Hsu M C, Chen S, Shih J C
Division of Biological Sciences, School of Pharmacy, University of Southern California, Los Angeles, Calif., U.S.A.
Neurochem Int. 1987;10(2):167-72. doi: 10.1016/0197-0186(87)90123-9.
Previous work from this laboratory has shown that 4-fluoro-3-nitrophenyl azide (FNPA) is an effective photoaffinity labeling probe for MAO-B (Chen et al., Biochem. Pharmac.34, 781-785, 1985). The FNPA binding sites have been further studied by using [(3)H]FNPA. When [(3)H]FNPA was photolyzed with purified beef liver MAO, then subjected to tryptic and chymotryptic digestion, three radioactive peaks were observed after Sephadex G-25 column chromatography procedure. The extent of [(3)H]FNPA incorporation varied directly with [(3)H]FNPA concentration. They could be protected by the presence of the substrate (phenylethylamine) or inhibitors (pargyline and trans-phenylcyclopropylamine) of MAO-B during photolysis. These protections were concentration dependent. Furthermore, the decrease in [(3)H]FNPA labeling in the presence of inhibitors paralleled the decrease in MAO catalytic activity. These results suggest that the FNPA binding sites were related to the active site of MAO-B. Under the same conditions, the separation profiles of [(3)H]FNPA labeled and [(3)H]pargyline labeled tryptic-chymotryptic peptides after Sephadex G-25 column chromatography are distinctly different. This result suggests that FNPA labeling sites may be different from the pargyline binding site. Since pargyline binds to the prosthetic group(-FAD) of MAO, [(3)H]FNPA may label different domains of the active site. This probe may be useful for the characterization of the active site of MAO-B.
该实验室之前的研究表明，4-氟-3-硝基苯基叠氮化物（FNPA）是一种用于单胺氧化酶B（MAO-B）的有效光亲和标记探针（Chen等人，《生物化学与药理学》34，781-785，1985）。已通过使用[³H]FNPA对FNPA结合位点进行了进一步研究。当用纯化的牛肝MAO对[³H]FNPA进行光解，然后进行胰蛋白酶和糜蛋白酶消化时，在葡聚糖凝胶G-25柱色谱过程后观察到三个放射性峰。[³H]FNPA的掺入程度与[³H]FNPA浓度直接相关。在光解过程中，它们可受到MAO-B的底物（苯乙胺）或抑制剂（帕吉林和反式苯环丙胺）的保护。这些保护作用具有浓度依赖性。此外，在存在抑制剂的情况下[³H]FNPA标记的减少与MAO催化活性的降低平行。这些结果表明，FNPA结合位点与MAO-B的活性位点有关。在相同条件下，[³H]FNPA标记的和[³H]帕吉林标记的胰蛋白酶-糜蛋白酶肽段在葡聚糖凝胶G-25柱色谱后的分离图谱明显不同。这一结果表明，FNPA标记位点可能与帕吉林结合位点不同。由于帕吉林与MAO的辅基（-FAD）结合，[³H]FNPA可能标记活性位点的不同结构域。该探针可能有助于表征MAO-B的活性位点。
