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R M Philpot

Publications and source records attributed to R M Philpot.

At least 55 records · Page 3Linked to original sources
PubMed1994

Prochiral sulfoxidation as a probe for multiple forms of the microsomal flavin-containing monooxygenase: studies with rabbit FMO1, FMO2, FMO3, and FMO5 expressed in Escherichia coli.

Multiple forms of the microsomal flavin-containing monooxygenase (FMO) exist in rabbit tissues. In order to better understand the catalytic properties of these isoforms, we have expressed rabbit FMO1, FMO2, FMO3, and FMO5 in Escherichia coli and examined their kinetic parameters and prochiral selectivities for the sulfoxidation of methyl-, ethyl-, n-propyl-, and n-butyl-substituted p-tolyl sulfides. FMO1 and FMO2 exhibited high affinities for these substrates (Km < 10 microM), in contrast to the low-affinity FMO3 form for which Km values ranged between 100 and 280 microM. FMO5 did not form quantifiable levels of sulfoxide metabolites at the concentrations used. The individual stereochemical metabolite profiles generated by FMO1, FMO2, and FMO3 were unique and served to distinguish among these three cDNA-expressed isoforms. To investigate the relationship between the kinetic parameters for the cDNA-expressed enzymes and the native microsomal enzymes, we examined the kinetics and stereoselectivity of metabolism of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes. We analyzed these data with respect to FMO1 and FMO3, the two predominant hepatic isoforms. Sulfoxidation of methyl p-tolyl sulfide by FMO1 and FMO3 solubilized from E. coli microsomes proceeded with apparent Kms of 18 and 270 microM, respectively. FMO1 was essentially stereospecific for formation of (R)-methyl p-tolyl sulfoxide, whereas FMO3 generated this metabolite with little prochiral selectivity. Sulfoxidation of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes was best described by a two-enzyme model, with apparent Km values of 11 and 340 microM. The enantiomeric purity of the (R)-methyl p-tolyl sulfoxide metabolite, generated by detergent-solubilized rabbit liver microsomes, decreased progressively with increasing substrate concentration, from a high of 96% enantiomeric excess at a substrate concentration of 5 microM to a low of 63% enantiomeric excess at a substrate concentration of 2 mM. The kinetic and stereochemical properties of the high-affinity and low-affinity components of detergent-solubilized rabbit liver microsomes were similar to those exhibited by cDNA-expressed FMO1 and FMO3, respectively. Therefore, methyl p-tolyl sulfide, used at the appropriate substrate concentrations, is useful for discriminating between FMO1- and FMO3-mediated catalysis in rabbit liver microsomal preparations.

Alkylation↗
PubMed1994

The mammalian flavin-containing monooxygenases: molecular characterization and regulation of expression.

The flavin-containing monooxygenase (FMO) has been characterized in several mammalian species, including human. The FMO forms a stable NADP(H)- and oxygen-dependent 4 alpha-hydroperoxy flavin enzyme intermediate in the absence of an oxygenatable substrate. As such, substrate specificity appears to be controlled by access to this stabilized intermediate, resulting in this enzyme's ability to metabolize a wide variety of xenobiotics. These include tertiary and secondary alkyl- and arylamines, many hydrazines, thiocarbamides, thioamides, sulfides, disulfides, thiols, and other soft nucleophiles. Although some of these compounds are oxidized to less active derivatives, several examples of metabolic activation to potentially toxic intermediates also exist. Mercapto-pyrimidines and thiocarbamides, for example, appear to be activated predominantly by FMO. Thus, this enzyme system may play an important role in the early steps of chemical toxicity. Often, the contribution of FMO to the metabolism of a given compound can be assessed by its unique stereoselectivity relative to other oxygenases. For example, the cytochromes P450 oxidize (S)-nicotine to a mixture of cis- and trans-N-1'-oxides. In contrast, (S)-nicotine is oxidized by human FMO3 exclusively to the trans-N-1'-oxide. With the purification and cloning of FMO from multiple tissues and species it became apparent that more than one FMO exists. Further, there are considerable tissue- and species-specific differences in FMO expression that likely contribute to observed differences in detoxication competency and toxicant susceptibility.

