Inhibitory sites in enzymes: zinc removal and reactivation by thionein.
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Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction. (+info)
Stabilization of L-ascorbic acid by superoxide dismutase and catalase.
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The effects of superoxide dismutase (SOD) and catalase on the autoxidation rate of L-ascorbic acid (ASA) in the absence of metal ion catalysts were examined. The stabilization of ASA by SOD was confirmed, and the enzyme activity of SOD, which scavenges the superoxide anion formed during the autoxidation of ASA, contributed strongly to this stabilization. The stabilization of ASA by catalase was observed for the first time; however, the specific enzyme ability of catalase would not have been involved in the stabilization of ASA. Such proteins as bovine serum albumin (BSA) and ovalbumin also inhibited the autoxidation of ASA, therefore it seems that non-specific interaction between ASA and such proteins as catalase and BSA might stabilize ASA and that the non-enzymatic superoxide anion scavenging ability of proteins might be involved. (+info)
Comparison of effects of acetaminophen on liver microsomal drug metabolism and lipid peroxidation in rats and mice.
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Studies were conducted to determine the in vivo effect of acetaminophen (AAP) on the lipid peroxidation, drug metabolizing enzyme activity and microsomal electron transfer system of rat and mouse liver. AAP was found to inhibit ethylmorphine N-demethylase activity in the presence of NADPH and this inhibition of the enzyme was due to decrease in cytochrome P-450 content, but not due to change in lipid peroxidation in liver microsomes. Kinetical data showed that AAP administration had no effect on Km values of ethylmorphine N-demethylase, however, a decrease in the Vmax values was seen in rats and mice. There was no significant effect of AAP on both NADPH-cytochrome c reductase and the content of cytochrome b5 3 hours after this administration to rats and mice. On the other hand, AAP induced a significant decrease in NADH-ferricyanide reductase in mice, but not in rats. The greatest decrease in cytochrome P-450 observed among the components of the liver microsomal electron transfer system of rats and mice. (+info)
Oxidative bioactivation of the lactol prodrug of a lactone cyclooxygenase-2 inhibitor.
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The lactol derivative of a lactone cyclooxygenase-2 inhibitor (DFU) was evaluated in vivo and in vitro for its potential suitability as a prodrug. DFU-lactol was found to be 10 to 20 times more soluble than DFU in a variety of aqueous vehicles. After administration of DFU-lactol at 20 mg kg-1 p.o. in rats, a Cmax of 7.5 microM DFU was reached in the plasma. After oral administration, the ED50s of DFU-lactol in the carrageenan-induced paw edema and lipopolysaccharide-induced pyresis assays in rats are comparable with the ED50s observed when dosing with DFU. Incubations of DFU-lactol with rat and human hepatocytes demonstrated that the oxidation of DFU-lactol can be mediated by liver enzymes and that a competing pathway is direct glucuronidation of the DFU-lactol hydroxyl group. Assays with subcellular fractions from rat liver indicated that most of the oxidation of DFU-lactol occurs in the cytosolic fraction and requires NAD(P)+. Human liver cytosol can also support the oxidation of DFU-lactol to DFU when NAD(P)+ is added to the incubations. Fractionation of human liver cytosolic proteins showed that at least three enzymes are capable of efficiently effecting the oxidation of DFU-lactol to DFU. Incubations with commercially available dehydrogenases suggest that alcohol and hydroxysteroid dehydrogenases are involved in this oxidative process. These data together suggest that lactols may represent useful prodrugs for lactone-containing drugs. (+info)
Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway.
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Escherichia coli DsbB has four essential cysteine residues, among which Cys41 and Cys44 form a CXXC redox active site motif and the Cys104-Cys130 disulfide bond oxidizes the active site cysteines of DsbA, the disulfide bond formation factor in the periplasm. Functional respiratory chain is required for the cell to keep DsbA oxidized. In this study, we characterized the roles of essential cysteines of DsbB in the coupling with the respiratory chain. Cys104 was found to form the inactive complex with DsbA under respiration-defective conditions. While DsbB, under normal aerobic conditions, is in the oxidized state, having two intramolecular disulfide bonds, oxidation of Cys104 and Cys130 requires the presence of Cys41-Cys44. Remarkably, the Cys41-Cys44 disulfide bond is refractory to reduction by a high concentration of dithiothreitol, unless the membrane is solubilized with a detergent. This reductant resistance requires both the respiratory function and oxygen, since Cys41-Cys44 became sensitive to the reducing agent when membrane was prepared from quinone- or heme-depleted cells or when a membrane sample was deaerated. Thus, the Cys41-Val-Leu-Cys44 motif of DsbB is kept both strongly oxidized and strongly oxidizing when DsbB is integrated into the membrane with the normal set of respiratory components. (+info)
Bcl-2 alters the balance between apoptosis and necrosis, but does not prevent cell death induced by oxidized low density lipoproteins.
