Solubilized Erythrocyte Membrane Research Articles

Pleurotolysin, a Novel Sphingomyelin-specific Two-component Cytolysin from the Edible Mushroom Pleurotus ostreatus, Assembles into a Transmembrane Pore Complex

Self-assembling, pore-forming cytolysins are illustrative molecules for the study of the assembly and membrane insertion of transmembrane pores. Here we purified pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the basidiocarps of Pleurotus ostreatus and studied the pore-forming properties of the cytolysin. Pleurotolysin consisted of non-associated A (17 kDa) and B (59 kDa) components, which cooperatively caused leakage of potassium ions from human erythrocytes and swelling of the cells at nanomolar concentrations, leading to colloid-osmotic hemolysis. Hemolytic assays in the presence of poly(ethylene glycol)s with different hydrodynamic diameters suggested that pleurotolysin formed membrane pores with a functional diameter of 3.8-5 nm. Pleurotolysin-induced lysis of human erythrocytes was specifically inhibited by the addition of sphingomyelin-cholesterol liposomes to the extracellular space. Pleurotolysin A specifically bound to sphingomyelin-cholesterol liposomes and caused leakage of the internal carboxyfluorescein in concert with pleurotolysin B. Experiments including solubilization of pleurotolysin-treated erythrocytes with 2% (w/v) SDS at 25 degrees C and SDS-polyacrylamide gel electrophoresis/Western immunoblotting showed that pleurotolysin A and B bound to human erythrocytes in this sequence and assembled into an SDS-stable, 700-kDa complex. Ring-shaped structures with outer and inner diameters of 14 and 7 nm, respectively, were isolated from the solubilized erythrocyte membranes by a sucrose gradient centrifugation. Pleurotolysin A and B formed an SDS-stable, ring-shaped complex of the same dimensions on sphingomyelin-cholesterol liposomes as well.

NAD Glycohydrolase Activities and ADP-Ribose Uptake in Erythrocytes From Normal Subjects and Cancer Patients

Erythrocytes from cancer patients exhibited up to fivefold higher NAD glycohydrolase activities than control erythrocytes from normal subjects and also similarly increased [14C] ADP-ribose uptake values. When [adenosine-14C] NAD was used instead of free [14C] ADP-ribose, the uptake was dependent on ecto-NAD glycohydrolase activity. This was reflected in the inhibition of ADP-ribose uptake from [adenosine-14C] NAD by Cibacron Blue. ADP-ribose uptake in erythrocytes appeared to be complex: upon incubation with free [14C] ADP-ribose, the radiolabel associated with erythrocytes was located in nearly equal parts in cytoplasm and plasma membrane. Part of [14C] ADP-ribose binding to the membrane was covalent, as indicated by its resistance to trichloroacetic acid-treatment. A preincubation with unlabeled ADP-ribose depressed subsequent erythrocyte NAD glycohydrolase activity and binding of [14C] ADP-ribose to erythrocyte membrane; but it failed to inhibit the transfer of labeled ADP-ribose to erythrocyte cytoplasm. On the other hand, incubation with [adenosine-14C] NAD did not result in a similar covalent binding of radiolabel to erythrocyte membrane. In line with this finding, a preincubation with unlabeled NAD was not inhibitory on subsequent NAD glycohydrolase reaction and ADP-ribose binding. ADP-ribose binding and NAD glycohydrolase activities were found also in solubilized erythrocyte membrane proteins and, after size fractionation, mainly in a protein fraction of around 45kDa-molecular weight.

Binding characteristics of purified Escherichia coli K88ab fimbriae to guinea pig erythrocyte membrane

The characteristics of the binding of biotinylated E. coli K88ab fimbriae to guinea pig erythrocyte membranes, as a possible model of the target host cell were studied. Binding showed sigmoidal dependence, with an apparent saturation at about 0.8 ng of fimbriae. Hill coefficient values ( h) were about 2–2.4, which indicated that the receptor population showed positive cooperativity with at least three binding sites. Apparent binding constants to the first and third binding sites ( 1K 3 and 3K 3) were determined. Three K88ab binding proteins, of 67, 63 and 48 kDa, were identified on solubilized erythrocyte membranes and were recovered mainly in a detergent phase, suggesting a possible integral localization of the receptors.

