Ghost Preparations Research Articles

Indices of Membrane Alterations in β-Thalassemic Erythrocytes

Analytical protocols for the study of thalassemic erythrocyte membrane alterations are described. Denatured hemoglobin derivatives and aggregated band 3 are separated from detergent membrane extracts by gel-filtration as high-molecular-weight aggregates and quantitated spectrophotometrically. Membrane-bound, low-molecular-weight iron is measured on SDS-solubilized ghosts by a ferrozine-based colorimetric test. We adapted these methods for micro-scale preparation and analysis of erythrocyte ghosts in order to have suitable tools to estimate oxidative membrane damage in human samples. Data from 11 β-thalassemia intermedia patients and from 10 normal controls are reported as an example of the application of these methods.

ImagingPlasmodium falciparum-Infected Ghost and Parasite by Atomic Force Microscopy

Atomic force microscopy was used to image the membrane cytoskeleton network of normal andP. falciparum-infected ghosts. The membrane cytoskeleton network was examined in air-dried ghost preparations from normal and infected cells. We found that the spectrin network was changed in infected ghosts. The thickness of the normal red cell membrane was about 15.05 ± 2.27 nm, while the thickness of theP. falciparum-infected membrane was found to be 22.97 ± 3.84 nm. The ghost containing ring stage parasites exhibited areas of particle-like protrusions ranging in size from 0.2 to 0.7 μm. The surface of theP. falciparumparasite was also imaged in air-dried samples, showing the existence of a large protrusion extending from the parasite surface.

Reaction of Ozone with Enzymes of Erythrocyte Membranes

Ozone is a widespread component of polluted air. It is the cause of many adverse effects on the lung such as decreased athletic performance and exacerbation of asthma. Ozone inactivated acetylcholine esterase (AChE) both in intact washed human erythrocytes and in ghosts prepared from the erythrocytes. This is consistent (a) with the location of AChE on the outer face of the membrane and (b) with the change in structure of AChE when amino acids were oxidized. The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) of intact washed erythrocytes was unaffected by ozone. However, ozone severely inactivated G3PDH of ghosts, much more severely than AChE in ghosts. This result raised questions about the relative permeability of intact erythrocytes and ghosts and also about the inherent susceptibility of the two enzymes. Inhibition of the ozone-treated erythrocyte AChE with the competitive inhibitor trimethyl-(p-aminophenyl) ammonium chloride was measured. The inhibited enzyme had a higherKMand slightly lowerVmaxthan the control. Ozone did not affect theKMof the uninhibited enzyme but decreased theKMof the inhibited enzyme. Ozone decreased theVmaxof both the inhibited and the uninhibited enzyme. TheKIwas unchanged by the treatment with ozone. This suggested that the active site of the enzyme was not affected by ozone, but other features of the protein were changed by ozone. The effects of products of lipid ozonolysis [hydrogen peroxide, nonanal, and 1-palmitoyl-2-(9-oxononanyl)-sn-3-glycerophosphorylcholine (PN1PC)] were tested on the ghost preparations. The ozonolysis products were tested at concentrations equivalent to calculated amounts that could have been produced by ozone. Hydrogen peroxide had no effect on the G3PDH and AChE. Nonanal slightly increased the permeability of the ghost membrane, as judged by the increase in rate of G3PDH in the absence of Triton X-100, but did not inhibit enzyme activity. PN1PC increased the permeability of the ghosts, as judged by the increase in rate of G3PDH in the absence of Triton X-100. There was also an increase in the activity of G3PDH in the presence of Triton X-100. AChE was not inhibited by ozone in the presence or absence of Triton X-100.

Phosphatidylethanol stimulates the plasma-membrane calcium pump from human erythrocytes.

