Involvement of thiol transferase- and thioredoxin-dependent systems in the protection of ''essential'' thiol groups of ornithine decarboxylase.

Ornithine decarboxylase (ODC), an enzyme with 'essential' thiol group(s), may be inactivated in vitro by removal of thiol reducing agents and re-activated by soluble factors from rat liver in the presence of NADPH or GSH. The NADPH- and GSH-dependent reducing systems were separated and resolved into three components, called factors A, B1 and B2, by chromatographic techniques. Factor B1 (Mr 12,000) could reactivate ODC in the presence of GSH and co-purified with thiol transferase activity. Factor B2 (Mr 12,000) and factor A (Mr approx. 110,000) were both needed to re-activate ODC in the presence of NADPH, and co-purified with thioredoxin and thioredoxin reductase activity respectively. In an attempt to investigate the physiological role of the 'essential' thiol group(s) of ODC, erythroleukaemia cells were incubated with NN-bis-(2-chloroethyl)-N'-nitrosourea, t-butyl hydroperoxide and vinblastine, which are known to increase the cellular GSSG/GSH ratio, azelaic acid, an inhibitor of thioredoxin reductase, and sodium arsenite, a strong inhibitor of the ODC-re-activating factors. All these compounds were able to decrease significantly the ODC activity induced in these cells. These results suggest that the thiol transferase- and thioredoxin-dependent systems may be physiologically relevant in maintaining ODC in the active, reduced, state.     

Properties of cytosolic components activating rat hepatic 5'' [corrected]-deiodination in the presence of NADPH.

The effects of cytosol, NADPH and reduced glutathione (GSH) on the activity of 5'-deiodinase were studied by using washed hepatic microsomes from normal fed rats. Cytosol alone had little stimulatory effect on the activation of microsomal 5'-deiodinase. NADPH had no stimulatory effect on the microsomal 5'-deiodinase unless cytosol was added. 5'-deiodinase activity was greatly enhanced by the simultaneous addition of NADPH and cytosol (P less than 0.001); this was significantly higher than that with either NADPH or cytosol alone (P less than 0.001). GSH was active in stimulating the enzyme activity in the absence of cytosol, but the activity of 5'-deiodinase with 62 microM-NADPH in the presence of cytosol was significantly higher than that with 250 microM-GSH in the presence of the same concentration of cytosol (P less than 0.001). The properties of the cytosolic components essential for the NADPH-dependent activation of microsomal 5'-deiodinase independent of a glutathione/glutathione reductase system were further assessed using Sephadex G-50 column chromatography to yield three cytosolic fractions (A, B and C), wherein A represents pooled fractions near the void volume, B pooled fractions of intermediate Mr (approx. 13 000), and C of low Mr (approx. 300) containing glutathione. In the presence of NADPH (1 mM), the 5'-deiodination rate by hepatic washed microsomes is greatly increased if both A and B are added and is a function of the concentrations of A, B, washed microsomes and NADPH. A is heat-labile, whereas B is heat-stable and non-dialysable. These observations provide the first evidence of an NADPH-dependent cytosolic reductase system not involving glutathione which stimulates microsomal 5'-deiodinase of normal rat liver. The present data are consistent with a deiodination mechanism involving mediation by a reductase (other than glutathione reductase) in fraction A of an NADPH-dependent reduction of a hydrogen acceptor in fraction B, followed by reduction of oxidized microsomal deiodinase by the reduced acceptor (component in fraction B).     

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).     

