Investigation of structure and rate of synthesis of ornithine decarboxylase protein in mouse kidney.
Investigation of structure and rate of synthesis of ornithine decarboxylase protein in mouse kidney.
复制标题
小鼠肾脏鸟氨酸脱羧酶蛋白的结构和合成速率的研究。
DOI：
10.1021/bi00311a033
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
Pegg,AE
中科院分区：
文献类型：
--
作者：
Persson,L;Seely,JE;Pegg,AE
Lo Persson, 1 James E. Seely, 5 and Anthony E. Pegg**摘要：采用免疫印迹技术研究雄激素诱导小鼠肾脏中鸟氨酸脱羧酶的形态。检测到的两种形式在等电点上略有不同，但在亚单位分子量（~ 55000）上没有差异。这两种形式都具有酶活性，可以通过与放射性a-（二氟甲基）鸟氨酸（一种酶激活的不可逆抑制剂）反应来标记。在肾脏粗匀浆或部分纯化的鸟氨酸脱羧酶制剂的储存中，酶蛋白被降解到更小的尺寸（Mr~ 53000），但酶活性没有实质性的损失。通过腹腔注射[35S]蛋氨酸标记和免疫沉淀，利用单克隆抗体和多克隆抗体研究鸟氨酸脱羧酶蛋白的合成和降解。雌性小鼠经睾酮处理后，肾脏鸟氨酸脱羧酶所代表的总蛋白合成比例增加了至少25倍，而在完全诱导的雄激素处理的雌性小鼠中，这一比例约为1.1%。两种形式的酶在体内都被快速标记，暴露于环己亚胺4小时后，免疫可沉淀的鸟氨酸脱羧酶蛋白几乎完全丢失，直接证实了这种酶的快速周转。用1,3 -二氨基丙烷处理，已知会导致鸟氨酸脱羧酶活性大大降低，但没有很大地选择性抑制酶的合成。然而，1,3 -二氨基丙烷确实增加了鸟氨酸脱羧酶的降解速率和蛋白质合成的普遍减少。因此，这两个因素似乎是对1,3 -二氨基丙烷反应的鸟氨酸脱羧酶活性和蛋白质损失的原因。由于鸟氨酸脱羧酶在多胺生物合成途径中的第一步催化作用具有显著的诱导性，因此对哺乳动物细胞中的鸟氨酸脱羧酶进行了许多研究（Janneet al., 1978; Russell, 1980; McCann, 1980; Pegg & McCann, 1982）。直到最近，这些研究大多局限于酶活性的测量，因为即使在最大程度的诱导后，哺乳动物细胞中也存在少量的鸟氨酸脱羧酶蛋白，而且研究蛋白质本身的方法不敏感或不可用。因此，尽管一些研究小组提出这种酶有多种形式，或者它可能是调节性翻译后修饰的底物（Richards et al., 1981; Kuehn & Atmar, 1982; Mitchell & Mitchell, 1982; Russell, 1983; Bullock et al., 1983），但尚未进行明确的实验来测试这些可能性。此外，基于免疫亲和层析的使用，有确凿的证据表明，酵母鸟氨酸脱羧酶在细胞中作为一个比纯化后获得的更大的蛋白质存在，这是由于在粗细胞提取物中发生的快速蛋白水解裂解（Tyagi等人，1982）。在目前的实验中，对小鼠鸟氨酸脱羧酶的单特异性抗体已被用于检查大小和可能存在的多种形式
Lo Persson, 1 James E. Seely, 5 and Anthony E. Pegg** abstract: An immunoblottingtechnique was used to study the forms of ornithine decarboxylase present in androgen-in-duced mouse kidney. Two forms were detected which differed slightly in isoelectric point but not in subunit molecular weight (~ 55 000). Both forms were enzymatically active and could be labeled by reaction with radioactive a-(difluoromethyl)-ornithine, an enzyme-activated irreversible inhibitor. On storage of crude kidney homogenates or partially purified preparations of ornithine decarboxylase, the enzyme protein was degraded to a smaller size (Mr~ 53 000) without sub-stantial loss of enzyme activity. Thesynthesis and degradation of ornithine decarboxylase protein were studied by labeling the protein by intraperitoneal injection of [35S] methionine and immunoprecipitation using both monoclonal and polyclonal antibodies. The fractionof total protein synthesis represented by renal ornithine decarboxylase was increased at least 25-fold by testosterone treatment of female mice and was found to be about 1.1% in the fully induced androgen-treated female. Both forms of the enzyme were rapidly labeled in vivo, and the immunoprecipitable ornithine decarboxylase protein was almost completely lost after 4-h exposure to cycloheximide, confirming directly the very rapid turnover of this enzyme. Treatment with 1, 3-diaminopropane which is known to cause a great reduction in ornithine decarboxylase activity didnot greatly selectively inhibit thesynthesis of the enzyme. How-ever, 1, 3-diaminopropanedid produce an increase in therate of degradation of ornithinedecarboxylase and a general re-duction in protein synthesis. These two factors, therefore, appear to be responsible for the loss of ornithine decarboxylase activity and protein in response to 1, 3-diaminopropane. ere have been many studies of ornithine decarboxylase in mammalian cells because of the remarkable inducibility of the activity of this enzyme which catalyzes the first step in the polyamine biosynthetic pathway (Janneet al., 1978; Russell, 1980; McCann, 1980; Pegg & McCann, 1982). Until recently, most of these investigations were limited to measurements of enzyme activity because of the small amount of ornithine decarboxylase protein present in mammalian cells even after maximal induction and the insensitivity or unavailability of methods for studying the protein itself. Therefore, although a number of groups have proposed that there are multiple forms of this enzyme or that it may be a substrate for regu-latory posttranslational modifications (Richards et al., 1981; Kuehn & Atmar, 1982; Mitchell & Mitchell, 1982; Russell, 1983; Bullock et al., 1983), definitive experiments to test these possibilities have not been carried out. Also, there is solid evidence based on the use of immunoaffinitychromatography that yeast ornithine decarboxylase is present in the cell as a larger protein than that obtained after purification owing to a rapid proteolytic cleavage which occurs in crude cell extracts (Tyagi et al., 1982). In thepresent experiments, monospecific antibodies to mouse ornithine decarboxylase have been used to examine the sizeand possible existence of multiple forms