acid residues corresponding to Mr = 134,000 and some complex type N-linked oligosaccharide chains. A fraction (15-20%) of the gp155 is recovered in nonionic detergent-extracted ghosts along with 25-30% of band 3 and other cytoskeletal proteins and is completely released into solution by extraction with 1 M KCl. Immunoprecipitation with anti-gp155 and anti-ankyrin antibodies of detergent-solubilized membranes separated on a gel permeation chromatography column showed that a part of the gp155 is tightly linked to band 3 with a molar ratio of 1:2 to 1:3. This gp155-band 3 complex in turn is associated to ankyrin through the binding of band 3 to ankyrin. These data indicate that, in native erythrocyte membranes, as well as in detergent solution, gp155 could play a physiological role in controlling cellular integrity and elasticity by forming the gp155-band 3-ankyrin complex. Partial amino acid sequences of the tryptic peptides are also determined.     

Effects of intracellular Ca2+ and proteolytic digestion of the membrane skeleton on the mechanical properties of the red blood cell membrane

Intracellular Ca2+ at concentrations ranging from 0 to 10 mumol/l increases the shear modulus of surface elasticity (mu) and the surface viscosity (eta) of human red blood cells by 20% and 70%, respectively. K+ selective channels in the red cell membrane become activated by Ca2+. The activation still occurs to the same extent when the membrane skeleton is degraded by incorporation of trypsin into resealed red cell ghosts, suggesting that the channel activation is not controlled by the proteins of the membrane skeleton and is independent of mu and eta. Incorporation of trypsin at concentrations ranging from 0 to 100 ng/ml into red cell ghosts leads to a graded digestion of spectrin, a cleavage of the band 3 protein and a release of the binding proteins ankyrin and band 4.1. These alterations are accompanied by an increase of the lateral mobility of the band 3 protein which, at 40 ng/ml trypsin, reaches a plateau value where the rate of lateral diffusion is enhanced by about two orders of magnitude above the rate measured in controls without trypsin. Proteolytic digestion by 10-20 ng/ml trypsin leads to a degradation of more than 40% of the spectrin and increases the rate of lateral diffusion to about 20-70% of the value observed at the plateau. Nevertheless, mu and eta remain virtually unaltered. However, the stability of the membrane is decreased to the point where a slight mechanical extension, or the shear produced by centrifugation results in disintegration and vesiculation, precluding measurements of eta and mu in ghosts treated with higher concentrations of trypsin. These findings indicate that alterations of the structural integrity of the membrane skeleton exert drastically different effects on mu and eta on the one hand and on the stability of the membrane on the other.     

The structural basis of ankyrin function. II. Identification of two functional domains

Human erythrocyte ankyrin was cleaved by restricted proteolysis at 0 degrees C into two distinct chemical domains. The site on ankyrin that binds spectrin was found to be within a 55,000-dalton domain by spectrin affinity chromatography and co-sedimentation with spectrin in a sucrose gradient. A 32,000-dalton fragment of this domain was prepared (tryptic digest, 0 degrees C, 24 h), separated by gel filtration, and shown to inhibit spectrin binding to the membrane. By comparison with previous two-dimensional peptide maps, the spectrin-binding site was located within this 32,000-dalton fragment near the end of the molecule. The band 3-binding site was identified within an 82,000-dalton domain by binding to a band 3 affinity column. Gel electrophoresis in the absence of detergents confirmed these results and demonstrated that a peptide from the cytoplasmic portion of band 3 retained the capacity to bind the 82,000-dalton domain. The binding properties of the structural domains of ankyrin were correlated with a determination of the affinity constant of the intact molecule. Ankyrin bound with a high affinity to the cytoplasmic portion of band 3 (KD = 8 X 10(-8) M) and to spectrin tetramer (KD = 1 X 10(-7) M) but less so to spectrin dimer (KD = 1 X 10(-6) M). These findings are summarized in a preliminary structural and functional model of ankyrin's role in linking spectrin to the membrane.     