Animals↗
PubMed1994

Catalytic selectivity and mechanism-based inactivation of stably expressed and hepatic cytochromes P450 2B4 and 2B5: implications of the cytochrome P450 2B5 polymorphism.

Cytochrome P450 (P450) 2B5 was recently found to be functionally distinct from three other rabbit P450 2B forms, based on androstenedione hydroxylase activities. In this investigation, we examined the frequency of the P450 2B5-null phenotype and the functional consequences of polymorphic P450 2B5 expression in hepatic microsomes from phenobarbital-treated rabbits. Four of the 10 animals examined did not have detectable levels of P450 2B5 mRNA and exhibited much lower microsomal androstenedione 15 alpha- and 16 alpha-hydroxylase activities. The 15 alpha-hydroxylase activity was found to correlate (r = 0.91) with liver P450 2B5 mRNA. P450 2B4 and 2B5 were stably expressed in human kidney 293 cells to further characterize substrate specificities and to investigate mechanism-based inactivation by phencyclidine. P450 2B4 was 4-16-fold more active than 2B5 towards benzphetamine, 7-ethoxycoumarin, methylenedioxybenzene, and pentoxyresorufin. Benzyloxyresorufin O-debenzylase activity was 160-fold higher for P450 2B4 than P450 2B5. Anti-P450 2B4 IgG inhibited benzyloxyresorufin O-debenzylation nearly completely in untreated and phenobarbital-induced liver microsomes. Phencyclidine selectively inactivated P450 2B4, compared with 2B5, in both human kidney 293 cell and liver microsomes. Poor inactivation of P450 2B5 by phencyclidine was found to be a result of its low maximal rate constant. Results of this study establish the idea that the metabolic consequences of phenobarbital induction depend on the potential of animals to express functionally variant P450 2B forms. Furthermore, we conclude that one or more of the 11 amino acid differences between these highly related P450 forms are critical to their substrate specificities and selective inactivation.

Androstenedione↗
PubMed1994

N-aralkylated derivatives of 1-aminobenzotriazole are potent isozyme- and lung-selective mechanism-based inhibitors of guinea pig cytochrome P-450 in vivo.

1-Aminobenzotriazole (ABT) and its N-benzyl-1-aminobenzotriazole (BBT) and N-alpha-methylbenzyl (alpha-MB) derivatives were compared as isozyme-selective, lung-selective (vs. liver) mechanism-based inhibitors of cytochrome P-450 (P450) in noninduced, beta-naphthoflavone-induced and phenobarbital-induced guinea pigs 4 hr after i.v. administration. Isozyme-selective monooxygenase activities for lung P450 1A1, 2B4 and 4B1 orthologues (7-ethoxyresorufin O-deethylation for guinea pig P450 1A1, 7-pentoxyresorufin O-depentylation for P450 2Bx and 4-aminobiphenyl N-hydroxylation for P450 4Bx, respectively) were determined in pulmonary and hepatic microsomes. BBT and alpha-MB inactivated pulmonary P450 in an isozyme-selective manner; in non- and phenobarbital-induced animals the order of inactivation was 2Bx > 1A1 >>> 4Bx. In beta-naphthoflavone-induced animals, alpha-MB specifically inhibited 2Bx in the lung (>90% inactivation at 0.075 mumol/kg, whereas a 100-fold higher dose did not inhibit 4Bx or 1A1). BBT and alpha-MB also were highly selective for the inactivation of pulmonary vs. hepatic P450. In each case at least one of the doses administered caused marked inactivation of pulmonary 2Bx (>80% with alpha-MB and 50-70% with BBT) without inhibiting the hepatic monooxygenase activities. In contrast, ABT displayed little isozyme-selectively and little tissue-selectivity. The differences in tissue-selectivity of the inhibitors are due to BBT and alpha-MB being much more potent (100- to 1000-fold) inactivators of pulmonary P450 2Bx than ABT consistent with BBT and alpha-MB, but not ABT, serving as substrates for the lipophilic aromatic amine uptake system in the lung. In summary, BBT and alpha-MB, at appropriate doses, are isozyme-selective/specific (P450 2Bx), lung-specific inhibitors of P450 in guinea pig in vivo.

