Release of galactosyltransferase from peritoneal macrophages during acute inflammation

Peritoneal cells harvested from mice injected with Salmonella enteritidis or thioglycollate released large amounts of galactosyltransferase (GT), but not sialyltransferase, into their culture supernatants. Maximum release of GT (using ovalbumin as acceptor) occurred from cells harvested 2-4 days after primary injection, but little GT was released from cells elicited by a secondary injection of salmonella or ovalbumin in sensitised mice or during intraperitoneal allogeneic reactions. Enzyme release in culture did not parallel GT levels in serum. Most enzyme was released by large, poorly adherent, macrophage-enriched, Fc receptor-bearing peritoneal cells of low density. Normal monocytes, bone marrow cells, and platelets also produced large amounts, and normal spleen cells or polymorphonuclear leukocytes moderate amounts, of GT. Lymphocytes, dead cells, mast cells, red blood cells, or whole populations of lymph node and thymus cells released very low levels of enzyme. Very little GT was bound to the cell surface and was not passively absorbed from serum or platelets. Release of GT was prevented at 4 degrees C but was not markedly affected by a variety of metabolic inhibitors except pretreatment of the cells with thrombin, which increased release and trypsin which decreased release.     

Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane

The hemoglobin binding sites on the inner surface of the erythrocyte membrane were identified by measuring the fraction of hemoglobin released following selective proteolytic or lipolytic enzyme digestion. In addition, binding stoichiometry to and fractional hemoglobin release from inside-out vesicle preparations of human and rabbit membranes were compared since rabbit membranes differ significantly from human membranes only in that they lack glycophorin. Our results show that rabbit inside-out vesicles bind about 65% less human or rabbit hemoglobin under conditions of optimal and stoichiometric binding, despite being otherwise similar in composition. We suggest that this difference is either directly or indirectly due to the absence of glycophorin in rabbit membranes. Further supportive evidence includes demonstrating (a) that neuraminidase treatment of human membranes did not affect hemoglobin binding and (b) that reconstitution of isolated glycophorin into phospholipid vesicles increased the hemoglobin binding capacity in a manner proportional to the fraction of glycophorin molecules oriented with their cytoplasmic sides exposed to the exterior of the vesicle. Proteolysis of human inside-out vesicles either before or after addition of hemoglobin reduced the binding capacity by about 25%. This is consistent with the known proportion of total hemoglobin binding sites involving band 3 protein and the selective lability of the cytoplasmic aspect of band 3 protein to proteolysis. Phospholipid involvement in hemoglobin binding was determined using various phospholipase C preparations which differ in their reactivity profiles. Approximately 38% of the bound hemoglobin was released upon cleavage of phospholipid headgroups. These results suggest that the predominant sites of binding for hemoglobin on the inner surface of the red cell membrane are the two major integral membrane glycoproteins.     

Ceramide channels increase the permeability of the mitochondrial outer membrane to small proteins

Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that C(2)- and C(16)-ceramide, but not dihydroceramide, form large channels in planar membranes (Siskind, L. J., and Colombini, M. (2001) J. Biol. Chem. 275, 38640-38644). Here we show that ceramides do not trigger a cytochrome c secretion or release mechanism, but simply raise the permeability of the mitochondrial outer membrane, via ceramide channel formation, to include small proteins. Exogenously added reduced cytochrome c was able to freely permeate the mitochondrial outer membrane with entry to and exit from the intermembrane space facilitated by ceramides in a dose- and time-dependent manner. The permeability pathways were eliminated upon removal of C(2)-ceramide by bovine serum albumin, thus ruling out a detergent-like effect of C(2)-ceramide on membranes. Ceramide channels were not specific to cytochrome c, as ceramides induced release of adenylate kinase, but not fumerase from isolated mitochondria, showing some specificity of these channels for the outer mitochondrial membrane. SDS-PAGE results show that ceramides allow release of intermembrane space proteins with a molecular weight cut-off of about 60,000. These results indicate that the ceramide-induced membrane permeability increases in isolated mitochondria are via ceramide channel formation and not a release mechanism, as the channels that allow cytochrome c to freely permeate are reversible, and are not specific to cytochrome c.     