Interactions of gold with cytosolic selenium-containing proteins in rat kidney and liver

Rats injected with aurothioglucose (ATG) for 5 days were subsequently injected with [75Se]selenious acid and killed after 3 days. Kidney and liver cytosols were chromatographed on Sephadex G-150. 75Se in kidney was associated with high molecular weight (HMW), 85,000 Mr, 26,000 Mr, and 10,000 Mr proteins and with a nonprotein fraction. The elution profile of liver cytosol was similar to that of kidney, but without a 26,000 Mr protein. ATG injection increased the association of 75Se with all fractions of kidney cytosol except the 85,000 Mr fractions, which contained Se-glutathione peroxidase (SeGSHPx) activity; 75Se in liver was increased only in HMW fractions. Unfractionated kidney cytosolic SeGSHPx activity was decreased 14% by ATG injection, but liver enzyme activity was not changed. However, Sephadex G-150 chromatography showed that total and specific activities, respectively, were decreased 28 and 23% in kidney and 25 and 16% in liver. Au coeluted with HMW and 10,000 Mr 73Se-containing kidney proteins; the latter contained 50% of the Au eluted from the column. DEAE Sephacel chromatography of the 10,000 Mr kidney protein showed that both Au and 75Se were tightly associated with metallothionein-like proteins. This study demonstrates the interaction of Au with rat liver and kidney 75Se-containing proteins.     

Protection against lipid peroxidation by a microsomal glutathione-dependent labile factor

Glutathione (GSH) protects rat liver microsomes against ascorbic acid (0.2 mM)/ferrous iron (10 microM)-induced lipid peroxidation for some time. The inhibitory effect of GSH is concentration-dependent (0.1-1.0 mM). Our data suggest that GSH acts by preventing initial radical formation rather than via radical scavenging or GSH--peroxidase activity. A labile GSH-dependent factor is involved in the inhibition of microsomal lipid peroxidation by GSH, inasmuch as heating the microsomes abolishes the GSH effect. We found that besides heating, lipid peroxidation also destroys the GSH-dependent factor. Consequently, continuous radical stress will produce lipid peroxidation, despite the presence of GSH. Moreover, a detrimental effect of in vivo-induced lipid peroxidation (CCl4-treatment) on the GSH-dependent factor was observed. The implications of the present data for the genesis of and the protection against peroxidative damage are discussed.     

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.     

Cytosol components from human placenta and rat liver in iodothyronine 5- and 5'-deiodination

Using either human placental microsomal 5-deiodinase as enzyme (5-DI) and thyroxine as substrate or rat liver (RL) microsomal 5'-deiodinase (5'DI) as enzyme and reverse [(3'- or 5'-)-125I]triiodo-L-thyronine ([125I]rT3) as substrate, activation of 5'-DI in the presence of NADPH was observed using either human placental or rat liver cytosolic components, but there was no activation of 5-DI. Both could be activated by DTT, with higher concentrations being required for 5-DI than for 5'-DI. Iopanoic acid, dicumarol, and sodium arsenite inhibited 5'-DI and 5-DI activated by DTT. In the presence of DTT, 1 mM 6-propyl-2-thiouracil had no effect on 5-DI but inhibited 5'DI. Thus, human placental and rat liver cytosolic components are interchangeable in activating hepatic 5'-DI in the presence of NADPH. However, if an endogenous cofactor system involved in the activation of human placental 5-DI exists, it probably differs from the activator of liver 5'-DI.     

Purification and characterization of a cytosolic protein enhancing GSH-dependent microsomal iodothyronine 5'-monodeiodination

A protein has been purified from rat liver cytosol which promoted GSH-responsive iodothyronine 5'-deiodinase activities in rat kidney microsomes. The factor behaved as a basic protein with an Mr of 11,000. It was active as a GSH-disulfide transhydrogenase with beta-hydroxyethyl disulfide as an acceptor and was also active in stimulating calf thymus ribonucleotide reductase with one-third the potency of native calf thymus glutaredoxin. Another basic protein, which degraded iodothyronines oxidatively, was also identified in the cytosolic preparations; this co-purified with soluble protein factor in the earlier purification stages and was partially separated from this factor by CM-cellulose chromatography. The glutaredoxin-like protein present in rat liver and kidney cytosol could provide a physiologic regulatory mechanism for GSH-dependent 5'-monodeiodination of iodothyronines.     

Reduction of disulphide bonds in proteins mixed disulphides catalysed by a thioltransferase in rat liver cytosol.

