Liver microsomal epoxide hydrase.

1. The substrate specificity of membrane-bound and purified epoxide hydrase from rat liver microsomes has been studied. Both enzyme preparations catalyzed the hydration of a variety of alkene oxidase as well as arene oxides of several polycyclic aromatic hydrocarbons. 2. Unlike the membrane-bound enzyme, the rate of hydration for most of the substrates catalyzed by the purified epoxide hydrase was constant for only 1 or 2 min. The addition of dilauroyl phosphatidylcholine or heated microsomes to the incubation mixture extended the linearity of the reaction. 3. When rat liver microsomes were used as the source of the enzyme, the apparent Km values for many of the substrates were dependent on the amount of microsomes used. When purified epoxide hydrase was used as the enzyme source and benzo(a)pyrene 11,12-oxide as substrate, the apparent Km for benzo(a)pyrene 11,12-oxide was independent of enzyme concentration but dependent on added lipid concentration. Thus, in the absence of added dilauroyl phosphatidylcholine or in the presence of this lipid at a concentration below its critical micelle concentration, the observed Km for benzo(a)pyrene 11,12-oxide remained constant. However, when the lipid concentration was greater than the critical micelle concentration, the apparent Km value increased linearly with lipid concentration. These results are consistent with a model based on the partition of lipid-soluble substrate between the lipid micelle and the aqueous medium.     

Hepatic microsomal epoxide hydrase. Involvement of a histidine at the active site suggests a nucleophilic mechanism

The effects of a wide variety of chemical modification reagents on the activity of purified rat liver microsomal epoxide hydrase have been investigated. Alkylating agents, such as the phenacyl bromides and benzyl bromide are potent inhibitors of epoxide hydrase. 2-Bromo-4'-nitroacetophenone (p-nitrophenacyl bromide) specifically and irreversibly inactivates epoxide hydrase. Pseudo-first order kinetics of inhibition is observed at higher inhibitor/enzyme ratios. The rate of inactivation is controlled by a group on the enzyme with an apparent pKa of 7.6. Inactivation of the enzyme with 14C-labeled 2-bromo-4'-nitroacetophenone leads to the incorporation of approximately 1 mol of radioactive inhibitor/mol of protein. Epoxide hydrase can be protected against this inactivation by the substrate phenanthrene-9,10-oxide. These results are consistent with the interpretation that 2-bromo-4'-nitroacetophenone acts as an active site-directed inhibitor. The site of alkylation by 2-bromo-4'-nitroacetophenone is a histidine residue of epoxide hydrase. The N-alkylated histidine derivative has been identified as 1-(p-nitrophenacyl)-4-histidine. A possible mechanism for the enzymatic hydration catalyzed by epoxide hydrase is discussed which involves a histidine residue of the enzyme serving as a general base catalyst for the nucleophilic addition of water.     

Hepatic microsomal epoxide hydratase of bluegill.

Hepatic microsomal epoxide hydratase of the bluegill fish shows characteristics similar to those of the marine fish. The bluegill hepatic microsomal epoxide hydratase activity towards styrene oxide is higher (4n-mole/min per mg protein) and that of mixed-function oxidase towards aldrin epoxidation is lower (0.7n-mole/min per mg protein) than the corresponding enzymes of the male mouse (1.90 and 2.0n-mole/min per mg protein, respectively, for epoxide hydratase and aldrin epoxidase).     

