A simple method for purification of epoxide hydratase from rat liver.
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ABSTRACT:
SUBMITTER: Knowles RG
PROVIDER: S-EPMC1164707 | biostudies-other | 1977 May
REPOSITORIES: biostudies-other
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Project description:Epoxide hydratase was solubilized from human liver microsomal fractions and purified to an extent where the specific activity was 40-fold greater than that of the liver homogenate. Combination of homogenate and purified preparation showed that the increase in activity was not due to the removal of an inhibitor. Monosubstituted oxiranes with a lipophilic substituent larger than an ethyl group (isopropyl, t-butyl, n-hexyl, phenyl) readily interacted as substrates or inhibitors with this purified human epoxide hydratase, whereas those with a small substituent (methyl, ethyl, vinyl) were inactive, probably reflecting greater affinity of the former epoxides owing to lipophilic binding sites near the active site of the enzyme. In a series of oxiranes having a lipophilic substituent of sufficient size (styrene oxides), monosubstituted as well as 1,1- and cis-1,2-disubstituted oxiranes readily served as substrates or inhibitors of the enzyme, but not the trans-1,2-disubstituted, tri- or tetra-substituted oxiranes. trans-Substitution at the oxirane ring apparently prevents access of the oxirane ring to the active site by steric hindrance. Epoxide hydratase was also solubilized from microsomal fractions of rat and guinea-pig liver and purified by the same procedure. Structural requirements for effective interaction of substrates, inhibitors and activators were qualitatively identical for epoxide hydratase from the three sources. However, several quantitative differences were observed. Thus human hepatic epoxide hydratase seems to be very similar to, although not identical with, the enzyme from guinea pig or rat. Studies with epoxide hydratase from the latter two species therefore appear to be significant with respect to man. In addition, knowledge of structural requirements for epoxides to serve as substrates for human epoxide hydratase may prove useful for drug design. Compounds which need aromatic or olefinic moieties for their desired effect would not be expected to lead to accumulation of epoxides if their structure was such as to allow for a metabolically produced epoxide to be rapidly consumed by epoxide hydratase.
| S-EPMC1166253 | biostudies-other
Project description:Rat hepatic microsomal cytochrome b5 was purified to homogeneity by solubilization with the detergent Lubrol 12-A9 and chromatography on Fractogel TSK DEAE-650(S). The protein was obtained in high yield (52-87%) in 8 h, and only one polypeptide band, of Mr 16 600, was visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.
| S-EPMC1144712 | biostudies-other
Project description:Microsomal epoxide hydrolase was purified from rat liver, and different fractions of the purified enzyme, which varied in their contents of phospholipid, were obtained by ion-exchange chromatography. One fraction (A), which did not bind to CM-cellulose, had a high phospholipid content, and a second fraction (B), which was eluted from CM-cellulose at high ionic strength, had a low phospholipid content. Removal of most of the phospholipid from fraction A altered its chromatographic behaviour. When the delipidated material was re-applied to CM-cellulose, most of the enzyme bound to the cation-exchanger. The specific activities of all the fractions described (with styrene epoxide [(1,2-epoxyethyl)benzene] as substrate) were altered by adding the non-ionic detergent Lubrol PX or phospholipid. Lubrol PX inhibited enzyme activity, and phospholipid reversed this inhibition. The various enzyme fractions isolated appeared to be different forms of the same protein, as judged by their minimum Mr values and immunochemical properties. These results indicate that different fractions of epoxide hydrolase isolated by ion-exchange chromatography probably are not different isoenzyme forms.
| S-EPMC1153070 | biostudies-other
Project description:1. The effect of oxalomalate on the oxidation of citrate and cis-aconitate in rat liver mitochondria, and on the activity of mitochondrial and cytoplasmic aconitate hydratase, has been investigated. 2. Oxalomalate that was added to intact rat liver mitochondria at high concentrations (2mm) produced complete inhibition of citrate and cis-aconitate oxidation, but lower concentrations (0.1-0.25mm) inhibited oxidation of citrate more than that of cis-aconitate. 3. Aconitate hydratase that was either extracted from mitochondria or soluble in the cytoplasm, was strongly inhibited by low concentrations of oxalomalate (0.01-0.2mm), the mitochondrial enzyme being more sensitive than the soluble one. 4. Oxalomalate, when added together with citrate, produced competitive inhibition; the K(i) values calculated were 1x10(-6)m for the mitochondrial and 2.5x10(-6)m for the cytoplasmic enzyme. 5. With both the enzymic preparations oxalomalate added together with the substrates inhibited the initial rate of the reaction citrate-->cis-aconitate more than that of the reaction isocitrate-->cis-aconitate. 6. After 2min of preincubation of the inhibitor with either of the enzymic preparations the inhibition increased tenfold and became irreversible; under these conditions both the reactions were inhibited to the same extent. 7. The inhibition by oxalomalate of aconitate hydratase appeared to be similar in many respects to that produced by fluorocitrate on the same enzyme.
