Intermediate Mr cytosolic components potentiate hepatic 5''-deiodinase activation by thiols.

The role in the activation of microsomal 5'-deiodinase (5'-DI) of rat hepatic cytosolic components of Mr approx. 13,000 (Fraction B) was studied in the presence of various concentrations of thiol compounds such as dithiothreitol (DTT), dihydrolipoamide (DHLA), GSH, and 2-mercaptoethanol (2-ME). Although Fraction B (which was prepared by gel filtration to exclude GSH and GSSG) had no intrinsic 5'-DI activity, could not stimulate microsomal 5'-DI activity in the absence of added thiol and did not contain GSH as a mixed disulphide, it could produce a 3-fold increase in the maximal deiodinase activity achievable with DTT as well as other thiols, with the order being the same as the activation potency of these thiols in the absence of Fraction B (i.e. DHLA greater than DTT greater than 2-ME greater than GSH). These observations suggest that: a component of cytosolic Fraction B, designated 'deiodination factor B' (DFB), operates as an efficient intermediary to enhance activation of microsomal 5'-DI by thiols through a mechanism independent of GSH; thiols may participate in a non-specific thiol-disulphide exchange with inactive (oxidized) DFB to convert it into an active form that contains one or more thiol groups and is more effective than GSH or other thiols in facilitating the re-activation of inactive (oxidized) microsomal 5'-DI thiol (ESI) to its active state (ESH).     

Nonenzymatic decarboxylation of pyruvate

Triton X-100, retinol, retinoic acid, retinal, hexane, dithiothreitol, mercaptoethanol, and some other commercially available chemicals caused nonenzymatic decarboxylation of pyruvate and alpha-ketoglutarate. "Lipids" obtained from human or pigeon liver homogenates using isopropanol/hexane also had very high nonenzymatic decarboxylating activity on these two alpha-ketoacids; most of this activity could be traced to the hexane (Eastman) used in the extraction. Optimum pH of the reaction with dithiothreitol and mercaptoethanol was 7-8 and with the other chemicals around 10, but considerable activity was present at pH 7-8. Liver homogenates had a scavenger effect on the decarboxylating activity of Triton X-100 and of dithiothreitol. Dithiothreitol and mercaptoethanol at high concentrations (greater than 1 mM) also had a scavenger effect on the decarboxylating activity of the "lipids." Pretreatment of Triton X-100, dithiothreitol, retinol, and the "lipids" with catalase markedly decreased the decarboxylating activity, while treatment with boiled catalase failed to do so. The results suggest that these compounds contain oxidizing contaminants, perhaps peroxide derivatives. Powerful oxidizing impurities have been reported in Triton X-100 from various sources by Y. Ashani and G. N. Catravas (1980, Anal. Biochem 109, 55-62). Such peroxide derivatives may cause nonenzymatic decarboxylation of pyruvate and alpha-ketoglutarate, presumably by a mechanism similar to the well-known nonenzymatic decarboxylation of alpha-ketoacids by hydrogen peroxide. In the absence of catalase and/or other protective agents against reactive oxygen derivatives, these chemicals would interfere in the assays of pyruvate dehydrogenase, pyruvate dehydrogenase complex, and alpha-ketoglutarate dehydrogenase complex which depend on the release of 14CO2 from alpha[1-14C]ketoacids.     

Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE

The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative stress. Our findings indicate that Grx2 plays a central role in the response of mitochondria to both redox signals and oxidative stress by facilitating the interplay between the mitochondrial glutathione pool and protein thiols.     

Radioprotection of DNA by thiols: relationship between the net charge on a thiol and its ability to protect DNA

Release of free bases from calf thymus DNA upon irradiation in aerated 0.1 mol dm-3NaClO4 at pH 7 has been measured by HPLC and shown to be markedly influenced by the presence of thiols during irradiation. The ability of thiols to protect DNA was shown to depend upon the net charge (Z) at pH 7 in the order WR 1065 (Z = +2) greater than cysteamine (Z = +1) greater than 2-mercaptoethanol (Z = 0) approximately equal to dithiothreitol (Z = 0) greater than GSH (Z = -1) approximately equal to 2-mercaptoethanesulfonic acid (Z = -1) approximately equal to 2-mercaptosuccinate (Z = -2). A similar dependence of protection upon net charge was found for disulfides: cystamine (Z = +2) greater than 2-mercaptoethyl disulfide (Z = 0) greater than GSSG (Z = -2). Protection by WR 1065, but not by 2-mercaptoethanol or GSH, was found to decrease significantly with increasing ionic strength. Protection by WR 1065 and GSH was not markedly dependent upon pH between pH 6 and 8. The results are explained in terms of electrostatic interaction of the thiols with DNA, leading to high concentrations of cations near DNA, which allow them to scavenge hydroxyl radicals and repair DNA radicals effectively and to low concentrations of anionic thiols near DNA, which limit their effectiveness as protectors. Poly(dG,dC) and calf thymus DNA exhibited comparable release of G and C upon changing from 0.1 to 0.7 mol dm-3 MgSO4. Since this change causes poly(dG,dC), but not calf thymus DNA, to undergo a change from the B-form to the Z-form of DNA, both forms must have a comparable susceptibility to radiation-induced base release.     