Comparison of nuclear and microsomal epoxide hydrase from rat liver

The specific activities of hydration of nine arene and alkene oxides by purified nuclei prepared from the livers of 3-methylcholanthrene-pretreated rats were found to fall within the range of 2.2 to 9.1% of the corresponding microsomal values. Pretreatment with phenobarbital enhanced both the nuclear and microsomal hydration of phenanthrene-9,10-oxide, benzo(a)pyrene-11,12-oxide, and octene-1,2-oxide. 3-Methylcholanthrene pretreatment enhanced the nuclear hydration of these three substrates by 30–60% but had no significant effect on microsomal hydration. An epoxide hydrase modifier, metyrapone, stimulated the hydration of octene-1,2-oxide by the two organelles to quantitatively similar extents, but affected the nuclear and microsomal hydration of benzo(a)pyrene-4,5-oxide differentially. Cyclohexene oxide also exerted differential effects on nuclear and microsomal epoxide hydrase which were dependent both on the substrate and on the organelle. The inhibition by this agent of nuclear and microsomal epoxide hydrase was quantitatively similar only for a single substrate, benzo(a)anthracene-5,6-oxide. When purified by immunoaffinity chromatography, nuclear and microsomal epoxide hydrases from 3-methylcholanthrene-pretreated rats were shown to have identical minimum molecular weights (? 49,000) on polyacrylamide gels in the presence of sodium dodecyl sulfate. These findings support the assertion that microsomal metabolism can no longer be considered an exclusive index of the cellular activation of polycyclic aromatic hydrocarbons.     

Hepatic cytochrome P-448 and epoxide hydrase: enzymes of nuclear origin are immunochemically identical with those of microsomal origin.

Nuclear and microsomal sources of hepatic cytochrome P-448 and epoxide hydrase were compared using antibodies made against the pure antigens isolated from rat liver microsomes. Both antigens were easily detected in detergent-solubilized nuclei and microsomes from rats using the Ouchterlony double-diffusion technique. Epoxide hydrase from either whole nuclei or nuclear envelope was immunochemically identical with the enzyme isolated from microsomes. Similarly, in rats pretreated with 3-methylcholanthrene, the cytochrome P-448 of nuclear origin was immunochemically indistinguishable from the enzyme derived from microsomes. These results establish the immunochemical identity of these hepatic nuclear and microsomal enzymes and provide a firm basis for applying the knowledge gained with the microsomal system of metabolism to the nuclear system.     

A practical radiochromatographic assay for cholesterol epoxide hydrase.

A method for the assay of cholesterol epoxide hydrase activity is described. The assay involves the thin-layer chromatographic separation and quantitation of radiolabeled cholestan-3β,5α,6α-epoxide and its major hydration product, cholestan-3β,5α,6β-triol. Radiochromatographic scanning is employed to quantitate the reaction. The procedure is sensitive, rapid, and nondestructive.     

Partial amino acid sequence of human thyroxine-binding globulin. Further evidence for a single polypeptide chain.

The NH₂-terminal amino acid of highly purified thyroxine-binding globulin has been identified by dansyl chloride, cyanate and Edman degradation methods. All three gave alanine as the only amino terminal residue. Carbamylation and Edman degradation of the denatured protein yielded 0.86 and 0.98 – 1.05 mole of alanine per mole of protein, respectively. These data further indicate that thyroxine-binding globulin is composed of a single polypeptide chain. Automated Edman degradation gave the partial sequence as: Ala-Ser-Pro-Glu-Gly-Lys-Val-Thr-Ala-Asp-Ser-Ser-Ser-Gln-(Pro)-X-Ala-(Ser)-Leu-Tyr- A computer search revealed no homology of the NH₂-terminal segment of thyroxine-binding globulin with human prealbumin. The NH₂-terminal portion of prealbumin contains part of the thyroxine binding site.     

Purification of human liver microsomal epoxide hydrase. Differences in the properties of the human and rat enzymes.

Human liver microsomal epoxide hydrase has been highly purified to a specific activity (570 to 620 nmol/min/mg of protein) comparable to that of the rat enzyme using styrene oxide as substrate. Like the purified rat liver microsomal epoxide hydrase, the human enzyme has a minimum molecular weight of 49,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and exhibits broad substrate specificity toward a variety of alkene and arene oxides. Despite these similarities, the human and rat enzymes are different proteins as judged by their immunochemical properties as well as their relative catalytic activities toward certain substrates.     

A rapid and sensitive liquid chromatographic assay for epoxide hydrase.

