Activation of rat liver microsomal glutathione S-transferase by reduced oxygen species

The effect of enzymatically generated reduced oxygen metabolites on the activity of hepatic microsomal glutathione S-transferase activity was studied to explore possible physiological regulatory mechanisms of the enzyme. Noradrenaline and the microsomal cytochrome P-450-dependent monooxygenase system were used to generate reduced oxygen species. When noradrenaline (greater than 0.1 mM) was incubated with rat liver microsomes in phosphate buffer (pH 7.4), an increase in microsomal glutathione S-transferase activity was observed, and this activation was potentiated in the presence of a NADPH-generating system; the glutathione S-transferase activity was increased to 180% of the control with 1 mM noradrenaline and to 400% with both noradrenaline and NADPH. Superoxide dismutase and catalase inhibited partially the noradrenaline-dependent activation of the enzyme. In the presence of dithiothreitol and glutathione, the activation of the glutathione S-transferase by noradrenaline, with or without NADPH, was not observed. In addition, the activation of glutathione S-transferase activity by noradrenaline and glutathione disulfide was not additive when both compounds were incubated together. These results indicate that the microsomal glutathione S-transferase is activated by reduced oxygen species, such as superoxide anion and hydrogen peroxide. Thus, metabolic processes that generate high concentrations of reduced oxygen species may activate the microsomal glutathione S-transferase, presumably by the oxidation of the sulfhydryl group of the enzyme, and this increased catalytic activity may help protect cells from oxidant-induced damage.     

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).     

Activation of rat liver microsomal glutathione S-transferase by hydrogen peroxide: role for protein-dimer formation.

The mechanism of oxygen radical-dependent activation of hepatic microsomal glutathione S-transferase by hydrogen peroxide was studied. Glutathione S-transferase activity in liver microsomes was increased 1.5-fold by incubation with 0.75 mM hydrogen peroxide at 37 degrees C for 10 min, and the increase in activity was reversed by incubation with dithiothreitol. Purified glutathione S-transferase was also activated by hydrogen peroxide after incubation at room temperature, and the increase in the activity was also reversed by dithiothreitol. Immunoblotting with anti-microsomal glutathione S-transferase antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hydrogen peroxide-treated microsomes or purified glutathione S-transferase revealed the presence of a glutathione S-transferase dimer. These results indicate that the hydrogen peroxide-dependent activation of the microsomal glutathione S-transferase is associated with the formation of a protein dimer.     

Melatonin and N-acetyl serotonin inhibit selectively enzymatic and non-enzymatic lipid peroxidation of rat liver microsomes

Melatonin (N-acetyl-5-methoxytryptamine) and its immediate precursor N-acetyl serotonin in the metabolism of tryptophan are free radical scavengers that have been found to protect against non-enzymatic lipid peroxidation in many experimental models. By contrast, little is known about the antioxidant ability of these indoleamines against NADPH enzymatic lipid peroxidation. The light emission produced by rat-liver microsomes, expressed as total cpm during 180 min of incubation at 37 degrees C, was two-fold greater in the presence of ascorbate (0.4mM) when compared with NADPH (0.2 mM). Maximal peaks of light emission produced by microsomes lipid peroxidized with ascorbic-Fe(2+) or NADPH and expressed as cpm were 354,208 (at 60 min) and 135,800 (at 15 min), respectively. During non-enzymatic lipid peroxidation a decrease of total chemiluminescence (inhibition of lipid peroxidation) was observed when increasing concentrations of melatonin were added to liver microsomes. The protective effect was concentration-dependent. The inhibition observed in light emission was coincident with the protection of the most PUFAs. Preincubation of microsomes with N-acetyl serotonin reduced these changes very dramatically. Thus, in the presence of both antioxidants (0.36, 0.75, 1.5 mM), light emission percent inhibition during non-enzymatic (ascorbate-Fe(2+)) lipid peroxidation of rat liver microsomes was for melatonin: 6.12, 16.20, 34.88 and for N-acetyl serotonin: 85.10, 88.48, 84.4 respectively. The incubation of rat liver microsomes in the presence of NADPH (0.36, 0.75, 1.5 mM) produce a sudden increase of chemiluminescence that gradually increased and reached a maximal value at about 15 min; however, N-acetyl serotonin reduced these changes very efficiently.     

