Cancer-associated fibroblasts-derived FMO2 as a biomarker of macrophage infiltration and prognosis in epithelial ovarian cancer.

OBJECTIVE: Recent molecular profiling revealed that cancer-associated fibroblasts (CAFs) are essential for matrix remodeling and tumor progression. Our study aimed to investigate the role of flavin-containing monooxygenase 2 (FMO2) in epithelial ovarian cancer (EOC) as a novel CAF-derived prognostic biomarker. METHODS: Primary fibroblasts were isolated from EOC samples. Microdissection and single-cell RNA sequencing (scRNA-seq) datasets (including TCGA, GSE9891, GSE63885, GSE118828 and GSE178913) were retrieved to determine the expression profiles. Gene set enrichment analysis (GSEA) was used to explore the correlation between FMO2 and stromal activation as well as immune infiltration. The predictive value of FMO2 and combined macrophage infiltration level was verified in an independent EOC cohort (n = 113). RESULTS: We demonstrated that FMO2 was upregulated in tumor stroma and correlated with fibroblast activation. Besides, FMO2 had the predictive power for worse clinical outcome of EOC patients. In the mesenchymal subtype of EOC, the FMO2-defined signature revealed that FMO2 contributed to infiltration of tumor-infiltrating lymphocytes. Moreover, we confirmed the positive correlation between FMO2 and CD163+ cell infiltration level in EOC tissues, and showed that combination of FMO2 expression with CD163+ cell infiltration level in the tumor stroma could predict poor overall survival (HR = 3.63, 95% CI = 1.93-6.84, p = 0.0008). Additionally, FMO2 also predicted the prognosis of patients with ovarian cancer based on the expression of immune checkpoints (such as PD-L1 and PD1). CONCLUSION: Our results address the tumor-supporting role of FMO2 in EOC and its association with immune components, and it might be a prospective target for stroma-oriented therapies against EOC.

Evidence of Trained Immunity in a Fish: Conserved Features in Carp Macrophages.

Trained immunity is a form of innate immune memory best described in mice and humans. Clear evidence of the evolutionary conservation of trained immunity in teleost fish is lacking. Given the evolutionary position of teleosts as early vertebrates with a fully developed immune system, we hypothesize that teleost myeloid cells show features of trained immunity common to those observed in mammalian macrophages. These would at least include the ability of fish macrophages to mount heightened responses to a secondary stimulus in a nonspecific manner. We established an in vitro model to study trained immunity in fish by adapting a well-described culture system of head kidney-derived macrophages of common carp. A soluble NOD-specific ligand and a soluble ß-glucan were used to train carp macrophages, after which cells were rested for 6 d prior to exposure to a secondary stimulus. Unstimulated trained macrophages displayed evidence of metabolic reprogramming as well as heightened phagocytosis and increased expression of the inflammatory cytokines il6 and tnf-α. Stimulated trained macrophages showed heightened production of reactive oxygen and nitrogen species as compared with the corresponding stimulated but untrained cells. We discuss the value of our findings for future studies on trained immunity in teleost fish.

NHR-49 Transcription Factor Regulates Immunometabolic Response and Survival of Caenorhabditis elegans during Enterococcus faecalis Infection.

Immune response to pathogens is energetically expensive to the host; however, the cellular source of energy to fuel immune response remains unknown. In this study, we show that Caenorhabditis elegans exposed to pathogenic Gram-positive and Gram-negative bacteria or yeast rapidly utilizes lipid droplets, the major energy reserve. The nematode's response to the pathogenic bacterium Enterococcus faecalis entails metabolic rewiring for the upregulation of several genes involved in lipid utilization and downregulation of lipid synthesis genes. Genes encoding acyl-CoA synthetase ACS-2, involved in lipid metabolism, and flavin monooxygenase FMO-2, involved in detoxification, are two highly upregulated genes during E. faecalis infection. We find that both ACS-2 and FMO-2 are necessary for survival and rely on NHR-49, a peroxisome proliferator-activated receptor alpha (PPARα) ortholog, for upregulation during E. faecalis infection. Thus, NHR-49 regulates an immunometabolic axis of survival in C. elegans by modulating breakdown of lipids as well as immune effector production upon E. faecalis exposure.

