Interrelation between the charge isoforms of mammalian ornithine decarboxylase.
Interrelation between the charge isoforms of mammalian ornithine decarboxylase.
复制标题
哺乳动物鸟氨酸脱羧酶电荷亚型之间的相互关系。
DOI：
10.1016/0003-9861(88)90485-7
复制
发表时间：
1988
影响因子：
3.9
通讯作者：
Hicks,MF
中科院分区：
文献类型：
--
作者：
Mitchell,JL;Rynning,MD;Chen,HJ;Hicks,MF
从多种组织中分离的鸟氨酸脱羧酶(ODC)已用DEAE离子交换层析分离成多个活性峰，这些峰似乎与该酶的稳定性控制有关。目前文献中关于这些电荷异构体的报道通常不清楚这些异构体是否代表共价的翻译后修饰，或者仅仅是结构构象或结合的改变。在这项研究中，我们研究了这种形式的分离与酶的聚合度，与其他蛋白质和缓冲成分的相互作用，以及该酶的多种等电形式的尿素变性浓度的关系。高效层析技术被用来证明两种主要的酶形式，ODC I和II，实际上是该酶的单体，而在ODC II之后经常观察到的次要活性峰包含各种二聚体酶状态。在由I和II单体组成的分离酶制剂中加入0.05 mM的5‘-磷酸吡哆醛，可诱导I和II二聚体以及I-II混合二聚体的形成。观察到这三种二聚体都是新分离的粗细胞匀浆的天然成分。在存在8M尿素的离子交换层析过程中，发现单体I和II之间的电荷差异保持不变，而在9M尿素存在的等电聚焦凝胶上，酶的异构体显示出明显的条带。因此，虽然哺乳动物细胞匀浆的离子交换层析鉴定的多种鸟氨酸脱羧酶形式中的一些与酶构象有关，但两种主要形式是明显带电的蛋白质状态，可以用高纯度样品的二维凝胶电泳法观察到。
Ornithine decarboxylase (ODC) isolated from a variety of tissues has been separated, using DEAE ion-exchange chromatography, into multiple peaks of activity that appear to be related to control of this enzyme stability. Reports of these charge isoforms in current literature are generally unclear as to whether these represent a covalent post-translational modification or merely an alteration in structural conformation or association. In this study we investigated the relationship of this form separation to the degree of enzyme polymerization, interaction with other proteins and buffer components, and the multiple isoelectric forms of this enzyme noted in denaturing concentrations of urea. High-performance chromatography techniques were used to demonstrate that two of the major enzyme forms, ODC I and II, are really monomers of the enzyme, while minor peaks of activity frequently observed to elute after ODC II contain various dimeric enzyme states. Pyridoxal 5′-phosphate (0.05 mm) added to isolated enzyme preparations composed of I and II monomers induced the formation of I and II dimers as well as a mixed I–II dimer. All three dimer forms were observed to be natural components of freshly isolated crude cell homogenates. The charge distinction between the monomer forms I and II was found to be maintained during ion-exchange chromatography in the presence of 8murea, and the enzyme isoforms demonstrated distinct bands on isoelectric focusing gels run in the presence of 9murea. Thus, although some of the multiple ornithine decarboxylase forms identified by ion-exchange chromatography of crude mammalian cell homogenates are related to enzyme conformation, the two major forms are distinctly charged protein states that can be visualized using two-dimensional gel electrophoresis of highly purified samples.