Phosphatidylethanol is formed by "transphosphatidylation' of phospholipids with ethanol catalysed by phospholipase D and can be accumulated in the plasma membrane of mammalian cells after treatment of animals with ethanol. In the present work we show that phosphatidylalcohols, such as phosphatidylethanol and phosphatidylbutanol, produced a twofold stimulation of the Ca(2+)-ATPase activity of human erythrocytes. This stimulation occurs with the purified, solubilized enzyme as well as with ghost preparations, where the enzyme is in its natural lipidic environment and is different to that obtained with other acidic phospholipids such as phosphatidylserine. Addition of either phosphatidylserine, phosphatidylethanol or phosphatidylbutanol to the purified Ca(2+)-ATPase, or to ghosts preparations, increased the affinity of the enzyme for Ca2+ and the maximal velocity of the reaction as compared with controls in the absence of acidic phospholipids. However, in contrast with what occurs with phosphatidylserine, simultaneous addition of phosphatidyl-alcohols and calmodulin increased the affinity of the enzyme for Ca2+ to a greater extent than each added separately. When ethanol was added to either the purified erythrocyte Ca(2+)-ATPase or to erythrocyte-ghost preparations in the presence of acidic phospholipids, an additive effect was observed. There was an increase in the affinity for Ca2+ and in the maximal velocity of the reaction, well above the values obtained with ethanol or with the acidic phospholipids tested separately. These findings could have pharmacological importance. It is conceivable that the decrease in the intracellular Ca(2+) concentration that has been reported in erythrocytes as a result of ethanol intoxication could be due to the stimulation of the Ca(2+)-ATPase by the accumulated phosphatidylethanol, to a direct effect of ethanol on the enzyme or to an additive combination of both.

Lipidic characterization of full-grown amphibian oocytes and their plasma membrane-enriched fractions.

The content and composition of neutral lipids and phosphoglycerides from full-grown prophase-arrested Bufo arenarum Hensel oocytes and from their ghost preparations were studied. The ghosts obtained are highly enriched in plasma membrane as suggested by the activity of 5'-nucleotidase, a marker enzyme, and the level of typical membrane components such as sphingomyelin, phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidic acid. In whole oocytes, triacylglyceride (TAG) comprises about 60% of the total lipids followed by phosphatidylcholine (PC), cholesterol, and phosphatidylethanolamine (PE). TAG and diacylglycerides have a similar unsaturation index. PC and PE account for about 80% of the phosphoglycerides in the whole oocyte and in their plasma membrane-enriched fractions. Arachidonic acid (20:4n-6), 18:0, and 16:0 make up about 80 mol% of the total fatty acids in PI in whole oocytes and ghost fractions. The unsaturation index in PS is higher in intact oocytes than in ghost preparations, probably owing to the significant amount of 20:4n-6 which comprises 23 mol% of the total fatty acids in whole oocytes. The fatty acid profile in phosphatidic acid from whole oocytes is rather different from that in ghosts. Sphingomyelin contains mainly saturated and monounsaturated fatty acids, 24:1 being the principal very long chain unsaturated fatty acid in both oocytes and ghosts.

Interaction of Ascorbate and α-Tocopherol in Resealed Human Erythrocyte Ghosts: TRANSMEMBRANE ELECTRON TRANSFER AND PROTECTION FROM LIPID PEROXIDATION

A role for ascorbate-derived electrons in protection against oxidative damage to membrane lipids was investigated in resealed human erythrocyte ghosts. Incubation of resealed ghosts with the membrane-impermeant oxidant ferricyanide doubled the ghost membrane concentration of F2-isoprostanes, a sensitive marker of lipid peroxidation. Incorporation of ascorbate into ghosts during resealing largely prevented F2-isoprostane formation due to extravesicular ferricyanide. This protection was associated with a rapid transmembrane oxidation of intravesicular ascorbate by extravesicular ferricyanide. Transmembrane electron transfer, which was measured indirectly as ascorbate-dependent ferricyanide reduction, correlated with the content of alpha-tocopherol in the ghost membrane in several respects. First, ascorbate resealed within ghosts protected against ferricyanide-induced oxidation of endogenous alpha-tocopherol in the ghost membrane. Second, when exogenous alpha-tocopherol was incorporated into the ghost membrane during the resealing step, subsequent ferricyanide reduction was enhanced. Last, incubation of intact erythrocytes with soybean phospholipid liposomes, followed by resealed ghost preparation, caused a proportional decrease in both the membrane content of alpha-tocopherol and in ferricyanide reduction. Incorporation of exogenous alpha-tocopherol during resealing of ghosts prepared from liposome-treated cells completely restored the ferricyanide-reducing capacity of the ghosts. These results suggest that the transmembrane transfer of ascorbate-derived electrons in erythrocyte ghosts is dependent in part on alpha-tocopherol and that such transfer may help to protect the erythrocyte membrane against oxidant stress originating outside the cell.

