Hemolysis and membrane lipid changes induced by xanthine oxidase in vitamin E deficient red cells

A procedure to induce homolysis by the hypoxanthine-xanthine oxidase reaction was developed and applied to vitamin E deficient red blood cells (RBCs) in rats. The reaction system was as follows: 0. 16 mM hypoxanthine, 0.05 U/ml xanthine oxidase in 2.5% RBC suspensions with an isotonic buffer (pH 7.4) containing 10 mM phosphate buffer and 125 mM saline (227 mOsm). Hemolysis was observed to depend on the vitamin E concentrations in the RBCs. Hemolysis was inhibited by catalase but not by SOD. After the reaction with vitamin E deficient RBCs, an increase in TBARS in the aqueous phase of the reaction mixture was observed. This accompanied the increase in fluorescent substances in the lipid extracts, in associatioon with a significant decrease in the PE and PS of the RBCs, and a decrease in arachidonic acid in membrane lipids. The above changes were almost completely inhibited by tocopherol incorporated into vitamin E deficient RBCs.     

Crosslinking of membrane proteins in red blood cells from vitamin E-deficient lead-poisoned rats

Red blood cells from vitamin E-deficient rats lose their filterability after incubation in vitro and concurrent lead poisoning of the rats accelerates this decline. This decreased red cell filterability is associated with an increased crosslinking of red cell membrane proteins. Previous reports by others suggested an association between red cell glutathione levels and filterability, but we found no such association in red cells treated with N-ethylmaleimide. Increased aggregation of red cell membrane proteins may play a role in the discocyte/ spherocyte shape change that accounts for the impaired filterability of red blood cells from vitamin E-deficient lead-poisoned rats.     

Changes in membrane properties of rat red blood cells induced by cadmium accumulating in the membrane fraction

When rat red blood cells were incubated in a cadmium (Cd)-free medium following 1-h pretreatment with 0.5 mM CdCl2, incorporated Cd was retained in the cell during 14-h incubation and progressively accumulated in the membrane fraction, especially in the cytoskeleton fraction. In parallel to this accumulation, red cell filterability decreased and echinocytic cells increased, although intracellular ATP was maintained at the control level. The echinocytic shape was maintained in ghosts and cytoskeletons prepared from the Cd-loaded cells. In addition, the association of bands 2.1, 3, 4.2, and 4.5 with cytoskeletons increased and dissociation of cytoskeletal networks at low ionic strength decreased as the incubation time increased. Pretreatment of red blood cells with Cd also induced a release of small vesicles. These vesicles contained hemoglobin but were depleted of spectrin and actin, showing a phospholipid composition similar to that of red cell ghosts. These results suggest that the organization of cell membranes, especially cytoskeletal networks, is altered by Cd accumulation in the cytoskeleton fraction, which results in acceleration of age-related changes of red blood cells such as shape change and decreased filterability.     

Visible chemiluminescence induced by t-butyl hydroperoxide in red blood cell suspensions

Red blood cells from Wistar rats were exposed to milimolar concentrations of t-butyl hydroperoxide. Extensive hemoglobin oxidation (methemoglobin formation), t-butyl hydroperoxide cleavage (t-butanol formation) and peroxidation (measured by oxygen consumption and thiobarbituric acid reactive substances) was observed. Significant chemiluminescence was emitted by the system. Hemoglobin oxidation and t-butanol production were independent of oxygen pressure and free radical scavengers, however, luminescence was enhanced as oxygen pressure increased and it was reduced by addition of free radical scavengers. The spectral distribution of the light emitted suggests that the luminescence detected is not due to singlet oxygen dimol emission. The results are in agreement with a lipid peroxidative mechanism initiated by t-butoxy radicals produced in the interaction of hemoglobin and t-butyl hydroperoxide.     

Nonlinear kinetics of the xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase

The xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase has been shown to give nonlinear kinetics of the type which has been identified as substrate activation. When a very wide range of substrate (pteridine) concentrations were studied, it was found that a downward deflection in reciprocal plots (substrate activation) occurs in the high region and an upward deflection in the very low region. When product (isoxanthopterin) was included in reaction mixtures, the upward deflection was enhanced and shifted to higher substrate concentration ranges. In addition, reciprocal plots with a second substrate (oxygen) and a product (isoxanthopterin) were nonlinear.     

Anoxia/induced membrane changes in human red blood cells.