Mechanism of human erythrocyte hemolysis induced by short-chain phosphatidylcholines and lysophosphatidylcholine

The incorporation and accumulation of a certain amount of short-chain phosphatidylcholine or lysophosphatidylcholine into lipid bilayers of erythrocyte membranes is the first step causing membrane perturbation in the process of hemolysis. Accumulation of dilauroylglycerophosphocholine into membranes makes human erythrocytes "permeable cells"; Ions such as Na+ or K+ can permeate through the membrane, though large molecules such as hemoglobin can not. The "pore" formation was partially reproduced in liposomes prepared from lipids extracted from human erythrocyte membranes; C12:0PC induced the release of glucose from liposomes but did not significantly induce the release of dextran. It was suggested that the phase boundary between dilauroylglycerophosphocholine and the host membrane bilayer or dilauroylglycerophosphocholine rich domain itself behaves as "pores." Erythrocytes could expand to 1.5 times the original cell volume without any appreciable hemolysis when incubated with C12:0PC at 37 degrees C. The capacity of the erythrocytes to expand was temperature dependent. The capacity may play an important role in the resistance of the cells against lysis. The "permeable cell" stage could be hardly observed when erythrocytes were treated with didecanoylglycerophosphocholine and lysophosphatidylcholine. Perturbation induced by accumulation of didecanoylglycerophosphocholine or lysophosphatidylcholine may cause non specific destruction of membranes rather than formation of a kind of "pore."     

Membrane transformation during malaria parasite release from human red blood cells

Three opposing pathways are proposed for the release of malaria parasites from infected erythrocytes: coordinated rupture of the two membranes surrounding mature parasites; fusion of erythrocyte and parasitophorus vacuolar membranes (PVM); and liberation of parasites enclosed within the vacuole from the erythrocyte followed by PVM disintegration. Rupture by cell swelling should yield erythrocyte ghosts; membrane fusion is inhibited by inner-leaflet amphiphiles of positive intrinsic curvature, which contrariwise promote membrane rupture; and without protease inhibitors, parasites would leave erythrocytes packed within the vacuole. Therefore, we visualized erythrocytes releasing P. falciparum using fluorescent microscopy of differentially labeled membranes. Release did not yield erythrocyte ghosts, positive-curvature amphiphiles did not inhibit release but promoted it, and release of packed merozoites was shown to be an artifact. Instead, two sequential morphological stages preceded a convulsive rupture of membranes and rapid radial discharge of separated merozoites, leaving segregated internal membrane fragments and plasma membrane vesicles or blebs at the sites of parasite egress. These results, together with the modulation of release by osmotic stress, suggest a pathway of parasite release that features a biochemically altered erythrocyte membrane that folds after pressure-driven rupture of membranes.     

Mechanism of release of integral proteins from rat liver microsomal membranes

The release of three integral enzymatic activities (NADH- and NADPH-cytochrome c reductase and 5'-nucleotidase) and total protein from washed rat liver microsomal membranes, upon simple incubation at 37 degrees C in aqueous media, was investigated. Release does not depend on contaminating proteases and is enhanced by alkaline pH. Total protein and enzyme release is consistent with a loss of phospholipids which are not recovered in the soluble phase. Following incubation at pH 9.0 large amounts of free fatty acids were recovered in the soluble phase, accounting for a ratio of 1/1 (w/w) with released protein. This evidence, together with the data available about densities (1.07-1.08 g/ml) and molecular weights (1 700 000-700 000) of the released enzymes, suggests that they are solubilized from microsomal membranes in the form of mixed micelles mostly formed by free fatty acids and integral proteins, probably owing to the activity of endogenous phospholipases on membrane lipids. Release of total protein and enzymatic activities is decreased by Ca2+, whose possible role in the phenomenon is discussed.     