Animals↗
PubMed1994

Unique distribution profiles of glutathione S-transferases in regions of kidney, ureter, and bladder of rabbit.

BACKGROUND: Glutathione S-transferases detoxify a broad range of exogenous compounds, but are important also in the metabolism of endogenous compounds. Physiologically relevant substrates are the endoperoxide and hydroperoxide metabolites of arachidonic acid that play important roles in many tissues including the kidney. EXPERIMENTAL DESIGN: We used immunohistochemical and immunoblotting techniques in a systematic study of renal localization of four rabbit enzymes that represent three major mammalian cytosolic glutathione S-transferase classes, alpha, pi, and mu. RESULTS: The two alpha-class enzymes (rbGST alpha I, rbGST alpha II) were distributed discretely in kidney, ureter, and bladder, while pi and mu were widely distributed in the renal system. Immunohistochemical localization in paraffin sections with antibodies specific for rbGST alpha I or rbGST alpha II demonstrated that no compartment of the renal system contained both enzymes. Collecting ducts of the inner medulla and all epithelial cells of the kidney pelvis, ureter, and bladder contained rbGST alpha I. All cells lining proximal tubules contained rbGST alpha II. No other compartment of the renal system exhibited immunoreactivity with anti-rbGST alpha II. Antibody specific for pi reacted with cells lining nephrons, ureter, and bladder and with endothelial cells throughout the renal system. Localization of pi was most prominent in the collecting ducts of medulla and in the epithelial cells lining the kidney pelvis, ureter, and bladder. As anti-mu did not react in tissue sections, distribution of mu was determined by immunoblotting. Immunoblots of cytosolic preparations from whole kidney, cortex, medulla, and epithelia of ureter, bladder, and kidney pelvis were prepared and tested with each of the 4 antibodies. This second localization method confirmed the distribution data from tissue sections for rbGST alpha I, rb GST alpha II, and pi; also, it demonstrated that the staining observed in tissue was specifically for each enzyme. mu was detected in all the renal cytosolic preparations except those from the epithelium of the kidney pelvis. CONCLUSIONS: The discrete renal distribution of rbGST alpha I and rbGST alpha II and their distinct catalytic activities with prostaglandin substrates suggest important roles for these enzymes in prostaglandin-dependent renal functions.

Animals↗
PubMed1994

Elevated susceptibility to 4-ipomeanol cytotoxicity in immature Clara cells of neonatal rabbits.