| S-EPMC1178092 | biostudies-other
Project description:1. The trans-epoxide of diethylstilboestrol and its pinacolone were synthesized chemically and the pinacolone shown to be formed from the epoxide by a non-enzymic process. 2. [14C]Diethylstiboestrol epoxide was converted by rat liver microsomal fraction into 4'-hydroxypropiophenone by a new type of cleavage reaction involving mono-oxygenase. Conditions for the formation of this metabolite and also water-soluble products were investigated together with the effect of inhibitors. A sex-difference in the conversion of diethylstilboestrol epoxide into 4'-hydroxypropiophenone and to polar and water-soluble products was observed. 3. Diethylstilboestrol epoxide was found to be a relatively stable compound that did not form a glutathione conjugate readily without further microsomal activation. A purified preparation of epoxide hydratase did not enhance its rate of conversion into the pinacolone. 4. Diethylstilboestrol epoxide was found to have about one-tenth the oestrogenic activity of diethylstilboestrol as measured by the increase in uterine weight or the induction of peroxidase in immature rat uteri. It was inactive as a mutagen when tested for its ability to inhibit bacteriophage phi X174 DNA viral replication. 5. The possible role of diethylstilboestrol epoxide as an intermediate in the metabolism of diethylstilboestrol and in mediating the harmful effects of this synthetic estrogen is discussed.
| S-EPMC1161277 | biostudies-other
Project description:1. The effects of synthetic fluorocitrate were studied on: (a) the oxidation of citrate and cis-aconitate by rat liver mitochondria; (b) the activity of the aconitate hydratase found in the liver cell sap; (c) the activity of the aconitate hydratase solubilized from liver mitochondria. 2. Fluorocitrate was found to be a potent inhibitor of oxidation of citrate but only a weak inhibitor of oxidation of cis-aconitate: 6.7mum-fluorocitrate (containing 4% of the inhibitory isomer) caused 94% inhibition of the oxidation of citrate (2mm) whereas 1.0mm-fluorocitrate was necessary to provoke the same inhibition when cis-aconitate (2mm) was the substrate. The degree of inhibition varied in relation to the respiratory state of mitochondria when fluorocitrate was added. The inhibition could be partially reversed by cis-aconitate. 3. The aconitate hydratase extracted from the mitochondria was much less inhibited by fluorocitrate than was the mitochondria-bound enzyme, and the aconitate hydratase found in the cell sap was even less sensitive. 0.3mm-Fluorocitrate was required to cause 50% inhibition of the reaction citrate-->cis-aconitate, catalysed by the aconitate hydratase extracted from the mitochondria, and 1.2m-fluorocitrate for the extramitochondrial enzyme. For both enzymes the reaction citrate-->cis-aconitate was 2-3 times more sensitive to fluorocitrate than was the reaction isocitrate-->cis-aconitate. The inhibition was of the competitive type for both reactions.
| S-EPMC1184776 | biostudies-other
Project description:Kievitone hydratase catalyzes the addition of water to the double bond of the prenyl moiety of plant isoflavonoid kievitone and, thereby, forms the tertiary alcohol hydroxy-kievitone. In nature, this conversion is associated with a defense mechanism of fungal pathogens against phytoalexins generated by host plants after infection. As of today, a gene sequence coding for kievitone hydratase activity has only been identified and characterized in Fusarium solani f. sp. phaseoli. Here, we report on the identification of a putative kievitone hydratase sequence in Nectria haematococca (NhKHS), the teleomorph state of F. solani, based on in silico sequence analyses. After heterologous expression of the enzyme in the methylotrophic yeast Pichia pastoris, we have confirmed its kievitone hydration activity and have assessed its biochemical properties and substrate specificity. Purified recombinant NhKHS is obviously a homodimeric glycoprotein. Due to its good activity for the readily available chalcone derivative xanthohumol (XN), this compound was selected as a model substrate for biochemical studies. The optimal pH and temperature for hydratase activity were 6.0 and 35°C, respectively, and apparent Vmax and Km values for hydration of XN were 7.16 μmol min-1 mg-1 and 0.98 ± 0.13 mM, respectively. Due to its catalytic properties and apparent substrate promiscuity, NhKHS is a promising enzyme for the biocatalytic production of tertiary alcohols.
| S-EPMC5805349 | biostudies-literature
Project description:1. The effect of biologically synthesized and purified fluorocitrate on the metabolism of tricarboxylate anions by isolated rat liver mitochondria was investigated, in relation to the claim by Eanes et al. (1972) that this fluoro compound inhibits the tricarboxylate carrier at concentrations at which it has little effect on the aconitate hydratase activity. 2. That the inhibitory action of fluorocitrate is at the level of the aconitate hydratase and not at the level of the tricarboxylate carrier is indicated by the following findings. Although the oxidation of citrate and cis-aconitate, but not that of isocitrate, was inhibited by fluorocitrate, the exchange of internal citrate for external citrate or l-malate was not. Had the tricarboxylate carrier been affected, these latter exchange reactions would have been inhibited. 3. By using aconitate hydratase solubilized from mitochondria it was found that with citrate as substrate the inhibition by fluorocitrate was partially competitive (K(i)=3.4x10(-8)m), whereas with cis-aconitate as substrate the inhibition was partially non-competitive (K(i)=3.0x10(-8)m).
| S-EPMC1177802 | biostudies-other
Project description:Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.
| S-EPMC3858352 | biostudies-literature
Project description:Plant-viroid interactions represent a valuable model for delineating structure-function relationships of noncoding RNAs. For various functional studies, it is desirable to minimize sample variations by using DNA, RNA, and proteins co-purified from the same samples. Currently, most of the co-purification protocols rely on TRI Reagent (Trizol as a common representative) and require protein precipitation and dissolving steps, which render difficulties in experimental handling and high-throughput analyses. Here, we established a simple and robust method to minimize the precipitation steps and yield ready-to-use RNA and protein in solutions. This method can be applied to samples in small quantities, such as protoplasts. Given the ease and the robustness of this new method, it will have broad applications in virology and other disciplines in molecular biology.
| S-EPMC6521142 | biostudies-literature