The cation-osmotic hemolysis was studied in human red blood cells incubated under anoxic conditions. In relation to the time course of anoxia, two phases of hemolysis were distinguished. A significant decrease of hemolysis was found between 3 and 24 h of incubation. On the other hand, hemolysis was significantly increased after prolonged incubation (48-72 h). Using the method of cation-osmotic hemolysis, the properties of two membrane constituents, spectrine membrane skeleton and membrane bilayer, were studied. The relation between cation-osmotic hemolysis and erythrocyte deformability is being discussed.     

Detergent-amplified chemiluminescence of lucigenin for determination of superoxide anion production by NADPH oxidase and xanthine oxidase

The detergent-induced amplification of lucigenin-dependent chemiluminescence of O2-, generated by xanthine oxidase or microsomal NADPH oxidase was studied. An assay system is described which is at least 10 times more sensitive than normal lucigenin-dependent chemiluminescence due to the amplification by high concentrations of octylphenylpolyethylene glycol (Triton X-100). Compared to the superoxide dismutase-sensitive reduction of acetylated cytochrome c, a 3750-fold lower amount of microsomal protein was necessary to produce an O2- signal 10-fold above the background. In contrast to cytochrome c reduction, detergent-amplified chemiluminescence of lucigenin was completely inhibited by superoxide dismutase and therefore more selective for O2-. The membrane-bound and Triton X-100-solubilized NADPH oxidase from microsomes of macrophages was activated by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid and inhibited by Ca2+ and sodium dodecyl sulfate. The membrane-bound enzyme showed a Km value of 1.35 microM, which decreased to 0.95 microM after the addition of 12% (g/g) Triton X-100. The Km and Vmax values of soluble xanthine oxidase were not influenced by Triton X-100, indicating that the enzyme activities were not impaired by the high concentrations of detergent.     

Functional and structural alterations induced by copper in xanthine oxidase

Xanthine oxidase （XO）, a key enzyme in purine metabolism, produces reactive oxygen species causing vascular injuries and chronic heart failure. Here, copper＇s ability to alter XO activity and structure was investigated in vitro after pre-incubation of the enzyme with increasing Cue＋ concentrations for various periods of time. The enzymatic activity was measured by following XO-catalyzed xanthine oxidation to uric acid under steady-state kinetics conditions. Structural alterations were assessed by electronic absorption, fluorescence, and circular dichroism spectroscopy. Results showed that Cu^2＋ either stimulated or inhibited XO activity, depending on metal concentration and preincubation length, the latter also determining the inhibition type. Cu^2＋-xo complex formation was characterized by modifications in XO electronic absorption bands, intrinsic fluorescence, and α-helical and β-sheet content. Apparent dissociation constant values implied high- and low-affinity Cu^2＋ binding sites in the vicinity of the enzyme＇s reactive centers. Data indicated that Cu^2＋ binding to high-affinity sites caused alterations around XO molybdenum and flavin adenine dinucleotide centers, changes in secondary structure, and moderate activity inhibition; binding to low affinity sites caused alterations around all XO reactive centers including FeS, changes in tertiary structure as reflected by alterations in spectral properties, and drastic activity inhibition. Stimulation was attributed to transient stabilization of XO optimal conformation. Results also emphasized the potential role of copper in the regulation of XO activity stemming from its binding properties.     

Role of xanthine oxidase and xanthine dehydrogenase systems in thymocyte apoptosis, induced by papaverine

It has been established that papaverine as well as other xenobiotics (dexamethasone and nitrosodimethylamine) [figure: see text] provoked the thymocyte death like apoptosis. The increase of the quantity of double-strand, single-strand DNA breaks and low molecular weight fragments of DNA preceded cell death. In papaverine-induced process of thymocyte apoptosis the total activity of xanthine oxidase in thymocytes strongly elevated long before their death, the conversion of xanthine dehydrogenase (D-form) to xanthinoxidase (O-form) and accumulation of O-form in the cultural medium took place. Direct stimulating effect of papaverine on O-form of enzyme in thymocyte lysate was revealed. The used digitonin thymocytes were divided into cytoplasmic and structural component fractions. It was shown that about 80% of total xanthinoxidase activity was concentrated in cytoplasma while only 20% of its activity was found in structural components. More higher ratio of xanthinoxidase/xanthindehydrogenase (XO/XDH) was observed and papaverine-induced changes of these enzyme forms activities were expressed more brightly in the structural components, than in the thymocyte cytoplasma. During the process of developing thymocytes apoptosis caused by papaverine the reaction of lipid peroxidation was intensified. XO-hypoxanthin system displaying prooxidant influence on cells increased the cytotoxic effect of papaverine but the presence of allopurinol or catalase and superoxidedismutase decreased it. Besides, cytotoxic action on thymocytes of allopurinol itself as well as hypoxanthin itself was revealed.     