The bronchiolar Clara cell is one of the primary targets in adult mammals for environmental contaminants metabolized by cytochrome P450 (CYP) monooxygenases. Previous studies show that the onset of CYP expression in Clara cells occurs during postnatal lung development. This study was designed to determine whether differentiating Clara cells are susceptible to CYP-activated cytotoxicants and whether these substances can influence subsequent cytodifferentiation. Adult and neonatal (5-9 days of age) rabbits were given a single dose of 4-ipomeanol (IPO) i.p. and sacrificed 2 or 7 days later. Their lungs were removed and assessed morphologically, immunohistochemically or for CYP activity. Treatment with 10 mg/kg of IPO (0.25 of the LD50 for adults) killed 6 of 10 neonatal rabbits. At a dose of 5 mg/kg of IPO, most terminal bronchiolar cells were destroyed in the neonatal rabbits. The basal lamina of terminal bronchioles was either bare or lined by squamous or low cuboidal epithelium and macrophages. Terminal bronchiolar epithelium in neonates was minimally affected by a dose of 1 mg/kg of IPO. The terminal bronchioles in adults appeared nearly unaffected by either 1 or 5 mg/kg of IPO. Interalveolar septa were unaffected in all treated animals. Lung microsomal enzymes from neonatal rabbits metabolized IPO to reactive intermediates at less than one-third the rate in the lungs of adults. Seven days (15 days of age) after IPO treatment, CYP activity (as measured by pentoxyresorufin O-dealkylation) was one-half that of age-matched controls after a dose of 5 mg/kg but equaled control activity after 1 mg/kg. Immunohistochemical analysis, using antibodies to CYP2B4, CYP4B and CYP reductase, indicated that the decrease in activity seen with a dose of 5 mg/kg of IPO was the result of a loss of immunoreactive CYP proteins from the cuboidal cells of terminal bronchioles. It was concluded that, in neonatal animals, differentiating Clara cells are more susceptible to injury by bioactivated cytotoxicants than are differentiated cells in adults, despite the neonate's lower levels of CYP monooxygenases. Furthermore, IPO-induced injury impairs the normal pattern of postnatal Clara cell differentiation.

Aging↗
PubMed1993

Cloning, sequencing, and functional studies of phenobarbital-inducible forms of cytochrome P450 2B and 4B expressed in rabbit kidney.

Expression of several forms of cytochrome P450 2B and of P450 4B1 in rabbit kidney was investigated by cloning from cDNA libraries constructed with renal mRNA from animals treated with phenobarbital. Isolation and sequencing of several cDNAs demonstrated that: (i) cytochrome P450 2B-B0 can be found in rabbit kidney along with a newly discovered form of P450 2B termed "P450 2B-Bx." P450 2B-Bx differs from P450 2B-B0 at 25 nucleotide positions and at four positions in the derived sequence of 491 amino acids. Two previously identified forms of cytochrome P450 2B, 2B-B1 and 2B-B2, are not detected in rabbit kidney. cDNA encoding cytochrome P450 4B1 was also cloned from the kidney library and found to be identical in sequence to cDNAs cloned from rabbit hepatic and pulmonary libraries. Analysis of renal mRNA indicates that forms of cytochrome P450 2B and P450 4B1 are expressed in a number of species but induced by phenobarbital in rabbit only (4B1) or rabbit and hamster (2B). Relatively high levels of mRNA related to P450 4B1 were detected in samples from untreated and phenobarbital-treated mice. Analysis of protein by immunoblotting was less sensitive but produced results consistent with those obtained by analysis of mRNA; protein related to cytochrome P450 2B was detected in renal microsomal samples from rabbit and hamster (phenobarbital > untreated), and protein related to P450 4B1 in samples from rabbits (phenobarbital > untreated) and mice (phenobarbital = untreated). The four forms of cytochrome P450 2B were expressed in COS-7 cells, and their activities were evaluated with androstenedione, testosterone, and 7-ethoxycoumarin as substrates. Three of the P450 2B forms, B0, B1, and Bx, metabolize these substrates in a manner characterized by preference for 16 beta-hydroxylation of androstenedione, low testosterone 16-hydroxylation, and high ethoxycoumarin O-deethylation. The fourth form, P450 2B-B2, is catalytically distinct from the others, with activities characterized by high androstenedione 16 alpha- and 15 alpha-hydroxylation and high testosterone 16-hydroxylation. Since P450 2B-B2 is catalytically distinct from the other forms, the metabolic profiles of phenotypes that include P450 2B-B2 might differ significantly from those of phenotypes that lack P450 2B-B2.

Amino Acid Sequence↗
PubMed1993

Cloning, sequencing, distribution, and expression in Escherichia coli of flavin-containing monooxygenase 1C1. Evidence for a third gene subfamily in rabbits.

