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R M Philpot

Publications and source records attributed to R M Philpot.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1996

Xenobiotic biotransforming enzymes in the central nervous system: an isoform of flavin-containing monooxygenase (FMO4) is expressed in rabbit brain.

The flavin-containing monooxygenase (FMO, EC 1.14.13.8) is involved in the metabolism of a number of important xenobiotics including many which affect the central nervous system (CNS). Recently, reports in the literature concerning the amount, activity, location, and isozyme characteristics of this enzyme in the brain have presented conflicting evidence. In order to resolve some of the controversy surrounding FMO in the brain, a highly sensitive method for the detection of flavin-containing monooxygenase (FMO) mRNA in whole brain was employed. A poorly conserved region of FMO transcripts was used to design five sets of oligonucleotide primers. Each primer set was specific for one of the five currently known isoforms of FMO. Four and five isoforms, respectively, are expressed in rabbit liver and kidney, as determined by reverse transcription-polymerase chain reaction. However, only one set of primers amplified a specific rabbit brain cDNA fragment. The sequence of the amplification produced affirmed its identity as a segment of FMO4 cDNA. Thus, the FMO of rabbit brain may consist of a single, as yet uncharacterized isozyme and, contrary to several recent reports, is likely to be expressed at low levels.

Animals↗
PubMed1996

Structural characteristics of flavin-containing monooxygenase genes one and two (FMO1 and FMO2).

As a first step in understanding the regulation of the expression of flavin-containing monooxygenases (FMOs), we have isolated the FMO genes from the rabbit and characterized the gene for FMO1. Probes based on the 3', middle and 5' regions of the cDNAs encoding FMO1, FM02, FM03, and FM05 were generated by polymerase chain reaction. A mixture of the 5' probes was used to screen a genomic library, and isolated clones were identified by hybridization with individual 5' probes. The complete gene for FM01 was isolated as three overlapping clones and found to span approximately 40 kb. The gene contains eight introns, ranging in size from 1.4 to 10 kb and nine exons ranging in size from 73 to 747 bases. The gene for FMO1 seems to have multiple transcription start sites. A genomic clone containing a 5' segment of the FM02 gene was isolated and found to contain intron 1, exon 1, and part of intron 2. The first intron of FM02 is considerably smaller than that of FMO1 (0.3 vs. 3.8 kb), and its 3' junction is 52 bases to the 5' of the start codon, compared with 6 bases in the case of FM01. In contrast, the 5' junction of intron 2 is the same distance from the start codon in both genes. The 5'-flanking regions of the FMO1 and FM02 genes contain several putative glucocorticoid responsive elements.

Amino Acid Sequence↗
PubMed1995

4-Ipomeanol and 2-aminoanthracene cytotoxicity in C3H/10T1/2 cells expressing rabbit cytochrome P450 4B1.

In the present study, retroviral vectors were used to stably transfer and express the cDNA encoding rabbit CYP4B1 in mouse C3H/10T1/2 cells. The replication defective retroviral vector was packaged in the ecotropic packaging cell line, GP+E-86, with infectious titer of approximately 1 x 10(6) cfu/mL. Infection, followed by selection with G418, showed an infection efficiency of approximately 70% for the recipient C3H/10T1/2 cells. Analysis of ten G418 resistant clones showed that the number of vector inserts ranged from 4 to 13 copies per cell genome. Each clone was positive for microsomal CYP4B1 protein as determined by immunoblotting. Cytochrome P450 4B1 activity was assessed by the cytotoxicity of 4-ipomeanol, a known substrate for P450 4B1 and a model compound for chemical-induced injury to the lung. The initial clonigenic assays showed that 100% toxicity occurred in all the clones after a 96-hr exposure to 250 microM 4-ipomeanol. Parental C3H/10T1/2 cells were resistant to 4-ipomeanol at concentrations as high as 1 mM. Two clones, designated No. 2 and No. 19, differing in levels of P450 4B1 protein, were characterized further for 4-ipomeanol and other chemical toxicities. A concentration-response study indicated 50% cytotoxicity at 4-ipomeanol concentrations of 1.5 micrograms/mL for clone No. 2 and 2.5 micrograms/mL for clone No. 19. A panel of agents representing the aromatic amines, some of which are known or suspected P450 4B1 substrates, were tested for cytotoxicity in clone No. 2. These agents included 2-aminoanthracene, 2-aminonaphthalene, 2-aminofluorene, 2-acetylaminofluorene and 4-aminobiphenyl. Only 2-aminoanthracene gave a clear cytotoxic response reducing the survival fraction of clone No. 2 to 50% at 0.2 micrograms/mL while affecting parental cells minimally. In vitro expression of CYP4B1 provides a new experimental system for further elucidating the cytotoxic and mutagenic effects of P450 4B1 substrates.

