Ghost Membranes Research Articles

Effects of chronic and acute lead treatments on the biophysical properties of erythrocyte membranes, and a comparison with model membranes

Rat erythrocytes, or erythrocyte membrane ghosts, have been subjected to either chronic (drinking water containing 15 mM lead acetate for 3 months) or acute (10−9–10−2 M lead acetate for 1 h) Pb2+ treatments and subsequent changes in membrane properties have been measured. Pb2+ concentration in chronically treated rat plasma was 1.8 μM, which is one order of magnitude above normal values. Membrane permeability, or hemolysis, was increased in both cases. A comparative study using liposomes, in the form of large unilamellar vesicles, also indicated an increase in membrane permeability. Membrane microviscosity, or acyl chain molecular order, measured as DPH fluorescence polarization, showed an increased order in the acute treatments, at least below 700 μM Pb2+, and a similar increase in chronically treated rats. The correlation between acute and chronic treatments, and between cell and model membranes, suggests that the present observations may be relevant in the pathogenesis of lead intoxication in humans.

An in vitro spectrometric method for determining the partition coefficients of non-steroidal anti-inflammatory drugs into human erythrocyte ghost membranes

Usefulness of second derivative spectrophotometry for determining the partition coefficients (Kps) of four non-steroidal anti-inflammatory drugs (NSAIDs) between human erythrocyte ghost (HEG) membranes and buffer at simulated physiological conditions (pH=7.4, 37°C) has been adequately emphasized. In the absorption spectra for each of the investigated NSAIDs, λmax was red-shifted in presence of HEG membranes, indicating that NSAIDs have the nature of metachromasy between lipid bilayer and water. Further quantitative spectral data for calculating Kps could not be obtained from the absorption spectra because of the presence of background signal impacts of HEG lipid bilayers. Second derivative spectra were calculated from absorption spectra and fortunately showed three isosbestic derivative points for each NSAID, indicating without doubt that the background signals were entirely eliminated. From the relation between the derivative intensity change (ΔD) induced by addition of HEG membranes, Kps were calculated and obtained with RSD of below 6%. Fractions of partitioned NSAIDs are in well-harmony with that derived from the experimental values. Moreover, validity of the proposed method was confirmed. Conclusively, the second derivative spectrometry has proven to be a facile, reliable and more expeditious method to obtain in vitro Kps of drugs to HEG without previous separation.

Proteomic Analysis of ERK1/2-Mediated Human Sickle Red Blood Cell Membrane Protein Phosphorylation.

Abstract 2120In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human erythrocytes. ERK1/2 is involved in activation of ICAM-4-mediated sickle red blood cell (SSRBC) adhesion to the endothelium. However, other effects of the ERK1/2 activation in SSRBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are still unknown. To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2 activation, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Consistent with other RBC membrane phosphorylation studies, the phosphoproteins of SSRBC membrane ghosts with the highest number of uniquely phosphorylated peptides (>10), were ankyrin-1 of the ankyrin complex (n=33), spectrin β chain of the cytoskeleton network (n=15), and proteins of the junctional complex involved in binding integral membrane proteins to cytoskeletal proteins, including α- and β-adducins (n=22 and n=18, respectively), dematin (n=16) and protein 4.1 (n=17). In addition, several other phosphoproteins with >5 unique phosphorylated peptides, affecting RBC shape, flexibility, anion transport and protein trafficking, and adhesion, all of which contribute to the pathophysiology of SCD, were also observed. However, the MEK1/2 inhibitor U0126 was able to significantly down-regulate 37 unique RBC membrane phosphopeptides (from 21 unique phosphoproteins) in SSRBCs. We found that MEK1/2-dependent ERK1/2 activation in SSRBCs affected membrane-bound proteomes of both the junctional and ankyrin complexes, including dematin, α-adducin, β-adducin with phosphorylation of residues within the ERK1/2 consensus motif, and glycophorin A. MEK1/2/ERK1/2 signaling in SSRBCs induced changes within the actins/spectrins network as well by affecting phosphorylation of β-spectrins. Furthermore, the peptide metabotropic glutamate receptor 7 (mGlu7) also underwent serine phosphorylation at the ERK consensus motif. This could explain the rate of active glutamate transport in these cells. Significant changes only in membrane ghosts prepared from SSRBCs treated with U0126 or after addition of exogenous active ERK2 to these membrane ghosts, were also observed in the status of leucine-rich repeats and immunoglobulin-like domains protein 2, leucine-zipper-like transcriptional regulator 1, glucose transporter 1, and adenylyl cyclase-associated protein 1 (CAP1), which may potentially disturb degradation of misfolded glycoproteins and receptor ubiquitination, protein transcription, glucose transport and cAMP production, respectively. These data also suggest that a negative regulatory mechanism might exist in normal cells to prevent activation of ERK1/2-dependent phosphorylation of these membrane proteins. Among all these phosphorylated proteomes, glycophorin A was the most affected protein in SSRBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreased in both anion transport by band 3 and band 3 trafficking. The abundance of thirteen of the thirty-seven phosphopeptides was subsequently increased in normal RBCs co-incubated with recombinant ERK2, and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2. These findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD. Disclosures:No relevant conflicts of interest to declare.

