Stephen Gene Sullivan

Effects of H2O2 on protein tyrosine phosphatase activity in HER14 cells

Oxidative stress has been implicated in protein phosphorylation and dephosphorylation in cells. In our current studies, H2O2 was shown to reversibly inhibit protein tyrosine phosphatase (PTPase) activity in HER14 cells. H2O2 (150 mM) resulted in 40% inhibition of PTPase activity by 15 min and recovery from inhibition was nearly complete by 60 min. H2O2-induced inhibition or recovery of PTPase activity was not affected by cycloheximide, a protein synthesis inhibitor. L-Buthionine-[S,R]-sulfoximine (BSO), an inhibitor of glutathione synthesis, had no effect on H2O2-induced inhibition of PTPase activity but retarded the recovery of activity. Epidermal growth factor (EGF) and EGTA, a Ca2+ chelator, did not influence H2O2-induced inhibition or recovery of PTPase activity. These results suggest that at least 40% of fibroblast PTPase activity can be regulated by cellular redox activity.

Oxidative reactivity of the tryptophan metabolites 3-hydroxyanthranilate, cinnabarinate, quinolinate and picolinate

The oxidative reactivities of four tryptophan metabolites in the kynurenine pathway were examined as a potential mechanism for their reported neurotoxicities and carcinogenicities. Neither quinolinic acid, a neurotoxin, nor its monocarboxylic analogue, picolinic acid, auto-oxidized over a wide pH range. However, 3-hydroxyanthranilic acid (3-HAT), a carcinogen, readily auto-oxidized and the reaction rate increased exponentially with increasing pH. 3-HAT auto-oxidation likely involves two steps: auto-oxidation of 3-HAT to the semiquinoneimine (anthranilyl radical) which oxidizes to the quinoneimine, followed by condensation and oxidation reactions to yield a second carcinogen, cinnabarinic acid. 3-HAT auto-oxidation to cinnabarinate required molecular oxygen and generated superoxide radicals and H2O2. Superoxide dismutase (SOD) accelerated 3-HAT auto-oxidation 4-fold, probably by preventing back reactions between superoxide and either the anthranilyl radical or the quinoneimine formed during the initial step of auto-oxidation. Catalase did not accelerate 3-HAT auto-oxidation, but it did prevent destruction of cinnabarinate by H2O2. Interconversion between oxyhemoglobin and methemoglobin occurred during 3-HAT auto-oxidation, although neither form of hemoglobin altered rates of 3-HAT auto-oxidation. Mn2+, Mn3+ and Fe3+-EDTA did not directly catalyze cinnabarinate formation in the absence of O2, but they did accelerate cinnabarinate formation under aerobic conditions.

Lipid peroxidation and hemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide: Dependence on glucose metabolism and hemoglobin status

Changes in hemoglobin status and lipid peroxidation were followed in red cells containing either oxy-met-, or carbonmonoxyhemoglobin, incubated with t-butyl hydroperoxide in a medium with or without glucose. Loss of intact hemoglobin (the sum of oxyhemoglobin and methemoglobin) was inversely proportional to the degree of lipid peroxidation in red cells containing either oxy- or methemoglobin. When glucose was added to the medium, lipid peroxidation increased while there was a decreased loss of intact hemoglobin in red cells containing either oxy- or methemoglobin, while both lipid peroxidation and changes in hemoglobin decreased in red cells containing carbonmonoxyhemoglobin. Methemoglobin formation and loss of intact hemoglobin were directly proportional to the degree of lipid peroxidation in red cells containing carbonmonoxyhemoglobin. The greatest amount of lipid peroxidation occurred in red cells containing carbonmonoxyhemoglobin, incubated without glucose. These results indicate that methemoglobin and non-intact hemoglobin may protect the membrane against lipid peroxidation. We propose that, depending on the availability of glucose and the liganded state of hemoglobin, lipid peroxidation and hemoglobin alterations represent extremes of a spectrum of oxidative damage.

Effects of superoxide dismutase and catalase on catalysis of 6-hydroxydopamine and 6-aminodopamine autoxidation by iron and ascorbate