The in vitro NADPH-dependent inhibition by CCl4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl3.radical

The hepatotoxicity of CCl4 is mediated through its initial reduction by cytochrome P-450 to the CCl3.radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca2+ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCl4 (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca2+ uptake from 28% without CCl4 to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals (Dambrauskas, T., and Cornish, H. H. (1970) Toxicol. Appl. Pharmacol. 17, 83-97; Long, R.M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306) and are toxic to cultured hepatocytes (Long, R. M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306). The inhibition of Ca2+ uptake was due both to a decrease in the Ca2(+)-dependent ATPase and to an enhanced release of Ca2+ from the microsomes. The NADPH-dependent CCl4 inhibition was greater under N2 and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl4 prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl4, the CCl4 metabolism was stopped, and then the Ca2+ uptake was determined; GSH reversed the CCl4 inhibition of Ca2+ uptake. This reversal showed saturation kinetics for GSH with two Km values of 0.315 and 93 microM when both the preincubation and the Ca2+ uptake were performed under air, and 0.512 and 31 microM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl4 inhibition of Ca2+ uptake. CCl4 increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove 14C bound to samples preincubated with the 14CCl4. Although EDTA (100 microM) decreased the CCl4 inhibition, the metal-complexing agents deferoxamine (100 microM) and diethyldithiocarbamate (100 microM) had no effect on the inhibition of the pump. Similarly, the reactive oxygen scavengers catalase (65 micrograms/ml), superoxide dismutase (15 micrograms/ml), mannitol (10 mM), and dimethyl sulfoxide (50 mM) also had no effect. Our results suggest that the initial toxicity of CCl4 for the Ca2+ pump results from the metabolism of CCl4 to the CCl3. radical. This radical then directly oxidizes the Ca2+ pump, leading to decreased Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of cAMP-dependent protein kinase and ATP on N1-oxidation of 9-benzyladenine by animal hepatic microsomes

N1-Oxidation is a major metabolic pathway for 9-benzyladenine (BA) catalyzed by the cytochrome P450 system in animal hepatic microsomes. After normal hamster hepatic microsomes or phenobarbital induced rabbit hepatic microsomes were preincubated in the presence of cyclic AMP-dependent protein kinase catalytic subunit (PKA), MgCl2 and ATP, BA-N1-oxidation was significantly decreased. However, further investigation indicated that the decrease of BA-N1-oxidation seemed to be a combination of the effects of PKA and ATP, as ATP alone showed a biphasic regulatory effect on BA-N1-oxidation when microsomes were preincubated in the presence of various concentrations of ATP. In the lower ATP concentration range (0.5-2.5mM), BA-N1-oxidation increased along with the increase of ATP concentration; whereas BA-N1-oxidation decreased when the ATP concentration was higher (>5mM). The biphasic regulatory effects of ATP on BA-N1-oxidation seem dependent on the incubation process, as preincubation markedly strengthened the effects. When microsomes were incubated at 37 degrees C for different time lengths in the absence or presence of ATP (2.5 or 20mM), the activity of BA-N1-oxidase decreased at similar rates in all groups, but the activity levels of BA-N1-oxidase were different among the groups. The cytochrome P450 content was not changed parallel to the variation of BA-N1-oxidation when microsomes were incubated in the presence of ATP, indicating that the effects of ATP on BA-N1-oxidation were not mediated by affecting CYP stability. In addition, the activity of NADPH-cytochrome P450 reductase was not markedly affected by ATP without incubation. The result implied that ATP did not inhibit the reductase directly. After microsomes were incubated in the presence of low ATP concentration (2.5mM), the reductase was slightly inhibited, whilst high ATP concentration (20mM) showed marked inhibition (83% of control). This may partially contribute to the down-regulatory effect of ATP on BA-N1-oxidation. Furthermore, it was found that the presence of magnesium ions during preincubation weakened the up-regulatory effect of ATP (2.5mM) on BA-N1-oxidation, but showed no effect on the down-regulatory effect of ATP (20mM). Since these observed phenomena are not readily explained, a possible mechanism, i.e. phosphorylation and dephosphorylation of cytochrome P450, is suggested.     