The reduction of mixed disulphides of some proteins and GSH [Protein(-SSG)n] is accomplished with GSH as a reductant and a thioltransferase from rat liver as a catalyst, thus: See article. The spontaneous reaction is negligible in comparison with the enzymic reaction in vivo, and any direct reduction with glutathione reductase is not detectable with the substrates used. The reduction is only indirectly dependent on NADPH, which is required for the regeneration of GSH from GSSG. Other protein disulphides apparently are reduced via analogous GSH-dependent reactions     

4-Hydroxy-2,3-trans-nonenal stimulates microsomal lipid peroxidation by reducing the glutathione-dependent protection

Glutathione (GSH) protects liver microsomes against lipid peroxidation. This is probably due to the reduction of vitamin E radicals by GSH, a reaction catalyzed by a membrane-bound protein. Pretreatment of liver microsomes with 0.1 or 1mM 4-hydroxy-2,3-trans-nonenal (HNE), a major product of lipid peroxidation, reduces the GSH-dependent protection. GSH and vitamin E concentrations are not affected by this pretreatment. Pretreatment with 0.1 mM N-ethyl maleimide (NEM), a synthetic sulfhydryl reagent, resulted in a reduction similar to that with HNE of the GSH-dependent protection against lipid peroxidation. The reduction of the GSH-dependent protection by HNE and NEM is probably the result of inactivation of the membrane-bound protein by covalent binding to an essential SH group on the protein. If the GSH-dependent protection would proceed via the microsomal GSH transferase, pretreatment with NEM, which activates the microsomal GSH transferase, should enhance the GSH-dependent protection. Actually a decrease in the GSH-dependent protection is found. Apparently the GSH-dependent protection does not proceed via the microsomal GSH transferase. Also the microsomal phospholipase A2 is not involved, since addition of 0.1 mM mepacrine, an inhibitor of phospholipase A2, did not preclude the GSH-dependent protection. Once the process of lipid peroxidation, either in vivo or in vitro, has started, the protection of liver microsomes by GSH is less effective. This might be the result of formed HNE. In this way an endproduct of lipid peroxidation stimulates the process that generates this product.     

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and beta-mercaptoethanol, with concentrations of 10 mM inhibiting by approximately 40%. DTT's inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [(3)H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.     

Characterization and physiological function of glutathione reductase in Euglena gracilis z.

The purified glutathione reductase was homogeneous on polyacrylamide-gel electrophoresis. It had an Mr of 79,000 and consisted of two subunits with a Mr of 40,000. The activity was maximum at pH 8.2 and 52 degrees C. It was specific for NADPH but not for NADH as the electron donor; the reverse reaction was not observed. The Km values for NADPH and GSSG were 14 and 55 microM respectively. The enzyme activity was markedly inhibited by thiol inhibitors and metal ions such as Hg2+, Cu2+ and Zn2+. Euglena cells contained total glutathione at millimolar concentration. GSH constituted more than 80% of total glutathione in Euglena under various growth conditions. Glutathione reductase was located solely in cytosol, as were L-ascorbate peroxidase and dehydroascorbate reductase, which constitute the oxidation-reduction cycle of L-ascorbate [Shigeoka et al. (1980) Biochem. J. 186, 377-380]. These results indicate that glutathione reductase functions to maintain glutathione in the reduced form and to accelerate the oxidation-reduction of L-ascorbate, which scavenges peroxides generated in Euglena cells.     

The heat-stable cytosolic factor that promotes glucocorticoid receptor binding to DNA is neither thioredoxin nor ribonuclease

Treatment of rat liver cytosol containing temperature-transformed [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). However, transformed receptors that are treated with MMTS and then separated from low Mr components of cytosol by passage through a column of Sephadex G-50 have very little DNA-binding activity when DTT is added to regenerate sulfhydryl moities. The receptors will bind to DNA if whole liver cytosol or boiled liver cytosol is added in addition to DTT. The effect of boiled cytosol is mimicked by purified rat thioredoxin or bovine RNase A in a manner that does not reflect the reducing activity of the former or the catalytic activity of the latter. This suggests that the reported ability of each of these heat-stable peptides to stimulate DNA binding by glucocorticoid receptors is not a biologically relevant action. We suggest that stimulation of DNA binding of partially purified receptors by boiled cytosol does not constitute a reconstitution of a complete cytosolic system in which the dissociated receptor must associate with a specific heat-stable accessory protein required for DNA binding, as has been suggested in the "two-step" model of receptor transformation recently proposed by Schmidt et al. (Schmidt T.J., Miller-Diener, A., Webb M.L. and Litwack G. (1985) J. biol. Chem. 260, 16255-16262).     

