Epoxide Hydratase Activity Research Articles

Variable effects of cigarette smoking on aryl hydrocarbon hydroxylase, epoxide hydratase and UDP-glucuronosyltransferase activities in rat lung, kidney and small intestinal mucosa

Variable effects of cigarette smoking on aryl hydrocarbon hydroxylase, epoxide hydratase and UDP-glucuronosyltransferase activities in rat lung, kidney and small intestinal mucosa

Latency of epoxide hydratase and its relationship to that of udpglucuronyltransferase

Epoxide hydratase activity in liver microsomal preparations from adult male rats is latent to a slight extent. Maximal activations with neutral or anionic detergents were 30–60% whilst UDPglucuronyltransferase was maximally activated by 160–830% by the same detergents. Activation of microsomal epoxide hydratase requires much higher amounts of neutral or anionic detergents than activation of microsomal UDPglucuronyltransferase. High concentrations of inorganic salt, sonication or freeze-thawing which activate microsomal UDPglucuronyltransferase have no influence on microsomal epoxide hydratase activity. From this it appears that the activation which may involve either removal of a permeability barrier or release from conformational restraint occurs more easily for UDPglucuronyltransferase than for epoxide hydratase and that the activation of microsomal epoxide hydratase requires breakage of some hydrophobic bonds between the enzyme and membrane components(s).

A rapid assay for epoxide hydratase activity with benzo(a)pyrene 4,5-(K-region-)oxide as substrate

A rapid radiometric assay for epoxide hydratase activity has been developed using the highly mutagenic [ 3H]benzo(a)pyrene 4,5-(K-region-)oxide as substrate. By addition of dimethylsulfoxide after the incubation, conditions were found where the unreacted substrate could be separated from the product benzo(a)pyrene-4,5-dihydrodiol( trans) simply by extraction into petroleum ether. The product is then extracted into ethyl acetate and, radioactivity is measured by scintillation spectrometry. This assay allows a rapid measurement of epoxide hydratase activity with an epoxide derived from a carcinogenic polycyclic hydrocarbon as substrate and is at the same time sensitive enough for accurate determination of epoxide hydratase activity in preparations with extremely low enzyme levels such as rat skin homogenate (8–14 pmol of product/mg of protein/min).

A specific gas chromatographic method for the determination of microsomal styrene monooxygenase and styrene epoxide hydratase activities

A gas chromatographic (GC) method for the determination of the metabolite resulting from the activities of microsomal styrene monooxygenase (epoxide synthetase) and epoxide hydratase using styrene or styrene epoxide as substrates has been developed. The determination of the activities of both enzymes is based on the GC determination of phenylethylene glycol after its esterification with n-butylboronic acid. Kinetic parameters for both enzymes are given.

Differential control of rat microsomal "aryl hydrocarbon" monooxygenase and epoxide hydratase.

A growing body of evidence implicates epoxide metabolites of mutagenic and carcinogenic polycyclic hydrocarbons as either the only species, or one of the contributing species responsible for these adverse effects. Selective induction of epoxide hydratase(s) catalyzing the transformation of epoxides to electrophilically unreactive dihydrodiols, under conditions not leading to increases in monooxygenase(s) responsible for epoxide formation would, therefore, be of interest. All inducers of rat hepatic epoxide hydratase (determined with [7-3H]styrene oxide as substrate) which have been discovered also induced monooxygenase (determined with benzo(a)pyrene as substrate) suggesting a possible common biosynthetic control of these enzymes. The enzyme levels observed in different sexes and at different stages of the ontogenetic development, possibly dependent on endogenous inducers, strengthened this view. No sex difference is epoxide hydratase activity was observed in young rats (1 to 5 days old) while epoxide hydratase levels were about 3-fold higher in adult males than in females, which was remarkably similar to the behavior of monooxygenase. Moreover, the prenatal development of epoxide hydratase and monooxygenase appeared to be similar--although the low enzyme levels precluded accurate determinations of the latter. Although different types of known monooxygenase inducers all led to epoxide hydratase induction in adult rat liver, their effect of epoxide hydratase and monooxygenase could be dissociated by transplacental treatment. Dissociation was clearest with inducers of the polycyclic hydrocarbon type which led to great induction of monooxygenase while epoxide hydratase remained unchanged. The increases in monooxygenase activity were very different when determined by two methods based on different principles, demonstrating that at least two monooxygenases are involved in oxidative metabolism of benzo(a)pyrene, and that the control of epoxide hydratase is not under common control with either of them.

Properties and amino acid composition of pure epoxide hydratase

1. Introduction Rat liver epoxide hydratase [EC 4.2.1.631 which catalyses the conversion of epoxides to trurans-dihydro- diols has been purified to apparent homogeneity as determined by three independent criteria [l] . The preparation obtained was capable of catalysing the hydration of both styrene oxide and the 4,5- (K- region)epoxide of benzo(a)pyrene [ 11. Epoxides of polycyclic hydrocarbons have been implicated as the agents responsible for the cytotoxic and carcinogenic properties of such compounds (for reviews see [2-41). A detailed knowledge of the properties of epoxide hydratase may, therefore, contribute towards an understanding of the mechanisms of cytotoxicity and carcinogenesis. We report here the results of initial investigations with the pure enzyme. 2. Materials and methods Anhydrous hydrazine was obtained from Pierce Chem. Co., USA. Carboxypeptidases A and B were isopropylphosphate treated preparations and were obtained from Worthington Biochemical Co. Carboxy- peptidase Y (from Yeast) and Carboxypeptidase P (from Penicillium) were kindly donated by Dr R. Hayashi from Kyoto University, Japan, and Dr E. Ichishima, Tokyo Noko University, Japan. Epoxide hydratase activity was assayed under the conditions 296 described in detail [5], specifically in the absence of Tween 80. 2.1. Amino acid analysis Samples containing 250 ,ug protein were dialysed against 0.2 M borate buffer, pH 9.0, free ammonia was removed by placing samples in a boiling water bath for 5 min and samples were dried in vacua over H2S04. Samples were hydrolysed under reduced pressure at 108 + 1°C [6] in 1 ml of twice distilled 5.7 N HCl for 24 h and 144 h in evacuated sealed tubes. The hydrolysates were dried under vacuum with a rotary evaporator at 80°C. The dried residues were divided into five portions and one portion (50 pg) was applied to one column. The analysis of the hydrolysates was carried out with a Beckman amino acid analyser model 4255, equipped with a 5 cm column (for basic amino acids) and 50 cm column (for acidic and neutral amino acids) at a flow rate of 30 ml/h according to Spackmann et al. [7]. Trypto- phan was analysed using 5% thioglycolic acid [8]. Tyrosine and tryptophan residues were also determined spectrophotometrically in 0.1 N NaOH according to Goodwin Morton [9]. Cyst(e)ine methionine residues were also determined after performic acid oxidation of the protein [lo] N-terminal analysis was performed using [‘“Cl - dinitrofluorobenzene [ 1 l] and the Edman degrada- tion methods [ 121. C-terminal analysis was performed by hydrazinolysis [ 131 and by carboxypeptidase