The reaction of aralkyl sulphate esters with glutathione catalysed by rat liver preparations.
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ABSTRACT: 1. Rat liver supernatant preparations catalyse the reactions of some aralkyl sulphate esters with GSH to yield S-aralkylglutathione derivatives. 2. A glutathione S-transferase that catalyses these reactions has been purified 16-fold. 3. The purified enzyme preparation catalyses the release of sulphate ions from benzyl sulphate, 1-menaphthyl (naphth-1-ylmethyl) sulphate and phenanthr-9-ylmethyl sulphate only in the presence of GSH. It does not cause the release of sulphate ions from prop-1-yl sulphate, l-serine O-sulphate, phenyl sulphate or oestrone 3-sulphate even when GSH is added. 4. The stability and specificity of the enzyme and its response to inhibitors and to changes of pH were studied. 5. The activity of the preparation was compared with the activities of glutathione S-transferases described previously.
Project description:1. The glutathione S-transferase that catalyses the reaction of 1-menaphthyl (naphth-1-ylmethyl) sulphate with GSH was purified 76-fold from rat liver. 2. The properties of the purified enzyme were studied by gel filtration and isoelectric focusing. 3. The initial-velocity pattern in the absence of products and the product-inhibition pattern have been determined. These are consistent with an Ordered Bi Bi mechanism in which the GSH adds to the enzyme before 1-menaphthyl sulphate and the products are released in the order SO(4) (2-) followed by S-(1-menaphthyl)glutathione. 4. Dead-end-inhibition studies with p-aminobenzoic acid, which has been shown to be competitive with GSH and non-competitive with 1-menaphthyl sulphate, support the suggestion that an Ordered Bi Bi mechanism is operative. 5. Values were determined for some of the dissociation and Michaelis constants for the reaction of the substrates and products with the enzyme. 6. It appears that S-(1-menaphthyl)glutathione activates the enzyme when the concentration of GSH is saturating and that of 1-menaphthyl sulphate is low (of the order of its Michaelis constant).
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.
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.
Project description:N-Heterocyclic carbene catalysed redox isomerisation with reduction about the carbonyl has been developed in the transformation of trienyl esters to tetrasubstituted benzaldehydes. The reaction proceeds in good to excellent yield, and in cases that provide 2,2'-biaryls, enantioselectivity is observed. Mechanistic studies demonstrate the intermediacy of a cyclohexenyl β-lactone, while implicating formation of the homoenolate as turnover limiting.
Project description:1. The distribution of rat-liver polyribosomes in sucrose density gradients has been investigated with regard to the effects of the preparative procedures and the physiological and pathological condition of the animal. 2. By using carefully defined conditions, three principal polyribosomal fractions have been isolated with S(20,w) values of 340, 275 and 225s in addition to the dimerized 120s and single 80s ribosomes. 3. The polyribosomes were very sensitive to treatment with ribonuclease and to mechanical stresses. 4. Incubation of dispersed hepatic cells and also cell-free preparations with puromycin in the presence of ATP and phosphoenolpyruvate caused rapid partial degradation of the polyribosomes. Treatment of the dispersed cells with actinomycin D also degraded the polyribosomes. 5. The liver polyribosomes of rats not raised under pathogen-free conditions and possibly of rats with an arthritic syndrome may be more fragile than those of healthy pathogen-free animals. 6. Treatment of pathogen-free rats with drugs stimulating liver anabolism profoundly affected the distribution of polyribosomes in sucrose density gradients.
Project description:Adult male rat liver and kidney preparations were incubated with (2-hydroxyoestradiol-1-yl)[(35)S]glutathione. The glutamic acid and glycine residues were removed by enzymes present in the kidney microsomal fraction; the liver preparations had no effect. The resulting 2-hydroxyoestradiol-cysteine conjugate was acetylated at the alpha-amino group by both liver and kidney homogenates fortified with acetyl-coenzyme A, but not significantly in the absence of this coenzyme, or by liver or kidney slices. These results suggest that an oestrogen-glutathione conjugate, if formed in vivo, would be converted into the corresponding mercapturic acid before excretion.