Erythrocyte membrane fluidity in adult polycystic kidney disease: difference between intact cells and ghost membranes.

In adult polycystic kidney disease (APCKD) the cytoskeleton of renal tubular cells is abnormal. In erythrocytes the cytoskeleton affects the fluidity of membrane lipids. The authors determined fluorescence anisotropy in intact erythrocytes and erythrocyte ghosts in 12 APCKD patients and 12 normal subjects. In APCKD whole erythrocytes had a much lower core-region anisotropy, which indicated higher membrane fluidity than normal (mean 0 center dot 175 vs. 0 center dot 224, P < 0 center dot 01). This abnormality was not detected in erythrocyte ghosts, which suggests that preparation of ghosts altered membrane lipid organization. This could be directly due to ghosting or secondary to the loss of cytoskeletal effects, which may be abnormal in APCKD.

Diffusion Across the Nuclear Envelope Inhibited by Depletion of the Nuclear Ca 2+ Store

Intact, isolated nuclei and a nuclear membrane (ghost) preparation were used to study regulation of the movement of small molecules across the Xenopus laevis oocyte nuclear membrane. In contrast to models of the nuclear pore complex, which assume passive bidirectional diffusion of molecules less than 70 kilodaltons, diffusion of intermediate-sized molecules was regulated by the nuclear envelope calcium stores. After depletion of nuclear store calcium by inositol 1,4,5-trisphosphate or calcium chelators, fluorescent molecules conjugated to 10-kilodalton dextran were unable to enter the nucleus. Dye exclusion after calcium store depletion was not dependent on the nuclear matrix because it occurred in nuclear ghosts lacking nucleoplasm. Smaller molecules and ions (500-dalton Lucifer yellow and manganese) diffused freely into the core of the nuclear ghosts and intact nuclei even after calcium store depletion. Thus, depletion of the nuclear calcium store blocks diffusion of intermediate-sized molecules.

The Instability of the Membrane Skeleton in Thalassemic Red Blood Cells

The thalassemias are a heterogeneous group of disorders characterized by accumulation either of unmatched alpha or beta globin chains. These in turn cause the intramedullary and peripheral hemolysis that leads to varying anemia. A partial explanation for the hemolysis came our of our studies on material properties that showed that beta-thalassemia (beta-thal) intermedia ghosts were very rigid but unstable. A clue to this instability came from the observation that the spectrin/band 3 ratio was low in red blood cells (RBCs) of splenectomized beta-thal intermedia patients. The possible explanations for the apparent decrease in spectrin content included deficient or defective spectrin synthesis in thalassemic erythroid precursors or globin chain-induced membrane changes that lead to spectrin dissociation from the membrane during ghost preparation. To explore the latter alternative, samples from different thalassemic variants were obtained, ie, beta-thal intermedia, HbE/beta-thal, HbH (alpha-thal-1/alpha-thal-2), HbH/Constant Spring (CS), and homozygous HbCS/CS. We searched for the presence of spectrin in the first lysate of the standard ghost preparation. Normal individuals and patients with autoimmune hemolytic anemia, sickle cell anemia, and anemia due to chemotherapy served as controls. Using gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, no spectrin was detected in identical aliquots of the supernatants of normals and these control samples. Varying amounts of spectrin were detected in the first lysate supernatants of almost all thalassemic patients. The identification of spectrin was confirmed by Western blotting using an affinity-purified, monospecific, rabbit polyclonal antispectrin antibody. Relative amounts of spectrin detected were as follows in decreasing order: splenectomized beta-thal intermedia including HbE/beta-thal; HbCS/CS; nonsplenectomized beta-thal intermedia, HbH/CS; and, lastly, HbH. These findings were generally confirmed when we used an enzyme-linked immunosorbent assay technique to measure spectrin in the first lysate. Subsequent analyses showed that small amounts of actin and band 4.1 also appeared in lysates of thalassemic RBCs. Therefore, the three major membrane skeletal proteins are, to a varying degree, unstably attached in severe thalassemia. From these studies we could postulate that membrane association of abnormal or partially oxidized alpha-globin chains has a more deleterious effect on the membrane skeleton than do beta-globin chains.