Calcium sequestration activity in rat liver microsomes. Evidence for a cooperation of calcium transport with glucose-6-phosphatase

Mechanisms regulating the energy-dependent calcium sequestering activity of liver microsomes were studied. The possibility for a physiologic mechanism capable of entrapping the transported Ca2+ was investigated. It was found that the addition of glucose 6-phosphate to the incubation system for MgATP-dependent microsomal calcium transport results in a marked stimulation of Ca2+ uptake. The uptake at 30 min is about 50% of that obtained with oxalate when the incubation is carried out at pH 6.8, which is the pH optimum for oxalate-stimulated calcium uptake. However, at physiological pH values (7.2-7.4), the glucose 6-phosphate-stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. The Vmax of the glucose 6-phosphate-stimulated transport is 22.3 nmol of calcium/mg protein per min. The apparent Km for calcium calculated from total calcium concentrations is 31.9 microM. After the incubation of the system for MgATP-dependent microsomal calcium transport in the presence of glucose 6-phosphate, inorganic phosphorus and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. In the system for the glucose 6-phosphate-stimulated calcium uptake, glucose 6-phosphate is actively hydrolyzed by the glucose-6-phosphatase activity of liver microsomes. The latter activity is not influenced by concomitant calcium uptake. Calcium uptake is maximal when the concentration of glucose 6-phosphate in the system is 1-3 mM, which is much lower than that necessary to saturate glucose-6-phosphatase. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose-6-phosphatase multicomponent system. The physiological implications of such a cooperation are discussed.     

The mechanism of calcium uptake by liver microsomes: effect of anions and ionophores

The mechanism of calcium uptake by liver microsomes was investigated using various anions and ionophores. Calcium uptake was shown to be specific to microsomes and unlikely to be due to contamination by plasma membranes by correlation of calcium uptake to the marker enzymes specific for these two fractions. Under the conditions employed, phosphates, sulfate, chloride, acetate, nitrate, thiocyanate, maleate, succinate and oxalate all stimulated calcium uptake by microsomes, but to different degrees. The greatest effect (4-6-fold) was observed with phosphate. On the contrary, phosphate is the only anion that stimulates the plasma membrane calcium uptake to any significant degree. Treatment of isolated microsomes with 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) resulted in inhibition of ATP- and anion-dependent calcium uptake. A lipid-permeable organic acid such as maleate retained its ability to promote calcium uptake in DIDS-treated microsomes. However, a lipophilic anion, such as nitrate, stimulated calcium uptake only in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). In addition, 2 microM valinomycin, when added in the absence or presence of 10 to 100 mM K+, had no stimulatory effect on calcium uptake. These results appear to be consistent with a model in which the active uptake of calcium into microsomes involves electroneutral Ca2+-nH+ exchange.     

Metabolic activation of hexamethylmelamine and pentamethylmelamine by liver microsomal preparations

Incubation of [¹⁴C]-ring labeled hexamethylmelamine and pentamethylmelamine with rat and mouse liver microsomal preparations results in metabolic activation of both drugs as measured by covalent binding of radiolabel to acid-precipitable microsomal macromolecules. Covalent binding is dependent on viable microsomes, NADPH, and molecular oxygen. Binding of HMM (280 pmol/mg protein/15 min) was approximately 5 times greater than that observed for PMM (60 pmol/mg protein/15 min), and represents 0.22% of incubated material. Similar results were found with [¹⁴C]-methyl labeled substrates. Pretreatment with phenobarbital increased covalent binding while addition of SKF 525-A, addition of glutathione, or incubation in an 80% carbon monoxide atmosphere reduced covalent binding.     