Direct measurement of the equilibrium between glutathione and dithiothreitol by high performance liquid chromatography

The equilibrium constant between reduced glutathione (GSH), oxidized glutathione (GSSG), reduced dithiothreitol (DTTSHSH), and oxidized dithiothreitol (DTTSS) has been directly measured by high performance liquid chromatography analysis of equilibrium mixtures. The equilibrium constant at 25 degrees C for the reaction GSSG + DTTSHSH in equilibrium 2GSH + DTTSS varies from approximately 200 M, below pH 8, to approximately 2800 M, above pH 11. The observed pH dependence is generally consistent with published values of acid dissociation constants of these thiols.     

Thiol/disulfide redox equilibrium and kinetic behavior of chicken liver fatty acid synthase

Chicken liver fatty acid synthase is rapidly inactivated and cross-linked at pH 7.2 and 8.0 by incubation with low concentrations of common biological disulfides including glutathione disulfide, coenzyme A disulfide, and glutathione-coenzyme A-mixed disulfide. Glutathione disulfide inactivation of the enzyme is accompanied by the oxidation of a total of 4-5 enzyme thiols per monomer. Only one glutathione equivalent is incorporated per monomer as a protein-mixed disulfide, and its rate of incorporation is significantly slower than the rate of inactivation. The formation of protein-SS-protein disulfides results in significant cross-linking of enzyme subunits. The inactive enzyme is rapidly and completely reactivated, and the cross-linking is completely reversed by incubation of the enzyme with thiols (10-20 mM) including dithiothreitol, mercaptoethanol, and glutathione. In a glutathione redox buffer (GSH + GSSG), disulfide bond formation comes to equilibrium. The enzyme activity at equilibrium is dependent both on the ratio of glutathione to glutathione disulfide and on the total glutathione concentration. The equilibrium constant for the redox equilibration of fatty acid synthase in a glutathione redox buffer is 15 mM (Ered + GSSG in equilibrium Eox + 2GSH). The formation of at least one protein-protein disulfide per monomer dominates the redox properties of the enzyme while the formation of one protein-mixed disulfide with glutathione (Kmixed = 0.45) has little effect on activity. The oxidation equilibrium constant suggests that there would be no significant cycling between the reduced and the oxidized enzyme in response to likely physiological variations in the hepatic glutathione status. The possibility that changes in the concentration of cellular glutathione may act as a mechanism for metabolic control of other enzymes is discussed.     

Chromatographic and mass spectrometric analysis of glutathione in biological samples

Biological thiol compounds are classified into high-molecular-mass protein thiols and low-molecular-mass free thiols. Endogenous low-molecular-mass thiol compounds, namely, reduced glutathione (GSH) and its corresponding disulfide, glutathione disulfide (GSSG), are very important molecules that participate in a variety of physiological and pathological processes. GSH plays an essential role in protecting cells from oxidative and nitrosative stress and GSSG can be converted into the reduced form by action of glutathione reductase. Measurement of GSH and GSSG is a useful indicator of oxidative stress and disease risk. Many publications have reported successful determination of GSH and GSSG in biological samples. In this article, we review newly developed techniques, such as liquid chromatography coupled with mass spectrometry and tandem mass spectrometry, for identifying GSH bound to proteins, or for localizing GSH in bound or free forms at specific sites in organs and in cellular locations.     

