Hydroxylase Assay Research Articles

Human pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha. Characterization and kinetic analysis of wild-type and mutant enzymes.

Pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor-1 alpha is a protein with two different functions. We have overexpressed and purified the human wild-type protein, and its Cys81Ser and Cys81Arg mutants. The Cys81Arg mutant has been proposed to be causative in a hyperphenylalaninaemic patient [Citron, B. A., Kaufman, S., Milstien, S., Naylor, E. W., Greene, C. L. & Davis, M. D. (1993) Am. J. Hum. Genet. 53, 768-774]. The dehydratase behaves as a tetramer on gel filtration, while cross-linking experiments showed mono-, di-, tri-, and tetrameric forms, irrespective of the presence of the single Cys81. Sulfhydryl-modifying reagents did not affect the activity, but rather showed that Cys81 is exposed. Various pterins bind and quench the tryptophan fluorescence suggesting the presence of a specific binding site. The fluorescence is destroyed upon light irradiation. Wild-type and the Cys81Ser protein enhance the rate of the phenylalanine hydroxylase assay approximately 10-fold, a value similar to that of native dehydratase from rat liver; the Cys81Arg mutant, in contrast, has significantly lower activity. This is compatible with the hypothesis that the dehydratase is a rate-limiting factor for the in vivo phenylalanine hydroxylase reaction. The three proteins enhance the spontaneous dehydration of the synthetic substrate 6,6-dimethyl-7,8-dihydropterin-4a-carbinolamine approximately 50-70-fold at 4 degrees C and pH 8.5. The results are discussed in view of the recently solved three-dimensional structure of the enzyme [Ficner, R., Sauer, U. W., Stier, G. & Suck, D. (1995) EMBO J. 14, 2032-2042].

Synthesis of [N‐C3H3]‐trans‐(1R,3S)‐(−)‐1‐Phenyl‐3‐N,N‐dimethylamino‐1,2,3,4‐tetrahydronaphthalene (H2‐PAT)

AbstractSubsequent to the discovery that the (+)‐benzomorphan sigma receptor ligands, (+)‐pentazocine and (+)‐N‐allylnormetazocine, stimulated tyrosine hydroxylase activity and dopamine synthesis in rat striatum in vitro, we reported a similar effect on a structurally similar series of 1‐phenyl‐3‐aminotetrahydronaphthalenes (phenylaminotetralins, PAT's). Both racemic 1‐phenyl‐3‐dimethylamino‐6‐chloro‐7‐hydroxytetralin (CI,OH‐PAT) and racemic 1‐phenyl‐3‐dimethylaminotetralin (H2‐PAT) stimulated tyrosine hydroxylase with an EC50 of approximately 0.1 μM. The former was also found to have a non‐specific dopamine releasing effect while the latter was devoid of such activity affording it the less complicated pharmacological profile of the two analogs. We previously reported the synthesis of tritium labeled Cl,OH‐PAT to be used in radioreceptor and autoradiography studies and found that it labeled a sigma‐like site in guinea pig brain with an apparent Kd of ∼50 pM and with a pharmacological profile unique from other known CNS receptors. Here we report the synthesis of high specific activity tritium labeled trans‐(1R,3S)‐(−)‐H2‐PAT as this enantiomer was found to be more active in the tyrosine hydroxylase assay and possessed approximately 45 fold greater affinity for the novel neuromodulatory sigma‐like receptor.

Benzo(a)Pyrene Hydroxylase Activity in Yeast Is Mediated by P450 Other Than Sterol 14α-Demethylase

Benzo(a)pyrene hydroxylation has been observed in Saccharomyces cerevisiae and the role of sterol 14α-demethylase (CYP51A1) in this activity has been examined by using a strain which contains a gene disruption of CYP51A1. This strain still contained P450 protein(s) with a Soret absorption maximum at 448nm in reduced carbon monoxide difference spectra of the microsomal fraction. On addition of benzo(a)pyrene to this microsomal extract a typical Type I substrate-binding spectrum was obtained and was also observed for the isogenic sister strain containing no CYP51A1 gene disruption. Microsomal extracts of both strains had equivalent activity in the aryl hydrocarbon hydroxylase assay. These results indicate a yeast benzo(a)pyrene hydroxylase activity distinct from sterol 14α-demethylase P450.