Hydroxyurea up-Regulates Voltage-Dependent Anion Channels in Human Sickle Cell Erythrocytes.

AbstractAbstract 2119 Background:Hydroxyurea (HU) is clinically effective in reducing the frequency of pain crises in adults and children with sickle cell disease (SCD). In addition to increased fetal hemoglobin production, HU is known to induce macrocytosis in sickle cell erythrocytes and prolong red cell survival. However, the precise mechanism by which HU produces its varied effects is unknown. Isoelectric focusing has been applied to characterize changes in the red cell membrane following exposure to HU, but this technique has limitations in its ability to identify differences in specific membrane proteins. Mass spectrometry (MS) provides another proteomic approach in analyzing red cell proteins in SCD. Methods:Red blood cells were obtained from patients with SCD on HU therapy. After multiple wash and lysis steps, ghost membranes were obtained following ultracentrifugation. The membrane samples were then analyzed via tandem mass spectrometry and western immunoblot assay. Results:In comparison to a normal control, patients with SCD on HU therapy were found to have significantly elevated levels of voltage-dependent anion channels (VDAC) by MS, using a false discovery rate of less than five percent. The application of a stringent threshold initially identified 346 protein candidates. Out of these 346 proteins, 125 contained multiple peptides which all passed quantitation data filters according to quality parameters. Within the set of 125 proteins, 4 proteins were significantly up-regulated (up to 50-fold) in patients with SCD on HU therapy. VDAC2 and VDAC3 are members of the eukaryotic mitochondrial porin family which form channels through the mitochondrial outer membrane allowing diffusion of small hydrophilic molecules. VDAC1 can be found in both the mitochondrial outer membrane and the plasma membrane, where it is involved in cell volume regulation and apoptosis. Conclusions:Up-regulation of VDAC proteins may play an important role in altering the intracellular osmotic composition of the sickle cell erythrocyte resulting in decreased sickling and improved red cell survival in patients with SCD on HU therapy. Disclosures:No relevant conflicts of interest to declare.

A label-free proteome analysis strategy for identifying quantitative changes in erythrocyte membranes induced by red cell disorders