Feeding Mechanisms in Extracellular Babesia microti and Plasmodium lophurae*†

SYNOPSIS. Although large hemoglobin inclusions are observed in intraerythrocytic Babesia microti parasites, they are absent from parasites freed of hamster red cells by immune lysis with antihamster erythsocyte serum. Babesia microti has no cytostome. This parasite, therefore, does not appear to feed by phagocytosis of large boluses of hemoglobin, as does Plasmodium. To determine whether Babesia can pinocytose protein, free parasites were fed ferritin in an in vitro system. Ferritin was taken up from the entire cell surface into narrow channels within 15 min at 37 C. Only merozoites, with their pellicular complex, failed to take up the protein. By 60 min, the ferritin was highly concentrated in many channels and vesicles, which formed interconnecting stacks. The ferritin-containing channels became associated with membrane whorls of the multimembranous structure. Membrane whorls were also observed in the process of extrusion in samples incubated for longer times. These events may represent steps in the digestion and excretion of the pinocytosed protein. Empty channels formed when Babesia was fed albumin. The diaminobenzidine reaction for hemoprotein was positive for the channels in both free and intraerythrocytic babesias. The staining reaction was completely inhibited by cyanide, but not at all by aminotriazole. These results further suggest that Babesia pinocytoses hemoglobin in vivo. Plasmodium lophurae parasites freed of red cells by immune lysis are surrounded by 2 membranes and apparently can ingest ferritin only through the cytostome. Extracellular cytostomal feeding involves both membranes, as it does in vivo. Ferritin was found in food vacuoles, some of which contained hemoglobin ingested before parasite isolation, connected to or near the cytostome. In both Plasmodium and Babesia low temperature inhibited ferritin uptake.     

Interaction of plasma lipoproteins with erythrocytes. I. Alteration of erythrocyte morphology.

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL2 and HDL3, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

Approximate dimensions of membrane lesions produced by streptolysin S and streptolysin O

Membrane lesions produced by the streptococcal membranolysins streptolysin S and streptolysin O were investigated. Escape of labeled marker molecules of various sizes from resealed sheep erythrocyte ghosts treated with the toxins for 30 min allowed estimation of the sizes of the primary channels formed. Streptolysin S formed lesions ranging in size up to 45 A in diameter, and even high toxin concentrations did not result in larger channels. The lesions produced by streptolysin O exceeded 128 A in diameter. Kinetics experiments demonstrated that the primary streptolysin O lesions were formed rapidly (1-2 min), but release of marker molecules from streptolysin S-treated vesicles began only after a 5-15-min lag period. Label release from large unilamellar liposomes treated with streptolysin S suggested that membrane fluidity does not affect the size of the streptolysin S lesions.     

A fluorimetric assay for the effects of cytolytic toxins on the transport properties of resealed erythrocyte ghosts.

We prepared resealed erythrocyte ghosts loaded with SPQ and chloride. We demonstrated that these membranes were still functional, as they were capable of exchanging anions, most probably through the band-3 protein. When cytolytic toxins (Escherichia coli hemolysin and Staphylococcus aureus alpha-toxin) were offered to the resealed ghosts, the internal SPQ was released. This could be attributed to the formation of toxin-induced ion channels into the ghost membrane that were so large that SPQ could escape through them. This release was actually independent of the anion-exchanging protein, since DIDS had no inhibitory effect on it. Due to their simplicity, and because they do not lyse, erythrocyte ghosts may serve as useful models to study the action of cytolytic pore-forming toxins. To assess the validity of these model membranes we compared results obtained using RBC and resealed erythrocyte ghosts as targets for the toxin, finding complete consistency. Pre-assembled toxin channels could also be studied on the ghosts. Applying different proteolytic enzymes to the external compartment after channel formation, we found that performed E. coli hemolysin pores were at least partially destroyed by enzymatic digestion.     