Microsomal glutathione-dependent protection against lipid peroxidation acts through a factor other than glutathione peroxidase and glutathione S-transferase in rat liver

Ascorbate-Fe3+-induced and NADPH-induced lipid peroxidation of rat liver microsomes were inhibited by glutathione (GSH). This inhibition was due to microsomal GSH-dependent factor. This factor was heat labile, and storage of microsomes at 4 degrees C for 1 week diminished the activity. GSH could not be substituted by other sulfhydryl compounds tested. Deoxycholate (1 mM) and bromosulfophthalein (0.1 mM) inhibited GSH-dependent protection but did not inhibit microsomal GSH peroxidase activity. Iodoacetate (10 mM) inhibited GSH-dependent protection but did not inhibit microsomal GSH S-transferase. N-Ethylmaleimide (0.1 mM) and oxidized glutathione (10 mM) inhibited GSH-dependent protection but activated microsomal GSH S-transferase activity. These results indicate the existence of a heat-labile, microsomal GSH-dependent protective factor against lipid peroxidation that acts through a factor other than GSH-peroxidase and GSH S-transferase.     

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and β-mercaptoethanol, with concentrations of 10 mM inhibiting by ∼40%. DTT’s inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [³H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.     

Separation and characterization of receptor-translocation inhibitors from AH 130 tumor cells

The objective of this investigation was to study the relationship between glucocorticoid resistance and macromolecular receptor-translocation inhibitors ( MTIs ). MTIs in various cytoplasmic preparations are known to inhibit the "activated" receptor-steroid complex association with isolated nuclei, chromatin, or DNA. It was found that the MTI in the cytosol of AH 130 tumor cells (glucocorticoid resistant cells) appeared to be about 5 times more inhibitory than crude MTI from rat liver. Another difference between these MTI preparations was that ATP decreased the inhibition by crude MTI from rat liver, but had little effect on that of MTI from the tumor cells. Both preparations gave three fractions of material with inhibitory activity on DEAE-cellulose chromatography. The first fraction (Peak I), eluted with about 0.1 M NaCl, was the largest fraction separated from the tumor cytosol, but a minor fraction of that from liver. In the presence of 5 mM ATP, Peak I from rat liver enhanced nuclear binding, but that from the tumor did not, suggesting that these fractions were qualitatively different. The other two fractions (Peak II and Peak III), eluted with about 0.2 M and 0.3 M NaCl, respectively, were comparable in the two preparations.     

Selective elution of rodent glutathione S-transferases and glyoxalase I from the S-hexyglutathione-Sepharose affinity matrix.

1. The major hepatic glutathione S-transferases (GSTs) from gerbil, guinea-pig, hamster, mouse and rat comprise Ya- (Mr 25,500-25,800), Yb- (Mr 26,100-26,400), Yc- (Mr 27,000-27,500) and Yf- (Mr 24,800) type subunits. 2. In all rodent species the GST subunits possess characteristic affinities for S-hexyglutathione-Sepharose and are eluted at distinct positions when a gradient of counter-ligand is employed to develop this affinity gel. The enzymes that bind to this matrix can be eluted, according to their subunit composition, in the order Ya-, Yc-, Yf- and Yb-containing GST; glyoxalase I, also retained by S-hexylglutathione-Sepharose, is eluted after the major GST YbYb peak. 3. Conditions are also described for the isocratic affinity elution of S-hexylglutathione-Sepharose that allow rat GST to be divided into four separate fractions (pools 1-4). A further fraction (pool 5) can be prepared from material that does not bind S-hexylglutathione-Sepharose and is obtained by chromatography on glutathione-Sepharose. 4. The sequential use of S-hexylglutathione-Sepharose and glutathione-Sepharose has facilitated the isolation of novel GSTs by enriching the various affinity-purified fractions with different subunits. This strategy allowed the Yk (Mr 25,000) and Yo (Mr 26,500) subunits from rat testis as well as Y1 (Mr 25,700) from rat kidney to be rapidly purified. 5. The binding properties of GST subunits for S-hexylglutathione-Sepharose have been compared with their Km values for GSH. The elution order from this matrix is inversely related to the Km value. The GSTs that do not bind to S-hexylglutathione-Sepharose have considerably higher Km values for GSH (i.e. greater than 2.0 mM) than do those enzymes that readily bind to the affinity gel (i.e. 0.13-0.77 mM). GST YkYk and YoYo, which have weak affinities for S-hexylglutathione-Sepharose, possess intermediate Km values for GSH of 1.0 and 1.2 mM respectively.     

