Biomedical subjects

R M Philpot

Publications and source records attributed to R M Philpot.

At least 19 recordsLinked to original sources

PubMed1999

Nonsubstrate recognition site residues are involved in testosterone hydroxylation by cytochrome P450 CYP 2C11.

We have previously characterized an allelic variant of cytochrome P450 CYP 2C11 from the Gunn rat that differs at three positions (amino acids 4, 116, and 187) from the predominant allele from Wistar rats and that displays a dramatically reduced testosterone hydroxylation activity. To assess the relative contribution of these mutations to the decrease in the enzymatic activity we constructed single and double mutants and coexpressed them with reductase. Testosterone metabolism was determined with a baculovirus/insect cell expression system. None of the identified positions alone is critical for the activity since the reversion of one of these mutations is unable to restore fully the Wistar-type activity. The activity of CYP 2C11 containing either the Asn116Ser substitution or the Phe187Leu represents congruent with30% of the activity of the CYP 2C11 Wistar-type protein. In contrast, the activity of the Val4Ala mutated protein is only 10% that of the Wistar-type protein, close to that of the Gunn-type protein. This study reevaluates the contribution of amino acid 4 to the catalysis by cytochrome P450 2C11 and points out the role of extra SRS residues.

Animals↗

PubMed1999

Repeated cocaine exposure: effects on catecholamines in the nucleus accumbens septi of periadolescent animals.

Substance abuse is a major issue in today's society, and is an issue of critical importance in the adolescent population. Research indicates that substance use is often initiated during the adolescent period, and that brain reward areas are still undergoing changes during this time. Despite this, little research has investigated the effects of repeated drug use on the reward mechanisms of periadolescent animals. For this reason, the present study examined the effects of repeated cocaine administration on the responsiveness of the nucleus accumbens septi (NAcc) to either cocaine or saline challenge. The data indicate that repeated exposure to cocaine produces temporal shifts in the dopaminergic (DAergic) activity of the NAcc, with peak activity occurring earlier. Importantly, following repeated injections of cocaine, saline injections alone elicit increases followed by a subsequent suppression in DA overflow in the NAcc. These results suggest that the context of cocaine administration produces fundamental changes in the way that neurochemical reinforcement mechanisms respond. The expectancy of the drug alone elicits reward-related activity within the NAcc, which may play a critical role in the development of addiction.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1999

Species and sex differences in expression of flavin-containing monooxygenase form 3 in liver and kidney microsomes.

Flavin-containing monooxygenase (FMO) 3 is the predominant FMO isoform in adult human liver; however, little is known about its expression in common laboratory species. Studies have shown FMO3 levels to be sex-dependent in mouse liver, but not in human liver. The current study was undertaken to determine the expression of FMO3 in liver and kidney microsomes from multiple species, and to determine whether the sex dependence seen in mouse liver extends to other species and/or tissues. FMO3 had previously been shown to be the major FMO involved in methionine S-oxidation in rat and rabbit liver microsomes. In this study, species differences in FMO3 levels were assessed in liver microsomes from humans, rats, dogs, mice, and rabbits, and in kidney microsomes from rats, dogs, mice, and rabbits, by comparing methionine S-oxidase activities. Species differences were noted in male liver microsomes, with rabbits having 3-fold higher methionine S-oxidase activity than mice and dogs and 1.5-fold higher activity than humans and rats. Species differences were also noted in male and female kidney microsomes, with rats exhibiting 2- to 6-fold higher methionine S-oxidase activity than the other species. Sex differences in FMO3 levels were assessed using methionine S-oxidase activity and immunoblotting, and were noted only in liver microsomes from mice and dogs, with females having higher levels than males. Results also show that FMO3 orthologs from multiple species are catalytically similar with regard to methionine, S-allyl-L-cysteine, and S-(1,2-dichlorovinyl)-L-cysteine S-oxidations.

Animals↗

PubMed1998

Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by FMO3.