Association State of Human Red Blood Cell Band 3 and its Interaction With Ankyrin

We have studied the association state of band 3, the anion channel and predominant transmembrane protein of the human red blood cell, and the anomalous stoichiometry and dynamics of its interaction with ankyrin, which acts as a link to the spectrin of the membrane skeletal network. Band 3 exists in benign nonionic detergent solutions as a dimer. Tetramer is formed irreversibly in the course of manipulations, particularly in ion-exchange chromatography. The dimer in solution binds ankyrin without self-associating. In ankyrin-free inside-out membrane vesicles and when incorporated into phosphatidylcholine liposomes, only some 10% to 15% of band 3 chains bind ankyrin at saturation. Moreover, in liposomes this was independent of protein:lipid ratio between 1:2 and 1:40. The bound fraction of band 3 remains with the detergent-extracted membrane cytoskeleton, but is released if the ankyrin has been cleaved with chymotrypsin before detergent treatment; thus, the attachment to the membrane cytoskeleton is entirely through ankyrin and not through other constituents such as protein 4.1. The ratio of band 3 to ankyrin in this complex implies that it consists of two chains of band 3 and one chain of ankyrin, at least after detergent extraction. The bound and free populations of band 3 exchange freely in the membrane. In the artificial liposome membrane binding of ankyrin to band 3 dimers cause association of the band 3 into higher aggregates, as seen in freeze-fracture electron microscopy. Successive manipulations of the red blood cell membrane, which are involved in the preparation of ghosts, of inside-out vesicles, and of inside-out vesicles stripped of peripheral proteins are accompanied by progressive aggregation of intramembrane particles, as judged by freeze-fracture electron microscopy. Thus the intramembrane particles are evidently stabilized in the intact cell by the peripheral protein network and the cytosolic milieu. Aggregation may be expected to limit the number of functional ankyrin binding sites. However, although extraneous ankyrin binds to the unoccupied binding site on the spectrin tetramers in intact ghost membranes, little or no ankyrin can bind to the unoccupied band 3 dimers in situ, perhaps by reason of occlusion of binding sites by the membrane skeletal network.

Chapter 43 Release of the Cytoskeleton and Flagellar Apparatus from Chlamydomonas

Publisher Summary Flagellar apparatus can be released from cells in a complex that includes the flagella, the basal bodies, the rootlet microtubules, the structures linking the basal bodies to the nucleus (nucleus-basal body connectors), and a chromatin-containing nuclear remnant. This complex is called the nucleoflagellar apparatus, or NFAp. Studies of isolated Chlamydomonas NFAps, in both wild-type and mutant organisms have led to a better understanding of the structures that makeup this basic unit of the basal apparatus and have allowed for detailed analysis of its components at the cytological and biochemical levels. The juxtaposition of mitochondria, nucleus, and basal body apparatus makes it problematic to determine, in a whole cell, whether a protein is localizing to the basal body apparatus, whereas the distinction can readily be made using isolated NFAps. Isolated NFAps greatly simplified protein composition as compared with whole cells. The cytoskeleton, the flagellar apparatus, and the nucleus exist as a physically integrated complex. Two strains have been used for most of the basal body apparatus isolations: the “cell wall”-less strain cw 92 (CC503) and the wild-type strain 137c (CC125). cw 92 cells are easily lysed with nonionic detergents, facilitating separation of the NFAps from the other cellular components, while detergent extraction of walled cells allows for the isolation of cell “ghosts” with the NFAps trapped inside. There are details of the solutions and the procedures—preparation of NFAps on glass slides for light microscopy and electron microscopy, immunofluorescence microscopy, isolation of NFAps, and preparation of cell ghosts containing NFAps for light or electron microscopy. Additional treatments like Flagella can be removed prior to sample preparation; removal of Mg 2+ from the nucleus buffer results in the preparation of flagellar apparatuses without attached nuclei possibly as a result of loss of the nucleus-basal body connectors, etcetera.