A rapid method for the assay of epoxide hydrase activity is described. 3-Methylcholanthrene-11,12-oxide is employed as substrate and high speed liquid chromatography is used to separate and quantitate trans-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene (product) formation. The determination of product at picomole levels can be obtained.     

Characterization of human thyroxine-binding globulin. Evidence for a single polypeptide chain.

Thyroxine-binding globulin (TBG) was purified from fresh human plasma by affinity, anion exchange, and gel filtration chromatography. The protein gave a single band in overloaded analytical disc gel electrophoresis. The molecular weight was 54,000 and E1%/1 cm at 280 nm, corrected for thyroxine (T4) absorbance, was 6.17. Six preparations of TBG contained from 0.09 to 0.64 mol of T4/mol; the TBG used in this study contained 0.19 mol of T4 and was able to bind an additional 0.85 mol. The carbohydrate composition was determined and accounted for 23% of the molecular weight. Four lines of chemical and physical evidence failed to demonstrate subunits. These included quantitative COOH-terminal amino acid analysis, peptide mapping and amino acid composition, treatment with sodium dodecyl sulfate, and denaturation of the reduced, alkylated protein with guanidine. From these data, we conclude that TBG is a single polypeptide chain.     

Colicin B consists of a single polypeptide chain

Abstract Colicin B was isolated in pure form from Escherichia coli Cl139 and was shown to consist of a single polypeptide chain with an apparent M _r of 70000. Therefore, it does not differ from other colicins where the toxic activity resides in one polypeptide.     

The immobilization antigen of Paramecium aurelia is a single polypeptide chain.

The immobilization antigen (i-antigen) fraction of Paramecium aurelia syngen 4 is shown to contain a protease that is activated by mercaptoethaneol. After the protease has been heat-inactivated, the molecular weight of the i-antigen (similar to 250,000 daltons) cannot be decreased by mercaptoethanol treatment. It is demonstrated that the i-antigen is a single polypeptide chain. Reasons are also given why low molecular weight subunits were previously reported by other authors.     

Isolation and incorporation of rabbit liver epoxide hydrase into phospholipid vesicles.

Methods are described for the incorporation into phospholipid vesicles of epoxide hydrase isolated from liver microsomes of phenobarbital-treated rabbits. Chromatography on a Sephadex G-50 column of epoxide hydrase and egg yolk phosphatidylcholine treated with sodium cholate yielded homogeneous vesicles with a diameter of about 25 nm and containing 80 to 85% of the protein applied. At high substrate concentrations, the vesicles catalyzed the hydration of benzo(a)pyrene-4,5-oxide and styrene-7,8-epoxide at a rate similar to that obtained with the enzyme in a soluble form. However, the kinetics of styrene glycol formation catalyzed by the vesicular or microsomal preparations were complex. Convex Lineweaver-Burk plots and concave Hill plots were obtained, whereas normal Michaelis-Menten kinetics characterized the hydration catalyzed by the enzyme in a soluble form. The results could be explained if reconstitution of the enzyme into the vesicles gives rise to low affinity high capacity sites for the substrate on the enzyme, or alternatively facilitates the interaction of the substrate with such sites already present. It is suggested that reconstituted liposomes containing both the liver microsomal hydroxylase system and epoxide hydrase may prove to be a good model system for evaluating substrate specificity and factors of importance in the formation of toxic and carcinogenic metabolites by these enzymes.     

Liver microsomal expoxide hydrase. Solubilization, purification, and characterization.

Epoxide hydrase was solubilized from liver microsomes of phenobarbital-treated rats by treatment with cholate and purified to apparent homogeneity by ammonium sulfate fractionation and column chromatography in the presence of the nonionic detergent Emulgen 911 on DEAE-cellulose and hydroxylapatite. The purified enzyme preparation had a single major band with a molecular weight of 53,000 to 54,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Other studies indicated that in the absence of sodium dodecyl sulfate, purified epoxide hydrase exists as high molecular weight aggregates. The preparation was essentially free of heme and flavin, but still contained small amounts of lipids and Emulgen 911.