The inhibition kinetics of yeast glutathione reductase by some metal ions

Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn2+ (up to 2 mM) and activated above this concentration. Ca2+ inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals.     

Inhibition by aldehydes as a possible further mechanism for glucose-6-phosphatase inactivation during CCl4-poisoning

Diffusable aldehydes are known to be produced during lipoperoxidative deterioration of unsaturated fatty acids. Malealdehyde (MLA) and 4-hydroxy-2,3-trans-penten-1-al (4-HPE) inhibit rat liver glucose-6-phosphatase activity in vitro. With MLA inhibition is significant at 0.25 mM concentration. With 4-HPE inhibition takes place at 0.5 mM. 1 mM MLA inhibited by about 89%, 6 mM 4-HPE by about 67%. Maximal inhibition is present as early as 5 min after addition of both aldehydes. Preincubation of aldehydes with 2 mM cysteine or glycine in the absence of microsomes almost completely prevents the inhibitory influence. Previous incubation of microsomes with 2 mM glutathione or 2 mM dithiothreitol or 2 mM cysteine affords a good protection towards the inhibitory action of the aldehydes; on the contrary, no protection is seen when microsomes are preincubated in the presence of either 2 mM glycine or asparagine. The total content of microsomes -SH groups is strongly decreased after incubation with 2mM malealdehyde.These results support the idea that the two aldehydes inhibit glucose-6-phosphatase mostly through interaction with protein -SH groups. The possibility that aldehydes derivated from the peroxidative decomposition of lipids may play a cooperative role in the inhibition of glucose-6-phosphatase occurring early after CCl₄-poisoning is discussed.     

Pyruvate protection against endothelial cytotoxicity induced by blockade of glucose uptake

We have previously demonstrated that the redox reactant pyruvate prevents apoptosis in the oxidant model of bovine pulmonary artery endothelial cells (BPAEC), and that the anti-apoptotic mechanism of pyruvate is mediated in part via the mitochondrial matrix compartment. However, cytosolic mechanisms for the cytoprotective feature of pyruvate remain to be elucidated. This study investigated the pyruvate protection against endothelial cytotoxicity when the glycolysis inhibitor 2-deoxy-D-glucose (2DG) was applied to BPAEC. Millimolar 2DG blocked the cellular glucose uptake in a concentration- and time-dependent manner with >85% inhibition at > or =5 mM within 24 h. The addition of 2DG evoked BPAEC cytotoxicity with a substantial increase in lipid peroxidation and a marked decrease in intracellular total glutathione. Exogenous pyruvate partially prevented the 2DG-induced cell damage with increasing viability of BPAEC by 25-30%, and the total glutathione was also modestly increased. In contrast, 10 mM L-lactate, as a cytosolic reductant, had no effect on the cytotoxicity and lipid peroxidation that are evoked by 2DG. These results suggest that 2DG toxicity may be a consequence of the diminished potential of glutathione antioxidant, which was partially restored by exogenous pyruvate but not L-lactate. Therefore, pyruvate qualifies as a cytoprotective agent for strategies that attenuate the metabolic dysfunction of the endothelium, and cellular glucose oxidation is required for the functioning of the cytosolic glutathione/NADPH redox system.     

