Allelic variants of human cytochrome P450 2C9: baculovirus-mediated expression, purification, structural characterization, substrate stereoselectivity, and prochiral selectivity of the wild-type and I359L mutant forms.

The purpose of the present studies was to define the role of the I359L allelic variant of CYP2C9 in the metabolism of the low therapeutic index anticoagulant warfarin, by performing in vitro kinetic studies with the two enantiomers of the drug. To obtain sufficient quantities of these variants to perform kinetic studies at physiologically relevant substrate concentrations, methodology was established for the high-level expression, purification, and structural characterization of wild-type CYP2C9 and CYP2C9V1 using the baculovirus system. Both forms were expressed at levels up to 250 nmol/liter and purified in 50-55% yield to specific contents of 13-14 nmol holoenzyme/mg protein. The purified preparations were characterized by Edman degradation and electrospray-mass spectrometry. Both forms of the enzyme metabolized the pharmacologically more potent (S)-enantiomer of warfarin with the same regioselectivity; however, CYP2C9V1 exhibited a fivefold lower Vmax and a fivefold higher Km compared to the wild-type enzyme for this substrate. Neither form of the enzyme formed significant quantities of the (R)-warfarin phenols. Additional studies performed with prochiral arylalkyl sulfides provided confirmation of the low turnover rates catalyzed by CYP2C9V1 and demonstrated further that sulfoxide product stereochemistry did not differ significantly between the two variants. Therefore, decreased catalytic efficiency rather than a gross alteration in substrate orientation appears to be the consequence of this putative active-site mutation. The greatly decreased catalytic efficiency of the I359L variant suggests that leucine homozygotes would eliminate (S)-warfarin, and probably many other CYP2C9 substrates, at much slower rates in vivo than individuals expressing the wild-type enzyme.

cDNA cloning and expression of a new form of human aryl sulfotransferase.

To date, four human cytosolic sulfotransferases have been cloned and characterised. The aim of the present study was to identify new forms of these enzymes using molecular cloning techniques. Two full length human aryl sulfotransferase (HAST) cDNAs were cloned from a lambda gt10 liver cDNA library. The COS cell expression system was used to express the cDNAs and to determine the ability of the encoded proteins to metabolise the model substrates p-nitrophenol and dopamine. The two cDNAs were 1036 bp (HAST4) and 1060 bp (HAST4v) in length, and encoded proteins that differed by two amino acids (Thr-7 to Ile and Thr-235 to Asn). The coding domains of HAST4 and HAST4v were 97 and 94% homologous to previously reported phenol (HAST1) and monoamine (HAST3) sulfonating forms of sulfotransferase, respectively. On expression of these cDNAs in COS cells the encoded proteins were capable of sulfonating p-nitrophenol with markedly different affinities: the K(m)s for HAST4 and HAST4v being 73.7 and 7.75 microM, respectively. For the same reaction HAST1 and HAST3 have K(m)s of 0.7 and 2200 microM, respectively. Unlike HAST1 and HAST3, the expressed HAST4/4v proteins could not sulfonate dopamine. In addition to having markedly different K(m)s for p-nitrophenol as a substrate, the expressed HAST4/4 proteins also differed significantly in their affinity for the cofactor 3'-phosphoadenosine-5'-phosphosulfate. This report on the functional dissimilarity between two allelic variants of HAST4 highlights that substitution at two residues, Thr-7 and -235, markedly alters their substrate specificities and provides insight into the domains that determine these characteristics.

Cytochromes P450, 1A2, and 2C9 are responsible for the human hepatic O-demethylation of R- and S-naproxen.

