The enzymic oxidation of glutathione in rat liver homogenates.
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ABSTRACT:
SUBMITTER: Jocelyn PC
PROVIDER: S-EPMC1179054 | biostudies-other | 1970 May
REPOSITORIES: biostudies-other
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Project description:GSH added to rat liver homogenates inhibited respiration and increased GSSG formation approximately proportionally to the amount of GSH added; the effect was increased by added magnesium chloride. Added NADPH and citrate decreased GSSG formation and increased respiration; 6.0mm-nicotinamide prevented GSSG formation and increased respiration. There was a negative correlation between GSSG formation and oxygen uptake. It is suggested that the decrease in oxygen uptake is mainly due to GSSG concentration and also that in vivo a GSH-GSSG steady state occurs.
| S-EPMC1184788 | biostudies-other
Project description:1. Changes in liver glutathione reductase and glutathione peroxidase activities in relation to age and sex of rats were measured. Oxidation of GSH was correlated with glutathione peroxidase activity. 2. Glutathione reductase activity in foetal rat liver was about 65% of the adult value. It increased to a value slightly higher than the adult one at about 2-3 days, decreased until about 16 days and then rose after weaning to a maximum at about 31 days, finally reaching adult values at about 45 days old. 3. Weaning rats on to an artificial rat-milk diet prevented the rise in glutathione reductase activity associated with weaning on to the usual diet high in carbohydrate. 4. In male rats glutathione peroxidase activity in the liver increased steadily up to adult values. There were no differences between male and female rats until sexual maturity, when, in females, the activity increased abruptly to an adult value that was about 80% higher than that in males. 5. The rate of GSH oxidation in rat liver homogenates increased steadily from 3 days until maturity, when the rate of oxidation was about 50% higher in female than in male liver. 6. In the liver a positive correlation between glutathione peroxidase activity and GSH oxidation was found. 7. It is suggested that the coupled oxidation-reduction through glutathione reductase and glutathione peroxidase is important for determining the redox state of glutathione and of NADP, and also for controlling the degradation of hydroperoxides. 8. Changes in glutathione reductase and glutathione peroxidase activities are discussed in relation to the redox state of glutathione and NADP and to their effects on the concentration of free CoA in rat liver and its possible action on ketogenesis and lipogenesis.
| S-EPMC1187646 | biostudies-other
Project description:The presence of the enzyme bilirubin oxidase, which degrades bilirubin in vitro, was demonstrated in the liver. Subcellular-fractionation experiments indicate that bilirubin oxidase is located in both the inner and outer membranes of the mitochondria. The mean rate of the reaction is 1.57 +/- 0.38 (S.D.) nmol of bilirubin degraded/min per mg of mitochondrial protein (munits/mg of protein). With respect to the overall breakdown of bilirubin, the enzyme has a Km' of 136 microM-bilirubin and a Vmax.' of 9.13 munits/mg of protein. Its activity is influenced by the ionic strength of the media and is inhibited by KCN, thiol reagents, NADH and albumin. The enzyme is aerobic, and between 1 and 1.5 mol of O2 are consumed per mol of bilirubin degraded. The products of the reaction include propentdyopents. The hepatic bilirubin oxidase activity of the jaundiced Gunn-rat liver is not significantly different from that of the Sprague-Dawley rat, and it is not induced by beta-naphthoflavone.
| S-EPMC1146892 | biostudies-other
Project description:1. A new method was used to diminish the autoxidation of GSH. 2. The oxidation of GSH by liver homogenates was studied with regard to concentration of homogenate, concentration of GSH, time, pH and anaerobiosis. 3. GSH was oxidized by recombinations of the supernatant with microsomes and with mitochondria. Each fraction alone caused little oxidation. 4. Proteins in the supernatant were required to obtain the effect, and low-molecular-weight compounds in the same fraction increased its effect. 5. GSH diminished the formation of malonaldehyde in homogenates. 6. GSH prevented a stimulating effect of the supernatant on the formation of malonaldehyde in microsomes and in mitochondria. 7. The malonaldehyde formation in microsomes together with the supernatant did not start until the concentration of endogenous low-molecular-weight thiols had decreased to a low level. 8. It is suggested that part of the oxidation of GSH in homogenates is coupled to a mechanism that counteracts the peroxidation of membrane lipids.