Possible existence of a CYP3A protein in liver microsomes from female rats.

CYP3A proteins are P450 monooxygenases involved in the metabolism of steroids, retinoic acid and several important drugs. In rats, the number of CYP3A genes and proteins, and therefore important aspects of their inducibility, developmental regulation and sex specificity are not known for certain. Using triacetyloleandomycin-metabolite complex formation, testosterone hydroxylase assays and immunoblots from peptide maps, we obtained results suggesting that in liver microsomes from mature rats, at least three, possibly four CYP3A proteins are expressed: one is present in females, another is male-specific, and one or two additional CYP3A proteins are inducible by phenobarbital, steroids, and triacetyloleandomycin.

Expression of hepatic microsomal cytochrome P450s as altered by uremia

The proportions of different hepatic microsomal cytochrome P450s expressed in uremic rats were studied with specific antibodies and with a steroid hydroxylase assay. In male uremic rats, the hepatic levels of P450 2C11, a male-specific form, and 3A2, a male-dominant form, were decreased to about 30% at 5 weeks after the induction of uremia. These changes were paralleled by decreases in the activities of testosterone 2α-, 16α-, and 6β-hydroxylation. The level of P450 2A1, abundant in immature rats, was increased 2-fold by uremia and accompanied by an increase in testosterone 7α-hydroxylation activity. The levels of P450 2C6 and 2E1 were not changed by uremia. The levels of male-specific and male-dominant forms such as P450 2C11 and 3A2 are affected by the serum level of testosterone, which was decreased in the male rats with uremia. Therefore, castrated rats were prepared to compare the effects of testosterone on hepatic cytochrome P450s in uremic rats with those in castrated rats. When testosterone was administered to the castrated rats, the decreased levels of both P450 2C11 and 3A2 returned to normal. However, the administration of testosterone to the uremic rats did not prevent the decrease in the levels of these P450s. In female rats, changes in the levels of cytochrome P450s were not as great during uremia as those in male uremic rats. The level of P450 2C12, a female-specific form, was not changed; the level of P450 2A1 was increased by 50%, that of 3A2 which is barely detected in female rats was increased by 60%, and that of 2E1 was increased by 25%. These results suggested that the changes in the hepatic levels of cytochrome P450s were affected by factors other than testosterone in uremic rats.

Effects of gentamicin on catecholamine levels in the striatum, hypothalamus, adrenal gland and vas deferens

The effects of gentamicin, an aminoglycoside antibiotic, on changes in the catecholamine levels in the rat striatum, hypothalamus, adrenal medulla and vas deferens were studied. When rats were i.v. injected with gentamicin (1.0 mg/kg), the catecholamine content of all tissues increased 2–4 h after injection. The increases in catecholamine levels induced by gentamicin were about 1.4- to 2.3-fold those induced by saline. The effect of gentamicin was observed at 0.1 or 0.5 mg/kg, and was maximal at 2.0 mg/kg. The activity of tyrosine hydroxylase, a rate-limiting enzyme in catecholamine biosynthesis, was markedly increased in all four tissues of rats treated with gentamicin (1.0 mg/kg, 4 h). However, direct addition of gentamicin to the tyrosine hydroxylase assay medium did not affect tyrosine hydroxylase activity. These data indicate that gentamicin administration to rats increases the catecholamine content of both central and peripheral catecholamine-containing tissues. The results also suggest that the effect of gentamicin is due to an indirect activation of tyrosine hydroxylase.

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses L-[carboxy-14C]tyrosine as the substrate, 14CO2 is released from the products of the hydroxylation and further metabolism of L-[carboxy-14C]tyrosine by incubation with ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.