A preliminary report implicated cytochrome P450 (CYP) 2C9 in the human liver microsomal O-demethylation of S-naproxen, suggesting that this pathway may be suitable for investigation of human hepatic CYP2C9 in vitro. Kinetic and inhibitor studies with human liver microsomes and confirmatory investigations with cDNA-expressed enzymes were undertaken here to define the role of CYP2C9 and other isoforms in the O-demethylation of R- and S-naproxen. All studies utilised a newly developed sensitive and specific HPLC assay that measured the respective O-desmethyl metabolites of R- and S-naproxen in incubations of human liver microsomes and in COS cell lysates. Microsomal R- and S-naproxen O-demethylation kinetics followed Michaelis-Menten kinetics, with respective mean apparent Km values of 123 microM and 143 microM. Sulfaphenazole, a specific inhibitor of CYP2C9, reduced the microsomal O-demethylation of R- and S-naproxen by 43% and 47%, respectively, and the CYP1A2 inhibitor furafylline decreased R- and S-naproxen O-demethylation by 38% and 28%, respectively. R,S-Mephenytoin was a weak inhibitor of R- and S-naproxen O-demethylation, but other CYP isoform specific inhibitors (e.g., coumarin, diethyldithiocarbamate, quinidine, troleandomycin) had little or no effect on these reactions. cDNA-expressed CYP2C9 and CYP1A2 were both shown to O-demethylate R- and S-naproxen. Apparent Km values (92-156 microM) for the reactions catalysed by the recombinant enzymes were similar to those observed for human liver microsomal R- and S-naproxen O-demethylation. The data demonstrate that CYP2C9 and CYP1A2 together account for the majority of human liver R- and S-naproxen O-demethylation, precluding the use of either R- or S-naproxen as a CYP isoform-specific substrate in vitro and in vivo.

Photoaffinity labeling of human recombinant sulfotransferases with 2-azidoadenosine 3',5'-[5'-32P]bisphosphate.

Photoaffinity labeling with 2-azidoadenosine 3', 5'-[5'-32P]bisphosphate was used to identify and characterize adenosine 3',5'-bisphosphate-binding proteins in human liver cytosol and recombinant sulfotransferase proteins. The sulfotransferases investigated in these studies were the human phenol sulfotransferases, HAST1, -3, and -4, dehydroepiandrosterone sulfotransferase, and estrogen sulfotransferase. The cDNAs for these enzymes have been previously cloned and expressed in COS-7 cells or Escherichia coli. Photoaffinity labeling of all proteins was highly dependent on UV irradiation, was protected by co-incubation with unlabeled adenosine 3',5'-bisphosphate and phosphoadenosine phosphosulfate, and reached saturation at concentrations above 10 microM. To verify that the 31 35-kDa photolabeled proteins were indeed sulfotransferases, specific antibodies known to recognize human sulfotransferases were used for Western blot analyses of photolabeled proteins. It was shown unequivocally that the proteins in the 31-35-kDa region recognized by the antibodies also photoincorporated 2-azidoadenosine 3',5'-[5'-32P]bisphosphate. This is the first application of photoaffinity labeling with 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for the characterization of recombinant human sulfotransferases. Photoaffinity labeling will be also useful in the purification and functional identification of other adenosine 3',5'-bisphosphate-binding proteins and to determine amino acid sequences at or near their active sites.

Human cytochrome P450 isoform specificity in the regioselective metabolism of toluene and o-, m- and p-xylene.

