ORNITHINE DECARBOXYLASE MODIFICATION AND INACTIVATION

鸟氨酸脱羧酶修饰和失活

• 批准号: 3283939
• 负责人: JOHN L A MITCHELL
• 金额: $ 11.04万
• 依托单位: NORTHERN ILLINOIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3283939
• 关键词: adenosine triphosphate; affinity chromatography; cell growth regulation; conformation; cytoplasm; disease /disorder model; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; enzyme structure; genetic translation; hepatocellular carcinoma; hormone regulation /control mechanism; isozymes; laboratory mouse; laboratory rabbit; laboratory rat; neoplastic cell; neoplastic cell culture for noncancer research; ornithine decarboxylase; peptidases; phosphorylation; polyamines; protein biosynthesis; protein kinase; proteolysis; spermidine; testosterone; tissue /cell culture

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.

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 亚精胺和精胺，它们是细胞生长所必需的。 这种酶在所有正常细胞中都受到严格的调节， ODC的异常调节与肿瘤的发生有关 因此，有些人认为它是一种 癌基因 这种生物合成途径的抑制剂很重要 不仅用于癌症化疗， 感染，如非洲昏睡病的锥虫 以及与艾滋病相关的卡氏肺孢子虫。 尽管一些 ODC活性的控制在合成水平发挥作用， 这种酶是最不寻常的，因为它表现出一种极其 快速的蛋白质周转率，允许非常快速地调节这种 酶活性 这种非常有效的特异性酶 失活似乎涉及初始酶失活， 其可被产物亚精胺和精胺刺激， 随后迅速选择性降解酶蛋白。 我们一直在研究翻译后电荷 与活性物质中磷酸盐去除相关的修饰 酶状态、酶的结构变化和ODC 与一种假定的调节蛋白，抗酶。 我们 建议继续这些调查， 将这些信息组合成一个统一的模型， 控制这种酶的失活。 特别是，我们将使用 大鼠肝癌细胞系（HTC），睾丸激素的肾脏- 刺激雄性小鼠和肾上腺皮质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 开发了一种非氧化的，ODC灭活系统， 来自HTC细胞的匀浆，其将用于分离和 纯化启动灭活/降解的蛋白酶 ODC，并表征其底物特异性和控制。 还将评价多胺、抗酶 在这种快速、特异性的蛋白质降解过程中产生能量。 蛋白质 磷酸酶将更稳定的ODC亚型转化为更稳定的ODC亚型。 不稳定形式也将被分离、纯化和表征， 底物特异性和对照组分。 预计 这种对涉及的机制的统一方法， 使这种重要的酶失活并降解， 了解ODC活动的增加，以应对各种 激素，致癌物质和病毒转化， 似乎与这种酶失活的改变有关 过程