Search PubMed⌕ Search

Biomedical subjects

J Palek

Publications and source records attributed to J Palek.

At least 73 records · Page 4Linked to original sources
PubMed1989

Separation of the lipid bilayer from the membrane skeleton during discocyte-echinocyte transformation of human erythrocyte ghosts.

The membrane skeleton, a protein lattice at the internal side of the red cell membrane, is principally composed of spectrin, actin and proteins 4.1 and 4.9. We have examined negatively stained red cell ghosts and demonstrated, on an ultrastructural level, a separation of the lipid bilayer from the membrane skeleton during echinocytic transformation. The electron micrographs of discoidal red cell ghosts suspended in hypotonic buffer revealed a filamentous reticulum that uniformly laminated the entire submembrane region. transformation of the discoidal ghosts into echinocytic form, as induced by incubation in isotonic buffer, resulted in a disruption of skeletal continuity underlying the surface contour of the membrane spicule. The submembrane reticulum extended into the base and the neck of the spiny processes of the crenated ghosts but was absent at the tip of these projections. In addition, membrane vesicles without a submembrane reticulum were detected either attached to the tips of the spicules or released into the supernatant from the echinocytic ghosts. Protein analysis revealed that the released vesicles were enriched in bands 3, 4.1 and 7 and contained very little of the membrane skeletal proteins, spectrin and actin. The data indicate that during echinocyte formation, parts of the lipid bilayer physically separate from the membrane skeleton, leading to a formation of skeleton-poor lipid vesicles.

Actins↗
PubMed1989

Viscoelastic properties of red cell membrane in hereditary elliptocytosis.

The viscoelastic properties of the RBC membrane are in part determined by a submembrane network of proteins consisting of spectrin alpha beta heterodimers (SpD) assembled head-to-head to form spectrin tetramers (SpT) and spectrin oligomers (SpO). SpT, in turn, are connected into a two-dimensional network by the linkage of distal ends of SpT to protein 4.1 and actin. With the micropipette technique, we determined the membrane viscoelastic properties of RBCs from a subset of patients with hereditary elliptocytosis (HE); these RBCs exhibit membrane skeletal instability, defective SpD self-association, and a molecular defect in the alpha I domain of spectrin, which is involved in the SpD-SpD contact (HE SpD alpha-SpD). The elastic modulus and viscosity of the membrane were significantly higher for the HE RBCs than for the control cells. Incubation of normal cells with N-ethyl-maleimide (NEM) produced a similar defective SpD self-association and a significant increase in the viscoelastic parameters of the membrane. The data provide evidence that the mode of assembly of membrane spectrin in the cytoskeletal protein network plays a major role in determining the rheologic behavior of erythrocyte membrane.

Elasticity↗
PubMed1988

Identification of the protein 4.1 binding site to phosphatidylserine vesicles.

Previous studies have shown that protein 4.1 is a multifunctional protein that binds to spectrin, actin, glycophorins, the anion channel protein, and phosphatidylserine (PS). In this report, we have characterized the binding of protein 4.1 and its major proteolytic fragments to phospholipid vesicles. Pure 125I-labeled protein 4.1 was incubated with PS liposomes, and the free protein 4.1 was separated by ultracentrifugation. Protein 4.1 bound to PS liposomes with a high affinity. At saturation, there was 9 X 10(-3) pmol of protein 4.1 bound/pmol of PS with a Kd of 3.3 X 10(-7) M. When the protein 4.1 containing liposomes were examined in an electron microscope, the protein 4.1 was found uniformly decorating the vesicles in a rosettelike fashion. Among peripheral membrane proteins tested (spectrin, actin, ankyrin, and protein 4.1), protein 4.1 showed the highest level of binding to PS. The binding of protein 4.1 to PS, one of the principal phospholipids of the inner half of the lipid bilayer, was considerably higher than the binding to phosphatidylcholine, that is principally located in the outer half of the lipid bilayer. To identify the structural domain of protein 4.1 involved in binding to the phospholipids, a mixture of proteolytic fragments of protein 4.1 was incubated with PS liposomes. The liposomes selectively retained the 30-kilodalton (kDa) basic domain of the protein, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/isoelectric focusing.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Proteins↗
PubMed1988

Spectrin-alpha I/61: a new structural variant of alpha-spectrin in a double-heterozygous form of hereditary pyropoikilocytosis.

