Spectrophotometric assay of the flavin-containing monooxygenase and changes in its activity in female mouse liver with nutritional and diurnal conditions

doi:10.1016/0003-9861(84)90600-3

Archives of Biochemistry and Biophysics

Volume 233, Issue 1, 15 August 1984, Pages 50-63

https://doi.org/10.1016/0003-9861(84)90600-3 Get rights and content

Abstract

A highly sensitive spectrophotometric assay was developed for measuring flavin-containing monooxygenase activity using methimazole (N-methyl-2-mercaptoimidazole) as the substrate. With the procedure described, flavin-containing monooxygenase activity can be accurately measured in whole cell homogenates without interference due to NADPH oxidase activities. The effects of detergents and octylamine on female mouse liver flavin-containing monooxygenase activity were characterized for whole homogenates and microsomes prepared under conditions which tend to cause or minimize microsomal aggregation. A small activation was observed with 0.2% (v/v) Emulgen 913 with nonaggregated microsomes; higher levels of detergents gave maximal activity with aggregated microsomes. Variations in the activity of the female mouse liver enzyme with nutritional state and time of day were evaluated. Higher specific activities were observed in homogenates and microsomes of livers from fed animals than from livers of 24-h starved animals, and higher specific activities were present in samples from livers of animals sacrificed in late afternoon than in the early morning. In the period where activity increased in fed animals (i.e., the AM to PM transition), a portion of flavin-containing monooxygenase was more resistant to thermal inactivation. Other properties are described which suggest structural differences for at least a portion of the flavin-containing monooxygenase. The possibility that these differences may be related to turnover of the flavin-containing monooxygenase is discussed.

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To this date, most of the scientific literature related to drug metabolism has focused on the characterization of cytochrome P450-mediated processes, with the other phase I drug metabolizing enzymes largely overlooked. However, an increasing volume of recent studies have highlighted the important contribution of non-P450 human drug-metabolizing enzymes in converting drugs/pro-drugs into either more hydrophilic compounds, non-toxic excretable metabolites or activated drugs.
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Protein content of haemolymph plasma and hemocyte lysate was quantified (Lowry et al., 1951) and used for enzyme assays. FMO assay was performed as described using methimazole (N-methyl-2-mercaptoimidazole) as substrate (Dixit and Roche, 1984). Absorbance was read at 412 nm for 5 min at 1 min interval.
Infestation of commercially important silkworm Bombyx mori by dipteran endoparasitoid Exorista bombycis causes ~20% revenue loss. Though invasion has induced hemocyte encapsulation and melanisation of invaded larva, strategies to compromise with host-defense system by dipteran endoparasitoids is unknown. In the present study variation in genome wide gene expression was analyzed by utilizing microarray data acquired from hemocytes collected at 6 h after invasion that showed significant (P < 0.05) up-regulation of 423 genes and down-regulation of 587 genes. Among them 11 detoxification genes and 34 immune genes down-regulated showing repressed defense reactions substantiated by reduced activity of detoxification enzymes esterase and FMO in early stage of infestation. The down-regulated genes were enriched after comparing with control and by filtering unreliable probes. GO annotation of significantly (P < 0.05) down-regulated genes was used for clustering and categorization of functionally related defense processes. Networking of interacting partners of down-regulated defense genes showed relatedness among the down- regulated biological processes viz., Wnt signalling, DNA repair, transcription, catabolism, glutamine metabolism, components of cytoskeletal structure and transport. These clusters perform immune responses, signalling, stress responses, cell redox homeostasis, cytoskeletal organization, glutamine, glycogen and chitin metabolism, protein, lipid and carbohydrate metabolism and transmembrane transport. Autophagy, apoptosis, cell adhesion, cell growth and cellular lipid metabolism also showed down-regulation independently. Down-regulation of defense genes, interacting components and low detoxification enzyme activity in early infestation revealed active suppression of host defense at genic, enzymic and interactome level which is an unknown anti-defense mechanism of dipteran endoparasitoids.
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Progress in structural biology and related disciplines has advanced our knowledge of the physical and mechanistic properties of flavoproteins. This article will focus on two classes of flavoproteins (i.e., monoamine oxidase A and B (MAO A and B) and the flavin-containing monooxygenases (FMOs)) that are involved in the biotransformation of chemicals, drugs, pesticides, and xenobiotics in both small and large animals. Emphasis will be placed on the human enzymes, but comparison with animal forms will highlight the differences. MAO and FMO enzymes have largely been associated with detoxication of chemicals to more polar and generally less toxic metabolites that are usually efficiently excreted. Sometimes, however, MAOs or FMOs convert less toxic chemicals to more toxic materials. This article will attempt to present a view of the balance between these dual roles of MAO and FMO flavoenzymes. Before a detailed discussion of each enzyme is considered, however, a few general comments about the manner that MAO A and B and the FMOs fit into the overall view of the family of flavoproteins that exist in nature will be presented.
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In order to develop a throughput colorimetric detection method for hFMO3 substrates or inhibitors, methimazole-dependent TNB (nitro-5-thiobenzoate) oxidation assay was chosen as the starting point. Methimazole is a known substrate of hFMO3 that is S-oxygenated by the enzyme and it has been widely employed in the past for FMO activity measurement using a colorimetric assay developed by Dixit and Roche [7]. The main advantages of this assay are: (a) the specificity of methimazole S-oxide (product of hFMO3 enzyme activity) for TNB and (b) its colorimetric nature leading to possible conversion into a high throughput screening assay.
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Spectrophotometric assay of the flavin-containing monooxygenase and changes in its activity in female mouse liver with nutritional and diurnal conditions

Abstract

Biochem. Biophys. Res. Commun

Arch. Biochem. Biophys

Arch. Biochem. Biophys

J. Biol. Chem

Biochem. Pharmacol

Arch. Biochem. Biophys

Arch. Biochem. Biophys

Arch. Biochem. Biophys

Trends Biochem. Sci

Anal. Biochem

Anal. Biochem

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Biochem. Biophys. Res. Commun

Biochem. Pharmacol

Biochem. Pharmacol

J. Biol. Chem