Inhibition of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine metabolic activity of porcine FAD‐containing monooxygenase by selective monoamine oxidase‐B inhibitors

Abstract

The MPTP metabolic activity of porcine FAD-containing monooxygenase (FMO) (EC 1.14.13.8) was inhibited considerably by deprenyl and pargyline, selective MAO-B inhibitors, and they showed typical competitive inhibition. Deprenyl and pargyline, amine derivatives were also examined as to whether they are substrates for the FMO. It was found that deprenyl and pargyline are excellent substrates for the FMO. The K i and K m values of deprenyl and pargyline for the FMO are 14 pM and 9 pM, and 14.3 pM and 11.6 pM, at pH 8.0 and 25°C, respectively.Key words." FAD-containing monooxygenase; Monoamine oxidase-B inhibitor; Deprenyl; Pargyline [6]. The optical spectra of FMO are shown in Fig. 1, and the purified FMO gave a single protein band on SDS-PAGE (Fig. 2). Enzyme assay and kinetic parametersThe enzymatic activity was determined optically and aerobically by measuring the decrease in absorbance at 340 nm due to the oxidation of NADPH in the presence of a substrate. The reaction mixture comprised FMO (0.2 nmol), 0.1 M K-phosphate buffer, pH 8.0, at 25°C, and appropriate amounts of NADPH and 10 ~M to 600 ¢tM substrate, in a final volume of 1 ml. The reaction mixture was put into a cuvette of 1 cm light path and then the endogenous rate of the absorbance change at 340 nm was recorded for 5 rain at 25°C before addition of the substrate. After the addition of the substrate, the rate of the absorbance change was measured for 5 rain at 25°C, the endogenous rate being subtracted. For investigation of a reaction with a sequential Bi Bi mechanism, the Km, Vmax and K~ values of FMO for deprenyl and pargyline can be determined by means of Lineweaver-Burk plots [9].

Chiral characterization of deprenyl‐N‐oxide and other deprenyl metabolites by capillary electrophoresis using a dual cyclodextrin system in rat urine

“…However, the dealkylated me- tabolites do not inhibit apoptosis [25]. Recently, the generation of another metabolite, deprenyl-N-oxide by flavincontaining monooxygenase (FMO) was proposed by Wu et al [26]. This compound was later identified in vitro in microsomal preparation (from F. Lévai et al, submitted) and in vivo in human urine [27,28].…”

In vitro formation of selegiline-N-oxide as a metabolite of selegiline in human, hamster, mouse, rat, guinea-pig, rabbit and dog

“…Less selegiline-NOs were 106.85 4 : Formation of selegiline-N-oxide in rat microsomal preparation in vitro in the presence of specific isoenzyme inhibitors metabolites, numerous minor metabolites, such as phydroxydesmethylselegiline, p-hydroxymethamphetamine, p-hydroxyamphetamine, ephedrine, norephedrine, pseudoephedrine, norpseudoephedrine and phenylacetone have been identified in man and/or in various animal species (17). The formation of selegiline-NO as a metabolite of selegiline has previously been postulated (25,34) and it was recently identified in human urine (35,36).…”

“…Selegiline is an excellent substrate for FAD-containing mono-oxygenase, which probably converts selegiline to its N-oxide (25).…”

Basic cell physiological activities (cell adhesion, chemotaxis and proliferation) induced by selegiline and its derivatives in Mono Mac 6 human monocytes

“…The cytochrome P450-mediated desalkylations (depropargylation and demethylation) are the main metabolic pathways of selegiline metabolism, lead to the formation of R-methamphetamine N-desmethyl-R-deprenyl and R-amphetamine (Reynolds et al 1978;Heinonen et al 1989). Furthermore, the flavin-containing monooxygenase (FMO) enzymes are also capable to N-oxidize the drug, forming R-deprenyl-N-oxide (Wu and Ichikawa 1995;Szök} o et al 2004). The effects of the metabolites (e.g.…”

“…The main metabolites are R-methamphetamine, R-amphetamine N-desmethyl-R-deprenyl (Reynolds et al 1978;Heinonen et al 1989; Barrett et al 1996b) and their corresponding para-hydroxylated products (Shin 1997). Flavin-containing monooxygenase (FMO) enzyme is responsible for the formation of deprenyl-N-oxide (DNO) metabolite, which has a new chiral center with positive charge on the tertiary nitrogen (Wu and Ichikawa 1995;Szök} o et al 2004). Among the metabolites N-desmethyl-Rdeprenyl, deprenyl-N-oxide preserved their propargyl groups, simultaneously with neuroprotective action.…”