Altered thiol group status in the heart ornithine decarboxylase inactivated following perfusion with t-butylhydroperoxide

1. The perfusion for 15 min of isolated rat hearts with 100 microM t-butylhydroperoxide leads to a 75% diminuition of the tissue GSH/GSSG ratio. 2. After t-butylhydroperoxide infusion, the isoproterenol-stimulated heart ODC was strongly inhibited. The addition of 2 mM DTT in the assaying buffer removed the ODC inactivation. 3. The inhibited ODC had an eluition profile similar to active ODC when chromatographed on a Sephacryl S-200 column; moreover, the ODC activity recovered after a thiol affinity chromatography as unbound fraction, was two times increased in the t-butylhydroperoxide perfused hearts in comparison to control. 4. The hearts perfused with 1 mM acetylcysteine after 15 min of perfusion with t-butylhydroperoxide recovered almost completely the initial ODC activity.     

FAD and GSH participate in macrophage synthesis of nitric oxide.

Following partial purification of macrophage nitric oxide (NO) synthase, enzyme activity requires L-arginine, NADPH, and constitutive cytosolic factors, one of which is tetrahydrobiopterin (BH4) (Kwon, N.S., Nathan, C.F. and Stuehr, D.J. [1989] J. Biol. Chem. 264, 20496). Here we identify FAD and GSH as two additional cofactors needed for full enzyme activity. With all defined cytosolic cofactors in excess, NO synthesis was linear over 3 h and was approximately 50% dependent on exogenous FAD, approximately 50% on glutathione (GSH), 84% on tetrahydrobiopterin (BH4), 95% on NADPH, and 98% on L-arginine. The concentrations of added FAD, GSH, and BH4 required for optimal activity were consistent with their levels in macrophage cytosol. Kinetic studies showed that GSH (or DTT) had little or no effect on the rate of NO generation over the first 20-30 min of the reaction, but prevented a subsequent dropoff in rate. This effect was distinct from thiol participation in BH4 regeneration. In contrast, exogenous FAD doubled the rate of NO synthesis throughout the assay period, consistent with a cofactor role. The role of NADPH was not to regenerate BH4, furnish NADP+, nor form reactive oxygen intermediates. These findings demonstrate NO synthesis by a partially purified enzyme in an otherwise defined system, and suggest that an NADPH-utilizing FAD flavoprotein may participate in the reaction.     

Possible pathways of lipid peroxide reduction in the liver and their intracellular localization

Lipid peroxidase activity in rat liver was studied. Rat liver cytosolic fraction was found to be capable of reducing lipid hydroperoxides. On the contrary, no lipid hydroperoxide reduction was observed in microsomes. It was found that at least two proteins in rat liver cytosol are capable of reducing phospholipid hydroperoxides. One of them is precipitated by 33-55% at (NH4)2SO4 saturation and requires reduced glutathione (GSH) as a hydrogen donor, while the other one is precipitated by 55-80% at (NH4)2SO4 saturation and reduces phospholipid hydroperoxides in the presence of a unidentified low molecular weight cytosolic factor, but not GSH or NADPH.