Amines↗
PubMed1995

CYP4 isozyme specificity and the relationship between omega-hydroxylation and terminal desaturation of valproic acid.

The cytochrome P450-dependent terminal desaturation of valproic acid (VPA) is of both toxicological and mechanistic interest because the product, 4-ene-VPA, is a more potent hepatotoxin than the parent compound and its generation represents a rather novel metabolic reaction for the cytochrome P450 system. In the present study, lung microsomes from rabbits were identified as a rich source of VPA desaturase activity. Monospecific polyclonal antibodies directed against CYP4B1 (anti-4B) inhibited 82% of 4-ene-VPA formation, whereas monospecific polyclonal antibodies directed against CYP2B4 (anti-2B) inhibited only 15% of 4-ene-VPA formation. Anti-4B also inhibited 95% of the 5-hydroxy-VPA formation, but only 42% of 4-hydroxy-VPA formation. These data suggest that CYP4B1 accounts for more than 80% of the 4-ene- and 5-hydroxy-VPA metabolites generated by rabbit lung microsomes. CYP4B1 expressed in HepG2 cells metabolized VPA with a turnover number of 35 min-1 and formed the 5-hydroxy-, 4-hydroxy-, and 4-ene-VPA metabolites in a ratio of 110:2:1, respectively. In contrast, the lauric acid omega-hydroxylases, CYP4A1 and CYP4A3, did not give rise to detectable levels of any of these VPA metabolites. Therefore, these studies demonstrate a new functional role for CYP4B1 in the terminal desaturation and omega-hydroxylation of this short, branched-chain fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Characterization of flavin-containing monooxygenase 5 (FMO5) cloned from human and guinea pig: evidence that the unique catalytic properties of FMO5 are not confined to the rabbit ortholog.

Several full-length clones encoding the human and guinea pig orthologs of flavin-containing monooxygenase 5 (FMO5) have been isolated from libraries constructed with hepatic mRNA. The clones were detected by hybridization with the cDNA encoding FMO5 expressed in rabbit. The human and guinea pig cDNAs encode for proteins of 533 amino acids that contain putative pyrophosphate binding domains characteristic of mammalian FMOs. The sequences derived for the human and guinea pig FMO5 proteins are 87% identical and are 85 and 82% identical, respectively, to the sequence of rabbit FMO5. As is the case with other FMOs, FMO5 in human and guinea pig is encoded by multiple transcripts. Rabbit FMO5 expressed in Escherichia coli was purified and used to elicit antibodies in goat. These antibodies detected FMO5 in samples from livers of adult humans, rabbits, and guinea pigs and fetal livers of humans. The human and guinea pig forms of FMO5 were expressed in E. coli and characterized. Neither enzyme effectively catalyzed the metabolism of methimazole, a general FMO substrate; however, both were active with n-octylamine. The responses of the human FMO5 and guinea pig FMO5 to detergent, ions and elevated temperature are all similar to the responses described for rabbit FMO5. These results indicate that the unique properties of FMO5 from rabbit are species-independent and that this form of the flavin-containing monooxygenase is not readily classified as a drug-metabolizing enzyme.

Amino Acid Sequence↗
PubMed1995

Pulmonary cytochrome P-450 monooxygenase system and Clara cell differentiation in rats.