Polyamine inhibition of transbilayer movement of plasma membrane phospholipids in the erythrocyte ghost.

The resting plasma membrane of circulating blood cells demonstrates an asymmetric distribution of the phospholipid classes across the bilayer that is altered during cellular activation. To better understand the mechanisms governing transbilayer distribution of phospholipids, studies were conducted using the erythrocyte ghost, in which plasma membrane leaflet distribution of phospholipids can be readily probed. Preparation of ghosts by hypotonic lysis at increasingly high dilution markedly enhanced (up to 10-fold) calcium-induced (50-500 microM) transbilayer movement of phospholipids. The enhanced transbilayer movement was assessed by translocation of exogenously added sn-2-[6[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl]-labeled phosphatidylcholine and phosphatidylserine from the plasma membrane outer leaflet to the inner leaflet and vice versa, as well as transbilayer movement of endogenous phosphatidylserine to the outer leaflet. It was found that phospholipid movement was bidirectional and also nonspecific with regard to polar head group. It was further demonstrated that preparation of ghosts at increasing dilution resulted in depletion of cellular polyamines and that physiologic replenishment of spermine, and to a lesser extent spermidine, resulted in significant inhibition (50 and 25%, respectively) of transbilayer movement of phospholipids. Replenishment of other di- and polyamines demonstrated that inhibition was not simply dependent on total cationic charge but rather on charge density and suggestive of specific interaction of the polyamines, particularly spermine, with the plasma membrane. As most cells demonstrate both a high degree of regulation in maintenance of polyamine levels and the means for facile shifts within cellular polyamine pools, it is suggested that loss of membrane asymmetry during cellular activation may be mediated in part through enhanced transbilayer movement of phospholipids due to altered polyamine-membrane associations.

Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane

Reversible binding among components of the cellular submembrane cytoskeleton and reversible binding of some of these components with the plasma membrane likely play a role in nonelastic morphological changes and mechanoplastic properties of cells. However, relatively few studies have been devoted to investigating directly the kinetic aspects of the interactions of individual components of the membrane skeleton with the membrane. The experiments described here investigated whether one component of the erythrocyte membrane cytoskeleton, protein 4.1, binds to its sites on the membrane reversibly and if so, whether the different 4.1-binding sites display distinct kinetic behavior. Protein 4.1 is known to stabilize the membrane and to mediate the attachment of spectrin filaments to the membrane. Protein 4.1 previously has been shown to bind to integral membrane proteins band 3, glycophorin C, and to negatively charged phospholipids. To examine the kinetic rates of dissociation of carboxymethyl fluorescein-labeled 4.1 (CF-4.1) to the cytofacial surface of erythrocyte membrane, a special preparation of hemolyzed erythrocyte ghosts was used, in which the ghosts became flattened on a glass surface and exposed their cytofacial surfaces to the solution through a membrane rip in a distinctive characteristic pattern. This preparation was examined by the microscopy technique of total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP). Four different treatments were employed to help identify which membrane binding sites gave rise to the multiplicity of observed kinetic rates. The first treatment, the control, stripped off the native spectrin, actin, 4.1, and ankyrin. About 60% of the CF-4.1 bound to this control binded irreversibly (dissociation time > 20 min), but the remaining approximately 40% binded reversibly with a range of residency times averaging approximately 3 s. The second treatment subjected these stripped membranes to trypsin, which presumably removed most of the band 3. CF-4.1 binded significantly less to these trypsinized membranes and most of the decrease was a loss of the irreversibly binding sites. The third treatment simply preserved the native 4.1 and ankyrin. CF-4.1 binded less to this sample too, and the loss involved both the irreversible and reversible sites. The fourth treatment blocked the gycophorin C sites on the native 4.1-stripped membranes with an antibody. CF-4.1 again binded less to this sample than to a nonimmune serum control, and almost all of the decrease is a loss of irreversible sites. These rest suggest that 1) protein 4.1 binds to membrane or submembrane sites at least in part reversibly ; 2) the most reversible sites are probably not proteinaceous and not glycophorin C, but possibly are phospholipids (especially phosphatidylserine); and 3) TIWRFRAP can successfully examine the fast reversible dynamics of cytoskeletal components binding to biological membranes.