Soluble complement receptor type 1 (CD35) is released from leukocytes by surface cleavage.

The soluble form of complement receptor type 1 in human plasma (sCR1) might correspond to the shedding of the receptor by proteolytic cleavage at the cell surface. A new enzyme-linked immunosorbent assay (ELISA) was established to specifically measure membrane-bound CR1 using a rabbit polyclonal antibody against a 19-amino acid peptide corresponding to the C-terminal sequence of the intracellular domain of CR1 (mCR1-ELISA). This ELISA measured CR1 from solubilized erythrocyte membranes, polymorphonuclear leukocytes (PMN), a B lymphocyte cell line and renal podocyte-derived urinary vesicles in a dose-dependent manner. In contrast, and similarly to recombinant soluble CR1 which lacks the intracellular domain of CR1, plasmatic sCR1 was not recognized, suggesting that sCR1 corresponds to an extracellular fragment of whole CR1. In vitro, PMN were shown to release a soluble form of CR1 which was also not recognized in the mCR1-ELISA, and whose size was smaller (5 kDa) than the CR1 of PMN cell membranes. The release of soluble CR1 was highest for PMN and HL60 cells, followed by U937 cells and three different B lymphocyte cell lines, whereas T lymphocyte cell lines did not release soluble CR1. The levels of CR1 gene expression were also higher in PMN compared to remaining blood leukocytes and the different cell lines tested above. Incubation of PMN with formyl-methionyl-leucyl-phenylalanine, tumor necrosis factor-alpha or lipopolysaccharide accelerated the release of soluble CR1, and incubation with granulocyte/macrophage colony-stimulating factor resulted in sustained CR1 gene expression and higher total soluble CR1 release. Our results suggest that soluble CR1 is produced by cleavage of cell surface CR1, and that a large fraction of human plasma sCR1 is cleaved from PMN. The release of sCR1 by leukocytes may play a role in the control of complement activation at sites of inflammation.

Isolation of a human erythrocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels.

Erythrocytes are poorly lysed by homologous complement, whereas they are readily lysed by heterologous complement. This phenomenon had been attributed to an interference by the cell surface with the action of complement components C8 and C9. To isolate the responsible membrane constituent, detergent-solubilized human erythrocyte (EH) membranes were subjected to affinity chromatography by using human C9-Sepharose. The isolated protein had a mass of 38 kDa and, incorporated into liposomes, was highly effective in inhibiting complement-mediated channel expression, including the C5b-8, membrane attack complex, and tubular polymer of C9 channels. Antibody produced to the 38-kDa protein caused a 20-fold increase in reactive lysis of EH by isolated C5b6, C7, C8, and C9. The antibody did not enhance C5b-7 uptake, but it affected C9 binding to the target cell membrane. Antibody to human decay-accelerating factor, used as a control, had no effect on reactive lysis of EH. Anti-38-kDa protein did not enhance the action on EH of C8 and C9 from other species, indicating that the action of this regulatory protein is species specific. It was therefore termed homologous restriction factor (HRF). Blood cells other than erythrocytes, such as polymorphonuclear leukocytes, also exhibited cell-surface HRF activity. In immunoblots of freshly isolated EH membranes, anti-38-kDa HRF detected primarily a 65-kDa protein, suggesting that the 38-kDa protein constitutes an active fragment of membrane HRF. Because of the specific binding reaction observed between HRF and C8 or C9, HRF was tested with anti-human C8 and anti-human C9. A limited immunochemical relationship of HRF to C8 and C9 could be established and solid-phase anti-C9 proved an efficient tool for the isolation of HRF from solubilized EH membranes.