Because a number of studies suggest that the developmental expression of cytochrome P-450s (CYP) in Clara cells is species specific, this study was designed to compare the developmental patterns of the isoform CYP2B and NADPH reductase protein expression and CYP2B activity with the time course of smooth endoplasmic reticulum (SER) formation in Clara cells of rat lung. Pulmonary CYP2B activity measured as pentoxyresorufin O-dealkylation in lung homogenates was not detectable before 7 days postnatal age, but was detectable at adult levels at 50 days postnatal age. In Clara cells, CYP2B and NADPH reductase were detected immunohistochemically at 4 days postnatal age and at adult levels at 10 days postnatal age. The volume density of SER in Clara cells of terminal bronchioles measured morphometrically increased significantly with postnatal age. We conclude that in the rat 1) CYP2B and NADPH reductase distribution and CYP2B activity are age dependent; 2) the increase in Clara cell SER precedes the expression of CYP2B protein; 3) cellular appearance of CYP2B protein precedes CYP activity; and 4) SER appearance and P-450 protein expression do not occur uniformly in differentiating Clara cells, even within the same bronchiole.

Animals↗
PubMed1995

The rabbit pulmonary cytochrome P450 arachidonic acid metabolic pathway: characterization and significance.

Cytochrome P450 metabolizes arachidonic acid to several unique and biologically active compounds in rabbit liver and kidney. Microsomal fractions prepared from rabbit lung homogenates metabolized arachidonic acid through cytochrome P450 pathways, yielding cis-epoxyeicosatrienoic acids (EETs) and their hydration products, vic-dihydroxyeicosatrienoic acids, mid-chain cis-trans conjugated dienols, and 19- and 20-hydroxyeicosatetraenoic acids. Inhibition studies using polyclonal antibodies prepared against purified CYP2B4 demonstrated 100% inhibition of arachidonic acid epoxide formation. Purified CYP2B4, reconstituted in the presence of NADPH-cytochrome P450 reductase and cytochrome b5, metabolized arachidonic acid, producing primarily EETs. EETs were detected in lung homogenate using gas chromatography/mass spectroscopy, providing evidence for the in vivo pulmonary cytochrome P450 epoxidation of arachidonic acid. Chiral analysis of these lung EETs demonstrated a preference for the 14(R),15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET enantiomers. Both EETs and vic-dihydroxyeicosatrienoic acids were detected in bronchoalveolar lavage fluid. At micromolar concentrations, methylated 5,6-EET and 8,9-EET significantly relaxed histamine-contracted guinea pig hilar bronchi in vitro. In contrast, 20-hydroxyeicosatetraenoic acid caused contraction to near maximal tension. We conclude that CYP2B4, an abundant rabbit lung cytochrome P450 enzyme, is the primary constitutive pulmonary arachidonic acid epoxygenase and that these locally produced, biologically active eicosanoids may be involved in maintaining homeostasis within the lung.

Animals↗
PubMed1995

Expression of xenobiotic-metabolizing enzymes in cultured rat tracheal epithelial cells.

Rat tracheal epithelial (RTE) cells were cultured on membrane support with and without retinoic acid (RA). In early (6-day-old) cultures, the epithelium is a monolayer or bilayer of undifferentiated cells and secretes little mucuslike product either in the absence or presence of RA. In late (12- to 15-day-old) cultures, the epithelium differentiates as a mucociliary epithelium in the presence of RA and as a squamous epithelium in the absence of RA. The purpose of our study was to determine whether a number of xenobiotic enzymes are expressed in these cultures and whether their expression depends on the state of differentiation. Enzyme expression was characterized by electrophoresis and immunoblotting as a function of time in culture and phenotypic differentiation. Cytochrome P450 1A1 was not expressed in freshly harvested RTE cells. This isoenzyme was induced in rats by gavage with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or by exposure of early RTE cell cultures to TCDD, provided RA was also added to the cultures. Cytochrome P450 2B1 was observed in freshly isolated RTE cells, but not in early or late RTE cultures. In contrast, expression of NADPH-cytochrome P450 reductase was decreased in early cultures, but was increased in well-differentiated cultures. Flavin-containing monooxygenase was detected in lung tissue, but not in freshly harvested or cultured RTE cells. Glutathione S-transferase (GST) mu and pi were expressed in freshly harvested RTE cells. GST pi was expressed in early and late cultures, whereas GST mu was expressed in late cultures, but could not be found in early cultured RTE cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Flavin-containing monooxygenase (FMO)-dependent metabolism of methionine and evidence for FMO3 being the major FMO involved in methionine sulfoxidation in rabbit liver and kidney microsomes.