Regulation of rat liver microsomal glutathione S-transferase activity by thiol/disulfide exchange

The regulation of purified glutathione S-transferase from rat liver microsomes was studied by examining the effects of various sulfhydryl reagents on enzyme activity with 1-chloro-2,4-dinitrobenzene as the substrate. Diamide (4 mM), cystamine (5 mM), and N-ethylmaleimide (1 mM) increased the microsomal glutathione S-transferase activity by 3-, 2-, and 10-fold, respectively, in absence of glutathione; glutathione disulfide had no effect. In presence of glutathione, microsomal glutathione S-transferase activity was increased 10-fold by diamide (0.5 mM), but the activation of the transferase by N-ethylmaleimide or cystamine was only slightly affected by presence of glutathione. The activation of microsomal glutathione S-transferase by diamide or cystamine was reversed by the addition of dithiothreitol. Glutathione disulfide increased microsomal glutathione S-transferase activity only when membrane-bound enzyme was used. These results indicate that microsomal glutathione S-transferase activity may be regulated by reversible thiol/disulfide exchange and that mixed disulfide formation of the microsomal glutathione S-transferase with glutathione disulfide may be catalyzed enzymatically in vivo.     

The inhibition kinetics of yeast glutathione reductase by some metal ions

Glutathione reductase (GR, type IV, Baker's yeast, E.C 1.6.4.2) is a flavoprotein that catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). In this study some metal ions have been tested on GR; lithium, manganese, molybdate, aluminium, barium, zinc, calcium, cadmium and nickel. Cadmium, nickel and calcium showed a good to moderate inhibitory effect on yeast GR. GR is inhibited non-competitively by Zn^{2 +} (up to 2 mM) and activated above this concentration. Ca^{2 +} inhibition was non-competitive with respect to GSSG and uncompetitive with respect to NADPH. Nickel inhibition was competitive with respect to GSSG and uncompetitive with respect to NADPH. The inhibition constants for these metals on GR were determined. The chelating agent EDTA recovered 90% of the GR activity inhibited by these metals.     

Functional coupling of the calcium pump and glucose 6-phosphatase activity in liver microsomes: preliminary results

The addition of G-6-Pi to the incubation system for MgATP-dependent calcium transport in liver microsomes results in a marked stimulation of Ca2+ uptake. At physiological pH values (7.2-7.4), the G-6-Pi stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. In the system for the G-6-Pi-stimulated calcium uptake, G-6-Pi is actively hydrolyzed by the glucose 6-phosphatase activity of liver microsomes. Such an activity is not influenced by the concomitant calcium uptake. After the incubation of the system for the MgATP-dependent microsomal calcium transport in the presence of G-6-Pi, Pi and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose 6-phosphatase multicomponent system.     

Diquat-induced oxidative damage in hepatic microsomes: effects of antioxidants.

The ability of the redox cycling compound, diquat, to induce lipid peroxidation and oxidative damage was investigated using hepatic microsomes. Antioxidants, with demonstrated efficacy in physical models of oxidative stress, were examined in a diquat model. Diquat (10 microM-3 mM) induced lipid peroxidation (TBARS) in hepatic microsomes prepared from Fischer 344 rats. Diquat (1 mM) also increased protein carbonyl formation, NADPH oxidation and superoxide anion radical production (acetylated cytochrome c reduction). The novel antioxidants U-74,006F, U-78,517G and the known antioxidant, DPPD, decreased diquat-induced lipid peroxidation to levels below that of the control. These antioxidants also decreased protein carbonyl formation caused by diquat. U-74,006F and U-78,517G reduced NADPH oxidation slightly; although this inhibition was statistically significant, the biological significance is questionable. DPPD had no effect on this parameter. U-78,517G inhibited the reduction of acetylated cytochrome c slightly, whereas the other antioxidants had little effect. Thus overall, the increase in NADPH oxidation and the production of superoxide anion by redox cycling of diquat were not substantially affected by antioxidants. Neither did the test compounds show evidence of activity as iron chelators. This leads to the suggestion that antioxidants are preventing diquat-induced oxidative damage by scavenging lipid peroxyl radicals and preventing the propagation of the lipid peroxidation process.     

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.     