Two full-length cDNA clones (2.2 kilobases) encoding a newly recognized form of mammalian flavin-containing monooxygenase (FMO) have been isolated from independent libraries constructed with mRNA from different rabbits. The cDNAs encode a polypeptide of 533 amino acids which contains two putative pyrophosphate binding domains and a hydrophobic carboxyl terminus characteristic of FMOs. This sequence is 52 and 57% identical to sequences of the rabbit "hepatic" and "pulmonary" FMOs, respectively, and 55% identical to the sequence of "liver form 2" published recently by Ozols (Ozols, J. (1991) Arch. Biochem. Biophys. 290, 103-115). cDNA for the new FMO (FMO 1C1) hybridizes with two species of mRNA, one of 2.6 kilobases and one of about 5.4 kilobases, from liver or kidney, but not lung. Guinea pig, hamster, rat, and mouse all express this form of FMO in liver, kidney, and lung. FMO 1C1 has been tentatively characterized following expression in Escherichia coli. It is inactive with methimazole as substrate but highly active with n-octylamine. The temperature lability, responses to ions and detergent, and pH optimum of FMO 1C1 are similar to values reported for hepatic FMO. Sequence comparisons and analysis of rabbit and human genomic DNA indicate that FMO 1C1, as well as the pulmonary and hepatic FMOs, comprise a single gene family made up of distinct gene subfamilies (A, B,C,D, ... N), each appearing to contain a single gene. A nomenclature, based on these interrelationships and following the same designations used for classifying cytochromes P-450, is proposed.

Amino Acid Sequence↗
PubMed1993

Expression of truncated forms of liver microsomal P450 cytochromes 2B4 and 2E1 in Escherichia coli: influence of NH2-terminal region on localization in cytosol and membranes.

The currently accepted model for the membrane topology of microsomal cytochrome P450 is that of a largely cytoplasmic domain bound by only one or two transmembrane segments at the NH2 terminus. However, as we have reported previously, P450 2E1 lacking the hydrophobic NH2-terminal signal peptide, like the full-length protein, is located in the inner cell membrane when expressed in Escherichia coli and is active with typical substrates. In the present study, additional variants of alcohol-inducible P450 2E1 as well as truncated forms of phenobarbital-inducible P450 2B4 were similarly expressed to determine the influence of the NH2-terminal region on the membrane-binding properties. After deletion of S1 (the NH2-terminal hydrophobic segment), or both S1 and L1 (the following hydrophilic region, expected to be lumenal or cytosolic), one-third of the resulting P450 2B4 (delta 2-20) and 2B4 (delta 2-27) remained membrane bound. Furthermore, the idea that the first two hydrophobic segments are required for attachment by a hairpin loop is not supported by the finding that after deletion of the S1, L1, and S2 segments about half of the P450 2E1 (delta 3-48) remained membrane bound. Since Na2CO3 treatment of the membrane fraction had no significant effect, the findings are apparently not attributable to a loose attachment or occlusion of the truncated proteins. The replacement of neutral amino acids by positively charged residues in positions 3 and 8 of P450 2E1 (delta 3-29) changed the amount in the cytosol from 35% to 50%, and the deletion of residues 2-20 or 2-27 from P450 2B4, which resulted in positive charges occurring in the NH2-terminal region, changed the amount in the cytosol from 27% to 67%. We conclude that alterations in the NH2-terminal region can change the location of the cytochrome from largely membranous to largely cytosolic and that the first two hydrophobic segments are not uniquely involved in membrane attachment.

Amino Acid Sequence↗
PubMed1993

Functional characterization of flavin-containing monooxygenase 1B1 expressed in Saccharomyces cerevisiae and Escherichia coli and analysis of proposed FAD- and membrane-binding domains.