A tyrosine hydroxylase assay in microwells using coupled nonenzymatic decarboxylation of Dopa

A radiometric assay for tyrosine hydroxylase employing a coupled nonenzymatic decarboxylation of l-[ 14C]Dopa formed from l-[ 14C]tyrosine has been adapted for performance in a 96 microwell culture plate. The method uses an easily manufactured plate holder to compress blotting paper impregnated with methylbenzethonium hydroxide against the top rim of each well. This forms isolated, airtight compartments in which 14CO 2 is evolved and quantitatively absorbed into the blotting paper. The method is sensitive enough to detect the production of less than 5 pmol of 14CO 2. A major advantage of this system is that cells can be grown in tissue culture and subsequently assayed for tyrosine hydroxylase activity in the same well. The method is more facile than previously devised procedures, allowing for the simultaneous assay of up to 96 samples totally contained in a single, compact, portable unit.

Hydroxylation of aspartic acid in domains homologous to the epidermal growth factor precursor is catalyzed by a 2-oxoglutarate-dependent dioxygenase.

3-Hydroxyaspartic acid and 3-hydroxyasparagine are two rare amino acids that are present in domains homologous to the epidermal growth factor precursor in vitamin K-dependent plasma proteins as well as in proteins that do not require vitamin K for normal biosynthesis. They are formed by posttranslational hydroxylation of aspartic acid and asparagine, respectively. The first epidermal growth factor-like domain in factor IX (residues 45-87) was synthesized with aspartic acid in position 64, replacing 3-hydroxyaspartic acid. It was used as substrate in a hydroxylase assay with rat liver microsomes as the source of enzyme and reaction conditions that satisfy the requirements of 2-oxoglutarate-dependent dioxygenases. The synthetic peptide stimulated the 2-oxoglutarate decarboxylation in contrast to synthetic, modified epidermal growth factor (Met-21 and His-22 deleted and Glu-24 replaced by Asp) and synthetic peptides corresponding to residues 60-71 in human factor IX. This indicates that the hydroxylase is a 2-oxoglutarate-dependent dioxygenase with a selective substrate requirement.

Feminization of rat hepatic P-450 expression by cisplatin. Evidence for perturbations in the hormonal regulation of steroid-metabolizing enzymes.

The biochemical basis for the complex effects of the anti-cancer drug cisplatin on hepatic cytochrome P-450 activity was studied in adult male rat liver using P-450 form-specific steroid hydroxylase assays and antibody probes. Cisplatin treatment of adult male rats resulted in a marked and prolonged feminization of the pattern of P-450 enzymes expressed in hepatic tissue. The adult male-specific cytochrome P-450 forms designated P-450 2c (P-450 gene IIC11), P-450 2a (gene IIIA2), and P-450 RLM2 were decreased by 70-90% after 7-14 days, with parallel decreases in their respectively associated microsomal steroid hydroxylase activities. Concomitantly, hepatic levels of the female-predominant enzymes P-450 3 (gene IIA1) and P-450j (gene IIE1) were elevated approximately 2-4-fold. The female-specific microsomal enzyme androstenedione 5 alpha-reductase was induced approximately 20-fold by cisplatin; however, no elevation of the female-specific P-450 2d was detected. The underlying hormonal basis for these effects of cisplatin was then examined. Serum testosterone levels were found to be depleted by cisplatin in a time- and dose-dependent manner which correlated with the observed changes in these hepatic enzymes. Furthermore, castration of adult rats altered the profile of these enzymes in a manner which resembled that observed with cisplatin treatment, suggesting that androgen depletion was the primary cause for the observed feminization of hepatic enzyme expression. Consistent with this possibility, the synthetic androgen methyltrienolone effectively blocked the changes in hepatic enzyme expression induced by cisplatin. Moreover, hepatic enzyme feminization was significantly reversed by chorionic gonadotropin, which fully restored serum testosterone levels in the cisplatin-treated rat. Luteinizing hormone-releasing hormone challenge experiments demonstrated that the responsiveness of the pituitary to this hypothalamic regulator of testicular androgen production was unimpaired by cisplatin treatment, indicating that hypothalamic production or secretion of luteinizing hormone-releasing hormone may be deficient in the cisplatin-treated animals. These studies establish that the effects of cisplatin on hepatic P-450 enzyme expression result from its interruption of the hypothalamic-pituitary stimulation of testicular androgen production and that this, in turn, leads to a depletion of circulating androgens required for maintenance of normal P-450 enzyme expression in adult male rats.