Inhibition of erythrocyte Ca2+-ATPase by activated oxygen through thiol- and lipid-dependent mechanisms

We have studied erythrocyte Ca2+-ATPase as a model target for elucidating effects of activated oxygen on the erythrocyte membrane. Either intracellular or extracellular generation of activated oxygen causes parallel decrements in Ca2+-ATPase activity and cytoplasmic GSH, oxidation of membrane protein thiols, and lipid peroxidation. Subsequent incubation with either dithiothreitol or glucose allows only partial recovery of Ca2+-ATPase, indicating both reversible and irreversible components which are modeled herein using diamide and t-butyl hydroperoxide. The reversible component reflects thiol oxidation, and its recovery depends upon GSH restoration. The irreversible component is largely due to lipid peroxidation, which appears to act through mechanisms involving neither malondialdehyde nor secondary thiol oxidation. However, some portion of the irreversible component could also reflect oxidation of thiols which are inaccessible for reduction by GSH, since we demonstrate existence of different classes of thiols relevant to Ca2+-ATPase activity. Activated oxygen has an exaggerated effect on Ca2+-ATPase of GSH-depleted cells. Sickle erythrocytes treated with dithiothreitol show a heterogeneous response of Ca2+-ATPase activity. These findings are potentially relevant to oxidant-induced hemolysis. They also may be pertinent to oxidative alteration of functional or structural membrane components in general, since many components share with Ca2+-ATPase both free thiols and close proximity to unsaturated lipid.     

Analysis of biological thiols: determination of thiol components of disulfides and thioesters

This report describes a method for using selective cleavage of thioesters to allow differentiation between thioesters and disulfides. The method identifies thiol components (including glutathione, coenzyme A, and cysteine) of low-molecular-weight thioesters and disulfides in cell extracts, as well as thiols bound to protein via thioester or disulfide links. Thioesters were cleaved with 200 mM hydroxylamine under a nitrogen atmosphere in the presence of monobromobimane (mBBr), which forms a fluorescent derivative with the released thiol. For analysis of disulfides, thioesters were cleaved with hydroxylamine in the presence of N-ethylmaleimide to block released thiols: disulfides were then reduced with 10 mM dithiothreitol and subsequently labeled with mBBr. The bimane derivatives were identified and quantified using previously described HPLC methods (G. L. Newton, R. Dorian, and R. C. Fahey, 1981, Anal. Biochem. 114, 383-387). Traditional methods using dithiothreitol and sodium borohydride to cleave disulfides can also cleave thioesters and thus should not be used for specific analysis of disulfides.     

Glutathione depletion and formation of glutathione-protein mixed disulfide following exposure of brain mitochondria to oxidative stress

t-Butyl hydroperoxide was utilized to alter the thiol homeostasis in rat brain mitochondria. Following exposure to t-butyl hydroperoxide (50-500 microM), intramitochondrial GSH content decreased rapidly and irreversibly with a major portion of the depleted GSH being accounted for as protein-SS-Glutathione mixed disulfide. Formation of GSSG was not observed nor was efflux of GSSG or GSH from the mitochondria detected in the incubation medium. The loss of intramitochondrial GSH was accompanied by loss of protein thiols. Unlike liver mitochondria, which can reverse t-butyl hydroperoxide induced formation of GSSG, addition of 50 microM t-butyl hydroperoxide resulted in irreversible loss; indicating greater susceptibility of brain mitochondria to oxidative stress than liver mitochondria.     

Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity

The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.     

Membrane thiol-disulfide status in glucose-6-phosphate dehydrogenase deficient red cells. Relationship to cellular glutathione

The behavior of glucose-6-phosphate dehydrogenase (G6PD)-deficient red cell membrane proteins upon treatment with diamide, the thiol-oxidizing agent (Kosower, N.S. et al. (1969) Biochem. Biophys. Res. Commun. 37, 593–596), was studied with the aid of monobromobimane, a fluorescent labeling agent (Kosower, N.S., Kosower, E.M., Newton, G.L. and Ranney, H.M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382–3386) convenient for following membrane thiol group status. In diamide-treated G6PD-deficient red cells (and in glucose deprived normal cells), glutathione (GSH) is oxidized to glutathione disulfide (GSSG). When cellular GSH is absent, membrane protein thiols are oxidized with the formation of intrachain and interchain disulfides. Differences in sensitivity to oxidation are found among membrane thiols. In diamidetreated normal red cells, GSH is regenerated in the presence of glucose and membrane disulfides reduced. In G6PD-deficient cells, GSSG is not reduced, and the oxidative damage (disulfide formation) in the membrane not repaired. Reduction of membrane disulfides does occur after the addition of GSH to these membranes. A direct link between the thiol status of the cell membrane and cellular GSH is thereby established. GSH serves as a reductant of membrane protein disulfides, in addition to averting membrane thiol oxidation.     

Proteolytic activation and solubilization of endoplasmic-reticulum cyclic AMP phosphodiesterase activity.