Recent biochemical studies have led to the identification of abnormal spectrins in the erythrocytes of patients with hereditary pyropoikilocytosis (HPP) and hereditary elliptocytosis (HE). In this report we describe the biochemical characterization of the erythrocytes from a proband with severe HPP who is doubly heterozygous for two mutant spectrins (Sp): Sp alpha I/74 and a new, previously undetected, mutant of alpha-spectrin designated Sp alpha I/61. The proband's erythrocytes are unstable when exposed to 45 degrees C, and her membrane skeletons exhibit instability to shear stress. The content of spectrin in the proband's erythrocyte membranes is decreased to 75% of control values. The amount of spectrin dimers in crude 4 degrees C spectrin extracts is increased (58%) as compared with control values (6% +/- 4%). Limited tryptic digestion reveals a marked decrease in the normal 80,000-dalton alpha I domain, an increase in the 74,000-dalton fragment that is characteristic of Sp alpha I/74, and an increase in a series of new fragments of 61,000, 55,000, 21,000, and 16,000 daltons. Both parents are asymptomatic, but they have increased amounts of spectrin dimers (17% to 25%). Limited tryptic digestion of the father's spectrin demonstrates the presence of a previously identified abnormal spectrin (Sp alpha I/74) that is characterized by a decrease in content of the 80,000-dalton peptide and an increase in concentration of the 74,000-dalton peptide. The mother's spectrin digests show a decrease in the amount of 80,000-dalton peptide and the formation of new peptides of 61,000, 55,000, 21,000, and 16,000 daltons. The data indicate that this severe form of HPP is due to the inheritance of two distinct abnormal spectrins, Sp alpha I/74 and a new spectrin mutant, Sp alpha I/61.

Anemia, Hemolytic, Congenital↗
PubMed1988

Detection of hemin release during hemoglobin S denaturation.

Sickle hemoglobin is relatively unstable upon oxidation or mechanical shaking. During denaturation, it generates oxygen radicals and hemichromes and ultimately precipitates in the form of micro-Heinz bodies. It is not clear, however, whether the degradation product hemin, which is a potent hemolytic agent and a potential perturbant to protein-protein interactions in the red cell membrane skeleton, is also generated during sickle hemoglobin denaturation. By specific absorption of hemin with Dowex AG 1-X8 anion-exchange resin at high-ionic strength conditions, we now separate hemin for quantitation from the bulk hemoglobin and its derivatives. We demonstrate that upon mechanical shaking oxyhemoglobin S denatures much faster than oxyhemoglobin A and that a considerably higher level of hemin is detected in the shaken hemoglobin S as compared with hemoglobin A. By using the same method to measure the hemin content in the hemolysate of fresh red cells from patients with sickle cell disease, we detect a three- to fivefold increase in the hemin content in these patients (0.4 to 0.75 mumol/L) as compared with normal individuals (0.1 to 0.15 mumol/L). These data suggest that the instability of sickle oxyhemoglobin leads to increased intracellular precipitation of hemoglobin and the release of hemin, which may play a role in the membrane lesion of sickle red cells.

Anemia, Sickle Cell↗
PubMed1987

Hereditary elliptocytosis, spherocytosis and related disorders: consequences of a deficiency or a mutation of membrane skeletal proteins.