Red blood cells have been extensively studied but many questions regarding membrane properties and pathophysiology remain unanswered. Proteome analysis of red cell membranes is complicated by a very wide dynamic range of protein concentrations as well as the presence of proteins that are very large, very hydrophobic, or heterogeneously glycosylated. This study investigated the removal of other blood cell types, red cell membrane extraction, differing degrees of fractionation using 1-D SDS gels, and label-free quantitative methods to determine optimized conditions for proteomic comparisons of clinical blood samples. The results showed that fractionation of red cell membranes on 1-D SDS gels was more efficient than low-ionic-strength extractions followed by 1-D gel fractionation. When gel lanes were sliced into 30 uniform slices, a good depth of analysis that included the identification of most well-characterized, low-abundance red cell membrane proteins including those present at 500 to 10,000 copies per cell was obtained. Furthermore, the size separation enabled detection of changes due to proteolysis or in vivo protein crosslinking. A combination of Rosetta Elucidator quantitation and subsequent statistical analysis enabled the robust detection of protein differences that could be used to address unresolved questions in red cell disorders. This article is part of a Special Issue entitled: Integrated omics.

Alterations of the erythrocyte membrane proteome and cytoskeleton network during storage – a possible tool to identify autologous blood transfusion

Mature red blood cells (RBCs) are the end-stage of a development process that starts in the bone marrow and continues to differentiate, through reticulocyte stage, entering into the circulation with a four-month lifespan. While stored, RBCs undergo different changes. The aim of this study was to evaluate changes occurring in RBC membranes during storage that could be used as possible markers to detect the misuse of blood transfusion in sports. Whole blood was collected from two volunteers in blood bags and stored for 42 days at 4°C. At different times (1, 7, 21, and 42 days of storage) whole blood was extracted under sterile conditions and submitted to RBC membrane ghost preparation and further analysis. Proteomic methods were applied using two strategies: protein oriented using 2-DE gels and peptide oriented using isobaric tags for relative and absolute quantitation (iTRAQ). In both approaches, the goal was to compare detectable changes in RBC membrane proteome before and after standard storage at different times. Some of the changes were confirmed with both methodologies employed, while with others only with one of them. Complementarities of the methods in this case showed to be an advantage. Changes were observed in two different protein complexes. In one of them, changes consisted of proteins decreasing, while increasing in the other during storage of RBCs. They are mostly located in cytoskeleton--spectrin β, band 4.2, ankyrin-1, tropomodulin-1, β adducin, band 4.9 (dematin), tropomyosin, while some changes were also observed in transmembrane proteins (glycophorin C, aquaporin-1, band 3).

Effect of Zinc on Carbonic Anhydrase IX Activity in MDA‐MB‐231 Breast Cancer Cells

Carbonic anhydrase IX (CAIX) is a zinc‐dependent, membrane‐bound glycoprotein that catalyzes the reversible hydration of carbon dioxide like other members of the carbonic anhydrase family: CO2 + H2O → H+ + HCO3−. CAIX is also considered a marker for hypoxic tumors (including those in breast cancers), a poor prognosticator for outcome, and associated with tumorigenesis. Based on previous studies with recombinant proteins that suggested that zinc activates CAIX independent of its role in catalysis, we sought to explore the effect of zinc on CAIX activity in a breast cancer model system, the triple negative MDA‐MB‐231 cell line. These cells express only CAIX and not other membrane‐bound CA family members. We directly analyzed CAIX activity in intact cells and membrane ghosts by using 18O exchange between CO2 and water as measured by membrane inlet mass spectrometry (MIMS). Using this system, we were unable to demonstrate any effect of zinc, over a wide range of concentrations, on hypoxia‐induced CAIX activity. Further, supplementing the growth medium with zinc did not alter either CAIX expression or activity in MDA‐MB‐231 cells. Thus, it appears that zinc is only involved in the catalytic process in a biological setting.

