Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.
Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.
复制标题
原卟啉原氧化酶：针对小鼠肝线粒体中抑制剂/除草剂结合位点的高亲和力四氢邻苯二甲酰亚胺放射性配体。
DOI：
10.1021/tx960074h
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Casida,JE
中科院分区：
文献类型：
--
作者：
Birchfield,NB;Casida,JE
原卟啉原氧化酶（protox）是血红素和叶绿素生物合成中最后一种常见的酶，是植物和哺乳动物中作为抑制剂的几类除草剂的靶标。（4-氯-2-氟-5-（炔丙氧基）苯基）-3，4，5，6-四氢邻苯二甲酰亚胺通过选择性催化还原3，6-二氢邻苯二甲酸酐（DHPA）与氚气反应，然后与4-氯-2-氟-5-（炔丙氧基）苯胺缩合，产率为45%。在DHPA的3和6个碳以及预期的4和5个碳处插入氚导致高比活度[3 H]THP（92 Ci/mmol）。这种放射性配体经历快速，特异性，饱和，和可逆结合的抑制剂/除草剂结合位点的原氧化物组分的胆酸盐溶解的小鼠肝线粒体的表观Kd为0.41 nM和Bmax为0.40 pmol/mg的蛋白质。在标准测定中，将小鼠制备物（150 μg蛋白质）和[3 H]THP（0.5 nM）在500 μL磷酸盐缓冲液（pH 7.2）中于25 ℃孵育15 min，然后加入硫酸铵并用玻璃纤维过滤器过滤。五种硝基二苯醚和两种除草剂作为[~ 3 H]THP结合抑制剂的效力与抑制protox活性的效力具有良好的相关性（r ~ 2 = 0.97，n = 7），从而验证了结合测定与酶抑制相关。用乙氧氟草醚4 mg/kg ip处理的小鼠，肝线粒体中[3 H]THP结合减少约50%，这也适用于测定体内阻断。这是第一次报告的protox在哺乳动物中的结合试验。[~ 3 H]THP的高亲和力和比活性有利于protox的定量，从而研究卟啉生物合成的敏感抑制位点。
Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals.N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydrophthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparentKdof 0.41 nM andBmaxof 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 μg of protein) and [3H]THP (0.5 nM) are incubated in 500 μL of phosphate buffer at pH 7.2 for 15 min at 25 °C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2= 0.97,n= 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determinein vivoblock as illustrated by an ∼50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.