| S-EPMC1265098 | biostudies-other
Project description:1. Rat liver mitochondria incubated in oxygen with glutathione and [(35)S]-thiosulphate produced labelled sulphate. 2. Inner-labelled thiosulphate (S.(35)SO(3))(2-) was converted into [(35)S]sulphate more rapidly than outer-labelled thiosulphate ((35)S.SO(3))(2-). 3. Thiosulphate labelled in both sulphur atoms was formed during ((35)S.SO(3))(2-) oxidation; the outer sulphur atom before oxidation to sulphate was incorporated into the inner position. 4. A thiosulphate cycle in the metabolic pathway of sulphate formation in animal tissues is discussed.
| S-EPMC1270484 | biostudies-other
Project description:1. The rate and stability to aging of the metabolism of propionate by sheep-liver slices and sucrose homogenates were examined. Aging for up to 20min. at 37 degrees in the absence of added substrate had little effect with slices, whole homogenates or homogenates without the nuclear fraction. 2. Metabolism of propionate by sucrose homogenates was confined to the mitochondrial fraction, but the mitochondrial supernatant (microsomes plus cell sap) stimulated propionate removal. 3. The rate of propionate metabolism by liver slices was higher in a high potassium phosphate-bicarbonate medium [0.88(+/-s.e.m. 0.16)mumole/mg. of N/hr.] than in Krebs-Ringer bicarbonate medium [0.44(+/-s.e.m. 0.13)mumole/mg. of N/hr.]. 4. Metabolism of propionate by sucrose homogenates freed from nuclei was dependent on the presence of oxygen, carbon dioxide and ATP. Propionate removal was stimulated 250% by Mg(2+) ions and 670% by cytochrome c. 5. In the complete medium 2.39(+/-s.e.m. 0.15)mumoles of propionate were consumed/mg. of N/hr. 6. The ratio of oxygen consumption to propionate utilization was sufficient to account for the complete oxidation of half the propionate consumed. 7. The only products detected under these conditions were succinate, fumarate and malate. Propionate had no effect on the production of lactate from endogenous sources and did not itself give rise to lactate. 8. Methylmalonate did not accumulate when propionate was metabolized and was not oxidized. It was detected as an intermediate in the conversion of propionyl-CoA into succinate. The rate of this reaction sequence was adequate to account for the rate of propionate metabolism by sucrose homogenates or slices, provided that the rate of formation of propionyl-CoA was not limiting. 9. The methylmalonate pathway was predominantly a mitochondrial function. 10. The metabolism of propionate appeared to be dependent on active oxidative phosphorylation.
| S-EPMC1214338 | biostudies-other
Project description:1. The aerobic loss of GSH added to the supernatant fraction from rat liver is much increased by including the microsome fraction, which both inhibits the concurrent reduction of the GSSG formed and also augments the net oxidation rate. 2. Oxidation occurs with a mixture of dialysed supernatant and a protein-free filtrate; the latter is replaceable by hypoxanthine and the former by xanthine oxidase, whereas fractions lacking this enzyme give no oxidation. 3. In all these instances augmentation occurs with microsomes, with fractions having urate oxidase activity and with the purified enzyme; uric acid and microsomes alone also support the oxidation. 4. Evidence implicating additional protein factors is discussed. 5. It is suggested that GSH oxidation by homogenate is linked through glutathione peroxidase to the reaction of endogenous substrate with supernatant xanthine oxidase and of the uric acid formed with peroxisomal urate oxidase.
