ORNITHINE DECARBOXYLASE MODIFICATION AND INACTIVATION

鸟氨酸脱羧酶(ODC；E.C.4.1.1.17)被认为是 多胺生物合成中的限速酶 对细胞生长至关重要的亚精胺和亚精胺。 这种酶在所有正常细胞中都受到严格的调控，而且 ODC调节异常与肿瘤相关 一些人认为它是一种 致癌基因。这一生物合成途径的抑制物很重要。 不仅在癌症化疗方面，而且在抗击寄生虫方面也是如此 非洲睡眠病的锥虫等感染 和与艾滋病有关的卡氏肺孢子虫。尽管有些人 在合成水平上对ODC的活性进行控制， 这种酶的最不寻常之处在于它展示了一种极其 快速的蛋白质周转速度，允许非常快速的调节 酶活性。这种非常有效的、特定的酶 失活似乎涉及到最初的酶失活， 它可以被亚精胺和精胺的产品刺激， 紧随其后的是酶蛋白的选择性降解。 我们一直在调查翻译后的费用 与活性磷酸盐去除相关的修饰 酶的状态、酶的结构变化和ODC 与一种假定的调节蛋白，抗酶相关。我们的 提案是继续这些调查路线，目的是 将这些信息组合成一个统一的模型，供 这种酶的受控失活。特别是，我们将使用 一种大鼠肝癌细胞系(HTC)，肾脏来自睾酮- 刺激雄性小鼠和肾上腺激素诱导的大鼠组织 研究体内产生的ODC蛋白的变化。我们有 在原油中开发了一种非氧化的ODC失活系统 HTC细胞的匀浆将被用来分离和 启动失活/降解的蛋白酶(S)的纯化 ODC，并鉴定它们的底物特异性和控制性。 还将对多胺、抗酶的作用进行评估 和能量在这种快速的、特定的蛋白质降解中。这种蛋白质 磷酸酶能将更稳定的ODC亚型转化为更多 不稳定的形式也将被分离、纯化和鉴定为 底物专一性和对照成分。这是意料之中的 对涉及到的机制的这种统一方法 使这种重要的酶失活和降解将有助于我们 了解ODC活动在应对各种变化时的增长 荷尔蒙，致癌物质和病毒转化，它们 似乎与这种酶失活的变化有关 进程。

Ornithine decarboxylase (ODC; E.C. 4.1.1.17) is held to be the rate limiting enzyme in the biosynthesis of the polyamines spermidine and spermine, which are essential for cell growth. This enzyme is stringently regulated in all normal cells, and abnormal regulation of ODC has been correlated with tumor formation such that some consider it to be the product of an oncogene. Inhibitors of this biosynthetic pathway are important not only in cancer chemotherapy but also in combating parasitic infections such as the Trypanosomes of African Sleeping Sickness and Pneumocystis carinii associated with AIDS. Although some control of the activity of ODC is exerted at the level of synthesis, this enzyme is most unusual in that it demonstrates an extremely fast protein turnover rate, allowing very rapid modulation of this enzyme activity. This very efficient, specific enzyme deactivation appears to involve an initial enzyme inactivation, which can be stimulated by the products spermidine and spermine, followed quickly by selective degradation of the enzyme protein. We have been investigating post-translational charge modifications associated with phosphate removal from active enzyme states, structural changes in the enzyme, and ODC association with a presumptive regulatory protein, antizyme. Our proposal is to continue these lines of investigation with the intent of assembling this information into a unified model for the controlled deactivation of this enzyme. In particular we will use a rat hepatoma cell line (HTC), kidneys from testosterone- stimulated male mice and adrenal hormone-induced rat tissues to study changes in ODC protein that are produced in vivo. We have developed a non-oxidative, ODC-inactivation system in crude homogenates from HTC cells that will be used to isolate and purify the protease(s) initiating the inactivation/degradation of ODC, and to characterize their substrate specificity and control. Evaluation will also be made of the role of polyamines, antizyme and energy in this rapid, specific protein degradation. The protein phosphatases that convert the more stable ODC isoform to a more labile form will also be isolated, purified and characterized for substrate specificity and control components. It is anticipated that this unified approach to the mechanisms involved in inactivating and degrading this important enzyme will help us understand increases in ODC activity seen in response to various hormones, and to carcinogens and viral transformation, which appear to relate to alterations in this enzyme deactivation process.