Dipole relaxation in erythrocyte membrane: Involvement of spectrin skeleton

Polarization of spectrin–actin undermembrane skeleton of red blood cell (RBC) plasma membranes was studied by impedance spectroscopy. Relatedly, dielectric spectra of suspensions that contained RBCs of humans, mammals (bovine, horse, dog, cat) and birds (turkey, pigeon, duck), and human RBC ghost membranes were continuously obtained during heating from 20 to 70°C. Data for the complex admittance and capacitance were used to derive the suspension resistance, R, and capacitance, C, as well as the energy loss as a function of temperature. As in previous studies, two irreversible temperature-induced transitions in the human RBC plasma membrane were detected at 49.5°C and at 60.7°C (at low heating rate). The transition at 49.5°C was evident from the abrupt changes in R, and C and the fall in the energy loss, due to dipole relaxation. For the erythrocytes of indicated species the changes in R and C displayed remarkable and similar frequency profiles within the 0.05–13MHz domain. These changes were subdued after cross-linking of membranes by diamide (0.3–1.3mM) and glutaraldehyde (0.1–0.4%) and at the presence of glycerol (10%). Based on the above results and previous reports, the dielectric changes at 49.5°C were related to dipole relaxation and segmental mobility of spectrin cytoskeleton. The results open the possibility for selective dielectric thermolysis of cell cytoskeleton.

Amelioration of oxidative stress in bio-membranes and macromolecules by non-toxic dye from Morinda tinctoria (Roxb.) roots

Plant dyes have been in use for coloring and varied purposes since prehistoric times. A red dye found in the roots of plants belonging to genus Morinda is a well recognized coloring ingredient. The dye fraction obtained from the methanolic extract of the roots of Morinda tinctoria was explored for its role in attenuating damages caused by H2O2-induced oxidative stress. The antioxidant potential of the dye fraction was assessed through DPPH radical scavenging, deoxyribose degradation and inhibition of lipid peroxidation in mice liver. It was subsequently screened for its efficiency in extenuating damage incurred to biomembrane (using erythrocytes and their ghost membranes) and macromolecules (pBR322 DNA, lipids and proteins) from exposure to hydrogen peroxide. In addition, the non-toxic nature of the dye was supported by the histological evaluation conducted on the tissue sections from the major organs of Swiss Albino mice as well as effect on Hep3B cell line (human hepatic carcinoma). The LC–MS confirms the dye fraction to be morindone. Our study strongly suggests that morindone present in the root extracts of M. tinctoria, in addition to being a colorant, definitely holds promise in the pharmaceutical industry.

Role of zinc in catalytic activity of carbonic anhydrase IX

The carbonic anhydrases (CAs) in the α class are zinc-dependent metalloenzymes. Previous studies have reported that recombinant forms of carbonic anhydrase IX (CAIX), a membrane-bound form of CA expressed in solid tumors, appear to be activated by low levels of zinc independent of its well-studied role at the catalytic site. In this study, we sought to determine if CAIX is stimulated by zinc in its native environment. MDA-MB-231 breast cancer cells express CAIX in response to hypoxia. We compared CAIX activity associated with membrane ghosts isolated from hypoxic cells with that in intact hypoxic cells. We measured CA activity directly using 18O exchange from 13CO2 into water determined by membrane inlet mass spectrometry. In membrane ghosts, there was little effect of zinc at low concentrations on CAIX activity, although at high concentration zinc was inhibitory. In intact cells, zinc had no significant effect on CAIX activity. This suggests that there is an appreciable decrease in sensitivity to zinc when CAIX is in its natural membrane milieu compared to the purified forms.

Near-field Quantification of Complement Receptor 1 (CR1/CD35) Protein Clustering in Human Erythrocytes

We investigate the distribution of the membrane protein complement receptor 1 (CR1/CD35) in human erythrocyte membrane ghosts using scanning near-field optical microscopy. Recent studies have demonstrated that levels of Aβ peptide, associated with Alzheimers disease (AD) and present in brain and peripheral blood, vary significantly when bound by complement C3b-dependent adherence to CR1. It is unknown why patients with AD have a markedly impaired ability to bind Aβ to CR1 via this mechanism, but one possibility is a defect in the localization and/or conformation of CR1 in the membrane. Scanning near-field optical microscopy does not require the harsh preparation of electron microscopy techniques and may therefore be better suited for measuring membrane protein distributions. The clustering phenomenon of CR1 identified in electron microscopy studies is confirmed and quantified. The standard deviation of the inter-cluster spacing of CR1 is constant (79 ± 9nm) across erythrocytes with between 61 and 124 clusters per membrane ghost.