The conversion of toluene and o-, m- and p-xylene to their respective side-chain and ring monohydroxylated metabolites by human liver microsomes was investigated. Methyl hydroxylation, to form a benzylalcohol, was the major metabolic pathway for all four methylbenzenes. With the exception of 2,4-dimethylphenol formation from m-xylene, ring hydroxylation accounted for < 5% of total metabolite formation. However, regioselectivity of ring hydroxylation was apparent, with hydroxylation occurring only at positions ortho and/or para to a methyl substituent. Toluene and each xylene isomer exhibited biphasic methylhydroxylation kinetics in human liver microsomes. The high-affinity component of each methylhydroxylation was selectively inhibited by diethyldithiocarbamate and correlated significantly with cytochrome P-4502E1 (CYP2E1) content and activities in a panel of human liver microsomes. cDNA-expressed CYP2E1 was shown to catalyze the formation of each benzylalcohol, with apparent Km values similar to those of the high affinity microsomal reactions. In contrast, the conversion of m-xylene to 2,4-dimethylphenol followed single enzyme Michaelis-Menten kinetics, was inhibited selectively by furafylline, and correlated significantly with known CYP1A2 catalyzed reactions. cDNA-expressed CYP1A2 converted m-xylene to 2,4-dimethylphenol, with an apparent Km similar to that of the microsomal reaction. Although CYP1A2 appears to be responsible for the formation of the minor (phenolic) metabolites of toluene and the xylene isomers, CYP2E1 catalyzed methylhydroxylation will be the major determinant of the clearance of these compounds in humans.

Human hepatic cytochrome P450 2C9 catalyzes the rate-limiting pathway of torsemide metabolism.

Tolyl methylhydroxylation is the rate-limiting step in the elimination of torsemide, a newly developed diuretic, in humans in vivo. Kinetic and inhibitor studies with human liver microsomes and complementary DNA-expressed enzyme were performed to identify the cytochrome P450 (CYP) isoform responsible for torsemide tolyl methylhydroxylation to predict factors that might alter clearance in patients receiving torsemide. As in vivo, tolyl methylhydroxylation was the major biotransformation pathway in human liver microsomes. Microsomal tolyl methyl-hydroxylation kinetics followed Michaelis-Menten kinetics, with the mean apparent Km for the reaction being 11.2 +/- 1.3 microM. The microsomal reaction was almost completely abolished by the specific CYP2C9 inhibitor sulfaphenazole and was inhibited competitively by the alternative CYP2C9 substrate tolbutamide. Torsemide tolyl methylhydroxylase activity in microsomes from 16 human livers correlated significantly (rs = .81-.88) with tolbutamide and phenytoin hydroxylation, both CYP2C9-mediated reactions. Complementary DNA-expressed CYP2C9 catalyzed torsemide tolyl methylhydroxylation with an apparent Km (23 microM) similar to that observed for human liver microsomes and the IC50 values for sulfaphenazole inhibition of the reaction were essentially identical for the two enzyme sources. Taken together, these data demonstrate that human hepatic torsemide tolyl methylhydroxylation is catalyzed predominantly, if not solely, by CYP2C9. The implications of this finding for the regulation of torsemide metabolism in vivo are discussed.

Functional characterization of two human sulphotransferase cDNAs that encode monoamine- and phenol-sulphating forms of phenol sulphotransferase: substrate kinetics, thermal-stability and inhibitor-sensitivity studies.

The present paper describes the functional characterization of two human aryl sulphotransferase (HAST) cDNAs, HAST1 and HAST3, previously isolated by us from liver and brain, respectively [Zhu, Veronese, Sansom, and McManus (1993) Biochem. Biophys. Res. Commun. 192, 671-676; Zhu, Veronese, Bernard, Sansom and McManus (1993) Biochem. Biophys. Res. Commun. 195, 120-127]. These appear to encode the two major forms of phenol sulphotransferase (PST) characterized in a number of human tissue cytosols, these being the phenolsulphating (P-PST) and monoamine-sulphating (M-PST) forms of phenol sulphotransferase. HAST1 and HAST3 cDNAs were functionally expressed in COS-7 cells and kinetically characterized using the model substrates for P-PST and M-PST, p-nitrophenol and dopamine (3,4-dihydroxyphenethylamine) respectively. COS-expressed HAST1 was shown to be enzymatically active in sulphating p-nitrophenol with high affinity (Km 0.6 microM), whereas dopamine was the preferred substrate for HAST3 (Km 9.7 microM). HAST1 could also sulphate dopamine, as could HAST3 sulphate p-nitrophenol, but the Km for these reactions were at least two orders of magnitude greater than for the preferred substrates. COS-expressed HAST1 and HAST3 displayed inhibition profiles with the ST inhibitor 2,6-dichloro-4-nitrophenol (DCNP), identical with human liver cytosolic P-PST and M-PST activities respectively. Thermal-stability studies with the expressed enzymes showed that HAST1 was considerably more thermostable (TS) than HAST3, which is consistent with P-PST being termed the TS PST and M-PST being termed the thermolabile (TL) PST. Western immunoblot analyses of the expressed PST proteins using an antibody generated to a bacterially expressed rat liver aryl/phenol ST showed that HAST1 and HAST3 migrated as single proteins with different electrophoretic mobilities (32 versus 34 kDa). This is consistent with the differences in electrophoretic mobilities observed for P-PST and M-PST in a variety of tissues reported by other workers. This report on the functional characterization of P-PST and M-PST cDNAs provides important information on the structural as well as functional relationships of human PSTs, which sulphate a vast array of exogenous and endogenous compounds.

Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms.

1. The primary metabolism of diazepam was studied in human liver microsomes in order to investigate the kinetics and to identify the cytochrome P450 (CYP) isoforms responsible for the formation of the main diazepam metabolites, temazepam and N-desmethyldiazepam. 2. The formation kinetics of both metabolites were atypical and consistent with the occurrence of substrate activation. A sigmoid Vmax model equivalent to the Hill equation was used to fit the data. The degree of sigmoidicity was greater for temazepam formation than for N-desmethyldiazepam formation, so that the ratio of desmethyldiazepam:temazepam formation increased as the substrate (diazepam) concentration decreased. 3. alpha-Naphthoflavone activated both reactions but with a greater effect on temazepam formation than on N-desmethyldiazepam formation. In the presence of 25 microM alpha-naphthoflavone the kinetics for both pathways were approximated by Michaelis-Menten kinetics. 4. Studies with a series of CYP isoform selective inhibitors and with an inhibitory anti-CYP2C antibody indicated that temazepam formation was carried out mainly by CYP3A isoforms, whereas the formation of N-desmethyldiazepam was mediated by both CYP3A isoforms and S-mephenytoin hydroxylase.

Heterologous systems for expression of mammalian sulfotransferases.

This paper describes the use of both mammalian and bacterial expression systems as tools to study the structural and functional relationships of proteins encoded by cDNAs to both rat and human aryl sulfotransferases. In particular, we describe the use of the mammalian COS cell system for functional expression studies, and the use of Escherichia coli for the expression and purification of a sulfotransferase fusion protein suitable as an antigen for the generation of sulfotransferase antibodies.

Identification of human liver cytochrome P450 isoforms mediating secondary omeprazole metabolism.

1. The in vitro metabolism of omeprazole was studied in human liver microsomes in order to define the secondary metabolic pathways and identify the cytochrome P450 (CYP) isoforms responsible for the formation of the secondary metabolites of omeprazole. 2. The major secondary omeprazole metabolite was the hydroxysulphone, which was formed during incubation with both hydroxyomeprazole and omeprazole sulphone. A second metabolite, tentatively identified as pyridine-N-oxide omeprazole sulphone, was also formed during incubation with omeprazole sulphone. The formation kinetics of these two metabolites from omeprazole sulphone were biphasic suggesting the involvement of multiple CYP isoforms in each case. In contrast, the formation kinetics of hydroxysulphone from hydroxyomeprazole were linear. 3. Inhibition studies, performed with omeprazole sulphone as substrate at concentrations at which the high affinity activities predominated, with a series of isoform selective inhibitors as well as with an anti-CYP2C3 antibody suggested a dominant role of S-mephenytoin hydroxylase in the formation of hydroxysulphone from omeprazole sulphone. By contrast, CYP3A activities were predominant in the formation of hydroxysulphone from hydroxyomeprazole as well as in the formation of pyridine-N-oxide omeprazole sulphone from omeprazole sulphone.