The membrane skeleton, a protein lattice that laminates the internal side of the red cell membrane, contains four major proteins: spectrin, actin, protein 4.1 and ankyrin. By mass, the most abundant of these proteins is spectrin, a fibre-like protein composed of two chains, alpha and beta, which are twisted along each other into a heterodimer. At their head region, spectrin heterodimers are assembled into tetramers. At their distal end, these tetramers are interconnected into a two dimensional network by their linkage to oligomers of actin. This interaction is greatly strengthened by protein 4.1. The skeleton is attached to the membrane by ankyrin, a protein that connects the spectrin beta chain to the major transmembrane protein band 3, the anion channel protein. Additional attachment sites are those of protein 4.1 with several glycoproteins, namely glycophorin A and C, as well as direct interactions between spectrin, protein 4.1 and the negatively charged lipids of the inner membrane lipid bilayer. Hereditary spherocytosis, elliptocytosis and pyropoikilocytosis represent a group of disorders that are due to deficiency or dysfunction of one of the membrane skeletal proteins (Fig. 1). Known deficiency states include that of spectrin, ankyrin and protein 4.1. Severe spectrin and ankyrin deficiencies (with decrease in spectrin and ankyrin contents to about 50% of the normal amount) are both rare disorders associated with severe autosomal recessive hereditary spherocytosis. On the other hand, mild spectrin deficiency is found in the majority of patients with autosomal dominant spherocytosis in which the degree of spectrin deficiency correlates with the clinical severity of the disease. Protein 4.1 deficiency, in contrast, is associated with hereditary elliptocytosis, which in certain populations constitutes about 20% of all such patients. Known skeletal protein dysfunctions include mutants of both alpha and beta spectrin that involve the spectrin heterodimer self-association site. These are clinically expressed as hereditary elliptocytosis (HE) and a closely related disorder, hereditary pyropoikilocytosis (HPP). At the level of protein function, this defect can be detected by analysis of the content of spectrin dimers and tetramers in 0 degrees C low ionic strength extracts of red cell membranes. Their structural identification is accomplished by limited proteolytic digestion of spectrin followed by two-dimensional tryptic peptide mapping.(ABSTRACT TRUNCATED AT 400 WORDS)

Elliptocytosis, Hereditary↗
PubMed1987

Isolation and characterization of cDNA clones for human erythrocyte beta-spectrin.

Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical alpha (Mr 240,000) and beta (Mr 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. We report here the isolation and characterization of a human erythroid-specific beta-spectrin cDNA clone that encodes parts of the beta-9 through beta-12 repeat segments. This cDNA was used as a hybridization probe to assign the beta-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte beta-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the beta-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities.

Amino Acid Sequence↗
PubMed1987

Visualization of the hexagonal lattice in the erythrocyte membrane skeleton.

The isolated membrane skeleton of human erythrocytes was studied by high resolution negative staining electron microscopy. When the skeletal meshwork is spread onto a thin carbon film, clear images of a primarily hexagonal lattice of junctional F-actin complexes crosslinked by spectrin filaments are obtained. The regularly ordered network extends over the entire membrane skeleton. Some of the junctional complexes are arranged in the form of pentagons and septagons, approximately 3 and 8%, respectively. At least five forms of spectrin crosslinks are detected in the spread skeleton including a single spectrin tetramer linking two junctional complexes, three-armed Y-shaped spectrin molecules linking three junctional complexes, three-armed spectrin molecules connecting two junctional complexes with two arms bound to one complex and the third arm bound to the adjacent complex, double spectrin filaments linking two junctional complexes, and four-armed spectrin molecules linking two junctional complexes. Of these, the crosslinks of single spectrin tetramers and three-armed molecules are the most abundant and represent 84 and 11% of the total crosslinks, respectively. These observations are compatible with the presence of spectrin tetramers and oligomers in the erythrocyte membrane skeleton. Globular structures (9-12 nm in diameter) are attached to the majority of the spectrin tetramers or higher order oligomer-like molecules, approximately 80 nm from the distal ends of the spectrin tetramers. These globular structures are ankyrinor ankyrin/band 3-containing complexes, since they are absent when ankyrin and residual band 3 are extracted from the skeleton under hypertonic conditions.

Actins↗
PubMed1987

Synthesis and assembly of membrane skeletal proteins in mammalian red cell precursors.