The role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response.

Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme.     

Relation between Ca2+-ATPase and endogenous calmodulin of human erythrocyte membranes

Short incubation of erythrocyte membranes with oleic acid releases Ca2+-independently bound endogenous calmodulin together with a minor fraction of membrane-associated proteins without destruction of the membranes. The released endogenous calmodulin is similar if not identical to cytosolic calmodulin reversibly bound to ghosts in a Ca2+-dependent manner. The release of endogenous calmodulin proceeds without affecting the activity of Ca2+-ATPase when ghosts are incubated with oleic acid in the presence of Ca2+ plus ATP and thereafter freed from oleic acid by washings with serum albumin. Kinetic parameters of Ca2+-ATPase of ghosts with and without endogenous calmodulin are identical as are amounts of exogenous calmodulin bound to these ghosts. Thus, endogenous calmodulin does not function as an essential part of Ca2+-ATPase.     

Effect of triorganotin compounds on membrane permeability

Organotin compounds are widely distributed toxicants. They are membrane-active molecules with broad biological toxicity. In this contribution, we study the effect of triorganotin compounds on membrane permeability using phospholipid model membranes and human erythrocytes. Tribultyltin and triphenyltin are able to induce the release of entrapped carboxyfluorescein from large unilamellar vesicles. The rate of release is similar for phosphatidylcholine and phosphatidylserine systems and the presence of equimolar cholesterol decreases the rate of the process. Release of carboxyfluorescein is almost abolished when a non-diffusible anion like gluconate is present in the external medium, and it is restored by addition of chloride. Tributyltin is able to cause hemolysis of human erythrocytes in a dose-dependent manner. Relative kinetics determination shows that potassium leakage occurs simultaneously with hemoglobin release. Hemolysis is reduced when erythrocytes are suspended in a gluconate medium. These results indicate that triorganotin compounds are able to transport organic anions like carboxyfluorescein across phospholipids bilayers by exchange diffusion with chloride and suggest that anion exchange through erythrocyte membrane could be related to the process of hemolysis.     

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL₂ and HDL₃, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

Sphingosine Forms Channels in Membranes That Differ Greatly from Those Formed by Ceramide

Ceramide channels formed in the outer membrane of mitochondria have been proposed to be the pathways by which proapoptotic proteins are released from mitochondria during the early stages of apoptosis. We report that sphingosine also forms channels in membranes, but these differ greatly from the large oligomeric barrel-stave channels formed by ceramide. Sphingosine channels have short open lifetimes and have diameters less than 2 nm, whereas ceramide channels have long open lifetimes, enlarge in size reaching diameters in excess of 10 nm. Unlike ceramide, sphingosine forms channels in erythrocyte plasma membranes that vary in size with concentration, but with a maximum possible channel diameter of 2 nm. In isolated mitochondria, a large proportion of the added sphingosine was rapidly metabolized to ceramide in the absence of externally added fatty acids or fatty-acyl-CoAs. The ceramide synthase inhibitor, fumonisin B1 failed to prevent sphingosine metabolism to ceramide and actually increased it. However, partial inhibition of conversion to ceramide was achieved in the presence of ceramidase inhibitors, indicating that reverse ceramidase activity is at least partially responsible for sphingosine metabolism to ceramide. A small amount of cytochrome c release was detected. It correlated with the level of ceramide converted from sphingosine. Thus, sphingosine channels, unlike ceramide channels, are not large enough to allow the passage of proapoptotic proteins from the intermembrane space of mitochondria to the cytoplasm.     

