Photoaffinity labeling of human placental monoamine oxidase-A by 4-fluoro-3-nitrophenyl azide.
Our previous work has shown that low concentrations of 4-fluoro-3-nitrophenyl azide (FNPA) (0.01-1 microM) photodependently inhibited only the type B monoamine oxidase in rat brain [Biochem. Pharmacol. 34:781-785 (1985)]. Evidence is presented in this paper indicating that higher concentrations of FNPA (15 microM) photodependently inhibit type A monoamine oxidase (MAO-A) from human placenta. FNPA acted as a competitive inhibitor for human placental MAO-A in the dark (Ki = 10 microM) when [14C]serotonin was used as the substrate. The inhibition of MAO-A activity by FNPA was concentration dependent and also irradiation time dependent. The specificity of the photodependent incorporation of FNPA to MAO-A was shown by the protective effect of serotonin during the irradiation. The kinetic analysis showed that the Vmax was decreased whereas the Km was not changed after FNPA was photolyzed with MAO-A. Furthermore, there was no recovery of MAO-A activity upon washing of the photolyzed FNPA-enzyme mixture. These results suggest that FNPA may be covalently bound to the substrate-binding site. Thus, under the present experimental conditions, FNPA is a suitable photoaffinity labeling probe for human placental MAO-A. This is the first photoaffinity label for MAO-A, which may be useful for characterizing the substrate-binding site of this enzyme.
Hsu
M C
MC
Division of Biological Sciences, School of Pharmacy, University of Southern California, Los Angeles 90033.
Shih
J C
JC
D000345:Affinity Labels / Q000494:pharmacology*; D001386:Azides / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D007700:Kinetics; D008027:Light; D008995:Monoamine Oxidase / Q000032:analysis*; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology; D012701:Serotonin / Q000494:pharmacology; D013997:Time Factors
D000345:Affinity Labels; D001386:Azides; D008996:Monoamine Oxidase Inhibitors; C007753:4-fluoro-3-nitrophenyl azide; D012701:Serotonin; D008995:Monoamine Oxidase