Hsu M C, Shih J C
Division of Biological Sciences, School of Pharmacy, University of Southern California, Los Angeles 90033.
Mol Pharmacol. 1988 Feb;33(2):237-41.
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.
我们之前的研究表明，低浓度的4-氟-3-硝基苯叠氮化物（FNPA）（0.01-1微摩尔）光依赖地仅抑制大鼠脑中的B型单胺氧化酶[《生物化学与药理学》34:781-785(1985)]。本文提供的证据表明，较高浓度的FNPA（15微摩尔）光依赖地抑制人胎盘A型单胺氧化酶（MAO-A）。当使用[14C]血清素作为底物时，FNPA在黑暗中作为人胎盘MAO-A的竞争性抑制剂（Ki = 10微摩尔）。FNPA对MAO-A活性的抑制呈浓度依赖性且也呈照射时间依赖性。血清素在照射期间的保护作用表明了FNPA光依赖地掺入MAO-A的特异性。动力学分析表明，在用MAO-A光解FNPA后，Vmax降低而Km未改变。此外，对光解的FNPA-酶混合物进行洗涤后，MAO-A活性没有恢复。这些结果表明FNPA可能与底物结合位点共价结合。因此，在当前实验条件下，FNPA是用于人胎盘MAO-A的合适光亲和标记探针。这是MAO-A的首个光亲和标记，可能有助于表征该酶的底物结合位点。