In vitro proguanil activation to cycloguanil by human liver microsomes is mediated by CYP3A isoforms as well as by S-mephenytoin hydroxylase.

1. The activation of proguanil to cycloguanil by human liver microsomes was studied to define the cytochrome P450 (CYP) isoforms involved in this reaction. 2. Apparent Km values for proguanil ranged from 35 microM to 183 microM with microsomes from four human livers. 3. There was a 6.3-fold range of activity with microsomes from seventeen human livers. Rates of proguanil activation correlated significantly with CYP3A activities (benzo[a]pyrene metabolism, caffeine 8-oxidation and omeprazole sulphone formation) and CYP3A immunoreactive content. There was also a highly significant correlation with rates of hydroxyomeprazole formation. Correlations with activities selective for CYP1A2, CYP2C9/10 and CYP2E1, and with immunoreactive CYP1A2 content were not significant. 4. Proguanil activation was inhibited by R,S-mephenytoin, troleandomycin and by inhibitory anti-CYP3A antiserum and anti-CYP2C IgG and was activated by alpha-naphthoflavone. Inhibitors selective for CYP1A2, CYP2E1, CYP2A6 or CYP2C9/10 had little or no effect on proguanil activation. The extents of inhibition by R,S-mephenytoin, troleandomycin and the two antibodies varied with the immunoreactive CYP3A content of the microsomes used. 5. It is concluded that proguanil activation to cycloguanil by human liver microsomes is mediated both by S-mephenytoin hydroxylase and isoforms of the CYP3A subfamily. This has implications for the use of proguanil as an in vivo probe for the S-mephenytoin poor metaboliser phenotype.

Caffeine metabolism by human hepatic cytochromes P450: contributions of 1A2, 2E1 and 3A isoforms.

Caffeine (CA) N1-, N3- and N7-demethylase, CA 8-hydroxylase and phenacetin O-deethylase activities were measured in microsomes from 18 separate human livers which had been characterized previously for a range of cytochrome P450 (CYP) isoform-specific activities and immunoreactive CYP protein contents. Correlations between the high affinity components of the three separate CA N-demethylations were highly significant (r = 0.77-0.91, P < 0.001) and each of the three high affinity CA N-demethylations correlated significantly (r = 0.64-0.93, P < 0.05-0.001) with the high affinity phenacetin O-deethylase, 2-acetylaminofluorene N-hydroxylation and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) mutagenicity (all predominantly CYP1A2-mediated reactions). Consistent with these observations, cDNA-expressed human CYP1A2 catalyzed the N1-, N3- and N7-demethylation of CA and apparent Km values were similar (0.24-0.28 mM) for all three reactions and comparable to those observed previously with human liver microsomes. The low affinity components of CA N1- and N7-demethylation correlated significantly (r = 0.55-0.85, P < 0.05-0.001) with immunoreactive CYP2E1 content and the CYP2E1-specific activities 4-nitrophenol and chlorzoxazone hydroxylation. Diethyldithiocarbamate, a selective inhibitor of CYP2E1, inhibited the low affinity CA N1- and N7-demethylation, with IC50 values of 23 microM and 11 microM, respectively. The apparent Km values for CA N1- and N7-demethylation by cDNA-expressed CYP2E1 (namely 28 and 43 mM, respectively) were of a similar order to those calculated for the low affinity microsomal activities. Significant correlations (r = 0.87-0.97, P < 0.001) were observed between CA 8-hydroxylation and immunoreactive CYP3A content and the CYP3A-mediated reactions benzo(a)pyrene hydroxylation, omeprazole sulfoxidation and aflatoxin B1 mutagenesis. Effects of alpha-naphthoflavone, erythromycin, troleandomycin and nifedipine on microsomal CA 8-hydroxylation were generally consistent with CYP3A involvement. Taken together with previous data, the results indicate a major involvement of CYP1A2 in the high affinity component of all three human hepatic CA N-demethylations. In contrast, CYP2E1 appears to be the main enzyme involved in the low affinity components of CA N1- and N7-demethylation while CA 8-hydroxylation is catalysed predominantly by a CYP3A isoform(s).

Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression and microsomal kinetic techniques.

The involvement of human cytochrome P450 (CYP) 2E1 in the hydroxylation of 4-nitrophenol (4NP) to 4-nitrocatechol (4NC) has been investigated using cDNA expression and liver microsomal kinetic and inhibitor techniques. 4NP hydroxylation by human liver microsomes and cDNA-expressed human CYP2E1 exhibited Michaelis-Menten kinetics; the respective apparent Km values were 30 +/- 7 and 21 microM. Mutual competitive inhibition was observed for 4NP and chlorzoxazone (CZ) (an alternative human CYP2E1 substrate) in liver microsomes, with close similarities between the calculated apparent Km and Ki values for each individual compound. 4NP and CZ hydroxylase activities in microsomes from 18 liver donors varied to a similar extent (3.3- and 3.0-fold, respectively) and 4NP hydroxylase activity correlated significantly (rs > or = 0.75, P < 0.005) with both CZ hydroxylation and immunoreactive CYP2E1 content. The prototypic CYP2E1 inhibitor, diethyldithiocarbamate, was a potent inhibitor of 4NC formation and decreased 4NP hydroxylation by cDNA-expressed CYP2E1 and human liver microsomes in parallel. Probes for other human CYP isoforms namely (alpha-naphthoflavone, coumarin, sulphaphenazole, quinidine, troleandomycin and mephenytoin) caused < 15% inhibition of liver microsomal 4NP hydroxylation. These data confirm that, as in animal species, 4NP hydroxylation is catalysed largely by CYP2E1 in human liver and 4NP may therefore be used as an in vitro substrate probe for the human enzyme.

Identification of human liver cytochrome P450 isoforms mediating omeprazole metabolism.

1 The in vitro metabolism of omeprazole was studied in human liver microsomes in order to define the metabolic pathways and identify the cytochrome P450 (CYP) isoforms responsible for the formation of the major omeprazole metabolites. 2 The four major metabolites identified in vitro, in tentative order of importance, were hydroxyomeprazole, omeprazole sulphone, 5-O-desmethylomeprazole, and an unidentified compound termed metabolite X. Omeprazole pyridone was also detected but could not be quantitated. Incubation of hydroxyomeprazole and omeprazole sulphone with human microsomes resulted in both cases in formation of the hydroxysulphone. The kinetics of formation of the four primary metabolites studied were biphasic suggesting the involvement of multiple CYP isoforms in each case. Further studies used substrate concentrations at which the high affinity activities predominated. 3 Formation of the major metabolite, hydroxyomeprazole, was significantly correlated with S-mephenytoin hydroxylase and with benzo[a]pyrene metabolism and CYP3A content. Inhibition studies with isoform selective inhibitors also indicated a dominant role of S-mephenytoin hydroxylase with some CYP3A contribution in the formation of hydroxyomeprazole. Correlation and inhibition data for the sulphone and metabolite X were consistent with a predominant role of the CYP3A subfamily in formation of these metabolites. Formation of 5-O-desmethylomeprazole was inhibited by both R, S-mephenytoin and quinidine, indicating that both S-mephenytoin hydroxylase and CYP2D6 may mediate this reaction in human liver microsomes and in vivo. 4 The Vmax/Km (indicator of intrinsic clearance in vivo) for hydroxyomeprazole was four times greater than that for omeprazole sulphone. Consistent with findings in vivo, the results predict that omeprazole clearance in vivo would be reduced in poor metabolisers of mephenytoin due to reduction in the dominant partial metabolic clearance to hydroxyomeprazole.

High-performance liquid chromatographic assay for human liver microsomal omeprazole metabolism.