A receptor for formaldehyde-treated serum albumin on human placental brush-border membrane

Formaldehyde-treated serum albumin (f-Alb) is known to be taken up and degraded by sinusoidal liver cells via receptor-mediated endocytosis. We report that 125I-labeled f-Alb (125I-f-Alb) binding to human placental brush-border membranes also occurs. This binding reached equilibrium within 40 min at 37 degrees C. Kinetic studies demonstrated the presence of saturable binding with an apparent Kd of 2.1 micrograms of f-Alb/ml and a maximal binding of 2.3 micrograms/mg of membrane protein at pH 7.5. Maximal binding was observed at between pH 7.5 and 8.0. 125I-f-Alb binding to the membranes was little inhibited by a 1000-fold molar excess of ovalbumin, human apo-transferrin and native bovine serum albumin. No binding was observed with membranes which had been pretreated with proteinase or trypsin. This f-Alb receptor was extremely heat-stable, since the binding was not abolished even by pretreatment of the membranes at 78 degrees C for 30 min. EDTA, Ca2+ and Mg/4 had no effect on 125I-f-Alb binding, so the binding was independent of divalent cations. These data suggest that a receptor specific for f-Alb exists on human placental brush-border membranes of syncytial trophoblasts.     

Interaction of plasma lipoproteins with erythrocytes. I. Alteration of erythrocyte morphology

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL₂ and HDL₃, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

The separation of proteins with heteropolyacids

The precipitation of proteins with heteropolyacids has been studied for the purpose of large scale primary purification. A precipitate will form if the pH of the reaction between purified ovalbumin, hemoglobin, trypsin, pepsin, bovine serum albumin, ovomucoid, gelatin or ribonuclease and tungstrophosphoric, tungstosilicic or molybdosilicic acid is close to the isoelectric point of the protein and does not cause the dissociation of the heteropolyacid. Below the isoelctric point, the percent precipitation depends on the conformational changes of the protein. The precipitation of ovalbumin with tungstophosphoric decreases as the ionic strength of the buffer increases and is independent, of the protein concentration. Mixtures of ovalbumin and bovine serum albumin, though having close isoelectric points, can be separated by varying the concentration of the precipitant. The electropositive groups which combine with the tungstophosphoric acid are guanidino, ε-amino and imidazole. No precipitation is given by the α-amino groups. Filtrates of microbial fermentations containing lactase, glucose aerode-hydrogenase, alkaline protease, amyloglucosidase, and transglucosylase have been purified by precipitation with heteropolyacids.     

Studies on the terminal stages of immune hemolysis. V. Evidence that not all complement-produced transmembrane channels are equal.

The inhibitory effects of 0.1 M EDTA on the lysis of E prepared by incubating EA with whole GPC was studied. At high end point lysis (greater than 70%) 0.1 M EDTA failed to prevent hemoglobin release whereas at lower end point (less than 60%) 0.1 M EDTA was effective. In all cases hemoglobin release was inhibited by 25% BSA. When E were prepared by incubating EAC1-8 with C9, similar results were obtained. In this system the difference in the ability of 0.1 M EDTA to inhibit hemoglobin release at high or low end point lysis could not be correlated with the low end point lysis could not be correlated with the number of lesions/cell but appeared to be related to the C9 to SAC1-8 ratio. With limiting SAC1-8 and excess C9, E were produced from which hemoglobin release could not be prevented by 0.1 M EDTA whereas at lower C9 to SAC1-8 ratios hemoglobin release was prevented by 0.1 M EDTA. These differences most probably reflect functionally different sized transmembrane channels that were produced at different C9 to SAC1-8 ratios.     

The M2 delta transmembrane domain of the nicotinic cholinergic receptor forms ion channels in human erythrocyte membranes

A synthetic peptide with the sequence of the M2 delta segment of the nicotinic acetylcholine receptor from Torpedo californica forms pores in human erythrocyte membranes as determined by hemoglobin and potassium release. This peptide forms a permeability pathway with an apparent cross-sectional diameter of 7-9 A. The M2 delta pore is oligomeric and a pentamer is the species that accounts for the properties of the permeation path. Peptides that mimic other identifiable segments of the Torpedo acetylcholine receptor, M1 delta and MIR, do not form channels in erythrocytes under the same conditions.