Development of an immunoassay for the kidney-specific protein myo-inositol oxygenase, a potential biomarker of acute kidney injury.

BACKGROUND: Acute kidney injury (AKI) affects 45% of critically ill patients, resulting in increased morbidity and mortality. The diagnostic standard, plasma creatinine, is nonspecific and may not increase until days after injury. There is significant need for a renal-specific AKI biomarker detectable early enough that there would be a potential window for therapeutic intervention. In this study, we sought to identify a renal-specific biomarker of AKI. METHODS: We analyzed gene expression data from normal mouse tissues to identify kidney-specific genes, one of which was Miox. We generated monoclonal antibodies to recombinant myo-inositol oxygenase (MIOX) and developed an immunoassay to quantify MIOX in plasma. The immunoassay was tested in animals and retrospectively in patients with and without AKI. RESULTS: Kidney tissue specificity of MIOX was supported by Western blot. Immunohistochemistry localized MIOX to the proximal renal tubule. Serum MIOX, undetectable at baseline, increased 24 h following AKI in mice. Plasma MIOX was increased in critically ill patients with AKI [mean (SD) 12.4 (4.3) ng/mL, n = 42] compared with patients without AKI [0.5 (0.3) ng/mL, n = 17] and was highest in patients with oliguric AKI [20.2 (7.5) ng/mL, n = 23]. Plasma MIOX increased 54.3 (3.8) h before the increase in creatinine. CONCLUSIONS: MIOX is a renal-specific, proximal tubule protein that is increased in serum of animals and plasma of critically ill patients with AKI. MIOX preceded the increases in creatinine concentration by approximately 2 days in human patients. Large-scale studies are warranted to further investigate MIOX as an AKI biomarker.

Phospholipid remodeling and eicosanoid signaling in colon cancer cells.

Phospholipid remodeling and eicosanoid synthesis are central to lipid-based inflammatory reactions. Studies have revealed that membrane phospholipid remodeling by fatty acids through deacylation/reacylation reactions increases the risk of colorectal cancers (CRC) by allowing the cells to produce excess inflammatory eicosanoids, such as prostaglandins, thromboxanes and leukotrienes. Over the years, efforts have been made to understand the lipid remodeling pathways and to design anti-cancer drugs targeting the enzymes of eicosanoid biosynthesis. Here, we discuss the recent progress in phospholipid remodeling and eicosanoid biosynthesis in CRC.

Deficiency of CD73 activity promotes protective cardiac immunity against Trypanosoma cruzi infection but permissive environment in visceral adipose tissue.

Damaged cells release the pro-inflammatory signal ATP, which is degraded by the ectonucleotidases CD39 and CD73 to the anti-inflammatory mediator adenosine (ADO). The balance between ATP/ADO is known to determine the outcome of inflammation/infection. However, modulation of the local immune response in different tissues due to changes in the balance of purinergic metabolites has yet to be investigated. Here, we explored the contribution of CD73-derived ADO on the acute immune response against Trypanosoma cruzi parasite, which invades and proliferates within different target tissues. Deficiency of CD73 activity led to an enhanced cardiac microbicidal immune response with an augmented frequency of macrophages with inflammatory phenotype and increased CD8+ T cell effector functions. The increment of local inducible nitric oxide (NO) synthase (iNOS)+ macrophages and the consequent rise of myocardial NO production in association with reduced ADO levels induced protection against T. cruzi infection as observed by the diminished cardiac parasite burden compared to their wild-type (WT) counterpart. Unexpectedly, parasitemia was substantially raised in CD73KO mice in comparison with WT mice, suggesting the existence of tissue reservoir/s outside myocardium. Indeed, CD73KO liver and visceral adipose tissue (VAT) showed increased parasite burden associated with a reduced ATP/ADO ratio and the lack of substantial microbicidal immune response. These data reveal that the purinergic system has a tissue-dependent impact on the host immune response against T. cruzi infection.

Supplementation of endogenous Ahr ligands reverses insulin resistance and associated inflammation in an insulin-dependent diabetic mouse model.