A cDNA encoding the flavin-containing monooxygenase of rabbit lung (FMO 1B1) was expressed in yeast and Escherichia coli and the recombinant enzymes characterized. A high copy, isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-inducible E. coli expression vector, pKKHC, was used for expression in E. coli strain JM109, and a galactose-inducible vector, YEp53, was used for expression in yeast strain 334. Following transcriptional induction with IPTG or galactose, subcellular fractions were prepared and analyzed immunochemically and catalytically. Antibodies to rabbit FMO 1B1 were used to detect the recombinant proteins in the 100,000 x g pellet prepared from the 10,000 x g supernatant fraction of yeast homogenates and the 2,000 x g supernatant fraction of E. coli homogenates. No FMO 1B1 was detected in cytosol. Mobilities of the recombinant proteins in SDS-polyacrylamide gel electrophoresis appeared identical to that of the native microsomal enzyme. Catalytic similarity to the native FMO 1B1 was demonstrated by the ability of the expressed enzymes to metabolize methimazole, thiourea, dimethylaniline, and cysteamine, but not chlorpromazine or imipramine. In addition, the recombinant enzymes exhibited a number of the unique physical properties associated with FMO 1B1, including stability to elevated temperature and activation by sodium cholate and magnesium chloride. Based on the specific content of FAD, the level of expression was estimated to be approximately 2% of the total protein in the E. coli 100,000 x g particulate fraction and 1% in the fraction from yeast. To demonstrate the utility of the E. coli expression system for studying structure/function relationships of the flavin-containing monooxygenase, two mutant FMOs were expressed and characterized. One mutant, formed by deletion of a putative membrane-anchoring peptide (the 26 carboxyl-terminal amino acids) was tested for membrane association. No difference in the subcellular distribution was found between the truncated and unmodified proteins, suggesting that the 26-residue COOH-terminal peptide is not important in membrane association. Catalytic analysis of the truncated FMO 1B1 established its functional similarity to the full-length protein, indicating that the COOH terminus does not contribute to any of the unique properties of the lung enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

CYP4B1 activates 4-ipomeanol in rat lung.

Inhibition of pulmonary CYP4B1 activity by pretreatment of rats with p-xylene decreased the ability of lung microsomes to N-hydroxylate 2-aminofluorene and prevented the lung damage normally seen after dosing with ipomeanol. The toxicity of ipomeanol, as assessed by acute lethality, was decreased by a factor of eight. In contrast, induction of CYP1A1 by Aroclor or beta-naphthoflavone, or inhibition of CYP2B1 by O,O,S-trimethyl-phosphorodithioate, as assessed by measurement of lung microsomal dealkylation of ethoxyresorufin or pentoxyresorufin, did not change ipomeanol toxicity. A polyclonal antibody raised against CYP4B1 prevented the covalent binding of [14C]-ipomeanol to lung microsomal protein in vitro. Antibodies raised against the other major P450 isozymes of rat lung, CYP2B1 and CYP1A1, had no effect on this binding. Aroclor, beta-naphthoflavone, and O,O,S-trimethylphosphorodithioate failed to affect binding of radiolabeled ipomeanol in vivo, but pretreatment with p-xylene resulted in a significant reduction in this binding. The CYP4B1 substrate 2-aminofluorene, when dosed to rats, caused a sixfold decrease in ipomeanol toxicity. These results indicate that in the rat, unlike the rabbit, pulmonary bioactivation of ipomeanol is predominantly dependent upon CYP4B1.

Animals↗
PubMed1993

Postnatal changes in the expression and distribution of pulmonary cytochrome P450 monooxygenases during Clara cell differentiation in rabbits.