Microsomal isoflavone 2′- and 3′-hydroxylases from chickpea (Cicer arietinum L.) cell suspensions induced for pterocarpan phytoalexin formation

Microsomal fractions derived from suspension-cultured chickpea (Cicer arietinum L.) cells induced for phytoalexin biosynthesis catalyzed the monohydroxylation of 4′-methoxyisoflavones (biochanin A and formononetin) in the 2′- and 3′-positions. The reactions depended on NADPH and molecular oxygen. Post-microsomal supernatants or microsomes from non-induced cells are without detectable activity in the hydroxylase assay. 4′-Hydroxyisoflavones (genistein and daidzein) were not hydroxylated to any significant extent. The occurrence of these hydroxylases proceeds concomitantly with the accumulation of two pterocarpan phytoalexins, medicarpin and maackiain, by induced cell cultures. The results are discussed with regard to the biosynthetic sequences in the conversion of isoflavones to pterocarpans.

Endogenous tyrosine hydroxylase activity in the developing chick heart: A possible source of extraneuronal catecholamines

The source of catecholamines in the developing chick heart was investigated by using catecholamine assays and tyrosine hydroxylase assays on hearts from normal and chemically-sympathectomized chick embryos. A biochemical index of sympathetic nerve development in the heart was obtained by monitoring the ability of sympathetic nerves in the atria to take up [3H]-norepinephrine in vitro. Specific neuronal uptake of [3H]-norepinephrine in atria was first detected on incubation day 11 and increased throughout the incubation period. High performance liquid chromatography with electrochemical detection was used to measure the norepinephrine concentration and content of embryonic hearts. The cardiac norepinephrine concentration fluctuated throughout the incubation period but was particularly low (0.01 +/- 0.005 ng/mg wet wt) on incubation days 10 to 13, coincident with the arrival of sympathetic nerves in the heart. The highest norepinephrine concentration was measured on incubation day 7 (2.09 +/- 0.50 ng/mg wet wt) prior to the arrival of sympathetic nerves in the heart. Sympathetic nerve axotomy produced by chronic treatment with 6-hydroxydopamine reduced [3H]-norepinephrine uptake in atria and norepinephrine concentration in whole hearts on incubation day 20 to 33 and 47% of control, respectively. Tyrosine hydroxylase activity was detected in normal hearts on incubation day 7, 3 to 4 days before the heart is innervated by sympathetic nerves. Tyrosine hydroxylase activity persisted in the heart on incubation day 20, despite treatment with 6-hydroxydopamine on incubation days 13 to 19. The tyrosine hydroxylase activity in 6-hydroxydopamine lesioned hearts was not significantly different from saline-treated controls. This data indicates that tyrosine hydroxylase activity is present in the immature chick heart prior to the arrival of sympathetic innervation and following chemical sympathectomy; hence, an extraneuronal source of tyrosine hydroxylase, the rate limiting enzyme for catecholamine biosynthesis, exists in the embryonic chick heart.

Determination of cytochrome P-448 activity in biological tissues.