Hormone sensitive calcium uptake by liver microsomes

The effects of glucagon and insulin on hepatic microsomal calcium uptake were investigated. Microsomes isolated from perfused rat liver accumulated calcium in the presence of ATP and oxalate. Addition of glucagon to the perfusate significantly increased calcium uptake by microsomes subsequently isolated. In contrast, addition of insulin to the perfusate resulted in a decreased microsomal calcium uptake and inhibition of the glucagon effect. Because the effects of glucagon and insulin on hepatic microsomal calcium uptake are opposite, as are the metabolic effects of these hormones, it is likely that the observed differences are of physiological importance.     

Heme Inhibition of [delta]-Aminolevulinic Acid Synthesis Is Enhanced by Glutathione in Cell-Free Extracts of Chlorella

In plants, algae, and many bacteria, the heme and chlorophyll precursor, [delta]-aminolevulinic acid (ALA), is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate and requiring ATP and NADPH as cofactors. In particulate-free extracts of algae and chloroplasts, ALA synthesis is inhibited by heme. Inclusion of 1.0 mM glutathione (GSH) in an enzyme and tRNA extract, derived from the green alga Chlorella vulgaris, lowered the concentration of heme required for 50% inhibition approximately 10-fold. The effect of GSH could not be duplicated with other reduced sulfhydryl compounds, including mercaptoethanol, dithiothreitol, and cysteine, or with imidazole or bovine serum albumin, which bind to heme and dissociate heme dimers. Absorption spectroscopy indicated that heme was fully reduced in incubation medium containing dithiothreitol, and addition of GSH did not alter the heme reduction state. Oxidized GSH was as effective in enhancing heme inhibition as the reduced form. Co-protoporphyrin IX inhibited ALA synthesis nearly as effectively as heme, and 1.0 mM GSH lowered the concentration required for 50% inhibition approximately 10-fold. Because GSH did not influence the reduction state of heme in the incubation medium, and because GSH could not be replaced by other reduced sulfhydryl compounds or ascorbate, the effect of GSH cannot be explained by action as a sulfhydryl protectant or heme reductant. Preincubation of enzyme extract with GSH, followed by rapid gel filtration, could not substitute for inclusion of GSH with heme during the reaction. The results suggest that GSH must specifically interact with the enzyme extract in the presence of the inhibitor to enhance the inhibition.     

Effect of divalent cations on NADH-dependent and NADPH-dependent cytochrome b5 reduction by hepatic microsomes

The effect of Ca2+ or Mg2+ on cytochrome b5 reduction by porcine liver microsomes was examined using trypsin-solubilized cytochrome b5 as a substrate. The reduction of exogenous cytochrome b5 by microsomes was low at 1.2 microM cytochrome b5 (3.9 or 2.7 nmol/min/mg protein, respectively, with NADH or NADPH). The addition of CaCl2 greatly enhanced either NADH-dependent or NADPH-dependent cytochrome b5 reduction. At 2 mM CaCl2, the reduction rate was increased to 23- or 18-fold of control, respectively with NADH or NADPH. The concentration for half-maximal effect (EC50) was 0.5 or 0.6 mM in the NADH or NADPH systems, respectively. MgCl2 also stimulated cytochrome b5 reduction with a EC50 value of 1.0 mM in the NADH system or 0.6 mM in the NADPH system. The comparison with the result with KCl indicated that the activation by CaCl2 or MgCl2 is caused mainly by their divalent cation moiety. The Km value for cytochrome b5 was decreased and the Vmax was increased by calcium with either the NADH- or the NADPH-dependent system. NADH-ferricyanide reductase activity was not affected by calcium, but NADPH-ferricyanide reductase activity was stimulated as well as NADPH-cytochrome c reductase activity. In the presence of Triton X-100, divalent cations were inhibitory in NADH-dependent cytochrome b5 reduction, and in contrast, stimulative in NADPH-dependent reaction. These findings suggest that the activation of cytochrome b5 reduction by divalent cations in the NADH system is mainly due to an increasing accessibility of the substrate, and in the NADPH system, in addition to this, a direct effect of divalent cations on NADPH-cytochrome P450 reductase is also involved.