Changes in morphology and cell cycle traverse induced in CHO cells by methyl isobutyl xanthine

Methyl isobutyl xanthine (MIX) added to the medium of CHO cells induced a transient elevation of intracellular cAMP concentration. Values which were maximal after 30 min incubation in MIX subsequently declined until after 4–5 h incubation cAMP levels in MIX-treated cells were the same as those of controls. Following addition of MIX to the medium, several perturbations of cell cycle traverse were observed, including a block early in G1 which delayed progress of the cell through subsequent phases of the cell cycle by approx. 1 h, inhibition of transport of exogenous thymidine, arrest of cells in G2 and early M and more rapid completion of mitosis when MIX was added to metaphase cells. None of these changes persisted during prolonged incubation in MIX and cell cycle parameters of cells growing continuously in 0.2 mM MIX were identical with those of their control counterparts. In contrast, conversion from epithelial to fibroblast morphology induced by MIX persisted as long as MIX was maintained in the medium and thus appeared to be independent of elevation of cAMP levels. Morphological conversion was not related to any of the modifications of cell cycle traverse induced by short exposure to MIX.     

Stabilizing effects of cholesterol on changes in membrane permeability and potential induced in red blood cells by lysolecithin.

Effects of cholesterol on permeability of K+ ion and on change in membrane potential induced by lysolecithin were studied. Cholesterol inhibited K+ release from rabbit red blood cells treated with lysolecithin (1.25 micrograms/ml), 3.3 X 10(-6) M of cholesterol being the optimum concentration for blocking K+ release. Changes in membrane potential, monitored by changes in intensity of fluorescence of cyanine dye, were induced by lysolecithin and inhibited by cholesterol. The inhibitory action on both K+ permeability and membrane potential varied with the cholesterol concentration. The observed effects are thought to be due to membrane-stabilizing activities such as decreasing membrane fluidity and hardening the membrane at the fluid-phase transition temperature. These properties of cholesterol may have significance in relation to transformed cells (tumor cells, lymphomed cells).     

Purification of xanthine oxidase from the fat-globule membrane of bovine milk by electrofocusing

Summary Xanthine oxidase was purified from bovine milk-fat-globule membrane by extraction with butan-1-ol, precipitation with ammonium sulphate, separation by preparative electrofocusing and chromatography on Concanavalin-A/Agarose. The enzyme had an A₂₈₀/A₄₅₀ ratio of 4.8 and a specific activity of 3.09. At least five to seven variants of the enzyme with isoelectric points from pH 6.9 to 7.6 were identified. Previously identified minor variants of the enzymes with apparently acidic isoelectric points (1) were shown to be the result of aggregation of enzyme with membrane sialoglycoproteins.Specific antibodies to xanthine oxidase were prepared by fractionating immune serum on a column of enzyme covalently bound to Sepharose 4B. A single immunoprecipitate was obtained when the purified antibodies were allowed to diffuse in agarose gels against either Triton-X-100-extracted membrane or purified xanthine oxidase. Immunoelectrophoresis of the enzyme against anti-sera to xanthine oxidase, however, revealed two precipitin lines, both of which were positive when histochemically stained for enzyme activity.The results are discussed with reference to previous purification schemes for xanthine oxidase and previous estimates for the isoelectric points of the enzyme. We also outline practical uses for the antibody prepared against the enzyme in this present study.Abbreviations SDS sodium dodecyl sulphate - PMSF phenylmethylsulphonyl fluoride     

Hydroxyl radical is not a product of the reaction of xanthine oxidase and xanthine. The confounding problem of adventitious iron bound to xanthine oxidase