Project description:BackgroundTissue hypoperfusion and inflammation in sepsis can lead to organ failure including kidney and liver. In sepsis, mortality of acute kidney injury increases by more than 50%. Which type of volume replacement should be used is still an ongoing debate. We investigated the effect of different volume strategies on inflammatory mediators in kidney and liver in an early sepsis model.Material and methodsAdult male Wistar rats were subjected to sepsis by cecal ligation and puncture (CLP) and assigned to three fluid replenishment groups. Animals received 30mL/kg of Ringer's lactate (RL) for 2h, thereafter RL (75mL/kg), hydroxyethyl starch (HES) balanced (25mL/kg), containing malate and acetate, or HES saline (25mL/kg) for another 2h. Kidney and liver tissue was assessed for inflammation. In vitro rat endothelial cells were exposed to RL, HES balanced or HES saline for 2h, followed by stimulation with tumor necrosis factor-α (TNF-α) for another 4h. Alternatively, cells were exposed to malate, acetate or a mixture of malate and acetate, reflecting the according concentration of these substances in HES balanced. Pro-inflammatory cytokines were determined in cell supernatants.ResultsCytokine mRNA in kidney and liver was increased in CLP animals treated with HES balanced compared to RL, but not after application of HES saline. MCP-1 was 3.5fold (95% CI: 1.3, 5.6) (p<0.01) and TNF-α 2.3fold (95% CI: 1.2, 3.3) (p<0.001) upregulated in the kidney. Corresponding results were seen in liver tissue. TNF-α-stimulated endothelial cells co-exposed to RL expressed 3529±1040pg/mL MCP-1 and 59±23pg/mL CINC-1 protein. These cytokines increased by 2358pg/mL (95% CI: 1511, 3204) (p<0.001) and 29pg/ml (95% CI: 14, 45) (p<0.01) respectively when exposed to HES balanced instead. However, no further upregulation was observed with HES saline. PBS supplemented with acetate increased MCP-1 by 1325pg/mL (95% CI: 741, 1909) (p<0.001) and CINC-1 by 24pg/mL (95% CI: 9, 38) (p<0.01) compared to RL. Malate as well as HES saline did not affect cytokine expression.ConclusionWe identified HES balanced and specifically its component acetate as pro-inflammatory factor. How important this additional inflammatory burden on kidney and liver function is contributing to the sepsis-associated inflammatory burden in early sepsis needs further evaluation.
Project description:Enantioselective synthesis of chiral isochromans bearing a terminal alkyne moiety has been accomplished by copper-catalysed enantioselective intramolecular propargylic substitution reactions of propargylic esters with alcoholic nucleophiles. This method represents the first successful example which directly introduced a terminal alkyne group into chiral isochromans.
Project description:Catalytic enantioselective transformations usually rely upon optimal enantioselectivity being observed in kinetically controlled reaction processes, with energy differences between diastereoisomeric transition state energies translating to stereoisomeric product ratios. Herein, stereoselectivity resulting from an unusual reversible Michael addition of an aryl ester to 2-benzylidene malononitrile electrophiles using an isothiourea as a Lewis base catalyst is demonstrated. Notably, the basicity of the aryloxide component and reactivity of the isothiourea Lewis base both affect the observed product selectivity, with control studies and crossover experiments indicating the feasibility of a constructive reversible Michael addition from the desired product. When this reversible addition is coupled with a crystallisation-induced diastereomer transformation (CIDT) it allows isolation of products in high yield and stereocontrol (14 examples, up to 95 : 5 dr and 99 : 1 er). Application of this process to gram scale, plus derivatisations to provide further useful products, is demonstrated.
Project description:The inhibitory effect of new chemical entities on rat liver P450 marker activities was investigated in a functional approach towards drug development. Treatment of colorectal cancer (CRC) and chemoprevention using salicylic acid has gained a lot of attention, mainly in the prevention of the onset of colon cancer. Thus, an in vitro inhibitory effect of salicylic acid on rat CYP2C11 activity was examined by using high performance liquid chromatography (HPLC). High performance liquid chromatography analysis of a CYP2C11 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 243 nm using 32% phosphate buffer (pH 3.36) and 68% methanol as a mobile phase. The CYP2C11 assay showed good linearity for all components (R2 > 0.999). Substrates and metabolites were found to be stable for up to 72 hours. Additionally, the method demonstrated good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80%-120%), and low detection (1.3501 µM and 3.2757 µM) and quantitation limit values (4.914 µM and 9.927 µM) for 16α-hydroxytestosterone and testosterone, respectively. Salicylic acid acts reversibly as a noncompetitive (weak) inhibitor with Ki = 84.582 ± 2.67 µM (concentration of inhibitor to cause 50% inhibition of original enzyme activity (IC50) = 82.70 ± 2.67 µM) for CYP2C11 enzyme activity. This indicates a low potential to cause toxicity and drug-drug interactions.