In the present study, we expressed human flavin-containing monooxygenase 1 (FMO1), FMO3, FMO4t (truncated), and FMO5 in the baculovirus expression vector system at levels of 0.6 to 2.4 nmol FMO/mg of membrane protein. These four isoforms, as well as purified rabbit FMO2, and eleven heterologously expressed human P450 isoforms were examined for their capacity to metabolize trimethylamine (TMA) to its N-oxide (TMAO), using a new, specific HPLC method with radiochemical detection. Human FMO3 was by far the most active isoform, exhibiting a turnover number of 30 nmol TMAO/nmol FMO3/min at pH 7.4 and 0.5 mM TMA. None of the other monooxygenases formed TMAO at rates greater than 1 nmol/nmol FMO/min under these conditions. Human fetal liver, adult liver, kidney and intestine microsomes were screened for TMA oxidation, and only human adult liver microsomes provided substantial TMAO-formation (range 2.9 to 9.1 nmol TMAO/mg protein/min, N = 5). Kinetic studies of TMAO formation by recombinant human FMO3, employing three different analytical methods, resulted in a Km of 28 +/- 1 microM and a Vmax of 36.3 +/- 5.7 nmol TMAO/nmol FMO3/min. The Km determined in human liver microsomes ranged from 13.0 to 54.8 microM. Therefore, at physiological pH, human FMO3 is a very specific and efficient TMA N-oxygenase, and is likely responsible for the metabolic clearance of TMA in vivo in humans. In addition, this specificity provides a good in vitro probe for the determination of FMO3-mediated activity in human tissues, by analyzing TMAO formation at pH 7.4 with TMA concentrations not higher than 0.5 mM.

Chromatography, High Pressure Liquid↗

PubMed1998

Modulation of human flavin-containing monooxygenase 3 activity by tricyclic antidepressants and other agents: importance of residue 428.

Human flavin-containing monooxygenase 3 (FMO3) is subject to modulation by tricyclic antidepressants and other agents. Imipramine activates FMO3-catalyzed metabolism of methimazole at all substrate concentrations tested. This distinguishes FMO3 from rabbit FMO1 and FMO2, which are activated at high substrate concentration and inhibited at low substrate concentration, and pig FMO1, which is inhibited at all substrate concentrations. The response of FMO3 is also unique in that chlorpromazine is markedly more effective as a modulator than is imipramine. n-Octylamine, MgCl2, and HgCl2 all inhibit FMO3, the first two in a biphasic manner. Substitution of lysine for threonine at position 428 significantly alters the response of FMO3 to modulators without changing the kinetic parameters for the metabolism of the substrate. Activation by imipramine and chlorpromazine is reduced or abolished and inhibition, most obvious at low substrate concentrations, is observed. This is consistent with elimination of self-activation in the metabolism of imipramine. The mutation at 428 also eliminates the biphasic nature of the inhibition by n-octylamine and MgCl2, but does not alter the effect of HgCl2. Our findings show that the activity of FMO3 can be modulated by large drug molecules as well as short-chain amines and metal ions. This modulation can be markedly altered by changing a single amino acid in the enzyme.

Amines↗

PubMed1998

New prodrug activation gene therapy for cancer using cytochrome P450 4B1 and 2-aminoanthracene/4-ipomeanol.

Vector-mediated transfer of prodrug-activating genes provides a promising means of cancer gene therapy. In a search for more selective and more potent bioactivating enzymes for gene therapy of malignant brain tumors, the toxicity-generating capacity of the rabbit cytochrome P450 isozyme CYP4B1 was investigated. Rabbit CYP4B1, but not rat or human isozymes, efficiently converts the inert prodrugs, 2-aminoanthracene (2-AA) and 4-ipomeanol (4-IM), into highly toxic alkylating metabolites. Toxicity of these two prodrugs was evaluated in culture in parental and genetically modified rodent (9L) and human (U87) glioma cell lines stably expressing CYP4B1, and in vivo in a subcutaneous 9L tumor model in nude mice. The most sensitive CYP4B1-expressing glioma clone, 9L4B1-60, displayed an LD50 of 2.5 microM for 2-AA and 4-IM after 48 h of prodrug incubation, whereas 20 times higher prodrug concentrations did not cause any significant toxicity to control cells. Substantial killing of control tumor cells by 2-AA was achieved by co-culturing these cells with CYP4B1-expressing cells at a ratio of 100:1, and toxic metabolites could be transferred through medium. In both CYP4B1-expressing cells and co-cultured control cells, prodrug bioactivation was associated with DNA fragmentation, as assayed by fluorescent TUNEL assays and by annexin V staining. Alkaline elution of cellular DNA after exposure to 4-IM revealed extensive protein-DNA crosslinking with single-strand breakage. Growth of 9L-4B1 tumors in nude mice was inhibited by intraperitoneal injection of 4-IM with minimal side effects. Potential advantages of the CYP4B1 gene therapy paradigm include: the low concentrations of prodrug needed to kill sensitized tumor cells; low prodrug conversion by human isozymes, thus reducing toxicity to normal cells; a tumor-killing bystander effect that can occur even without cell-to-cell contact; and the utilization of lipophilic prodrugs that can penetrate the blood-brain barrier.