Aryl-hydrocarbon receptor (Ahr) plays an important role in the regulation of intestinal homeostasis. Diabetes is characterized by vascular complications and intestinal dysfunction. We aimed at understanding the relationship between intestinal defense impairment and inflammation in diabetes and effects of Ahr ligands on diabetes-induced insulin resistance, endovascular inflammation, and intercellular adhesion molecule (ICAM) and flavin mono-oxygenase (FMO3) expression. Effects of Ahr ligands, such as tryptophan (Trp) and indole-3-carbinol (I3C) on intestinal barrier and inflammation of Ins2Akita mice were examined. Myeloid differentiation primary response 88 (MYD88) is the adaptor for inflammatory signaling pathways. Ins2Akita-MyD88-/- mice were used to study the role of MyD88. Ins2Akita mice demonstrated decreased Ahr and regenerating islet-derived 3-ß (Reg3ß) expression, and increased Klebsiella pneumoniae translocation. Ins2Akita mice demonstrated increased inducible nitric oxide synthase (iNOS) expression of intestine; ICAM, iNOS, interleukin 1 beta (IL-1ß), and FMO3 expression of liver; and ICAM, iNOS, and FMO3 expression in aorta. Trp and I3C decreased diabetes-induced translocation and increased Ahr and Reg3ß expression of intestine. Ahr ligands reduced diabetes-induced ICAM and FMO3 expression in liver and aorta; IL-6, tumor necrosis factor alpha (TNF-α), and iNOS expression in Kupffer cells; plasma IL-6 and TNF-α levels; dipeptidyl peptidase (DPP4) activity; and insulin insensitivity. Ins2Akita-MyD88-/- mice demonstrated decreased expression of p-NF-κB of liver and ICAM of aorta compared with Ins2Akita mice. Altogether, our data suggest that diabetes induces ICAM and FMO3 expression through the decrease in intestinal defense and MyD88. Ahr ligands reverse diabetes-induced intestinal defense impairment, insulin insensitivity, FMO3/ICAM expression, and systemic inflammation.

Differential localization of flavin-containing monooxygenase (FMO) isoforms 1, 3, and 4 in rat liver and kidney and evidence for expression of FMO4 in mouse, rat, and human liver and kidney microsomes.

Flavin-containing monooxygenases (FMOs) play significant roles in the metabolism of drugs and endogenous or foreign compounds. In this study, the regional distribution of FMO isoforms 1, 3, and 4 was investigated in male Sprague-Dawley rat liver and kidney using immunohistochemistry (IHC). Rabbit polyclonal antibodies to rat FMO1 and FMO4, developed using anti-peptide technology, and commercial anti-human FMO3 antibody were used; specificities of the antibodies were verified using Western blotting, immunoprecipitation, and IHC. In liver, the highest immunoreactivity for FMO1 and FMO3 was detected in the perivenous region, and immunoreactivity decreased in intensity toward the periportal region. In contrast, FMO4 immunoreactivity was detected with the opposite lobular distribution. In the kidney, the highest immunoreactivity for FMO1, -3, and -4 was detected in the distal tubules. FMO1 and FMO4 immunoreactivity was also detected in the proximal tubules with strong staining in the brush borders, whereas less FMO3 immunoreactivity was detected in the proximal tubules. Immunoreactivity for FMO3 and FMO4 was detected in the collecting tubules in the renal medulla and the glomerulus, whereas little FMO1 immunoreactivity was detected in these regions. The FMO1 antibody did not react with human liver or kidney microsomes. However, the FMO4 antibody reacted with male and female mouse and human tissues. These data provided a compelling visual demonstration of the isoform-specific localization patterns of FMO1, -3, and -4 in the rat liver and kidney and the first evidence for expression of FMO4 at the protein level in mouse and human liver and kidney microsomes.

LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase.