Biochemical Characterization of a Monoclonal Antibody to the H Type-2 Antigen: Comparison to Other ABH Antibodies

Monoclonal and polyclonal antibodies to the ABH blood group antigens were tested for their specificity to glycoproteins with ABH activity on immunoblots of solubilized erythrocyte membranes. Immunoblots were stained with monoclonal antibody G10 to the H type-2 carbohydrate structure or with commercially prepared monoclonal and polyclonal antibodies to A, B, and H blood group antigens. G10 antibody specifically stained antigens in the regions that contain the erythrocyte membrane bands 3 and 4.5; the staining was proportional to the expected H content of the erythrocytes (O greater than A2 greater than B greater than A2B greater than A1 greater than A1B). No specific staining was observed with membranes derived from Oh (Bombay) erythrocytes which lack the H type-2 structure. A commercially prepared monoclonal anti-H did not specifically stain erythrocyte membrane antigens. Monoclonal and polyclonal anti-A specifically stained bands from A and AB but not O, B, or Oh erythrocytes (A1 greater than A1B greater than A2 greater than A2B). Polyclonal anti-B serum specifically stained bands from B and AB but not O, A, or Oh erythrocytes (B greater than A2B greater than A1B). However, no specific staining was observed in tests with monoclonal anti-B. Monoclonal antibodies G10 and anti-A and polyclonal anti-A and -B blood typing sera will be useful in the further characterization of the molecular nature of the ABH antigens.

Identification of proteolytic activities in the cytosolic compartment of mature human erythrocytes.

Individual lysates from human erythrocyte suspensions, completely deprived of leucocytes and were assayed for a number of proteolytic activities using both naturally occurring and synthetic substrates. Removal of hemoglobin by batchwise DEAE-cellulose chromatography did not modify the complement of the various proteolytic activities which were then fractionated by means of chromatography on a column of DEAE-cellulose, followed by conventional techniques such as gel chromatography and preparative electrophoresis. This procedure allowed a number of proteinases to be identified in the erythrocyte cytosol while providing a tool for their selective though partial separation. The following peptidases were found to be present in the soluble fraction of mature human erythrocytes: (a) a neutral endopeptidase having an approximate molecular weight of 110 000; (b) three acidic endopeptidases, with pH optima between 2.5 and 3.5, showing molecular and functional properties almost identical with those of the three proteinases previously purified from solubilized erythrocyte membranes [Pontremoli et al. (1979) Biochem. J. 181, 559--568]; (c) two dipeptidylaminopeptidases whose molecular weights are around 80 000 and tentatively identified as dipeptidyl aminopeptidases II and III, respectively, on the basis of their substrate specificities and pH optima; (d) presumably two aminopeptidases, having an approximate molecular weight of 80 000 and classified as an aminopeptidase with broad substrate specificity and an aminopeptidase B, respectively. No evidence for any carboxypeptidase activity was found in the cytosolic compartment of mature human erythrocytes.

Isolation of an abnormally phosphorylated erythrocyte membrane band 3 glycoprotein from patients with myotonic muscular dystrophy

A fraction of erythrocyte Band 3 (Mr, 93,000) glycoprotein that demonstrates decreased autophosphorylation in membranes from myotonic muscular dystrophy patients is demonstrated. Sequential affinity chromatography of Triton X-100 solubilized erythrocyte membrane proteins separated three specifically retained glycoprotein fractions on a Ricin Communis I-Sepharose 4B column. One fraction contains a portion of the major sialoglycoprotein (apparent Mr, 78,000) and is specifically eluted from the column by 10 mM NaCl and 100 mM D-galactose (10/100). The two other glycoprotein fractions are eluted by 100 mM NaCl, 10 mM D-galactose (100/10) and 100 mM NaCl, 100 mM D-galactose (100/100). The composition of both fractions contains greater than 95% Band 3 (apparent Mr, 93,000 glycoprotein. The quantities of glycoprotein in each fraction obtained from erythrocytes of myotonic dystrophy patients did not differ from the quantities obtained from control erythrocytes. Following endogenous protein kinase incubations of ghosts with [gamma-32P]ATP, the specific [32P] phosphorylation of the 10/100 and 100/10 fractions are identical. The 100/100 fraction, which makes up approximately 3% of the total erythrocyte membrane protein, demonstrates a different pattern for myotonic dystrophy patients; specific phosphorylation was reduced by 50% relative to activity in control experiments. These findings are consistent with previous experiments that demonstrated decreased autophosphorylation of the glycoprotein portion of Band 3 (Roses & Appel, 1975, J. Membrane Biol 20:51) and are consistent with the autosomal dominant mode of inheritance in this disease.