Animals↗

PubMed1998

The effects of repeated alcohol exposure on the neurochemistry of the periadolescent nucleus accumbens septi.

Substance abuse is a major issue in today's society and is an issue of critical importance in the adolescent population. Research indicates that substance use is often initiated during the adolescent period and that brain reward areas are still undergoing changes during this time. Despite this, little research has investigated the effects of repeated drug use on the reward mechanisms of periadolescent animals. For this reason, the present study examined the effects of repeated ethanol (EtOH) administration on the responsiveness of the nucleus accumbens septi (NAcc) to either EtOH or saline challenge. The data indicate that repeated exposure to EtOH produces temporal shifts in the dopaminergic (DAergic) activity of the NAcc, with peak activity occurring earlier. Importantly, following repeated injections of EtOH, saline injections alone elicit DA increases in the NAcc suggesting that the context of alcohol administration produces fundamental changes in the way that neurochemical reinforcement mechanisms respond. The expectancy of the drug alone elicits reward-related activity within the NAcc.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1998

Identification of amino acid residues associated with modulation of flavin-containing monooxygenase (FMO) activity by imipramine: structure/function studies with FMO1 from pig and rabbit.

The activity of the flavin-containing monooxygenase (FMO) can be modulated by a number of nitrogen-containing compounds in a manner that is both isoform and modulator-dependent. We now show that the direction (activation or inhibition) and extent of modulation can also be dependent on substrate concentration. Imipramine activates methimazole metabolism catalyzed by rabbit FMO1 or FMO2 at methimazole concentrations greater than 50 or 100 microM, respectively, and inhibits at lower methimazole concentrations. The extent of the activation increases as the substrate concentration increases, and the extent of inhibition increases as the substrate concentration decreases. With either inhibition or activation, the magnitude of the effect shows a similar, direct dependency on imipramine concentration. In contrast, imipramine inhibits the metabolism of methimazole catalyzed by pig FMO1 at all substrate concentrations. The structural basis for this unique ortholog difference between the responses of rabbit and pig FMO1 to imipramine was studied by random chimeragenesis and site-directed mutagenesis. Results with chimeras indicated that modulation of FMO1 activity by imipramine is controlled to a great extent by two areas of the FMO primary structure (residues 381-432 and 433-465). Four amino acids in these regions (positions 381, 400, 420 and 433) and one additional residue (position 186) were identified by site-directed mutagenesis as primary determinants of the imipramine response. When the residues at these positions in rabbit FMO1 are exchanged for the corresponding residues of pig FMO1, a mutant with the functional properties of pig FMO1 is produced. Our results suggest that the response of FMO1 to imipramine involves a distribution between two sites that is regulated by structural features that do not alter the overall binding. The inhibition observed, although it appears to be competitive, likely does not involve competition for a binding site since alteration of imipramine metabolism has no effect on the parameters of methimazole metabolism.

Amino Acid Substitution↗

PubMed1998

Molecular cloning, sequence, and expression of mouse flavin-containing monooxygenases 1 and 5 (FMO1 and FMO5).