LKM-1 autoantibodies, which are associated with autoimmune chronic active hepatitis, recognize P450IID6, a cytochrome P-450 monooxygenase. The reactivities of 26 LKM-1 antisera were tested with a panel of deletion mutants of P450IID6 expressed in Escherichia coli. 22 sera recognize a 33-amino acid segment of P450IID6, and 11 of these recognize a shorter segment, DPAQPPRD. PAQPPR is also found in IE175 of herpes simplex virus type 1 (HSV-1). Antibodies for HSV-1 proteins were detected by ELISA in 17 of 20 LKM-1 sera tested. An immobilized, synthetic peptide, DPAQPPRDC, was used to purify LKM-1 antibodies. Affinity purified LKM-1 autoantibodies react on immunoblots with a protein in BHK cells after infection with HSV-1. 11 of 24 LKM-1 sera, including 3 that recognize DPAQPPRD, also exhibit antibodies to the hepatitis C virus (HCV) protein, C100-3. Affinity purified LKM-1 antibodies did not recognize C100-3. However, partial sequence identity was evident between portions of the immunopositive 33-amino acid segment of P450IID6 and other portions of the putative HCV polyprotein. Immune cross-recognition of P450IID6 and HCV or HSV-1 proteins may contribute to the occurrence of LKM-1 autoantibodies.

Mycobacterium tuberculosis MymA is a TLR2 agonist that activate macrophages and a TH1 response.

Cell wall of Mycobacterium tuberculosis (M.tb) is a major source of immunogenic proteins that can be tested as vaccine candidates. MymA (Rv3083), a 55 kDa M.tb flavin containing monooxygenase, is involved in modification of mycolic acids during acidic shock following M.tb internalization in macrophage. In this study, we have investigated the role of this cell wall associated protein in activation of macrophages by toll like receptor (TLRs) engagement and subsequent signaling. Our results showed that MymA stimulation of THP1 cells and human monocyte derived macrophages (MDM) lead to upregulation of TLR2 and co-stimulatory molecules CD40, CD80, CD86 and HLA-DR. This upregulation is partially reduced by TLR2 blocking antibodies. The activation of macrophage following MymA stimulation also resulted in release of proinflammatory cytokines, TNF-α and IL-12. Moreover, MymA also polarized the immune response towards TH1 as shown by an increased IFN-γ level in the supernatant of stimulated peripheral blood mononuclear cells (PBMC). In consensus with the TLR2 signaling involving MyD88 and NF-κB, we also observed several fold increase in mRNA for TLR2, MyD88 and NF-κB on MymA induction of THP-1 and MDM by qRT-PCR. The increased production of NF-κB following recognition of MymA by TLR2 was further confirmed by HEK-TLR2 reporter cell line colorimetric assay. In conclusion, immunological evaluation revealed that MymA is a TLR2 agonist that upregulates signaling via MyD88 and NF-κB in macrophages to stimulate the release of proinflammatory cytokines. The MymA protein should be investigated further for expression in recombinant BCG as a pre-exposure vaccine or as a post-exposure subunit vaccine candidate.

Purification of macaque liver flavin-containing monooxygenase: a form of the enzyme related immunochemically to an isozyme expressed selectively in adult human liver.

A microsomal flavin-containing monooxygenase (FMO) was purified 77-fold from macacque liver microsomes on the basis of its methyl p-tolyl sulfoxidase activity. Sequential chromatography on anion- and cation-exchangers, lauryl-Sepharose and 2',5'-ADP-Sepharose provided a purified preparation which exhibited an apparent molecular mass of 59 kDa and a pI of 8.3. N-terminal amino-acid sequencing revealed the characteristic Gly-X-Gly-X-X-Gly consensus sequence for the putative FAD-binding domain of microsomal FMO. In marked contrast to the well-characterized hepatic and pulmonary forms present in experimental animals, the macacque liver enzyme displayed stereoselectivity for sulfoxidation of p-tolyl methyl sulfide on the pro-S rather than the pro-R face of the substrate. Polyclonal antibodies raised against the macacque liver form exhibited little or no cross-reactivity with major purified forms of the enzyme isolated from rabbit liver, guinea-pig liver or rabbit lung. Anti-macacque liver FMO did not cross-react with human fetal liver or adult kidney microsomes, but did recognize a 59 kDa constituent of human adult liver microsomes. The intensity of this immunoreactive 59 kDa band correlated well with human liver microsomal N,N-dimethylaniline N-oxygenase activity. We conclude that human adult liver selectively expresses a microsomal FMO which is functionally and immunochemically distinct from the FMO form(s) present in human fetal liver and adult kidney, and from the major hepatic and pulmonary forms present in common laboratory animals.

Immunological characterization of flavin-containing monooxygenases from the liver of rainbow trout (Oncorhynchus mykiss): sexual- and age-dependent differences and the effect of trimethylamine on enzyme regulation.