The cholesterol to phospholipids ratio (C/PL) of the erythrocyte membrane in normotensive, hypertensive pregnant and in cord blood as assessed by a simple enzymatic method.

The study described was conducted to evaluate a simple enzymatic method for the study of the cholesterol/phospholipids ratio in erythrocyte membrane (C/PL) in a group of normal pregnant, of hypertensive pregnant, in non-pregnant controls and in cord blood. Subjects consisted of 28 normotensive pregnant women (NT), 14 women with pregnancy induced hypertension (PIH), 10 non-pregnant normotensive women (Non-Preg) and 14 samples obtained from umbilical cord (C) at delivery from normotensive pregnant. Red blood cells were isolated from heparinated blood samples. Lipids were extracted from erythrocytes by isopropanol/chloroform, without preparation of cell ghosts. Cholesterol content was evaluated by cholesterol oxidase and phospholipids were estimated as organic phosphorus in the total lipid extract. We found a significant difference of C/PL between the PIH group and the NT group (1.01, SD 0.11 vs. 0.76, SD 0.10, 95% CI 0.74-0.78; p < 0.001) and the Non-Preg group (0.83, SD 0.11, 95% CI 0.80-0.86; p < 0.001). Cord blood C/PL was significantly elevated with respect to NT (1.25, SD 0.13 vs. 0.76, SD 0.10; p < 0.001). The method was proven to be fast, reliable and of value for the study of the pathophysiology of the alteration of the lipid composition, i.e., the increased cholesterol content, of the red cell membrane found in hypertensive pregnant patients.

Direct labelling of hormone-sensitive phosphoinositides by a plasma-membrane-associated PtdIns synthase in turkey erythrocytes.

We have previously characterized phosphatidylinositol (PtdIns) synthase and PtdIns/myo-inositol-exchange enzyme activities in ghost membranes prepared by hypotonic lysis of turkey erythrocytes [McPhee, Lowe, Vaziri and Downes (1991) Biochem. J. 275, 187-192]. Here we show that PtdIns synthase activity is relatively enriched in plasma-membrane preparations of turkey erythrocytes and that inositol phospholipids labelled by both PtdIns synthase and PtdIns myo-inositol exchange enzymes are susceptible to hydrolysis by the receptor- and G-protein-regulated phospholipase C (PLC), which is present also in ghost preparations. Specific-radioactivity measurements of [3H]PtdIns from ghosts labelled to equilibrium under conditions favouring [3H]inositol incorporation by PtdIns synthase activity indicate that PtdIns synthase can directly access approx. 14% of the total erythrocyte ghost PtdIns. Approx. 16% of the [3H]PtdIns labelled by the PtdIns synthase reaction can be phosphorylated to polyphosphoinositides, which are then hydrolysed by the receptor- and G-protein-stimulated PLC. Since the mass of PtdIns declines to a similar extent as [3H]PtdIns during stimulation in the presence of guanine nucleotides and ATP, it is evident that both the labelled and unlabelled phosphoinositides are susceptible to hydrolysis by the relevant PLC. Phosphoinositides present in nuclei-free plasma membranes were also labelled by [3H]inositol under conditions favouring PtdIns synthase and PtdIns/myo-inositol-exchange enzyme activities respectively. These membranes lack PLC activity [Vaziri and Downes (1992) J. Biol. Chem. 267, 22973-22981], but the labelled lipids were sensitive to purinergic-receptor-stimulated hydrolysis in reconstitution assays using partially purified turkey erythrocyte PLC. The results strongly suggest that at least a portion of the PtdIns synthase in turkey erythrocytes is located in the plasma membrane and has direct access to an agonist-sensitive pool of inositol phospholipids.

