REGULATION OF HEME METABOLISM IN THE LIVER

血红素是肝血红素蛋白细胞色素P450的辅基部分 （P450）和色氨酸吡咯酶（TO），它们致力于 内源性和外源性物质的代谢/解毒（P450），和 氧化色氨酸代谢（TO）。 然而，并非所有P450介导的 反应是有益的。 某些P450通过一个电子氧化， 将一些底物转化为急性毒性自由基， 定位并引发急性/慢性病变。 其他P450可能会喜欢 相同底物的非自由基途径。 其他P450，在 这种氧化过程导致“自杀”，基于机理的失活， 并伴有血红素和/或脱辅基蛋白的破坏。 我们的研究 这表明不仅每种P450所支持的氧化途径不同， 而且由特定底物引起的自杀失活模式 不同的P450 因为所有P450的血红素是相同的， 暗示apoP 450活性位点结构决定了它们的催化活性， 喜好 在这方面，P450 IIIA 1是臭名昭著的，因为它的高度偏离， 催化行为及其明显的偏离，否则高度保守 活性部位结构 建议进行定点诱变研究， 确定这些自然突变对P450 IIIA 1催化的影响 自由基产生底物（DDEP和有机氢过氧化物），及其 不寻常的自杀失活通过不可逆的血红素结合到 脱辅基蛋白 研究还提出，以阐明是否其他假定 基于机制的失活（司可巴比妥引起的P450 IIIB 1， 螺内酯），实际上发生在P450活性位点，因此真正 有自杀倾向 因为在自杀性血红素破坏后， P450（P450 IIC 11）引起免疫化学可检测的丢失， hemin可预防和ATP依赖，建议其 血红素的剥离和丢失可能会促进它们的磷酸化和/或 泛素化作为其蛋白水解处置的前奏。 的确， 初步研究结果表明，DDEP-灭活的P450 IIC 11在分离的 大鼠肝细胞被磷酸化。 建议进行研究以确定 这是否是血红素剥离的P450 IIC 11的特征，以表征其 时间进程，识别磷酸化位点并确定 这一过程的血红素调节。 血红素介导的细胞凋亡调控的研究 大鼠肝脏TO将延长：显著的功能性TO丧失导致 从免疫化学检测的结构损失后，急性血红素 消耗，表明假体血红素损失使apoTO不稳定。 然而，在这方面， 不仅在急性血红素缺乏时TO丢失持续， 糖皮质激素也受损。 建议进行研究以确定 这种损伤是否反映了新合成的apoTO的不稳定性， 在没有血红素的情况下，或者与肝P450共同，血红素是否也 调节TO转录/翻译。 据信，集体， 这些研究将提供深入了解结构-功能 关系，使P450承诺的结构特征 解毒/解毒途径，从而影响外源性物质介导的 毒性，以及对这些物质的周转的血红素调节知之甚少， 重要的血红素蛋白。

Heme is the prosthetic moiety of the hepatic hemoproteins Cytochromes P450 (P450) and tryptophan pyrrolase (TO), which are committed to metabolism/detoxification of endobiotics and xenobiotics (P450s), and oxidative tryptophan metabolism (TO). However, not all P450-mediated reactions are beneficial. Certain P450s through one electron oxidations, convert some substrates to acutely toxic radicals that escape the active site and elicit acute/chronic pathologies. Other P450s may favor non-radical pathways for the same substrates. Yet other P450s, in the course of such oxidations incur "suicide", mechanism-based inactivations, with destruction of prosthetic heme and/or apoprotein. Our studies indicate that not only the oxidative pathways favored by each P450 differ, but also that the mode of suicide inactivation by a particular substrate differs among P450s. Because the heme of all P450s is identical, they implicate the apoP450 active site structure in dictating their catalytic preferences. In this, P450IIIA1 is notorious both for its highly deviant catalytic behaviour and its overt digressions in otherwise highly conserved active site structure. Site-directed mutagenesis studies are proposed to determine the influence of these natural mutations on P450IIIA1-catalyses of radical-producing substrates, (DDEP and organic hydroperoxides), and its unusual suicide inactivation via irreversible heme binding to the apoprotein. Studies are also proposed to elucidate whether other presumed mechanism-based inactivations (P450IIIB1 by secobarbital, P450IIC11 by spironolactone), actually occur at the P450 active site and thus truly qualify as suicidal. Because after suicidal heme destruction, certain P450s (P450IIC11) incur immunochemically detectable loss, which is hemin-preventable and ATP-dependent, it is proposed that their heme-stripping and loss might promote their phosphorylation and/or ubiquitination as a prelude to their proteolytic disposal. Indeed, preliminary findings indicate that DDEP-inactivated P450IIC11 in isolated rat hepatocytes is phosphorylated. Studies are proposed to determine whether this is a feature of heme-stripped P450IIC11, to characterize its time-course, identify the phosphorylation sites and determine the heme-regulation of this process. Studies on heme-mediated regulation of rat hepatic TO will be extended: The marked functional TO loss resulting from immunochemically detectable structural loss after acute heme depletion, suggests that prosthetic heme loss destabilizes apoTO. However, not only TO loss persists in acute heme deficiency, but TO induction by glucocorticoids is also impaired. Studies are proposed to determine whether this impairment reflects instability of the newly synthesized apoTO in the absence of heme, or whether, in common with hepatic P450s, heme also regulates TO transcription/translation. It is believed, that collectively, these studies will provide insight into the structure-function relationships, the structural features that commit P450s to toxification/detoxification pathways and thus influence xenobiotic-mediated toxicity, and the ill-understood heme regulation of the turnover of these important hemoproteins.