Effets du glycérol sur les interactions des cations monovalents avec la (Na +-K +)-ATPase des membranes d'érythrocytes humains solubilisées

Effets du glycérol sur les interactions des cations monovalents avec la (Na +-K +)-ATPase des membranes d'érythrocytes humains solubilisées

Analysis of Partially Degraded Proteins by Quantitative Immunoelectrophoresis

For the recognition of partially degraded proteins in quantitative immunoelectrophoresis, controlled proteolytic degradation of two model systems was investigated: human serum proteins and solubilized human erythrocyte membrane proteins. The following characteristic precipitates were found: 1) unending ('flying') precipitates, 2) precipitates splitting into several lines, 3) precipitation arcs showing shoulders or double peaks ('camels'), and 4) skew precipitation arcs. The same types of precipitates were also found for solubilized erythrocyte membrane proteins containing contaminating proteolytic enzymes and normal concentrated urine proteins.

Quantitative immunoelectrophoresis for comparative analysis of membrane proteins from various mammalian species

1. 1. Reference precipitation patterns obtained with crossed inimunoelectrophoresis of human and bovine erythrocyte membrane proteins, extensively solubilized with non-ionic detergent, using specific rabbit antibodies, were established. 2. 2. By tandem-crossed and crossed-line inimunoelectrophoresis those two precipitation patterns were correlated and corresponding proteins were identified. 3. 3. The antibodies against human membrane proteins also revealed precipitation lines in crossed immunoelectrophoresis of solubilized erythrocyte membrane proteins from ox, sheep, pig, dog, cat and rat. Using the same techniques these precipitation lines were correlated with the human reference precipitation pattern. 4. 4. Also, partial identity between the main proteins of bovine erythrocyte membranes and bovine milk fat globule membranes was demonstrated.

Antigenicity of Solubilized Erythrocyte Membrane Fractions through Sephadex G-150 Column against the Anti-Membrane Factor

Antigenicity of Solubilized Erythrocyte Membrane Fractions through Sephadex G-150 Column against the Anti-Membrane Factor

A comparative study of phospholipids bound to erythrocyte membrane proteins

1. 1. The phosphoprotein and total phosphoinositide contents of solubilized erythrocyte membrane proteins were similar in human, dog, rabbit and sheep blood. Diphosphoinositide, a prominent component of human preparations, was found in trace quantities in the other species. The converse was true for phosphatidylinositol. 2. 2. During storage of human blood the triphosphoinositide was partially converted to diphosphoinositide. In stored dog blood the triphosphoinositide was degraded to phosphatidylinositol while little change was detected in rabbit blood. 3. 3. Solubilized membrane protein preparations from all species exhibited the same amino acid composition but there were differences in carbohydrate content. 4. 4. The polyphosphoinositides and phosphoproteins were metabolically active in all species.

Reactivity of Anti-Membrane Factor with Solubilized Erythrocyte Membrane Fractions of Sepharose 4B Column

Reactivity of Anti-Membrane Factor with Solubilized Erythrocyte Membrane Fractions of Sepharose 4B Column

The phosphorus components of solubilized erythrocyte membrane protein.

Membrane protein preparations were obtained by n-butanol extraction of salt-free aqueous suspensions of human erythrocyte ghosts. The solubilized protein contained 4.5% carbohydrate, including glucosamine and galactosamine, 1.7% sialic acid, and 0.2% phosphorus. Gel electrophoresis indicated the presence of a large number of proteins in the solubilized fraction. Of the phosphorus present 15% could be extracted with chloroform–methanol (2/1) and was shown to consist of phosphatidylserine and some phosphatidylinositol. A further 65% of the phosphorus was extracted with chloroform–methanol–HCl (200/100/1) and this extract was shown to consist principally of diphosphoinositide and triphosphoinositide. The remaining protein-bound phosphorus, representing 0.03% of the protein, could not be separated from the protein. Following treatment with the organic solvents the protein was resolubilized. The carbohydrate and sialic acid concentrations and the gel electrophoretic pattern were not altered. Following incubation of erythrocytes with inorganic 32P, the polyphosphoinositides were rapidly labelled. The phosphoprotein was also rapidly labelled but to a lesser extent. The phosphatidylinositol and phosphatidylserine were very poorly labelled.