Polyclonal antibodies raised against flavin-containing monooxygenase (FMO) enzymes purified from pig liver and rabbit lung were used in conjunction with N,N-dimethylaniline (DMA) N-oxidase to better characterize FMO from the liver of rainbow trout (Oncorhynchus mykiss). Two proteins reacted with polyclonal antibodies raised against pig liver FMO (PL-1 and PL-2) and anti-rabbit lung FMO (RL-1 and RL-2). Although there was no difference in DMA N-oxidase observed between sexually mature male and female trout liver microsomes, RL-2 and PL-2 were significantly less than RL-1 and PL-1, respectively, in sexually mature females. FMO activity and protein content increased as fish aged. DMA oxidase and FMO isozymes were unaltered after pretreatment with the endogenous substrate trimethylamine. Since antibodies to the purified mammalian enzymes react with proteins of similar MW in trout, some forms of FMO appear to be structurally conserved through evolution.

Ribulose 1,5-bisphosphate carboxylase-oxygenase. I. Structural, immunochemical and catalytic properties.

Some structural, immunochemical and catalytic properties are examined for ribulose 1,5-bisphosphate carboxylase-oxygenase from various cellular organisms including bacteria, cyanobacteria, algae and higher plants. The native enzyme molecular masses and the subunit polypeptide compositions vary according to enzyme sources. The molecular masses of the large and small subunits from different cellular organisms, on the other hand, show a relatively high homology due to their well-conserved primary amino acid sequence, especially that of the large subunit. In higher plants, the native enzyme and the large subunit are recognized by the antibodies raised against either the native or large subunit, whereas the small subunit apparently cross-reacts only with the antibodies directed against itself. A wide diversity exists, however, in the serological response of the native enzyme and its subunits with antibodies directed against the native enzyme or its subunits from different cellular organisms. According to numerous kinetic studies, the carboxylase and oxygenase reactions of the enzyme with ribulose 1,5-bisphosphate and carbon dioxide or oxygen require activation by carbon dioxide and magnesium prior to catalysis with ribulose 1,5-bisphosphate and carbon dioxide or oxygen. The activation and catalysis are also under the regulation of other metal ions and a number of chloroplastic metabolites. Recent double-labeling experiments using radioactive ribulose 1,5-bisphosphate and 14CO2 have elucidated the carboxylase/oxygenase ratios of the enzymes from different organisms. Another approach, i.e., genetic experiments, has also been used to examine the modification of the carboxylase/oxygenase ratio.

Cerebral metabolism of imipramine and a purified flavin-containing monooxygenase from human brain.

Flavin-containing monooxygenase (FMO), previously reported both from hepatic and extrahepatic tissues, including brain, catalyze the oxidation of certain xenobiotics and drugs that contain a nucleophilic heteroatom. Psychoactive drugs, including the antidepressant imipramine, are substrates for the brain FMO. Since FMO-mediated metabolism of these drugs might contribute to local pharmacodynamic modulation within the human brain, the metabolism of imipramine by human brain FMO was studied in further detail. In the present study, the FMO activity was determined in human brain microsomes by estimating the actual amount of imipramine N-oxide formed. It was then compared with the corresponding activity measured using substrate (imipramine)-stimulated rates of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation, which was significantly higher than the activity estimated as the amount of N-oxide assayed using high-pressure liquid chromatography (HPLC). The brain FMO activity was measurable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The activity was inhibited by an antibody to rabbit pulmonary FMO, but an antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect indicating that cytochrome P-450 was not involved in the above metabolic pathway. The optimum pH for N-oxidation of imipramine was found to be 8.5; thermolability experiments indicated that the FMO activity was completely lost only after the incubation of brain microsomes at 45 degrees C for 20 minutes. An FMO purified to apparent homogeneity from a human brain had a molecular weight of 71,000 Da. The purified enzyme cross-reacted with the antibody to rabbit pulmonary FMO and efficiently catalyzed the metabolism of imipramine to its N-oxide. The human brain clearly contains an active FMO system, and it is conceivable that such enzymes are significantly involved in the local metabolism and modulation of pharmacological and/or toxic effects of certain xenobiotics, including psychoactive drugs.