Three enzymes used for the determination of cytochrome P-448 activity, namely aryl hydrocarbon hydroxylase, biphenyl 2-hydroxylase and ethoxyresorufin O-de-ethylase, were evaluated with respect to their specificity, sensitivity and inducibility. Purified cytochrome P-448 (LM4), but not cytochrome P-450 (LM2), catalysed the O-de-ethylation of ethoxyresorufin in a reaction that was markedly inhibited by 9-hydroxyellipticine. After the administration of 3-methylcholanthrene to rats all three activities were induced, the extent of induction being highest for ethoxyresorufin O-de-ethylase. Administration of very small doses of benzo[a]pyrene (50 micrograms/kg) to rats to induce cytochrome P-448 specifically increased only the O-de-ethylation of ethoxyresorufin. 3-Hydroxybenzo[a]pyrene, the major metabolite determined by the aryl hydrocarbon hydroxylase assay, undergoes further NADPH-dependent oxygenation leading to loss of fluorescence. On the basis of these observations and those by other workers, we conclude that ethoxyresorufin O-de-ethylase provides the most specific, sensitive and reproducible means of determining cytochrome P-448 activity.

Oocyte Destruction by Polycyclic Aromatic Hydrocarbons

Certain polycyclic aromatic hydrocarbons, ubiquitous environmental pollutants, destroy oocytes. Oocyte destruction by polycyclic hydrocarbons requires distribution of the parent hydrocarbon to the ovary where ovarian enzymes metabolize the compound to reactive intermediates responsible for ovotoxicity. Descriptive assays of polycyclic hydrocarbon metabolic activation such as the aryl hydrocarbon (benzo(a)pyrene) hydroxylase assay (AHH) are not good predictors of strain or species differences in sensitivity to polycyclic hydrocarbon ovotoxicity. Using benzo(a)pyrene as a probe of ovarian metabolic processing suggests that the rate of formation of metabolites along the metabolic pathway to the 7,8-dihydrodiol-9,10-epoxide may be the appropriate measure of the role of metabolic activation in strain or species differences in sensitivity to oocyte destruction. The multistep metabolic pathway involved in ovarian metabolic processing of benzo(a)pyrene may represent a useful model for exploring the roles of metabolic activation, detoxification, intrinsic sensitivity, and repair in reproductive toxicity.

Further characterization and applications of the 4-chlorobiphenyl hydroxylase assay.

Studies of the metabolism-mediated covalent binding of halobiphenyls to cellular macromolecules revealed that the in vitro metabolism of 4-chlorobiphenyl (4-CBP) by hepatic microsomes is greatly enhanced by in vivo pretreatment of rats with 3-methylcholanthrene (MC) but not phenobarbitone (PB) (Crawford and Safe, 1979). This differential response enables these two classes of inducers to be readily distinguished (Parkinson et al., 1980a).KeywordsHepatic MicrosomePolycyclic HydrocarbonAryl Hydrocarbon HydroxylaseMicrosomal Electron TransportHepatic Microsome Enzyme ActivityThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Studies on the in vitro metabolism of aflatoxin B 1 and G 1 in the liver of rats fed at various levels of vitamin C

Male and female weanling rats, divided into three groups, were maintained for 30, 60 and 90 days on an experimental diet without vitamin C. The drinking water containing or devoid of vitamin C was offered each day. The first group had no vitamin C; the second group or control had normal vitamin C and the third group had above-normal vitamin C in the water. Twenty-four hours after the last feeding, the animals were decapitated and the liver microsomes plus soluble fraction (9000 g) were prepared. This preparation was used for O-demethylase and the hydroxylase assays of aflatoxins B 1 and G 1 (AFB 1 and AFG 1). Results from O-demethylation in both control and experimental groups of both sexes, showed that O-demethylase activity is directly related to ascorbic acid content of the water. But the hydroxylase activity in male and female animals maintained on the same diet showed only slight differences. In the male, there was an increase with time of hydroxylase activity with AFB 1 up to 60 days and a decrease at 90 days. Hydroxylation assay revealed that in the female, metabolism of AFB 1 increased at 30 days but decreased at 60 and 90 days.

Novel variants in benzo(a)pyrene metabolism. Isolation by fluorescence-activated cell sorting.