| S-EPMC1179055 | biostudies-other
Project description:Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxoglutarate carriers (OGC; Slc25a11). To determine whether these carriers function similarly in liver mitochondria, we assessed the effect of competition with specific substrates or inhibitors on GSH uptake in isolated rat liver mitochondria. GSH uptake was uniphasic, independent of ATP hydrolysis, and exhibited K(m) and V(max) values of 4.08 mM and 3.06 nmol/min per mg protein, respectively. Incubation with butylmalonate and phenylsuccinate inhibited GSH uptake by 45-50%, although the individual inhibitors had no effect, suggesting in rat liver mitochondria, the DIC and OGC are only partially responsible for GSH uptake. H4IIE cells, a rat hepatoma cell line, were stably transfected with the cDNA for the OGC, and exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by H(2)O(2), methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver.
| S-EPMC3840356 | biostudies-literature
Project description:Oral administration of glutathione has been demonstrated to reduce exercise-induced fatigue and improve liver function, although glutathione can be synthesized in the liver. However, little is known about the underlying mechanism of this effect. To address this, the status of food-derived glutathione in the intestine, blood, and liver was examined. Glutathione-1-13C or N-acetyl-cysteine-1-13C (NAC) was orally administered to rats (50 mg/kg). Food-derived glutathione contents within tissues were estimated by subtracting endogenous glutathione-1-13C from the total glutathione-1-13C. Food-derived glutathione was present in rat intestines and livers (approximately 60 and 300 μmol/kg, respectively, 120 min after ingestion) in electrochemically reduced form, while all food-derived glutathione in the blood plasma was conjugated with proteins and low-molecular-weight thiol compounds. However, no significant amounts of NAC-derived glutathione were detected in the blood plasma. These findings indicate that food-derived glutathione is directly absorbed in its electrochemically reduced form in the intestine, is then transported in the blood in bound forms, and is finally deposited into the liver in reduced form. Therefore, upon entering the bloodstream, food-derived glutathione binds to thiol compounds and releases hydrogen atom; subsequently, it does the reverse upon incorporation into the liver, which might impact the physiological redox condition. With respect to food-derived glutathione and cysteine-containing peptides, this study provides new insights on their modes of transportation and mechanisms of action.
| S-EPMC6550177 | biostudies-literature
Project description:To assess whether the synthesis of haem can be studied in small amounts of human liver, we measured kinetics of the conversion of 5-aminolaevulinate into haem and haem precursors in homogenates of human livers. We used methods previously developed in our laboratory for studies of rat and chick-embryo livers [Healey, Bonkowsky, Sinclair & Sinclair (1981) Biochem. J. 198, 595-604]. The maximal rate at which homogenates of human livers converted 5-aminolaevulinate into protoporphyrin was only 26% of that for rat, and 58% of that for chick embryo. In the absence of added Fe2+, homogenates of fresh human liver resembled those of chick embryos in that protoporphyrin and haem accumulated in similar amounts, whereas fresh rat liver homogenate accumulated about twice as much haem as protoporphyrin. However, when Fe2+ (0.25 mM) was added to human liver homogenates, mainly haem accumulated, indicating that the supply of reduced iron limited the activity of haem synthase, the final enzyme in the haem-biosynthesis pathway. Addition of the potent iron chelator desferrioxamine after 30 min of incubation with 5-amino[14C]laevulinate stopped further haem synthesis without affecting synthesis of protoporphyrin. Thus the prelabelled haem was stable after addition of desferrioxamine. Since the conversion of 5-amino[14C]laevulinate into haem and protoporphyrin was carried out at pH 7.4, whereas the pH optimum for rat or bovine hepatic 5-aminolaevulinate dehydratase is about 6.3, we determined kinetic parameters of the human hepatic dehydrase at both pH values. The Vmax was the same at both pH values, whereas the Km was slightly higher at the lower pH. Our results indicate that the synthesis of porphyrins and haem from 5-aminolaevulinate can be studied with the small amounts of human liver obtainable by percutaneous needle biopsy. We discuss the implications of our results in relation to use of rat or chick-embryo livers as experimental models for the biochemical features of human acute porphyria.
| S-EPMC1144919 | biostudies-other