A flow EPR study of deformation and orientation characteristics of erythrocyte ghosts: A possible effect of an altered state of cytoskeletal network

Summary Using the flow EPR technique, we investigated the resealed ghost deformability in shear flow and the effects of the altered state of cytoskeletal network induced by hypotonic incubation of ghosts. Isotonically resealed ghosts in the presence of Mg-ATP, in which alteration of cytoskeletal network is not effected, have smooth biconcave discoid shapes, and show a flow orientation and deformation behavior similar to that of erythrocytes, except that higher viscosities are required to induce the same degrees of deformation and orientation as in erythrocytes. The flow behavior of resealed ghosts is Mg-ATP dependent, and the shape of the ghosts resealed without Mg-ATP is echinocytic. In contrast, the ghosts resealed by hypotonic incubation show a markedly reduced deformability even with Mg-ATP present. Nonreducing, nondenaturing polyacrylamide gel electrophoresis (PAGE) of the low ionic strength extracts from hypotonically resealed ghosts reveals a shift of the spectrin tetramer-dimer equilibrium toward the dimers. In the maleimide spin-labeled ghosts, the ratios of the weakly immobilized to the strongly immobilized EPR intensities are larger in hypotonically resealed ghosts than in the isotonically resealed ghosts, indicating an enhanced mobility in the spectrin structure in the former. Photomicrographs of hypotonically resealed ghosts show slightly stomatocytic transformations. These data suggest that the shape and the deformability loss in hypotonically resealed ghosts are related to an alteration of the spectrin tetramer-dimer equilibrium in the membrane. Thus, the shift of the equilibrium is likely to affect the regulation of the membrane deformability both in normal and pathological cells such as hereditary elliptocytes.     

Membrane protein phosphorylation during stomatocyte-echinocyte transformation of human erythrocytes

The normal, discoid shape of red blood cells represents an equilibrium between two opposing factors, i.e., stomatocytic and echinocytic transformations. Most stomatocytic agents were found to be inhibitors of calmodulin, a regulator of the phosphorylation of membrane proteins. We determined whether red cell shape transformations could be caused by changes in phosphorylation of membrane proteins, specifically the cAMP-dependent phosphorylation of ankyrin and band 4.1. Red blood cells were incubated with 32P and 100 microM chlorpromazine (stomatocytic transformation) or 30 mM sodium salicylate (echinocytic transformation) for various time intervals. Ghost membrane proteins were examined by polyacrylamide gel electrophoresis and autoradiography. Spectrin (beta-chain), ankyrin, band 3, band 4.1 and 4.9 were phosphorylated. No change was found in the degree and pattern of phosphorylation after stomatocytic transformation. Salicylate caused a reversible inhibition of transmembranous phosphate transport in both directions. The results indicate that the stomatocytic transformation induced by chlorpromazine and the echinocytic transformation induced by salicylate do not involve a change in phosphorylation, but that the echinocytic transformation induced by salicylate is associated with an inhibition of transmembranous transport of phosphate. Studies with salicylate suggest that the phosphorylation sites of band 3 are found mainly on the endofacial side of the membrane.     

Restriction of the lateral motion of band 3 in the erythrocyte membrane by the cytoskeletal network: dependence on spectrin association state