Association of a receptor and G-protein-regulated phospholipase C with the cytoskeleton

Approximately 98% of turkey erythrocyte phospholipase C (PLC) is cytosolic and is released by hypotonic lysis of the cells and extensive washing of the resultant erythrocyte ghosts. Well washed turkey erythrocyte ghosts retain a fraction of tightly associated PLC, which is activated by the P2y-purinergic receptor and G-protein present in ghost membranes. The particulate PLC is sufficient to couple to all the available purinergic receptor-regulated G-protein. In contrast to ghosts, turkey erythrocyte plasma membrane preparations contain no detectable PLC. To investigate the subcellular location of the ghost-associated PLC, cytoskeletons were prepared by Triton X-100 extraction of turkey erythrocyte ghosts. The ghost-associated PLC was quantitatively recovered in cytoskeleton preparations. Cytoskeleton-associated PLC was solubilized by sodium cholate extraction, partially purified, and shown to reconstitute with PLC-free plasma membrane preparations in an agonist and guanine nucleotide-dependent fashion, indicating that the cytoskeleton-associated PLC is G-protein-regulated. Dissociation of erythrocyte ghost cytoskeletons with the actin-binding protein DNase 1 resulted in a dose-dependent inhibition of agonist and guanine nucleotide-stimulated PLC responses in ghosts and caused release of PLC from ghost or cytoskeleton preparations. These data demonstrate the specific association of a receptor and G-protein-regulated PLC with a component of the detergent-insoluble cytoskeleton and indicate that the integrity of the actin cytoskeleton is important for localization and effective coupling of PLC to the relevant G-protein.

Water-phase palmitate concentrations in equilibrium with albumin-bound palmitate in a biological system.

The palmitate (PA) binding and transport capacity of human and bovine red cell membranes enables us to establish, in a biological system, the existence of a well-defined monomer concentration in equilibrium with PA bound to bovine serum albumin (BSA, 30 microM) inside the resealed red cell ghosts. Supernatants of suspensions of the [3H]PA-labeled ghosts contain a tiny quantity of dissolved binding capacities besides the monomer PA. This is demonstrated by linear regression of supernatant tracer concentrations versus ghost concentrations in a dilution series. The extrapolated value, corresponding to zero ghost concentration, is the monomer PA concentration in equilibrium with PA bound to BSA within the ghosts in molar ratio (nu). Measurements have been carried out for nu between 0.1 and 1.5 and at 0 degrees C, 10 degrees C, 23 degrees C and 38 degrees C. The important nu-dependent binding of PA to the ghost membrane itself enables us to use preparations of BSA-free ghosts in the same way, whereas this is impossible in the case of arachidonic acid. Within the physiological range of nu the PA monomer concentrations are accounted for by an apparent dissociation equilibrium constant (Kd) 3.4 10(-8) M at 38 degrees C calculated on basis of three equivalent binding sites per mol BSA. Kd depends on temperature with a well-defined enthalpy of 38.4 kJ/mol.

G-protein-mediated activation of turkey erythrocyte phospholipase C by beta-adrenergic and P2y-purinergic receptors.