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Oxidized low density lipoproteins (oxLDL) participate in atherosclerosis plaque formation, rupture, and subsequent thrombosis. Because oxLDL are toxic to cultured cells and Bcl-2 protein prevents apoptosis, the present work aimed to study whether Bcl-2 may counterbalance the toxicity of oxLDL. Two experimental model systems were used in which Bcl-2 levels were modulated: 1) lymphocytes in which the (high) basal level of Bcl-2 was reduced by antisense oligonucleotides; 2) HL60 and HL60/B (transduced by Bcl-2) expressing low and high Bcl-2 levels, respectively. In cells expressing relatively high Bcl-2 levels (lymphocytes and HL60/B), oxLDL induced mainly primary necrosis. In cells expressing low Bcl-2 levels (antisense-treated lymphocytes, HL60 and ECV-304 endothelial cells), the rate of oxLDL-induced apoptosis was higher than that of primary necrosis. OxLDL evoked a sustained calcium rise, which is a common trigger to necrosis and apoptosis since both types of cell death were blocked by the calcium chelator EGTA. Conversely, a sustained calcium influx elicited by the calcium ionophore A23187 induced necrosis in cells expressing high Bcl-2 levels and apoptosis in cells expressing low Bcl-2 levels. This suggests that Bcl-2 acts downstream from the calcium peak and inhibits only the apoptotic pathway, not the necrosis pathway, thus explaining the apparent shift from oxLDL-induced apoptosis toward necrosis when Bcl-2 is overexpressed. (+info)
Oxidized derivatives of 7-dehydrocholesterol induce growth retardation in cultured rat embryos: a model for antenatal growth retardation in the Smith-Lemli-Opitz syndrome.
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7-Dehydrocholesterol accumulates in fetuses affected by the Smith-Lemli-Opitz syndrome as a result of a deficit in the ultimate step of cholesterol synthesis catalyzed by Delta7 reductase. Rat embryos explanted at gestation day 10 and cultured for 48 h in the presence of the Delta7 reductase inhibitor AY 9944 were used as a model to discriminate between the beneficial effect of supplementation with cholesterol and the deleterious effect of supplementation with 7-dehydrocholesterol. Cholesterol supplementation in the form of mixed cholesterol/lecithin liposomes added to serum serving as the culture medium restores the growth of embryos which is markedly decreased in the presence of the inhibitor. 7-Dehydrocholesterol under identical conditions does not restore growth and impairs the beneficial effect of cholesterol added simultaneously. UV-photooxidation of 7-dehydrocholesterol-supplemented culture medium enhances its embryotoxicity, which suggests uptake by the embryo of toxic by-products formed from 7-dehydrocholesterol. By contrast photooxidation of cholesterol-supplemented culture medium does not induce embryotoxicity. alpha-Tocopherol reduces the toxicity of photooxidized 7-dehydrocholesterol supplementing the culture medium. We conclude that 7-dehydrocholesterol does not fulfill the cholesterol requirement of the developing embryos and exerts an additional embryotoxic effect probably via oxidized by-products. This could explain the antenatal growth retardation of SLOS by a blockage of the maternal compensatory cholesterol influx. (+info)
Mass spectral study of polymorphism of the apolipoproteins of very low density lipoprotein.
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New isoforms of apolipoprotein (apo)C-I and apoC-III have been detected in delipidated fractions from very low density lipoprotein (VLDL) using matrix-assisted laser desorption (MALDI) and electrospray ionization (ESI) mass spectrometry (MS). The cleavage sites of truncated apoC-III isoforms have also been identified. The VLDL fractions were isolated by fixed-angle single-spin ultracentrifugation using a self-generating sucrose density gradient and delipidated using a newly developed C18 solid phase extraction protocol. Fifteen apoC isoforms and apoE were identified in the MALDI spectra and the existence of the more abundant species was verified by ESI-MS. The relative intensities of the apoCs are closely correlated in three normolipidemic subjects. A fourth subject with type V hyperlipidemia exhibited an elevated apoC-III level and a suppressed level of the newly discovered truncated apoC-I isoform. ApoC-II was found to be particularly sensitive to in vitro oxidation. The dynamic range and specificity of the MALDI assay shows that the complete apoC isoform profile and apoE phenotype can be obtained in a single measurement from the delipidated VLDL fraction. (+info)