Previous studies have indicated that both cytodifferentiation of Clara cells and the onset of pulmonary cytochrome P450 activity are postnatal events. However, the relationship between these two events during lung development remains poorly understood. To determine how these events interrelate, we examined rabbit Clara cells during postnatal differentiation, with the following goals in mind: 1) to identify the patterns of intracellular expression of cytochrome P450 monooxygenase isozymes 2B and 4B and cytochrome P450 reductase, 2) to describe the biogenesis of the organelles with which these isozymes are associated, namely smooth and rough endoplasmic reticulum, and 3) to compare the patterns of expression with cytochrome P450 activity in the whole lung over the same period. Lungs of rabbits ranging in age from 24 days gestational age (DGA) to 25 weeks postnatally were studied. Ultrastructural morphometry showed that smooth endoplasmic reticulum averaged < 5% of the Clara cell volume in late gestational (24-30 DGA) and neonatal rabbits [0-7 days postnatally (DPN)], grew to 20-30% of the cell volume in 14-21-DPN animals, and approximated adult levels (> 40%) in 28-DPN rabbits. In contrast, rough endoplasmic reticulum decreased from > 10% of the cell volume at 27 DGA to < 5% in adults. All postnatal animals showed considerable heterogeneity in the abundance of smooth endoplasmic reticulum among individual cells. Immunohistochemistry revealed that cytochrome P450 reductase appeared in Clara cells earlier (28 DGA) than did either isozyme 2B or 4B (1 DPN). Each antigen was detected first in the apical borders of the cells, then throughout the cytoplasm in a few cells by 7 DPN, and finally in adult abundance by 28 DPN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that cytochrome P450 protein concentrations increased postnatally. Cytochrome P450 heme protein was not detected spectrophotometrically in the lungs of animals younger than 3 DPN but increased to approximately 70% of adult levels by 28 DPN. Likewise, cytochrome P450 activity (measured as ethoxy- and pentoxyresorufin O-dealkylation) was not detected in animals younger than 2 DPN but increased to approximately 75% of adult levels by 28 DPN.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Functional expression of renal organic anion transport in Xenopus laevis oocytes.

Secretion of organic anions by the kidney plays a critical role in the elimination of toxic agents from the body. Recent findings in isolated membranes and intact tissue have demonstrated the participation of multiple transport proteins in this process. As a first step toward molecular characterization of these proteins through expression cloning, the studies reported below demonstrate functional expression of both fumarate- and lithium-sensitive glutarate and probenecid-sensitive p-aminohippurate transport in Xenopus oocytes injected with rat kidney poly(A)+ RNA. Maximal increase in substrate uptake over buffer-injected controls was reached by 5 days after mRNA injection. Expression of size-fractionated mRNA indicated that the active species with respect to both transport activities were in the range of 1.8 to 3.5 kb.

Animals↗
PubMed1992

Guinea pig or rabbit lung flavin-containing monooxygenases with distinct mobilities in SDS-PAGE are allelic variants that differ at only two positions.

Both guinea pig and rabbit express two variants of the 'lung' flavin-containing monooxygenase (FMO), observed as three distinct phenotypes based on mobility differences in SDS-PAGE. Samples of messenger RNA prepared from lungs of the two homozygous phenotypes of the guinea pig were used for the construction of two cDNA libraries. The libraries were screened with a cDNA encoding the rabbit lung FMO, and positive clones for each guinea pig lung FMO variant were isolated and sequenced. A full length clone from each library was found to encode a protein of 535 amino acids containing two pyrophosphate binding sites. Comparison of the sequences of the guinea pig and rabbit lung FMOs shows that their primary structures are 86% identical. The coding region sequences of the guinea pig variants differ at only two positions, and both differences result in amino acid substitutions. Sequence analysis has also been completed on a partially characterized variant of the rabbit lung FMO. As with the guinea pig, the nucleotide and amino acid sequences of the rabbit variants differ at only two positions. The cDNAs encoding the guinea pig variants were expressed in yeast. The activities of the enzymes are characteristic of the lung FMO, and the mobilities of the expressed enzymes are the same as those observed for the variants present in guinea pig pulmonary microsomal preparations. Similar to findings for the rabbit, analysis of genomic DNA indicates that the guinea pig lung FMO is associated with a single gene. The results of cDNA sequence analysis, expression in yeast, and analysis of genomic DNA indicate that the multiple lung FMOs in guinea pig and rabbit are allelic variants whose mobilities in SDS-PAGE are markedly altered by minimal changes in primary structure.