Cell type-specific expression of beta-carotene 15,15'-mono-oxygenase in human tissues.

We studied the cell type-specific expression of human beta-carotene 15,15'-mono-oxygenase (BCO1), an enzyme that catalyzes the first step in the conversion of dietary provitamin A carotenoids to vitamin A. Immunohistochemical analysis using two monoclonal antibodies against different epitopes of the protein revealed that BCO1 is expressed in epithelial cells in a variety of human tissues, including mucosa and glandular cells of stomach, small intestine, and colon, parenchymal cells in liver, cells that make up the exocrine glands in pancreas, glandular cells in prostate, endometrium, and mammary tissue, kidney tubules, and in keratinocytes of the squamous epithelium of skin. Furthermore, BCO1 is detected in steroidogenic cells in testis, ovary, and adrenal gland, as well as skeletal muscle cells. Epithelia in general are structures that are very sensitive to vitamin A deficiency, and although the extraintestinal function of BCO1 is unclear, the finding that the enzyme is expressed in all epithelia examined thus far leads us to suggest that BCO1 may be important for local synthesis of vitamin A, constituting a back-up pathway of vitamin A synthesis during times of insufficient dietary intake of vitamin A.

Purification and comparison of liver microsomal flavin-containing monooxygenase from normal and streptozotocin-diabetic rats.

Liver microsomal flavin-containing monooxygenase (FMO) activity towards thiobenzamide is two-fold increased in streptozotocin diabetic (insulin deficient) rats and mice and, to a lesser degree in congenital insulin resistant Ob/Ob mice. No difference in thermal stability appears between microsomal FMOs from both normal and diabetic rats. FMO has been purified to homogeneity from these two sources, with a 50-fold increase of specific activity. Their apparent molecular weight is respectively 50,000 and 49,000 and a discrete modification appears in the HPLC profiles of tryptic peptides from purified FMOs. They appear immunochemically very similar and present in equal quantity in microsomal membranes from both normal and diabetic rats, so that the increased activity cannot be ascribed to an increased concentration of the enzyme protein.

The flavin-containing monooxygenase of mouse kidney. A comparison with the liver enzyme.

Flavin-containing monooxygenase (FMO; EC 1.14.13.8) was purified from mouse kidney microsomes and compared to that isolated from mouse liver microsomes. The purified enzymes from kidney and liver appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 58,000 daltons. On wide range (pH 3.5 to 9.0) isoelectric focusing, FMOs from kidney and liver resolved as a single band with an isoelectric point of 8.2. The enzymes from both kidney and liver have a pH optimum of 9.2. Thiobenzamide-S-oxidation catalyzed by both enzymes was sensitive to inhibition by the competitive inhibitors thiourea and methimazole. At an n-octylamine concentration of 3 mM, thiobenzamide-S-oxidation by the kidney FMO was increased by 122% and that by the liver FMO by 148%. Km and Vmax values were determined and compared between the two tissue enzymes for xenobiotic substrates containing nucleophilic nitrogen, sulfur or phosphorus atoms. In general, for most FMO substrates, Km and Vmax values were similar between kidney and liver FMO with only a few exceptions. The Km and Vmax values for fenthion for kidney were only half of those observed for liver FMO. Fonofos was unusual in having a low Km as well as a low Vmax for both tissue enzymes. Anti-sera developed to the FMO purified from kidney and liver showed cross-reactivity with each purified enzyme as well as with a protein with the same molecular weight as the purified FMO present in both kidney and liver microsomes. These bands showed equal intensity based on an equivalent amount of protein. Analysis of kidney and liver FMO by proteolytic digestion followed by visualization of peptides by silver staining or immunoblotting showed only minor differences between the enzymes of the two tissues. The amino acid composition of both mouse kidney and liver FMO was low in methionine and histidine and rich in aspartate/asparagine, glutamate/glutamine, leucine, valine and glycine. Edman degradation of the purified mouse kidney and liver FMO provided a single amino acid sequence of the NH2-terminus. This sequence matched exactly with the cDNA-deduced sequence reported for the pig and rabbit liver beginning with the fifth amino acid and contained the highly conserved FAD-binding domain Gly-X-Gly-X-X-Gly, commonly found in a number of other FAD-binding proteins. These studies indicate that the renal and hepatic forms of FMO from mouse are similar enzymes that are immunologically related and show only a few minor differences.