The synthesis of membrane skeletal proteins in avian nucleated red cells has been the subject of extensive investigation, whereas little is known about skeletal protein synthesis in bone marrow erythroblasts and peripheral blood reticulocytes in mammals. To address this question, we have isolated nucleated red cell precursors and reticulocytes from spleens and from the peripheral blood, respectively, of rats with phenylhydrazine-induced hemolytic anemia and pulse-labeled them with [35S]methionine. Pulse-labeling of nucleated red cell precursors shows that the newly synthesized alpha- and beta-spectrins are present in the cytosol, with a severalfold excess of alpha-spectrin over beta-spectrin. However, in the membrane-skeletal fraction, newly synthesized alpha- and beta-spectrins are assembled in stoichiometric amounts, suggesting that the association of alpha-spectrin with the membrane skeleton may be rate-limited by the amount of beta-spectrin synthesized, as has been shown recently in avian erythroid cells (Blikstad, I., W. J. Nelson, R. T. Moon, and E. Lazarides, 1983. Cell, 32:1081-1091). Pulse-chase experiments in the rat nucleated red cell precursors show that the newly synthesized alpha- and beta-spectrin of the cytosol turn over coordinately and extremely rapidly. In contrast, in the membrane-skeletal fraction, the newly synthesized polypeptides of spectrin are stable. In contrast to nucleated erythroid cells, in reticulocytes the synthesis of alpha- and beta-spectrins is markedly diminished compared with the synthesis and assembly of proteins comigrating with bands 2.1 and 4.1 on SDS gels. Thus, in nucleated red cell precursors, the newly synthesized spectrin may be attached to the plasma membrane before proteins 2.1 and 4.1 are completely synthesized and incorporated in the membrane.

Animals↗
PubMed1987

Clinical and laboratory study of two Caucasian families with hereditary pyropoikilocytosis and hereditary elliptocytosis.

Hereditary pyropoikilocytosis (HPP) is a severe, congenital hemolytic anemia occurring almost exclusively in black persons and characterized by extreme red blood cell anisopoikilocytosis. The authors report two unrelated white females with HPP. Both had severe hemolytic anemia at birth, red blood cell morphologic features characteristic for HPP, and increased thermal sensitivity of the red blood cells. Examination of the red blood cell membranes of both patients showed markedly unstable membrane skeletons when subjected to shear stress, spectrin dimer association defects with increased dimers, and partial spectrin deficiency. Limited tryptic digestion of the spectrin molecule from both patients yielded an abnormal pattern with a decrease in the normal 80,000-dalton alpha I domain and a concomitant increase of an abnormal 74,000-dalton peptide (Sp alpha 1/74). One parent and one sibling of one of the patients with HPP had hereditary elliptocytosis (HE) and the Sp alpha 1/74 defect. The other patient with HPP was different from others reported in that both parents were hematologically and biochemically normal. In addition, her daughter had HE and the Sp alpha 1/74 defect.

Adult↗
PubMed1987

Clinical expression of alpha spectrin mutants in hereditary elliptocytosis.

The group of disorders manifesting as hereditary elliptocytosis/pyropoikilocytosis (HE/HPP) represent a unique group of experiments of nature that result from molecular defects of alpha spectrin. At the level of protein structure, these alpha spectrins can be identified by analysis of peptides generated by limited tryptic digestion. Such an approach reveals that the peptide containing alpha spectrin self-association site (the alpha I domain, molecular mass of 80 daltons) is cleaved to peptides of smaller size, presumably due to changes in the primary structure that lead to increased susceptibility of existing cleavage sites or the opening of new sites. Based on the mass of these peptides, we designate these alpha spectrin (Sp) mutants, Sp alpha 1/74, Sp alpha 1/65, and Sp alpha 1/46. At the level of protein function, these mutant alpha spectrins are characterized by a defective self-association of spectrin heterodimers to tetramers, the major structural subunits of the skeleton. One of the most interesting features of this group of disorders is a variable severity of their clinical expression. Molecular determinants of disease severity include the percentage of unassembled, that is, dimeric spectrin in the membrane and the total spectrin content in the cells. Consequently, the most severely affected patients, manifesting as HPP, contain a high fraction of unassembled, dimeric spectrin in the membrane (55 +/- 7%) and are, in addition, partially deficient in spectrin. In contrast, HE individuals and asymptomatic carriers have a moderate (33 +/- 11) or mild (24 +/- 9) increase in spectrin dimers (normals 5 +/- 4%) and they contain normal amounts of spectrin in their membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Elliptocytosis, Hereditary↗
PubMed1987