The reaction of xanthine and xanthine oxidase generates superoxide and hydrogen peroxide. In contrast to earlier works, recent spin trapping data (Kuppusamy, P., and Zweier, J.L. (1989) J. Biol. Chem. 264, 9880-9884) suggested that hydroxyl radical may also be a product of this reaction. Determining if hydroxyl radical results directly from the xanthine/xanthine oxidase reaction is important for 1) interpreting experimental data in which this reaction is used as a model of oxidant stress, and 2) understanding the pathogenesis of ischemia/reperfusion injury. Consequently, we evaluated the conditions required for hydroxyl radical generation during the oxidation of xanthine by xanthine oxidase. Following the addition of some, but not all, commercial preparations of xanthine oxidase to a mixture of xanthine, deferoxamine, and either 5,5-dimethyl-1-pyrroline-N-oxide or a combination of alpha-phenyl-N-tert-butyl-nitrone and dimethyl sulfoxide, hydroxyl radical-derived spin adducts were detected. With other preparations, no evidence of hydroxyl radical formation was noted. Xanthine oxidase preparations that generated hydroxyl radical had greater iron associated with them, suggesting that adventitious iron was a possible contributing factor. Consistent with this hypothesis, addition of H2O2, in the absence of xanthine, to "high iron" xanthine oxidase preparations generated hydroxyl radical. Substitution of a different iron chelator, diethylenetriaminepentaacetic acid for deferoxamine, or preincubation of high iron xanthine oxidase preparations with chelating resin, or overnight dialysis of the enzyme against deferoxamine decreased or eliminated hydroxyl radical generation without altering the rate of superoxide production. Therefore, hydroxyl radical does not appear to be a product of the oxidation of xanthine by xanthine oxidase. However, commercial xanthine oxidase preparations may contain adventitious iron bound to the enzyme, which can catalyze hydroxyl radical formation from hydrogen peroxide.     

Polymerization of membrane components in aging red blood cells

The addition of malonyldialdehyde to red blood cells in vitro causes the formation of fluorescent chromolipids characteristics of those produced during the peroxidation of endogenous membrane phospholipids. Additionally, gel electrophoresis reveals that this agent also causes a decrease in bands 1 and 2 of spectrin as well as an increase in high molecular weight protein polymers. These same changes are observed in membranes of older cell populations fractionated from freshly drawn, untreated blood. The results obtained suggest that polymerization of membrane components, subsequent to the peroxidation of membrane lipids, may contribute to the altered biochemical and mechanical properties of aging cells and to their eventual sequestration.     

pH-jump studies at subzero temperatures on an intermediate in the reaction of xanthine oxidase with xanthine.

Xanthine oxidase is stable and active in aqueous dimethyl sulphoxide solutions of up to at least 57% (w/w). Simple techniques are described for mixing the enzyme in this solvent at--82 degrees C, with its substrate, xanthine. When working at high pH values under such conditions, no reaction occurred, as judged by the absence of e.p.r. signals. On warming to--60 degrees C, for 10 min, however, the Very Rapid molybdenum(V) e.p.r. signal was obtained. This signal did not change on decreasing the pH, while maintaining the sample in liquid nitrate reductase, caused its molybdenum(V) e.p.r. signal to change from the high-pH to the low-pH form. These findings are not compatible with the conclusions of Edmondson, Ballou, Van Heuvelen, Palmer & Massey [J. Biol. Chem. (1973) 248, 6135-6144], that the Very Rapid signal is in prototropic equilibrium with the Rapid signal, and should be important in understanding the mechanism of action of the enzyme. They emphasize the unique nature of the intermediate represented by the Very Rapid e.p.r. signal. The possible value of the pK for loss of an exchangeable proton from the Rapid signal is discussed.     

Postanoxic damage of microglial cells is mediated by xanthine oxidase and cyclooxygenase

Brain ischemia and the following reperfusion are important causes for brain damage and leading causes of brain morbidity and human mortality. Numerous observations exist describing the neuronal damage during ischemia/reperfusion, but the outcome of such conditions towards glial cells still remains to be elucidated.

Microglia are resident macrophages in the brain. In this study, we investigated the anoxia/reoxygenation caused damage to a microglial cell line via determination of energy metabolism, free radical production by dichlorofluorescein fluorescence and nitric oxide production by Griess reagent. Consequences of oxidant production were determined by measurements of protein oxidation and lipid peroxidation, as well. By using site-specific antioxidants and inhibitors of various oxidant-producing pathways, we identified major sources of free radical production in the postanoxic microglial cells. The protective influences of these compounds were tested by measurements of cell viability and apoptosis. Although, numerous free radical generating systems may contribute to the postanoxic microglial cell damage, the xanthine oxidase- and the cyclooxygenase-mediated oxidant production seems to be of major importance.