The effects of incubation of erythrocyte ghosts under various conditions (ionic strength or addition of ankyrin, diamines, or ATP) on the lateral motion of band 3 in the membranes were studied by using the fluorescence photobleaching recovery technique. Incubation of ghosts with exogenous ankyrin increased the immobile fraction of band 3, from 0.6 in intact ghosts to 0.8-0.9 when an average of 0.2 mol of extra ankyrin was bound per mole of band 3. Ankyrin-free band 3 proteins were mobile, but their mobility was governed by the spectrin association state in the cytoskeletal network. The diffusion constant was 5.3 X 10(-11) cm2 s-1 at a spectrin tetramer mole fraction of 0.3-0.4 in 10 mM NaCl/5 mM sodium phosphate, pH 7.8, and decreased 1 order of magnitude when the tetramer fraction increased to 0.5 in higher NaCl concentration (150 mM NaCl). A similar decrease was observed when the spectrin tetramer fraction was increased by 0.2 mM spermine in 10 mM NaCl/10 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.6. On the other hand, the rotational motion of band 3 in the membranes was not affected by the spectrin association state. Trypsin treatment of ghosts cleaved off the cytoplasmic domain of band 3 and caused a marked (8-fold) increase in the lateral mobility, D = 4.0 X 10(-10) cm2 s-1. These results indicate that the lateral mobility of ankyrin-free band 3 protein is restricted by interactions of their cytoplasmic domain with the cytoskeletal network. A model is presented that band 3 can pass the network when spectrins are in dissociated dimers and cannot pass when they are tetramers. The lateral diffusion constant is thus determined by the spectrin dimer population in the network.     

The cytoskeleton of isolated murine primitive erythrocytes

Summary Cytoskeletons of primitive erythrocytes have been isolated from the embryos of day 12 pregnant C57/Bl mice and examined by transmission electron microscopy, immunofluorescence microscopy, and SDS-polyacrylamide gel electrophoresis. Microtubules are the most prominent cytoskeletal component. They are found either singly or organized into loose bundles just under the plasma membrane, but do not form classical marginal bands in most cells. Immunofluorescence with a polyclonal tubulin antiserum confirms this distribution and further reveals numerous mitotic figures among the cells. Rhodamine-conjugated phalloidin and heavy meromyosin labeling reveal that actin is localized in the cortex of the primitive erythrocyte in the form of 6 nm filaments. Antibody directed against avian erythrocyte alpha spectrin demonstrates that spectrin is also found in the cortex. Occasional 10-nm intermediate filaments, observed in the primitve erythrocytes by electron microscopy, are believed to be of the vimentin class based on positive reaction of the cells with vimentin-specific antiserum. In addition, a band in erythrocyte cytoskeletons comigrates in SDS-polyacrylamide gels with vimentin isolated from mouse kidney. Spectrin and actin were also found to be associated with the membrane of primitive erythrocytes when membrane ghost preparations were analyzed by SDS-polyacrylamide gel electrophoresis.     

Mechanism of inhibition of activated protein C by protein C inhibitor

Protein C inhibitor isolated from human plasma inhibited thrombin, factor Xa, trypsin and chymotrypsin as well as activated protein C, but had very little effect on urokinase and plasmin. The inhibition constants (K1) of protein C inhibitor for activated protein C, thrombin and factor Xa were 5.6 X 10(-8) M, 6.7 X 10(-8) M and 3.1 X 10(-7) M, respectively. The second-order rate constant for inhibition of activated protein C by the inhibitor increased about 30-fold in the presence of an optimal heparin concentration (5-10 units/ml). The inhibition of activated protein C by plasma protein C inhibitor was also accelerated by heparin. When activated protein C (Mr = 62,000) was incubated with protein C inhibitor (Mr = 57,000), enzyme-inhibitor complexes with apparent Mr = 102,000 and 88,000 were observed in the nonreduced and the reduced samples, respectively, on SDS-polyacrylamide gel electrophoresis. In addition to these complexes, a band of unbound enzyme and a band with Mr = 54,000 were detected. When 125I-labeled protein C inhibitor was exposed to activated protein C, the inhibitor band was converted to bands with apparent Mr = 102,000 and 54,000 in the nonreduced samples, as determined by autoradiography after gel electrophoresis in SDS. The band with Mr = 54,000 also appeared when the inhibitor reacted with other serine proteases. The activated protein C was released from the inactive complex by treatment with 1 M ammonia or hydroxylamine. This phenomenon was found by SDS-polyacrylamide gel electrophoresis to represent the dissociation of the enzyme-inhibitor complex by ammonia or hydroxylamine into the free enzyme and the proteolytically modified inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)