Molecular determinants of clinical expression of hereditary elliptocytosis and pyropoikilocytosis.

The clinical severity of common hereditary elliptocytosis (HE) is highly variable, ranging from an asymptomatic carrier state to a severe hemolytic anemia. To elucidate the molecular basis of this variable clinical expression, we evaluated 56 subjects from 24 HE kindred, who carry alpha spectrin mutants characterized by a spectrin dimer (SpD) self-association defect related to a structural abnormality of the alpha I domain of spectrin. Twenty-nine subjects had common HE, 13 subjects have a closely related disorder, hereditary pyropoikilocytosis (HPP), and 14 are asymptomatic carriers. We compared the severity of hemolysis with the following biochemical parameters: (a) spectrin heterodimer self-association, as manifested by the percentage of SpD in the 4 degrees C low ionic strength spectrin extract; (b) spectrin structure, as examined by limited tryptic digestion of spectrin; and (c) spectrin content of the RBC membrane. Our analysis indicates that the severity of hemolysis may be correlated with quantitative differences in the percentage of SpD in the 4 degrees C spectrin extract, as well as the total spectrin content of the membrane. Thus, HPP subjects, who have the most severe hemolytic anemia, have the highest percentage of SpD as well as a decreased spectrin content. HE subjects and asymptomatic carriers, respectively, have a lower percentage of SpD and a normal spectrin content. Factors influencing these two determinants include functional differences between the individual spectrin mutants, the relative amounts of mutant spectrin present in the cells, the stability of mutant spectrin, and the possibility of a superimposed genetic defect involving spectrin synthesis.

Elliptocytosis, Hereditary↗
PubMed1986

Ultrastructural studies of the interaction of spectrin with phosphatidylserine liposomes.

Spectrin was shown previously to interact with phosphatidylserine and phosphatidylethanolamine, which are preferentially localized in the inner half of the membrane lipid bilayer, but this interaction is not well characterized. In the present study we used electron microscopy of rotary-shadowed platinum replicas of spectrin dimer-phosphatidylserine complexes to study the interaction of spectrin with phosphatidylserine vesicles. At a spectrin concentration of 0.6 mg/mL, 60% of spectrin dimers were associated with phosphatidylserine vesicles and at a spectrin concentration of 1.2 mg/mL, some vesicles were crosslinked by spectrin dimers. The length of the protruding segment of spectrin dimer from the liposome edge ranged from 400 to 960A degrees and the contact region to phosphatidylserine extended 272 +/- 144A degrees from either end of the molecule. Therefore, these data are consistent with multiple binding sites to phosphatidylserine throughout the spectrin dimer molecule. Spectrin tetramers, when bound to phosphatidylserine liposomes, extended 1804 +/- 79A degrees from the liposome edge and crosslinked liposomes, suggesting that some of the binding sites to phosphatidylserine vesicles is in the proximity of the tail end of spectrin. The association between spectrin dimers to phosphatidylserine was demonstrated by nondenaturing gel electrophoresis. The complexes were separated into multiple bands with molecular weight of 1.4 X 10(6), 1.8 X 10(6), and 2.3 X 10(6). These bands did not represent self-associated spectrin oligomers, since postincubation treatment with Triton-X-100 dissociated them into spectrin dimers. Furthermore, these spectrin high molecular weight bands, as visualized by Coomassie blue absorbance, closely corresponded to the 14C-phosphatidylserine distribution. These data provide ultrastructural and biochemical evidence that spectrin binds to phosphatidylserine at multiple sites including the tail end region.