Analysis of the Mobilities of Band 3 Populations Associated with Ankyrin Protein and Junctional Complexes in Intact Murine Erythrocytes

Current models of the erythrocyte membrane depict three populations of band 3: (i) a population tethered to spectrin via ankyrin, (ii) a fraction attached to the spectrin-actin junctional complex via adducin, and (iii) a freely diffusing population. Because many studies of band 3 diffusion also distinguish three populations of the polypeptide, it has been speculated that the three populations envisioned in membrane models correspond to the three fractions observed in diffusion analyses. To test this hypothesis, we characterized band 3 diffusion by single-particle tracking in wild-type and ankyrin- and adducin-deficient erythrocytes. We report that ∼40% of total band 3 in wild-type murine erythrocytes is attached to ankyrin, whereas ∼33% is immobilized by adducin, and ∼27% is not attached to any cytoskeletal anchor. More detailed analyses reveal that mobilities of individual ankyrin- and adducin-tethered band 3 molecules are heterogeneous, varying by nearly 2 orders of magnitude and that there is considerable overlap in diffusion coefficients for adducin and ankyrin-tethered populations. Taken together, the data suggest that although the ankyrin- and adducin-immobilized band 3 can be monitored separately, significant heterogeneity still exists within each population, suggesting that structural and compositional properties likely vary considerably within each band 3 complex.

Isolation of Hemoglobin from Bovine Erythrocytes by Controlled Hemolysis in the Membrane Bioreactor

In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system that provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts) was designed at a pilot scale. Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling, and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 88% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2-7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The hemoglobin purity of >80% was confirmed by SDS-PAGE. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min. The dynamics of both steps, (1) transport through the membrane of erythrocytes during process of hemolysis and (2) transport through the reactor filters, were evaluated.

Optimization of gradual hemolysis for isolation of hemoglobin from bovine erythrocytes

In this work, we describe an optimized procedure based on gradual hemolysis for the isolation of hemoglobin derived from bovine slaughterhouse erythrocytes in a membrane bioreactor. The membrane bioreactor system provided high yields of hemoglobin (mainly oxyhemoglobin derivate) and its separation from the empty erythrocyte membranes (ghosts). Ten different concentrations of hypotonic media were assessed from the aspect of the extent of hemolysis, hematocrit values of the erythrocyte suspensions, cell swelling and membrane deformations induced by decreased salt concentration. Effective gradual osmotic hemolysis with an extent of hemolysis of 83% was performed using 35 mM Na-phosphate/NaCl buffer of pH 7.2-7.4. Under these conditions most of the cell membranes presented the appearance of the normal ghosts under phase contrast microscope. The results show that isolation process yielded predominantly to oxyhemoglobin. Kinetic studies showed that maximal concentration of hemoglobin was reached after 40 min, but the process cycle at which recovery of 83% was achieved lasted for 90 min.

Effect of long dose exposure of Podophyllum hexandrum methanol extract on antioxidant defense system and body and organ weight changes of albino rats