Full-length cDNA clones encoding FMO1 and FMO5 have been isolated from a library constructed with mRNA from the liver of a female CD-1 mouse. The derived sequence of FMO1 contains 2310 bases: 1596 in the coding region, 301 in the 5'-flanking region, and 413 in the 3'-flanking region. The sequence for FMO5 consists of 3168 bases; 1599 in the coding region, 812 in the 5'-flanking region, and 757 in the 3'-flanking region. The sequence of FMO1 encodes a protein of 532 amino acids with a predicted molecular weight of 59.9 kDa and shows 83.3% identity to human FMO1 and 83-94% identity to other FMO1 homologs. FMO5 encodes a protein of 533 amino acids with a predicted molecular weight of 60.0 kDa and 84.1% identity to human FMO5 and 83-84% identity to other FMO5 orthologs. Two GxGxxG putative pyrophosphate binding domains exist beginning at positions 9 and 191 for FMO1, and 10 and 192 for FMO5. Mouse FMO1 and FMO5 were expressed in E. coli and show similar mobility to the native proteins as determined by SDS-PAGE. The expressed FMO1 protein showed activity toward methimazole, and FMO5 was active toward noctylamine. In addition, FMO1 was shown to metabolize radiolabeled phorate, whereas FMO5 showed no activity toward phorate.

Animals↗

PubMed1998

Structure-function of cytochromes P450 and flavin-containing monooxygenases: implications for drug metabolism.

This article is a report on a symposium held at Experimental Biology '98 in San Francisco, California. Recent developments in site-directed mutagenesis, computer-modeling, and mechanistic analysis of cytochromes P450 and flavin-containing monooxygenases are described. A unifying theme is the elaboration of general approaches for understanding and predicting the function of individual forms of these enzymes. A related goal is the production of soluble forms of mammalian cytochromes P450 for X-ray crystallography.

Animals↗

PubMed1997

Molecular cloning, sequencing, and expression in Escherichia coli of mouse flavin-containing monooxygenase 3 (FMO3): comparison with the human isoform.

The sequence of mouse flavin-containing monooxygenase 3 (FMO3) was obtained from several clones isolated from a mouse liver cDNA library. The nucleotide sequence of mouse FMO3 was 2020 bases in length containing 37 bases in the 5' flanking region, 1602 in the coding region, and 381 in the 3' flanking region. The derived protein sequence consisted of 534 amino acids including the putative flavin adenine dinucleotide and NADP+ pyrophosphate binding sites (characteristic of mammalian FMOs) starting at positions 9 and 191, respectively. The mouse FMO3 protein sequence was 79 and 82% identical to the human and rabbit FMO3 sequences, respectively. Mouse FMO3 was expressed in Escherichia coli and compared to E. coli expressed human FMO3. The FMO3 proteins migrated with the same mobility ( approximately 58 kDa) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The expressed FMO3 enzymes (mouse and human forms) were sensitive to heat and reacted in a similar manner toward metal ions and detergent. Catalytic activities of mouse and human FMO3 were high toward the substrate methimazole; however, in the presence of trimethylamine and thioacetamide, FMO-dependent methimazole oxidation by both enzymes was reduced by greater than 85%. Other substrates which inhibited methimazole oxidation were thiourea and thiobenzamide and to a lesser degree N,N-dimethylaniline. When probed with mouse FMO3 cDNA, FMO3 transcripts were detected in hepatic mRNA samples from female mice, but not in samples from males. FMO3 was detected in mRNA samples from male and female mouse lung, but FMO3 message was not detected in mouse kidney sample from either gender. Results of immunoblotting confirmed the tissue- and gender-dependent expression of mouse FMO3.

Amino Acid Sequence↗

PubMed1997

Quantitation and kinetic properties of hepatic microsomal and recombinant flavin-containing monooxygenases 3 and 5 from humans.