Abstract The effects of superoxide dismutase and catalase on autoxidation of 6-hydroxydopamine and 6-aminodopamine in several chemical environments were studied. Inhibition by superoxide dismutase of autoxidation of 6-hydroxydopamine to its p -quinone required the presence of metal chelators, EDTA or diethylenetriamine pentaacetic acid (DETAPAC). A “lag” period in 6-hydroxydopamine autoxidation in the presence of superoxide dismutase couid be prevented by carrying out autoxidation in a mixture of 6-hydroxydopamine and its p-quinone, conditions in which adequate levels of the semiquinone are available for reaction with O 2 . Catalase potentiated the inhibitory effect of superoxide dismutase in the presence of EDTA but had no effect in the presence of DETAPAC. Superoxide dismutase and catalase had no effect on the initial rate of 6-aminodopamine autoxidation to the p -quinone imine or on later intracyclization and polymer formation. Iron chelated by EDTA functioned as a catalyst in 6-hydroxydopamine autoxidation, making the reaction independent of superoxide as shown by the lack of effect of superoxide dismutase. the presence of iron-EDTA resulted in bleaching of the p -quinone product and the consumption of the H 2 O 2 formed during autoxidation. Catalase had no effect on the rate of 6-hydroxydopamine autoxidation but completely prevented bleaching of the p -quinone, probably by preventing formation of hydroxyl radical by a Fenton reaction between iron-EDTA and H 2 0 2 . Ethanol, which scavenges hydroxyl radical but not superoxide of H 2 O 2 , similarly prevented bleaching of the p -quinone with no other effects on autoxidation. Iron-EDTA also catalyzed 6-aminodopamine autoxidation with associated consumption of H 2 O 2 . Superoxide dismutase, catalase and ethanol had no effect on 6-aminodopamine autoxidation in the presence or absence of iron-EDTA, showing the independence of the kinetics of 6-aminodopamine autoxidation and polymerization from its products, superoxide, H 2 0 2 and hydroxyl radical. Chelation by DETAPAC prevented the effects of iron on 6-hydroxydopamine and 6-aminodopamine autoxidation. Autoxidation of 6-hydroxydopamine in the presence of ascorbate exhibited a lag phase followed by a linear phase of autoxidation. Superoxide dismutase, catalase and ethanol had no effect on 6-hydroxydopamine or 6-aminodopamine autoxidation in the presence of ascorbate. Autoxidation of 6-hydroxydopamine or 6-aminodopamine in the combined presence of iron-EDTA and ascorbate showed the full effects of both additions, except that 6-hydroxydopamine autoxidation exhibited no lag phase and the iron-catalyzed autoxidation of ascorbate occurred simultaneously with the 6-hydroxydopamine or 6-aminodopamine reactions.

Primaquine-mediated oxidative metabolism in the human red cell. Lack of dependence on oxyhemoglobin, H2O2 formation, or glutathione turnover.

Stimulation of the hexose monophosphate shunt by primaquine results from the oxidation of NADPH by primaquine. This conclusion was based on the observations that primaquine lowered cellular NADPH but not GSH and that, in red cells in which the GSH was unavailable for reaction, primaquine still stimulated the rate of the hexose monophosphate shunt. In a non-cellular system, primaquine interacted with NADPH, but not GSH, to produce H2O2. Stimulation of the hexose monophosphate shunt by primaquine does not primarily involve H2O2 accumulation since stimulation of the pathway by primaquine was also observed in red cells containing methemoglobin, a red cell preparation in which no H2O2 accumulates. Methemoglobin prevented the formation and/or accumulation of H2O2 in intact red cells incubated with primaquine as well as in a non-cellular system containing primaquine plus Fe2+-EDTA as an H2O2 source. Methemoglobin probably acts by scavenging reactive intermediates since oxyhemoglobin was formed from methemoglobin in the non-cellular experiments. In the red cell, primaquine stimulated glucose-dependent conversion of methemoglobin to oxyhemoglobin.

Inhibition of hemin-induced hemolysis by desferrioxamine: binding of hemin to red cell membranes and the effects of alteration of membrane sulfhydryl groups.

Hemin binds to red cell membranes during hemin-induced hemolysis but the precise mechanism of hemolysis has not been characterized. Desferrioxamine (DFO), an iron chelator, inhibited hemin-induced hemolysis. DFO partially prevented hemin binding to red cell membranes and partially removed previously bound hemin. Glutathione, an intracellular sulfhydryl compound, also inhibited hemin-induced hemolysis but was only about one tenth as potent as DFO. Decrease of membrane sulfhydryl groups by treatment of cells with either N-ethylmaleimide (NEM) or diamide (azodicarboxylic acid bis [dimethylamide]) enhanced hemin-induced hemolysis. Enhancement of hemin-induced hemolysis by NEM and diamide and inhibition of hemolysis by DFO were independent with no evidence of synergism or interference between the two processes. Red cell membranes were saturated with hemin at approximately 75 nmol per mg protein. DFO decreased the hemin saturation level to 25 nmol per mg protein. In the presence of DFO, hemin was bound as the DFO-hemin complex since membranes preferentially removed DFO-hemin complexes from mixtures of complexed and free hemin while free DFO was not bound by the membranes. Access to the inner surface of the membrane was required for binding of the DFO-hemin complex since DFO completely prevented hemin binding in intact cells but not in cells undergoing hemolysis or red cell ghosts. Approximately 50 x 10(6) molecules of hemin were bound to the membrane of one red cell following hemin-induced hemolysis.