ObjectiveThe present study was undertaken to investigate the effect of long dose administration of methanol rhizome extract of Podophyllum hexandrum and hydrogen peroxide on lipid peroxidation of erythrocytes, antioxidant enzyme status of rat liver, kidney, lung and brain tissue and body weight and organ weight changes of albino rats. MethodsThe body and organ weight was monitored with digital scale balance and lipid peroxidation of RBC ghost membrane was monitored by measuring malonaldehyde (MDA). Antioxidant enzymes were assayed by standard procedures. ResultsOur study showed that administration of H2O2 (0.1%) in drinking water of the rats for 25 weeks increased the malondialdehyde levels in erythrocytes of all the rats. However, rats receiving Podophyllum hexandrum extract and α-tocopherol had lower MDA levels in a dose dependent manner, which indicates decreased lipid peroxidation in these rats. Our results also showed decrease in the activity of glutathione reductase, glutathione-S-transferase and reduced glutathione levels in different organs of H2O2 treated rats. Rats receiving methanolic extract of Podophyllum hexandrum at the concentration of 5, 10 and 15mg% for 25 weeks increased the activity of glutathione reductase, glutathione-S-transferase and glutathione levels in different organs of the rats indicates the protective effect of the plant in combating oxidative stress undergone by the rats. No significant variation (P< 0.05) in the organ weights between the control and the treated groups was observed after 25 weeks of treatment. ConclusionsIn conclusion, this study presents strong evidence of the nontoxic effect of the methanol extract of Podophyllum hexandrum. The findings also demonstrate that Podophyllum hexandrum methanol extract increased the levels of antioxidant enzymes and decreased lipid peroxidation in albino rats and explained the extensive utilization of the plant in traditional medicine.

Actin-Dependent Chloroplast Anchoring is Regulated by Ca2+–Calmodulin in Spinach Mesophyll Cells

Chloroplasts are actively anchored at the appropriate intracellular regions to maintain advantageous distribution patterns under specific environmental conditions. Redistribution of chloroplasts is accompanied by their de-anchoring and re-anchoring, respectively, from and to the cortical cytoplasm. In spinach mesophyll cells, high-intensity blue light and Ca(2+) treatment induced the disappearance of the meshwork-like array of actin filaments surrounding chloroplasts, which was suppressed by a calmodulin antagonist. Regulatory mechanisms of chloroplast anchoring were investigated using plasma membrane (PM) ghosts, on which the cortical cytoplasm underlying the PM was exposed. Addition of an actin-depolymerizing reagent or > 1 µM Ca(2+) induced detachment of a substantial number of chloroplasts from the PM ghosts concomitant with disordered actin organization. Calmodulin antagonists and anti-calmodulin antibodies negated the effects of Ca(2+). In addition, Ca(2+)-induced detachment of chloroplasts was no longer evident on the calmodulin-depleted PM ghosts. We propose that chloroplasts are anchored onto the cortical cytoplasm through interaction with the actin cytoskeleton, and that Ca(2+)-calmodulin-sensitized de-anchoring of chloroplasts is a critical early step in chloroplast redistribution induced by environmental stimuli.

Erythrocytic enzymes and antioxidant status in people with type 2 diabetes: Beneficial effect of Syzygium cumini leaf extract in vitro

The aim of the present study was to investigate the effects of Syzygium cumini leaf extract (ASc), on Adenosine deaminase (ADA) and Acetylcholinesterase (AChE) activities, and also on oxidative stress parameters in erythrocytes hemolysates (RBCs) and erythrocytes membranes (ghosts) from type 2 diabetics patients (Type 2 DM) under in vitro conditions. Non protein thiol groups (NP-SH), AChE, Catalase (CAT) and Superoxide Dismutase (SOD) activities were measure in RBCs. Further, ADA activity, Thiobarbituric Acid-Reactive Substances (TBARS) levels and protein thiol groups (P-SH) were estimated in ghosts. Also, P-SH and Vitamin C (VIT C) were measure in plasma sample. The results demonstrated that ADA and AChE activities, besides TBARS levels were higher in erythrocytes of Type 2 DM, while SOD activity and NP-SH levels were decreased when compared to control group. ASc, in vitro, reduced ADA and AChE activities and some parameters of oxidative stress. Furthermore, we observed correlations between VIT C and P-SH levels, ADA activity and P-SH levels, as well as NP-SH and TBARS levels in diabetics. The results suggest that ASc in vitro is able to promote the reduction of inflammation and oxidative stress parameters, and act against biochemical changes occurring in Diabetes mellitus (DM).