Alleles↗
PubMed1992

Regiochemical differences in cytochrome P450 isozymes responsible for the oxidation of methylenedioxyphenyl groups by rabbit liver.

The cytochrome P450 isozymes catalyzing the oxidation of the methylenedioxyphenyl compounds methylenedioxybenzene (MDB) and methylenedioxyamphetamine (MDA) have been investigated in rabbit liver preparations. The aromatic ring in MDB undergoes both demethylenation to catechol and aromatic hydroxylation to sesamol, whereas that in MDA undergoes only demethylenation to dihydroxyamphetamine. Formation of catechol and sesamol from MDB in microsomal incubation mixtures was enhanced about 5- and 3-fold, respectively, by pretreatment of the rabbits with phenobarbital, which induced CYP2B4 and CYP4B1. The cytochrome P450 isozyme responsible for aromatic hydroxylation of MDB was induced by beta-naphthoflavone and was inhibited by alpha-naphthoflavone. Microsomal demethylenation of MDA was minimally sensitive to pretreatment of the rabbits with phenobarbital, beta-naphthoflavone, pyrazole, or rifampicin. However, MDA competitively inhibited the N-demethylation of erythromycin. Antibodies against CYP2B4, but not those against CYP4B1, caused a marked inhibition of the demethylenation and aromatic hydroxylation of MDB. Antibodies against CYP2C3 did not inhibit the demethylenation of MDA, nor did substrates or inhibitors of the CYP2D family except for bufuralol. MDB and MDA were both capable of forming metabolic intermediate complexes, and the rate of complex formation was accelerated by phenobarbital induction. Reconstitution experiments with CYP2B4 suggested that phenobarbital-inducible complex formation from MDA was not due to the carbene pathway involving the methylenedioxy group but was due to oxidation of the amino group. These results indicate that CYP2B4 oxidizes different regions of methylenedioxyphenyl compounds depending on their structure. MDB undergoes oxidation at the methylenedioxy group (major) and the benzene ring (minor). MDA is oxidized at the alkylamino side chain at the nitrogen and alpha-carbon. The results suggested that one or more constitutive isoforms (probably unknown) of cytochrome P450 present in rabbit liver microsomes are primarily responsible for MDA demethylenation but that CYP3A6 contributes slightly.

3,4-Methylenedioxyamphetamine↗
PubMed1992

Distribution of cytochrome P450 1A1 and NADPH-cytochrome P450 reductase in lungs of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin: ultrastructural immunolocalization and in situ hybridization.

Induction of cytochrome P450 1A1 (P450 1A1) in a variety of tissues is a well established consequence of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although localization of the induced protein within the lung has been described, the precise intracellular distribution of the enzyme is not clear. Analysis of tissue sections, microsomal proteins, and mRNA from lungs of treated and untreated rabbits established that P450 1A1 had been induced by treatment with TCDD. Rabbit lungs from animals treated with TCDD were examined with immunocytochemistry and in situ hybridization, to identify the cell types that contain P450 1A1 and those that contain mRNA encoding P450 1A1. Endothelial cells of the entire vascular bed of rabbit lung reacted markedly with anti-P450 1A1. Likewise, cells lining both arteries and veins, as well as capillary endothelial cells, reacted strongly with the cDNA probe for mRNA encoding P450 1A1. Clara cells at all levels of airway labeled prominently for both P-450 1A1 and P450 1A1 mRNA. In addition, type 2 cells, alveolar macrophages, and to a lesser degree, ciliated cells reacted with the cDNA probe. P450 reductase, which is required for P450 activity, has previously been identified in Clara cells, type 2 cells, and alveolar macrophages, but not in endothelium of rabbit lung. We have now obtained similar results for the localization of mRNA encoding P-450 reductase. This finding brings into question the function of P450 1A1 in endothelium.

Animals↗