Binding Sites↗
PubMed1986

Partial spectrin deficiency in hereditary pyropoikilocytosis.

Hereditary pyropoikilocytosis (HPP) is a severe hemolytic anemia in which an instability of the red cell membrane skeleton has been correlated with structural and functional defects of spectrin. We now report that 13 unrelated HPP subjects have approximately 30% less spectrin than normal as evidenced by a decreased spectrin/band 3 ratio. We also examine the role of spectrin degradation as an underlying cause of this partial spectrin deficiency. Our studies demonstrate that the reduced spectrin content of HPP red cells remains constant during in vivo aging of the cells in the peripheral blood, as well as during in vitro incubation. Furthermore, immunoblotting experiments using an affinity-purified antispectrin antibody indicate that there is no loss of spectrin during membrane preparation and also that neither whole HPP red cells nor ghosts nor cytosol contains any abnormal spectrin degradation products. These data suggest that spectrin is not degraded and that it is stable on the membrane of the circulating HPP red cell. In contrast, however, incubation of free spectrin with a lysate of nucleated erythroid precursor cells indicates that HPP alpha I/46 spectrin, but not HPP alpha I/74 spectrin, is more susceptible to proteolytic degradation than a control. These data imply that the decreased spectrin content of HPP is not due to a single defect but that a more complex mechanism is involved. In HPP Sp alpha I/46 subjects, an increased proteolytic degradation in bone marrow erythroid precursors of cytosolic spectrin, prior to its assembly on the membrane, could contribute toward the partial spectrin deficiency.

Anemia, Hemolytic↗
PubMed1985

Hemin-mediated dissociation of erythrocyte membrane skeletal proteins.

Spectrin tetramers and oligomers in normal erythrocytes are cross-linked by actin and protein 4.1 to form a two-dimensional membrane skeletal network. In the present study, we find that hemin, a breakdown product of hemoglobin, progressively (a) alters the conformation of spectrin as revealed by electron microscope studies and by the decreased resistance of spectrin to proteolytic degradation, (b) alters the conformation of protein 4.1 as revealed by the increased mobility of protein 4.1 on nondenaturing gel electrophoresis, (c) weakens spectrin dimer alpha beta-dimer alpha beta, spectrin alpha-spectrin beta, as well as spectrin-protein 4.1 associations as analyzed by nondenaturing gel electrophoresis, and (d) diminishes the structural stability of erythrocyte membrane skeletons (i.e. Triton-insoluble ghost residues) subjected to mechanical shearing. Since hemin may be liberated from oxidized or unstable mutant hemoglobin under pathological conditions, these hemin-induced effects on spectrin, protein 4.1, and membrane skeletal stability may play a role in the membrane lesion of these erythrocytes.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Sp alpha I/65: a new variant of the alpha subunit of spectrin in hereditary elliptocytosis.

Two molecular defects involving the spectrin heterodimer (SpD) contact site of the alpha chain (the alpha I domain) were previously identified using limited tryptic digestion followed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both are characterized by atypical peptide maps which reveal a marked decrease of the 80,000-dalton alpha I domain and a formation of new major peptides of either 74,000 (Sp alpha I/74) or 46,000 (Sp alpha I/46) daltons. We now report a third variant of the spectrin alpha chain, designated Sp alpha I/65, in three unrelated black families. In all three probands, the percentage of SpD in the low ionic strength (O degrees C) membrane extracts was increased to 19% to 32%. One- and two-dimensional electrophoretic separations of limited tryptic digests of spectrin from all three probands revealed a decrease of the alpha I domain of spectrin and the concomitant appearance of peptides at 65,000 daltons and isoelectric points ranging from 5.2 to 5.3. The abnormal 65,000-dalton peptides could be stained with an antiserum which had been raised against the alpha I domain, indicating that it was derived from the alpha I domain.

Adult↗