We have used the fluorescence-activated cell sorter to measure the metabolism of the carcinogenic polycyclic aromatic hydrocarbon, benzo(a)pyrene (BP), in individual, viable mouse hepatoma (Hepa 1c1c7) cells and to physically select variants with either markedly elevated or markedly decreased ability to metabolize to low, nontoxic concentrations of BP (25 nM) and measuring the rate of disappearance of BP fluorescence. We show by several criteria that the disappearance of BP fluorescence is due to the metabolism of the carcinogen. Cells sorted according to their rae of loss of BP fluorescence exhibit the predicted phenotypes in BP-metabolizing activity, as measured by the aryl hydrocarbon hydroxylase assay.

The utility of the microsomal 4-chlorobiphenyl hydroxylase enzyme assay in distinguishing between phenobarbitone- and 3-methylcholanthrene-induced microsomal monooxygenases

4-Chlorobiphenyl was used as a substrate for the in vitro determination of rat hepatic microsomal, cytochrome P-450-dependent monooxygenase activity. The 4-chlorobiphenyl hydroxylase assay was tested for its ability to distinguish between a variety of phenobarbitone- and 3-methylcholanthrene-type inducers. Two radiometric procedures were employed to investigate the metabolism of 4-chlorobiphenyl. First, the metabolite profile of 4-chlorobiphenyl was analyzed by radio-thin-layer chromatography. This procedure permitted an assessment of the effects of microsomal enzyme inducers on both the qualitative and quantitative aspects of 4-chlorobiphenyl metabolism. Second, the rate of 4-chlorobiphenyl metabolism was determined by a differential extraction procedure which separated unreacted starting material (hexane phase) from metabolites (base phase). This procedure provided a rapid measurement of the overall activity of 4-chlorobiphenyl hydroxylase. Irrespective of the animal pretreatment, the metabolite profile of 4-chlorobiphenyl was dominated by 4′-chloro-4-biphenyl. Unlike the qualitative aspects, the quantitative aspects of 4-chlorobiphenyl metabolism were markedly influenced by animal pretreatment. Specifically, 3-methylcholanthrene-type inducers (3-methylcholanthrene and 3,3′,4,4′-tetrachlorobiphenyl) enhanced the activity of 4-chlorobiphenyl hydroxylase at least 10 times more than phenobarbitone-type inducers (phenobarbitone, 2,2′,4,4′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl) enabling these two classes of inducers to be clearly distinguished. It is concluded that 4-chlorobiphenyl is preferentially metabolized by the 3-methylcholanthrene-inducible form(s) of cytochrome P-450 and that this class of microsomal enzyme inducers can be readily distinguished from phenobarbitone-type inducers by means of the 4-chlorobiphenyl hydroxylase assay.

Aryl hydrocarbon hydroxylase: Degradation of product due to contaminant in acetone

A contaminant present in reagent grade acetone causes degradation of fluorescent phenolic metabolites of benzo( a)pyrene as measured in the aryl hydrocarbon hydroxylase assay. Although the contaminant was not identified, its properties suggest that it is a relatively volatile organic material, possibly an oxidizing agent. The acetone may be readily purified by distillation.

Use of soluble, collagenous peptides from medium of chick calvaria cultures as substrate for prolyl hydroxylase

Underhydroxylated collagenous proteins accumulate in the media of embryonic chick calvaria cultured in the presence of α,α′-dipyridyl for 24 h. These soluble collagenous proteins, when labeled with radioactive proline, were shown to be a specific, stable, and highly efficient substrate for in vitro measurement of prolyl hydroxylase. The ability of the media proteins to serve as a substrate for prolyl hydroxylase was abolished by digestion with purified bacterial collagenase. This method of substrate preparation provides a soluble, efficient, economical substrate for routine prolyl hydroxylase assays, and permits the accumulation of sufficient quantities of substrate of one specific activity to serve for extended periods of time.