Dithiothreitol led to the activation and solubilization of the cyclic nucleotide phosphodiesterase activities associated with the smooth and various rough subfractions of rat liver endoplasmic reticulum. The activity in each of the subfractions exhibited somewhat different time courses, and sensitivities to dithiothreitol concentration, in respect of their solubilization and activation. Both activation and solubilization by dithiothreitol could be blocked by either thiol proteinase inhibitors or excess bovine serum albumin. Freeze-thaw solubilization was not blocked by the thiol proteinase inhibitor antipain and did not lead to the activation of the enzyme. After dithiothreitol-induced solubilization, all of the enzymes exhibited non-linear Lineweaver-Burk plots indicative of apparent negative co-operativity. In contrast, after freeze-thaw solubilization the enzyme in the smooth-endoplasmic-reticulum-plus-Golgi fraction still obeys Michaelis kinetics, as does the membrane-bound enzyme. It is possible to mimic the action of dithiothreitol in solubilizing and activating the enzyme by limited proteolysis with trypsin. Triton X-100 is highly efficient at solubilizing these enzymes, yet has little effect on their activities. Charged detergents exhibit highly selective effects on the enzymes as regards their solubilization and activity expressed.     

Radiosensitization, thiol oxidation, and inhibition of DNA repair by SR 4077

The mechanism of radiosensitization by diazenedicarboxylic acid bis(N),N-piperidide (SR 4077), a less toxic analog of diamide, was studied using Chinese hamster ovary cells. SR 4077 gave an average SER of 1.58 for postirradiation incubations of 0.5, 1.0, or 2.0 h. Intracellular GSH and protein thiols decreased rapidly following drug addition and GSSG increased. The GSH/GSSG ratio shifted to 1/1.6 after SR 4077 addition but returned to greater than 10/1 between 0.5 and 1.0 h. After 4 h, total intracellular GSH was only 58% of pretreatment level and extracellular GSSG increased. Protein thiols decreased to 18% of pretreatment values, recovered most rapidly between 0.5 and 1.0 h, and reached 87% of pretreatment level after 4 h. A decrease in DNA single-strand break repair as measured by alkaline filter elution rate over 0.5 h was seen, and the initial rate of repair was slower than in cells not treated with SR 4077. DNA double-strand break repair as measured by neutral filter elution rate was delayed during the first hour after irradiation when cells were treated with SR 4077. The times for maximum radiosensitization, GSH and protein thiol oxidation and recovery, and DNA strand break repair kinetics were closely linked. We propose that a protein thiol(s) required in repair processes was reversibly oxidized during SR 4077 treatment.     

Menadione-induced bleb formation in hepatocytes is associated with the oxidation of thiol groups in actin

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) or the thiol oxidant, diamide (azodicarboxylic acid bis(dimethylamide)), resulted in the appearance of numerous plasma membrane protrusions (blebs) preceding cell death. Analysis of the Triton X-100-insoluble fraction (cytoskeleton) extracted from treated cells revealed a dose- and time-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols. These changes were associated with the disappearance of actin and formation of large-molecular-weight aggregates, when the cytoskeletal proteins were analyzed by polyacrylamide gel electrophoresis under nonreducing conditions. However, if the cytoskeletal proteins were treated with the thiol reductants, dithiothreitol or beta-mercaptoethanol, no changes in the relative abundance of actin or formation of large-molecular-weight aggregates were detected in the cytoskeletal preparations from treated cells. Moreover, addition of dithiothreitol to menadione- or diamide-treated hepatocytes protected the cells from both the appearance of surface blebs and the occurrence of alterations in cytoskeletal protein composition. Our findings show that oxidative stress induced by the metabolism of menadione in isolated hepatocytes causes cytoskeletal abnormalities, of which protein thiol oxidation seems to be intimately related to the appearance of surface blebs.     

The behavior of calpain-generated N- and C-terminal fragments of talin in integrin-mediated signaling pathways.

Our previous results showed that the binding of an adhesive ligand to integrin alphaIIbbeta(3) on the surface of platelets triggers the activation of calpain and the limited proteolysis of talin by calpain. To explore the physiological significance of the calpain-mediated cleavage of talin, we analyzed the behavior of the calpain-generated fragments of talin (N-terminal 47 kDa and C-terminal 190 kDa) during platelet activation by biochemical and immunoelectron microscopic studies. Intact talin and micro-calpain translocate from the Triton X-100-soluble fraction to the insoluble fraction upon platelet stimulation by thrombin, and the limited proteolysis of talin occurs in the Triton X-100-insoluble fraction, the cytoskeletal fraction. The fully autolyzed 76-kDa micro-calpain (active form) is found predominantly in the Triton X-100-insoluble fraction in stimulated platelets. While the N-terminal 47-kDa fragment remains in the Triton X-100-insoluble fraction, the C-terminal 190-kDa fragment is released into the Triton X-100-soluble fraction in a time-dependent manner. Immunoelectron microscopic observations revealed that the 47-kDa fragment locates on the submembrane zone just beneath the plasma membrane, including the open canalicular systems, while most of the 190-kDa fragment exists diffusely in the cytoplasm in thrombin-stimulated platelets. These findings suggest that calpain may contribute to the reorganization of the cytoskeleton in an integrin-mediated signaling pathway through the redistribution of the functional domain of talin.     