Variable amounts of flavin-containing monooxygenase isoforms 3 and 5 (FMO3 and FMO5) are present in microsomal preparations from adult, male, human liver. Quantitation with monospecific antibodies and recombinant isoforms as standards showed levels of FMO3 and of FMO5 that ranged from 12.5 to 117 and 3.5 to 34 pmol/mg microsomal protein, respectively. The concentration of FMO3 was greater than that of FMO5 in all samples, but the ratio of FMO3 to FMO5 varied from 2:1 to 10:1. Human hepatic microsomal samples also showed variable activities for the S-oxidation of methimazole. This activity was associated totally with FMO3; no participation of FMO5 was apparent. This conclusion was supported by several lines of evidence: first, the catalytic efficiency of FMO3 with methimazole was found to be approximately 5000 times greater than that of FMO5; second, the rate of metabolism showed a direct, quantitative relationship with FMO3 content; third, the plot of the relationship between metabolism and FMO3 content extrapolated close to the origin. A second reaction, the N-oxidation of ranitidine, exhibited a much higher Km with recombinant FMO3 than did methimazole (2 mM vs. 35 microM). However, a direct relationship between this reaction and FMO3 content in human hepatic microsomal preparations was also apparent. This result shows that even with a high Km substrate, FMO3-catalyzed metabolism can account for the majority of the product formation with some drugs. Our findings demonstrate that the contribution of FMO isoforms to human hepatic drug metabolism can be assessed quantitatively on the basis of the characteristics of the enzymes expressed in Escherichia coli.

Adult↗

PubMed1997

Pulmonary cytochrome P450 monooxygenase and Clara cell differentiation in mice.

Various studies indicate that cytodifferentiation of Clara cells and development of pulmonary cytochrome P450 (CYP) monooxygenases occur postnatally. The timing of these events is species-specific. Neonatal mice are more susceptible than adult mice are to Clara cell injury by naphthalene, but little is known about the postnatal development of Clara cells and CYP in mice. This study was designed to determine the developmental pattern of Clara cell differentiation and CYP expression in mice. Lungs from mice aged 16 days gestation to 63 days postnatal (DPN) were studied. Clara cell secretory protein (CC10) expression in nonciliated cells was detected earlier in proximal airways than in distal airways, but reached adult levels at 14 DPN in all airway levels. Cilia-associated tubulin expression closely followed the onset of CC10 expression, as did expression of CYP reductase. CYP2B protein expression appeared and differentiated earlier in bronchi than in bronchioles and reached adult levels at 14 and 28 DPN, respectively. CYP2F2 expression appeared earlier in proximal airways, but did not reach adult levels of expression until after 28 DPN. CYP activity, measured by naphthalene metabolism, increased with age and corresponded to CYP2F2 protein expression. We conclude that in the mouse, (1) Clara cell maturation is a postnatal event, (2) Clara cell differentiation is complete at the same age in proximal and distal airways, (3) CYP reductase protein expression occurs at the same time as CC10 expression, but CYP2B and CYP2F2 lag behind, and (4) stereoselective naphthalene monooxygenase activity corresponds with CYP2F2 protein expression.

Animals↗

PubMed1997

Fetal alcohol syndrome: early olfactory learning as a model system to study neurobehavioral deficits.

The goal of basic research examining the deficits underlying fetal alcohol syndrome is to develop an animal model which allows investigation and assessment of the neural and cognitive impairments resulting from prenatal alcohol exposure. The following review focuses on animal models and their relationship to human deficits following prenatal alcohol exposure. In addition, this review examines a unique, well-established model system which may permit an increased understanding of the role of alcohol on the developing brain and cognitive behavior. Specifically, large metabolic, neurochemical, neuropharmacological, morphological and neurophysiological changes in young rats have been reported as a consequence of early olfactory preference conditioning, a form of learning that normally occurs during both human and rat development. This olfactory odor preference training paradigm can be used to assess changes in learning as well as the neural substrates underlying this learning. Olfactory preference training has been used to examine: 1) learning, as demonstrated by a behavioral preference for an odor previously paired with stimulation which mimics maternal care; 2) metabolism, by measuring 2-deoxyglucose uptake and distribution in response to the trained odor; 3) neurotransmitter levels, by using in vivo microdialysis, to examine changes in neurotransmitter levels in the olfactory bulb in response to a trained odor. Using in vivo microdialysis enables measurement of both baseline responsiveness of alcohol-exposed pups as well as learned responses at several different developmental ages. The established neural features of this olfactory model include an increase in behavioral preference for a trained odor, increases in 2-DG uptake in specific foci within the olfactory bulb in response to the odor, and increases in dopamine in response to olfactory preference training stimuli, as well as conditioned increases in norepinephrine following olfactory preference training. Using these known behavioral, metabolic and neurochemical indices in control pups allows identification of some of the neurotransmitter systems involved in deficits and the neurobiological basis for impairments induced by prenatal alcohol exposure.