CD38 expression as response of hematopoietic system to cancer

Erythrocyte and lymphocyte NAD(+) glycohydrolase levels were previously found to be elevated in cancer patients. These results were confirmed in an animal model. The administration of live Ehrlich ascites tumor cells to BALB/c mice led to increases in erythrocyte and lymphocyte NAD(+) glycohydrolase, along with tumor development. Serum samples, ascites fluid from mice with developed tumors, serum samples from cancer patients and Ehrlich cell supernatants had a similar stimulatory effect when administered to mice or when incubated with peripheric lymphocytes in culture. These increases were accompanied by the appearance of an anti-CD38 reactive band of 45 kDa in SDS-PAGE/Western blot analyses of erythrocyte ghost and lymphocyte membrane proteins. The results, supported by flow cytometry data, support previous clinical findings that an enhancement in CD38 expression occurs in the hematopoietic system during proliferative processes. Moreover, they suggest that CD38 expression is triggered at least in part by a certain cytokine(s) secreted by cancer cells. Finally, the results emphasize the prospective use of CD38 expression as a marker of tumor development and progression.

Catalysis and pH Control by Membrane-associated Carbonic Anhydrase IX in MDA-MB-231 Breast Cancer Cells

Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related enzyme whose expression is often considered a marker for hypoxia, an indicator of poor prognosis in the majority of cancer patients, and is associated with acidification of the tumor microenvironment. Here, we describe for the first time the catalytic properties of native CAIX in MDA-MB-231 breast cancer cells that exhibit hypoxia-inducible CAIX expression. Using (18)O exchange measured by membrane inlet mass spectrometry, we determined catalytic activity in membrane ghosts and intact cells. Exofacial carbonic anhydrase activity increases with exposure to hypoxia, an activity which is suppressed by impermeant sulfonamide CA inhibitors. Inhibition by sulfonamide inhibitors is not sensitive to reoxygenation. CAIX activity in intact cells increases in response to reduced pH. Data from membrane ghosts show that the increase in activity at reduced pH is largely due to an increase in the dehydration reaction. In addition, the kinetic constants of CAIX in membrane ghosts are very similar to our previous measurements for purified, recombinant, truncated forms. Hence, the activity of CAIX is not affected by the proteoglycan extension or membrane environment. These activities were measured at a total concentration for all CO(2) species at 25 mm and close to chemical equilibrium, conditions which approximate the physiological extracellular environment. Our data suggest that CAIX is particularly well suited to maintain the extracellular pH at a value that favors the survival fitness of tumor cells.

Catalysis of carbonic anhydrase IX and pH control in MDA‐MB‐231 breast cancer cells

Carbonic anhydrase IX (CAIX) is a membrane‐bound glycoprotein whose expression is considered a marker of tumor hypoxia and associated with tumor acidification. Our goal was to determine if the catalytic properties of native CAIX in MDA‐MB‐231 breast cancer cells are equivalent to those previously described in vitro and to evaluate its catalytic role in pH control. CAIX, like other CA family members, catalyzes the reversible hydration of carbon dioxide: CO2 + H2O ↔ H+ + HCO3−. We directly analyzed CAIX activity by using 18O exchange between CO2 and water as measured by membrane inlet mass spectrometry (MIMS). Data from membrane ghosts showed that the kinetic constants of CAIX catalysis in the membrane environment are very similar to those measured for purified, recombinant, truncated forms, suggesting that CAIX is not affected by the proteoglycan domain or the membrane environment. In intact cells, hypoxia increases CAIX activity compared to normoxia which is associated with increased expression. In the dehydration direction, the catalytic activity of CAIX increases as pH is decreased from pH 9 to pH 6.5. This suggests that at the pH of the tumor microenvironment (pH 6.8), CAIX uses protons to generate CO2. Thus, CAIX may contribute to both the development and maintenance of the acidic microenvironment of hypoxic tumors and thus to the survival fitness of tumor cells. BC073020 (SCF), DK45035 (SCF), and GM25254 (DNS)