Methionine was a substrate for cDNA-expressed rabbit flavin-containing monooxygenase (FMO) 1, FMO2, and FMO3, while incubations with membrane fractions containing cDNA-expressed FMO5 did not lead to the detection of methionine sulfoxide; Km values with FMO1, FMO2, and FMO3 were about 48.0, 30.0, and 6.5 mM, respectively. With FMO3 methionine d-sulfoxide was formed in nearly 8-fold higher concentrations than the l-diastereomer, whereas with FMO1 and FMO2, the d:l diastereomeric ratios were approximately 1.5:1 and 0.7:1, respectively. These results provide evidence for methionine being the first identified endogenous compound metabolized to diastereomeric sulfoxides by flavin-containing monooxygenases. The Km values for methionine sulfoxidation in rabbit liver and kidney microsomes (3.7 and 6.0 mM, respectively) were more comparable to the Km value obtained with FMO3 than FMO1 or FMO2. This result provides evidence that FMO3 is the major FMO isoform involved in methionine sulfoxidation in rabbit liver and kidney microsomes. Further evidence for this hypothesis is provided by the finding that methionine d-sulfoxide was also the preferred product in rabbit liver and kidney microsomes by nearly 8:1 and 6:1 over the l-diastereomer, respectively.

Animals↗
PubMed1994

Cloning and sequencing of flavin-containing monooxygenases FMO3 and FMO4 from rabbit and characterization of FMO3.

The flavin-containing monooxygenases (FMO) are a family of enzymes that contain putative FAD- and NADPH-binding domains within the first 200 residues of their N termini. The cDNAs encoding these enzymes contain an area of relatively high identity over the 5' half of the coding region. Rabbit genomic DNA was probed under low stringency conditions, with a mixture of 5' cDNA fragments encoding rabbit FMO1, FMO2, or FMO5. Bands associated specifically with FMO1, FMO2, or FMO5 were resolved by analysis at high stringency with individual probes. Several bands were detected that could not be assigned to FMO1, FMO2, or FMO5. The behavior of the 5' probes at low versus high stringency was used to facilitate the isolation of cDNAs corresponding to the unknown DNA bands. A cDNA library was constructed from rabbit liver mRNA and screened under low stringency hybridization conditions (30 degrees C, 50% formamide, 1 x SSC, 0.1% SDS) with the mixture of 5' FMO1, FMO2, and FMO5 cDNA probes. A total of 157 clones was detected. Of these, 117 clones remained under high stringency hybridization conditions (65 degrees C, 50% formamide, 0.1 x SSC, 0.1% SDS) and were identified as FMO1 (95 clones) or FMO5 (22 clones). Of the 40 remaining clones, 36 were characterized by sequence analysis as encoding FMO3, previously identified at the protein level by Ozols (Ozols, J. (1991) Arch. Biochem. Biophys. 290, 103-115) as a second rabbit liver FMO. Four clones were shown to encode an FMO not previously described for the rabbit, FMO4. No clones encoding FMO2 were isolated from the liver library. Sequence analysis revealed that FMO3 and FMO4 are 56% identical, and analysis of genomic DNA indicated that each is encoded by a single gene. Message distribution was tissue-, species-, and form-specific. The properties of FMO3 cDNA expressed in Escherichia coli were found to be more similar to those of FMO1 than FMO2, but to differ significantly from both. Rabbit genomic DNA was probed under conditions of low stringency with a mixture of 5' cDNA fragments encoding all five FMO forms and produced results consistent with the possibility of one additional FMO.

Amino Acid Sequence↗