Animals↗

PubMed1997

Oxidation of cysteine S-conjugates by rabbit liver microsomes and cDNA-expressed flavin-containing mono-oxygenases: studies with S-(1,2-dichlorovinyl)-L-cysteine, S-(1,2,2-trichlorovinyl)-L-cysteine, S-allyl-L-cysteine, and S-benzyl-L-cysteine.

Rabbit liver microsomes catalyzed the highly stereoselective, NADPH- and time-dependent S-oxidation of S-benzyl-L-cysteine (SBC), S-allyl-L-cysteine (SAC), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) to their respective sulfoxides. Methimazole, a flavin-containing mono-oxygenase (FMO) substrate, inhibited S-oxidation of all four conjugates. The cytochrome P450 inhibitor 1-benzylimidazole did not affect SAC, SBC, or DCVC S-oxidation but inhibited the S-oxidation of TCVC. Solubilization of microsomes also inhibited TCVC activity, whereas SBC, SAC, and DCVC activities were not affected. Because these results suggested that FMOs were the major catalysts of SBC, SAC, and DCVC sulfoxidations, the four conjugates were evaluated as substrates for cDNA-expressed rabbit FMO isoforms FMO1, FMO2, FMO3, and FMO5. At equimolar concentrations (10 mM), FMO1 S-oxidized SBC and SAC, but no sulfoxides were detected with DCVC or TCVC. FMO3 S-oxidized all four conjugates. Km values determined with FMO3 were comparable to Km values from rabbit liver microsomes. S-Oxidation by FMO2 was detected only with SAC, and no sulfoxides were detected in incubations with FMO5. These results show that FMO isoforms can catalyze cysteine conjugate S-oxidation and that the specific isoform involved depends on the structure of the cysteine conjugate. The cysteine conjugates with more nucleophilic sulfur atoms, SAC and SBC, were much better FMO substrates than those having the less nucleophilic sulfur atoms DCVC and TCVC. The sulfoxides of TCVC and DCVC were reactive toward GSH, whereas the sulfoxides of SBC and SAC were not reactive. These results provide evidence for different chemical reactivities of these sulfoxides.

Animals↗

PubMed1997

Baculovirus-mediated expression and purification of human FMO3: catalytic, immunochemical, and structural characterization.

The baculovirus expression vector system was used to overexpress human FMO3 in insect cells for catalytic, structural, and immunochemical studies. Membranes prepared from infected Trichoplusia ni cell suspensions catalyzed NADPH-dependent metabolism of methyl p-tolyl sulfide at rates 20 times faster than those obtained with detergent-solubilized human liver microsomes. Sulfoxidation of the methyl and ethyl p-tolyl sulfides by recombinant human FMO3 proceeded with little stereochemical preference, whereas sulfoxidation of the n-propyl and n-butyl homologs demonstrated increasing selectivity for formation of the (R)-sulfoxide. This chiral fingerprint recapitulated the metabolite profile obtained when detergent-treated human liver microsomes served as the enzyme source. Catalytically active human FMO3 was purified to apparent homogeneity by cholate solubilization and sequential column chromatography on Octyl-Sepharose, DEAE-Sepharose, and hydroxyapatite. Purified FMO3 exhibited the same electrophoretic mobility as native microsomal enzyme, and immunoquantitation showed that this isoform represents approximately 0.5% of human liver microsomal protein. Therefore, FMO3 is quantitatively a major human liver monooxygenase. LC/electrospray-mass spectrometry analysis of purified FMO3 identified >70% of the tryptic peptides, including fragments containing motifs for N-linked glycosylation and O-linked glycosylation. Although insect cells have the capacity for glycan modification, MS analysis of the tryptic peptides demonstrated that these sites were not modified in the purified, recombinant enzyme. Edman degradation of the recombinant product revealed that posttranslational modification of human FMO3 by insect cells was limited to cleavage at the N-terminal methionine, a process seen in vivo with animal orthologs of FMO3. These studies demonstrate the suitability of this eukaryotic system for heterologous expression of human FMOs and future detailed analysis of their substrate specificities.

