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Multiplicity of liver microsomal flavin-containing monooxygenase in the guinea pig: its purification and
Abstract:
Two distinct forms (FMO-I and FMO-II) of flavin-containing monooxygenase were purified from the liver microsomes of guinea pig. The minimum molecular weights estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 54,000 for FMO-I and 56,000 for FMO-II, respectively. Tryptic digestion of these enzymes gave different electrophoretic patterns, suggesting that FMO-I and -II have distinct amino acid sequences. The amino terminal sequence of FMO-II could not be estimated probably due to its blocking while that of FMO-I was determined to be highly homologous to the rabbit liver flavin-containing monooxygenase (J. Ozols, 1989, Biochem. Biophys. Res. Commun. 163, 49-55). Absorption maxima of FMO-I and -II were recorded at 368 and 440 nm and 381 and 456 nm, respectively. Molar ratios of FAD to both of these apoenzymes were shown to be one to one. Substrate specificity of FMO-I and -II was determined using 15 compounds as the substrate. The results showed two enzymes that exhibited overlapped but different specificity toward these substrates although FMO-I had lower activity than did FMO-II with all compounds except thiobenzamide. Of particular interest, only FMO-II showed considerably high activities for primary amines, n-octylamine, and n-decylamine. Immunoglobulin G raised against FMO-II could recognize FMO-I as well as FMO-II, but the reactivity of FMO-I toward the antibody was obviously lower than that of FMO-II. Electrophoresis followed by immunostaining revealed that microsomes of lung, kidney, urinary bladder, testis, and spleen contain the same protein as FMO-II and/or FMO-I. Only lung was shown to have an additional isozyme of FAD-monooxygenase with a molecular weight apparently higher than those of FMO-I and -II. These results strongly suggest that at least two forms of flavin-containing monooxygenases distinct from the lung-type isozyme are expressed in liver of guinea pigs.
Insights
Guinea pigs possess two distinct flavin-containing monooxygenase (FMO) forms, FMO-I and FMO-II, in their liver microsomes. These FMO enzymes exhibit unique molecular weights, amino acid sequences, and substrate specificities, indicating specialized biological roles.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Flavin-containing monooxygenases (FMOs) are crucial enzymes involved in the metabolism of xenobiotics and endogenous compounds.
- Understanding the diversity and characteristics of FMOs is essential for comprehending drug metabolism and detoxification pathways.
Purpose of the Study:
- To purify and characterize distinct forms of flavin-containing monooxygenase (FMO) from guinea pig liver microsomes.
- To investigate the molecular, catalytic, and immunological properties of these FMO forms.
- To determine the tissue distribution of FMOs in guinea pigs.
Main Methods:
- Purification of FMO-I and FMO-II from guinea pig liver microsomes.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular weight determination.
- Tryptic digestion and amino-terminal sequencing to assess amino acid sequence differences.
- Spectrophotometric analysis for absorption maxima and FAD content.
- Substrate specificity assays using 15 different compounds.
- Immunological studies using antibodies raised against FMO-II.
- Tissue distribution analysis by electrophoresis and immunostaining.
Main Results:
- Two distinct FMO forms, FMO-I (54,000 Da) and FMO-II (56,000 Da), were purified, differing in tryptic digestion patterns and amino acid sequences.
- FMO-I and FMO-II exhibited unique absorption maxima and substrate specificities, with FMO-II showing higher activity towards primary amines.
- Antibodies against FMO-II recognized both FMO-I and FMO-II, with higher reactivity towards FMO-II.
- FMO-I and FMO-II were detected in liver, lung, kidney, urinary bladder, testis, and spleen, with lung containing an additional higher molecular weight FAD-monooxygenase isozyme.
Conclusions:
- Guinea pigs express at least two distinct FMO forms (FMO-I and FMO-II) in their liver, with unique biochemical and catalytic properties.
- These FMO forms are differentially distributed across various tissues, suggesting specialized physiological roles.
- The findings highlight the molecular diversity of FMOs and their importance in xenobiotic metabolism and detoxification in guinea pigs.