Protein sulfhydryls are protected from irreversible oxidation by conversion to mixed disulfides.

Protein mixed thioselenides formed by reaction of sarcoplasmic reticulum (SR) with diselenide biradical spin labels were quantified by ESR. Whereas the reaction of SR membranes with the diselenide spin label led to a large ESR signal of the unbound monoselenide at equilibrium, treatment of the reaction mixture with a few millimolar hydrogen peroxide converted all of the nitroxides to protein-bound thioselenides. This technique of spin-labeling protein thiols avoids the need to remove unreacted spin labels. The bound spin labels were removable by reduction with excess mercaptoethanol, indicating a specific and reversible labeling of protein thiols. SR that had been extensively labeled with the diselenide spin label was resistant to ATPase inactivation by potent oxidants that arise when myoglobin reacts with hydroperoxides. Unmodified SR lost all activity within 10 min of exposure to either 1 mM tert-butyl hydroperoxide in the presence of 200 microM equine myoglobin or to 100 mM hydrogen peroxide in the absence of myoglobin. In both cases the loss of activity could not be reversed by subsequent treatment with mercaptoethanol. On the other hand, membranes that had been extensively treated with the diselenide spin label and were then subjected to these peroxide treatments were fully active after mercaptoethanol-mediated cleavage of the thioselenides. ESR analysis of spin-labeled SR showed no detectable oxidative cleavage of the thioselenide bonds. Sodium dodecyl sulfate gel electrophoresis showed that peroxide-mediated crosslinking of ATPase observed in unmodified SR membranes did not occur in the diselenide-modified SR membranes. Only limited protection was observed when SR pretreated with glutathione disulfide was incubated with hydroperoxides. In this case, however, the degree of protection was greatly increased when the reaction with glutathione disulfide was carried out in the presence of the supernatant of centrifuged rat liver homogenate, consistent with an acceleration of mixed disulfide formation by a factor tentatively identified as thiol transferase. It is concluded that conversion of protein thiol residues to either thioselenides or mixed disulfides confers protection against irreversible peroxide-dependent oxidation. We suggest that mixed disulfide formation by thiol transferase activity may help protect protein thiols from irreversible oxidation by heme-activated hydroperoxides.     

Effects of NADH and thiol compounds on wheat leaf nitrate reductase

The initial activity of wheat leaf nitrate reductase was depressed on inclusion of the following thiol compounds; dithiothreitol, dithioerythreitol or mercaptoethanol, but not cysteine and glutathione. This thiol effect simply resulted from an interference with the chemical determination of nitrite. Preincubation of the enzyme with NAD⁺ and these thiols enhanced the inhibition of nitrate reductase activity. This effect was mediated by NADH production by the thiol reduction of NAD⁺. The inactivation by NAD⁺ in the presence of thiol compounds which was enhanced by cyanide ions could be reversed by ferricyanide, as has been observed previously for NADH-mediated inactivation of nitrate reductase.     

Oxidative stress alters membrane sulfhydryl status, lipid and phospholipid contents of crossbred cattle bull spermatozoa

Ferrous ascorbate (FeAA: FeSO4+ascorbic acid) has been used in the past by different investigators to induce oxidative stress. The optimum dose of FeAA for inducing oxidative stress by affecting thiols [total thiols (TSH), glutathione reduced (GSH), glutathione oxidized (GSSG), redox ratio (GSH/GSSG)], total lipids and phospholipids has been ascertained in the local crossbred cattle bull spermatozoa. The fractions of spermatozoa suspended in 2.9% sodium citrate were subjected to three doses of FeAA (100 microM:500 microM, 150 microM:750 microM, 200 microM:1000 microM; FeSO4:ascorbic acid), and were assessed for various parameters. On increasing the concentration of FeAA, a gradual decrease in TSH, GSH, GSH/GSSG, lipid and phospholipid levels, but increase in GSSG content were observed. It is concluded that thiol groups play an important role in antioxidation and detoxification of ROS as well as maintaining intracellular redox status. Thiol groups, thus, serve as defense mechanisms of sperm cells to fight against oxidative stress. In addition, all doses of FeAA cause leakage of lipids and phospholipids from the bull sperm membranes.