Delaforge M, Andre F, Jaouen M, Dolgos H, Benech H, Gomis J M, Noel J P, Cavelier F, Verducci J, Aubagnac J L, Liebermann B
Université René Descartes, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS URA 400, Paris, France.
Eur J Biochem. 1997 Nov 15;250(1):150-7. doi: 10.1111/j.1432-1033.1997.00150.x.
The interaction between rat and human liver cytochrome P-450 with tentoxin, a natural phytotoxic cyclotetrapeptide having chlorotic properties, was studied by difference ultraviolet visible spectroscopy. Tentoxin interacted with rat liver microsomes and the difference spectrum was characteristic of binding to a protein site close to the heme. The intensity of this spectrum was clearly dependent on the amounts of P-450 3A in the microsomes and was optimal in dexamethasone-treated rat microsomes. Tentoxin exhibited a high affinity for P-450 3A (Ks approximately 10 microM). Similar results were observed with human P-450 isozymes expressed in yeast. Only P-450 3A4 and 3A5 were able to give spectral interactions with tentoxin. Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P-450 3A, were found to be particularly active for the oxidation of tentoxin, which occurs mainly on its Ala(Me) function leading to demethylation. Yeast-expressed P-450 3A also exhibited high activity to metabolize tentoxin. The metabolites were identified by their ultraviolet and mass spectra in fast atom bombardment and collision-activated dissociation modes. In addition to the major N-demethylated metabolite, other hydroxylated metabolites were formed. Preliminary analysis showed that as tentoxin, some metabolites were still efficient chloroplast ATPase inhibitors, while at least one of them exhibited even at low concentration stimulatory effects.
采用差示紫外可见光谱法研究了大鼠和人肝脏细胞色素P-450与滕毒素（一种具有褪绿特性的天然植物毒性环四肽）之间的相互作用。滕毒素与大鼠肝脏微粒体相互作用，其差示光谱具有与靠近血红素的蛋白质位点结合的特征。该光谱的强度明显取决于微粒体中P-450 3A的含量，在地塞米松处理的大鼠微粒体中最为明显。滕毒素对P-450 3A具有高亲和力（Ks约为10 microM）。在酵母中表达的人P-450同工酶也观察到了类似结果。只有P-450 3A4和3A5能够与滕毒素产生光谱相互作用。用P-450 3A的特异性诱导剂地塞米松预处理的大鼠肝脏微粒体对滕毒素的氧化特别活跃，氧化主要发生在其Ala(Me)官能团上导致去甲基化。酵母表达的P-450 3A对滕毒素的代谢也表现出高活性。通过快速原子轰击和碰撞激活解离模式下的紫外光谱和质谱对代谢产物进行了鉴定。除了主要的N-去甲基代谢产物外，还形成了其他羟基化代谢产物。初步分析表明，与滕毒素一样，一些代谢产物仍然是有效的叶绿体ATP酶抑制剂，而其中至少一种即使在低浓度下也表现出刺激作用。