Channel catfish liver monooxygenases. Immunological characterization of constitutive cytochromes P450 and the absence of active flavin-containing monooxygenases.

Multiple drugs and pesticides are used in the aquaculture of channel catfish in the Southeastern United States. However, little is known regarding the enzymatic metabolism of these chemicals in the fish. Western blots, utilizing polyclonal antibodies raised against five purified rainbow trout liver cytochrome P450 enzymes, revealed at least two protein bands that were approximately 50 kDa (CATL-1) and 53 kDa (CATL-2). Anti-trout LMC3 and LMC4 only hybridized with the 53 kDa protein, whereas anti-trout LMC1, LMC2, and LMC5 recognized both proteins. Cytochrome P450-catalyzed activities (testosterone and progesterone hydroxylases) associated with LMC1 and LMC5 were also found in catfish liver microsomes. These data suggest that at least two constitutive forms of cytochrome P450 are present in the liver of juvenile channel catfish. Western blots utilizing antibodies raised against rabbit-lung flavin-containing monooxygenases (FMO) showed hybridization with two proteins from rainbow trout liver microsomes, but no cross-reaction with microsomes from catfish liver. N,N,-Dimethylaniline N-oxidase and methimazole oxidase were observed in microsomes from trout, but were absent in catfish liver microsomes prepared in three different laboratories. Consequently, FMO do not appear to be present in liver microsomes from channel catfish or they are rapidly degraded during tissue homogenization.

Antibodies to a synthetic peptide that react with flavin-containing monooxygenase (HLFMO3) in human hepatic microsomes.

Flavin-containing monooxygenases (FMOs) catalyze the oxidation of a diverse array of xenobiotic compounds. The purpose of this investigation was to develop a specific immunological probe to human hepatic flavin-containing monooxygenase (HLFMO3). An oligopetide corresponding to amino acid residues 257-270 of HLFMO3 was coupled to keyhole limpet hemocyanin (KLH) through the sulfhydryl group of a cysteine residue added to the amino-terminus of the peptide. This peptide-KLH conjugate was used to generate a polyclonal antibody. The resulting immunoglobulin showed specific Western blot reactivity with HLFMO3 protein in human hepatic microsomes, the same protein that is recognized by a polyclonal antibody directed against macaque liver FMO. These findings demonstrate that an antibody directed against a synthetic peptide derived from HLFMO3 can be easily produced in large quantities and used in studies for the immunodetection and immunoquantification of HLFMO3. This is also the first antipeptide antibody directed against an FMO of any species.

Characterization of the purified microsomal FAD-containing monooxygenase from mouse and pig liver.

The FAD-containing monooxygenase (FMO) has been purified from both mouse and pig liver microsomes by similar purification procedures. Characterization of the enzyme from these two sources has revealed significant differences in catalytic and immunological properties. The pH optimum of mouse FMO is slightly higher than that of pig FMO (9.2 vs. 8.7) and, while pig FMO is activated 2-fold by n-octylamine, mouse FMO is activated less than 20%. Compounds, including primary, secondary and tertiary amines, sulfides, sulfoxides, thiols, thioureas and mercaptoimidazoles were tested as substrates for both the mouse and pig liver FMO. Km- and Vmax-values were determined for substrates representative of each of these groups. In general, the mouse FMO had higher Km-values for all of the amines and disulfides tested. Mouse FMO had Km-values similar to those of pig FMO for sulfides, mercaptoimidazoles, thioureas, thiobenzamide and cysteamine. Vmax-values for mouse FMO with most substrates was approximately equal, indicating that as with pig FMO, breakdown of the hydroxyflavin is the rate limiting step in the reaction mechanism. Either NADPH or NADH will serve as an electron donor for FMO, however, NADPH is the preferred donor. Pig and mouse FMOs have similar affinity for NADPH (Km = 0.97 and 1.1 microM, respectively) and for NADH (Km = 48 and 73 microM, respectively). An antibody, prepared by immunizing rabbits with purified pig liver FMO, reacts with purified pig liver FMO but not with mouse liver FMO, indicating structural differences between these two enzymes. This antibody inhibited pig FMO activity up to 60%.