Assays for the measurement of omeprazole metabolites in plasma and urine have been reported, but when applied to the determination of omeprazole metabolites formed by human liver microsomal incubations there were obvious limitations in sensitivity. The present high-performance liquid chromatographic (HPLC) assay, which comprises extraction, evaporation and reconstitution, is several-fold more sensitive with a limit of detection of approximately 2 pmol (2 nM in incubate) for omeprazole sulphone and 25 pmol (25 nM in incubate) for hydroxyomeprazole. Extraction efficiency is essentially quantitative and is highly reproducible (coefficient of variation = 2.1% for both metabolites). The assay is linear over a wide range of concentrations and the formation of the metabolites is linear with respect to both time (to 15 min) and protein concentration (to 1.5 mg/ml). Two minor metabolites, one of which was identified tentatively as 5-O-desmethylomeprazole, were also formed by human liver microsomes and could be determined by this method. Preliminary studies of the formation of omeprazole sulphone and hydroxyomeprazole showed that the formation kinetics in human liver microsomes were biphasic for both metabolites, suggesting that at least two different cytochrome P450 isoforms are involved in their formation.

Identification of two human brain aryl sulfotransferase cDNAs.

A 1,179 bp and a 1,424 bp full-length aryl sulfotransferase cDNAs were isolated from a human brain cDNA library. Their coding domains are 93% identical, each encoding a cytosolic protein of 295 amino acids. Their deduced amino acid sequences of these cDNAs are also 93% identical. The 1179 bp brain cDNA has an identical coding domain to a previously reported human liver aryl sulfotransferase cDNA but it has a different 5' noncoding sequence. Northern blot analysis using a probe specific for the 1,424 bp cDNA identified a 1500 bp band in mRNA of human liver, colon, kidney and lung. In a human hepatocellular carcinoma the same band plus an extra larger band was also recognised. An intron of the gene encoding the 1424 bp cDNA was also identified.

High-performance liquid chromatographic assay for 4-nitrophenol hydroxylation, a putative cytochrome P-4502E1 activity, in human liver microsomes.

A high-performance liquid chromatographic method which measures formation of product 4-nitrocatechol (4NC) has been developed and applied to the study of human liver microsomal 4-nitrophenol (4NP) hydroxylation. Following diethyl ether extraction, 4NC and the assay internal standard (salicylamide) were separated by reversed-phase (C18) liquid chromatography. Extraction efficiencies of 4NC and internal standard were both > 90%. The assay, which has a limit of detection of 15 pmol injected (or an incubation 4NC concentration of 0.25 microM), is accurate, reproducible and straightforward. With a chromatographic time of 12 min, 40-50 samples may be analyzed per day. Rates of 4NC formation were linear with time and protein concentration to 50 min and 0.5 mg/ml, respectively. Preliminary studies of 4NP hydroxylation showed that this reaction followed single enzyme Michaelis-Menten kinetics in human liver microsomes.

Molecular characterisation of a human aryl sulfotransferase cDNA.

A full-length aryl sulfotransferase cDNA was isolated from a human liver cDNA library. It was 1155 bp long containing a coding region of 885 basepairs encoding a cytosolic protein (M(r) 34178 Da) of 295 amino acids. This human cDNA shared 80% homology to the rat aryl sulfotransferase cDNA, 58% to the bovine and rat oestrogen sulfotransferase cDNAs, 53% to the rat hydroxysteroid sulfotransferase cDNA and 51% to the human liver dehydroepiandrosterone sulfotransferase cDNA over its whole 885 bp coding region. The deduced amino acid sequence of this human cDNA was 79% homologous to that of the rat aryl sulfotransferase cDNA and the putative common-substrate binding site motif GXXGXXK of the sulfotransferases has been conserved in this human amino acid sequence. At least two sizes of this human aryl sulfotransferase mRNA were detected in the human liver and lung.