Amino Acid Sequence↗

PubMed1996

Characterization of the methionine S-oxidase activity of rat liver and kidney microsomes: immunochemical and kinetic evidence for FMO3 being the major catalyst.

Methionine is oxidized to methionine sulfoxide by rat liver and kidney microsomes in an O2- and NADPH-dependent manner. In all microsomal assays, no methionine sulfone was detected. Use of a monoclonal antibody to rat liver cytochrome P-450 reductase, various cytochrome P-450 and peroxidase inhibitors, antioxidants, and competitive flavin-containing monooxygenase (FMO) substrates suggested that methionine sulfoxidation was exclusively mediated by FMOs. At 5 mM methionine, the d-isomer of methionine sulfoxide was preferentially detected over the l-isomer in both liver (ratio, 5:1) and kidney microsomes (ratio, 12:1); however, at 30 to 40 mM methionine concentrations, the diastereomeric ratio was reduced to approximately 3:1 in both tissues. The Vmax/K(m) ratios determined for the liver and kidney microsomes were similar. Because cDNA-expressed rabbit FMO3 and FMO1 were previously shown to preferentially catalyze methionine and S-benzyl-L-cysteine (SBC) sulfoxidations, respectively, these substrates were used to isolate two distinct S-oxidase activities from the same rat liver microsomal preparation. The purified activities have apparent molecular weights of approximately 55 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The findings that the methionine S-oxidase reacted intensely with antibodies raised against rabbit FMO3 and the SBC S-oxidase reacted intensely with antibodies raised against rabbit FMO1 provide evidence for these activities being associated with FMO3 and FMO1, respectively. The apparent methionine K(m) determined with the purified methionine S-oxidase was 3.4 mM, whereas the apparent methionine K(m) determined with the purified SBC S-oxidase was 48 mM. The methionine sulfoxide d:l diastereomeric ratio obtained with methionine S-oxidase was 15:1, whereas the diastereomeric ratio obtained with SBC S-oxidase was only 2:1. These results provide strong evidence for the expression of both FMO1 and FMO3 in rat liver microsomes and suggest that FMO3 is the major catalyst of methionine sulfoxidation in rat liver and kidney microsomes.

Animals↗

PubMed1996

Expression and characterization of a modified flavin-containing monooxygenase 4 from humans.

The inability to obtain flavin-containing monooxygenase 4 (FMO4) in heterologous systems has hampered efforts to characterize this isoform of the FMO gene family. Neither the human nor the rabbit ortholog of FMO4, each of which has been cloned and sequenced, has been expressed. Attempts to achieve expression of FMO4 have been made with Escherichia coli, baculovirus, yeast, and COS systems. The cDNAs encoding FMO4 have extended coding regions compared with those encoding other FMO isoforms. The derived amino acid sequences of FMO1, -2, -3, and -5 from all species examined contain about the same number of residues (531-535 residues), whereas the derived sequences of human and rabbit FMO4 contain 558 and 555 residues, respectively. We have investigated whether the elongation of the FMO4 coding region is related to the inability to achieve expression. The cDNA encoding human FMO4 has been modified by a single base change that introduces a stop codon at the consensus position. This modification allows for expression in E. coli. Lack of expression of intact FMO4 is caused by a problem that occurs following transcription, a problem that is overcome completely by relocation of the stop codon 81 bases to 5' of its normal position. Truncated FMO4 is expressed as an active enzyme with characteristics typical of an FMO isoform. Possible functional changes resulting from altering the 3'-end of an FMO were investigated with human FMO3. Elongation of the coding region of the FMO3 cDNA to the next available stop codon (FMO3*) resulted in the expression of an enzyme with properties very similar to those of unmodified FMO3. Elongation of FMO3 lowered the level of expression in E. coli but did not eliminate it. As with FMO4, the difference in expression levels between FMO3 and elongated FMO3 (FMO3*) appears to be related to translation rather than transcription. The functional characteristics of FMO3 and FMO3* are not significantly different.

Amino Acid Sequence↗