Desferrioxamine protects human red blood cells from hemin-induced hemolysis

Hemin binding to red cell membranes, its effect on red cell hemolysis, and it interaction with desferrioxamine (DFO) in these processes were investigated. DFO interacted with hemin via the iron moiety. Blockage of the binding groups in DFO prevented interaction of DFO with hemin, implying the importance of the hydroxamic acid groups in DFO-hemin interactions. Since hemolysis is a result of hemin association with the membrane components, its binding in the presence and absence of DFO was studied. DFO strongly inhibited hemin-induced lysis in a concentration-dependent manner. With 50 microM hemin, 1 mM DFO completely inhibited lysis. Preincubation of ghost membranes with DFO (1 mM) inhibited binding of hemin (50 microM) to membranes by 42%. After ghost membranes were preincubated with hemin (50 microM), the addition of DFO (1 mM) removed 20% of the membrane-bound hemin. It is suggested that DFO may have an important role in alleviating the hemin-induced deleterious effects on the red cell membrane, especially in hemolytic anemias associated with unstable, autoxidized hemoglobins.

Vanadate-mediated oxidation of NADH: Description of an in vitro system requiring ascorbate and phosphate

Oxidation of NADH has been observed in an in vitro system requiring NADH, vanadate, ascorbate, and phosphate. Similar results were observed with NADPH. Ascorbate provides the reducing equivalents necessary to reduce vanadate to vanadyl. Vanadyl autoxidizes producing superoxide which initiates a free radical chain reaction resulting in oxidation of NADH. Oxidation is inhibited by superoxide dismutase but not by catalase or ethanol. Ascorbate functions to initiate the free radical chain reaction but is not required in stoichiometric concentrations. At higher concentrations, ascorbate inhibits NADH oxidation. Inorganic phosphate was required for NADH oxidation. Dialysis of phosphate buffers against solutions containing apoferritin or conalbumin or addition of transition metal cations or chelators to the reaction medium did not alter dependence on phosphate. Phosphate and vanadate were interchangeable in their effects on kinetic parameters of NADH oxidation except that vanadate was 100 times more potent than phosphate. Vanadate participates directly in the initiating and propagating redox reactions of NADH oxidation. Phosphate may be important in lowering the energy of activation for the necessary transfer of hydronium ion and water in the transition state between vanadate anion and vanadyl cation.

Membrane protein changes induced by tert-butyl hydroperoxide in red blood cells

Red cells were incubated in the presence of t-butyl hydroperoxide and effects on red cell membrane proteins were studied by SDS-polyacrylamide gel electrophoresis. t-Butyl hydroperoxide caused diminution in intensity of all major cytoskeletal bands with the concomitant formation of high molecular weight material. Membrane glycoproteins were unaffected. t-Butyl hydroperoxide increased hemoglobin binding to ghosts. After dissolution in SDS and beta-mercaptoethanol, membrane-bound hemoglobin appeared on the gels in the form of monomers and crosslinked polymers of hemoglobin or globin chains. Crosslinking was partially prevented by metabolism of t-butyl hydroperoxide by the hexose monophosphate shunt except in methemoglobin-containing red cells where reaction with methemoglobin accounted for most of the consumption of t-butyl hydroperoxide. Metal chelators, deferoxamine mesylate and diethylenetriaminepentaacetic acid, had no effect on membrane protein changes. Butylated hydroxytoluene, diphenylamine and ascorbate, compounds that inhibit t-butyl hydroperoxide-induced red cell membrane lipid peroxidation, had no effect on t-butyl hydroperoxide-induced membrane protein changes. These results suggest that membrane proteins and membrane lipids have different mechanisms of peroxidant damage.

Effects of ascorbate on methemoglobin reduction in intact red cells

Abstract An endpoint of 75% HbO 2 /25% methemoglobin (MetHb) was approached in red cells incubated with a greater than physiologic concentration of ascorbate (10 m m ). The presence of glucose (5 m m ) with ascorbate shifted the endpoint to 90% HbO 2 /10% MetHb while lactate (2 m m ) plus pyruvate (0.1 m m ) had no effect. These endpoints were approached regardless of the HbO 2 MetHb ratio at zero time. No hemoglobin degradation was observed. When red cells containing 100% MetHb at zero time were used, analysis of the initial rate of HbO 2 formation in the presence of various substrates showed synergistic interaction between ascorbate (10 m m ) and glucose, additive activity with ascorbate and lactate, and less than additive activity with glucose and lactate. Incubation of red cells with a phsyiologic concentration of ascorbate (0.1 m m ) resulted in no significant HbO 2 formation in the absence of other additions. When red cells were incubated with glucose and/or lactate plus pyruvate, an endpoint of about 99% HbO 2 /1% MetHb was approached regardless of the HbO 2 /MetHb ratio at zero time or the presence or absence of physiologic ascorbate. Physiologic ascorbate slightly but consistently increased the rate of HbO 2 formation in red cells incubated with glucose but not with lactate. HbO 2 formation was not increased by ascorbate in red cells which contained more than about 90% HbO 2 at zero time. The results indicate that excess ascorbate functions stoichiometrically driving cellular chemistry to a steady state between HbO 2 and MetHb formation whereas physiologic ascorbate functions catalytically allowing electron transport from glucose to MetHb via the hexose monophosphate shunt.