Isoprenaline, previously known only to stimulate adenylate cyclase via the stimulatory G-protein, Gs, activates turkey erythrocyte ghost phospholipase C (PLC) in a dose-dependent manner when GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) is present. The effect is specific in that it is abolished by beta-adrenergic-receptor antagonists. Stimulation of adenosine receptors, which also couple to adenylate cyclase via Gs in turkey erythrocytes, does not activate PLC, indicating that the stimulation observed in the presence of isoprenaline is not due to Gs activation. Furthermore, the stimulation seen is independent of cyclic AMP production. Purified turkey erythrocyte PLC is activated in an adenosine 5'-[beta-thio]diphosphate (ADP[S]; a P2y-purinergic-receptor agonist)- or isoprenaline-regulated manner when reconstituted with turkey erythrocyte ghosts, demonstrating that a single species of PLC effector enzyme can be regulated by both the purinergic and the beta-adrenergic receptor populations present in turkey erythrocyte membranes. Pretreatment of intact turkey erythrocytes with the P2y agonist ADP[S] causes decreased PLC responsiveness of subsequent ghost preparations to ADP[S] stimulation, although responses to isoprenaline are unaffected (homologous desensitization). In contrast, pretreatment of intact erythrocytes with isoprenaline results in heterologous desensitization of both the P2y and the beta-adrenergic receptors. These effects occur at the level of receptor-G-protein coupling, since PLC stimulation by GTP[S] (which directly activates G-proteins) in the absence of agonists is unaffected.

Effect of Bothrops asper (Fer-De-Lance) snake venom on erythrocyte membrane. A comparative study

1. The effect of Bothrops asper venom on erythrocytes of different species, sheep erythrocyte ghost vesicles and liposomes made of phospholipids from sheep erythrocytes was studied. 2. B. asper venom had a direct hemolytic effect on mouse erythrocytes, but no lytic activity on goat, rabbit, horse, toad and sheep erythrocytes. Human erythrocytes were lysed only in the presence of bovine serum albumin and Ca 2+. 3. Although the phospholipase A 2 activity in the venom might be involved in the lytic effect on mouse erythrocytes, there is also evidence of direct lytic factor(s) acting in the absence of calcium ions. 4. Sheep erythrocytes were modified in order to study the basis of their resistance. Enhancement of membrane fluidity and variation of pH were of no consequence for the resistance of sheep erythrocytes to the action of venom. 5. The reorganization of membranes that takes place during ghost or liposome preparation made them susceptible to B. asper venom-induced disruption. Thus the resistance of sheep erythrocytes seems related to the accessibility of the target.

Are ethane and pentane evolution and thiobarbituric acid reactivity specific for lipid peroxidation in erythrocyte membranes?

Peroxidation of human erythrocyte membranes was followed in vitro with head space analysis of ethane and pentane and a thiobarbituric acid assay in a standardized system liberating free oxygen radicals. Simultaneously, the decrease of the membrane palmitic, linoleic, arachidonic and docosahexaenoic acid was monitored. The recoveries of the peroxidation products of the red cell ghost preparations were compared with those obtained by peroxidation of pure fatty acids. Experiments using purified fatty acids revealed that ethane was preferentially produced from docosahexaenoic and linolenic, and pentane from linoleic and arachidonic acids. Thiobarbituric acid-reactive material (TBAR) was produced from each unsaturated fatty acid tested, but the amount was dependent on the number of carbon chain double bonds. During peroxidation of the erythrocyte ghosts, 72% of ethane and 51% pentane were produced during the first 12 h of incubation, whereas TBAR was produced at a constant rate throughout the 36-h test period. Hydrocarbon and TBAR production were similarly inhibited by desferoxamine (at p less than 0.005 and p less than 0.0001, respectively). The total recoveries of ethane, pentane and TBAR exceeded the amount expected by 7.8-, 1.4- and 5.5-fold, respectively. It was concluded that measurement of pentane is a reliable method to monitor lipid peroxidation during oxidative damage of the erythrocyte membrane.