MITOCHONDRIAL ISOFORMS OF NUCLEOSIDE DIPHOSPHATE KINASE

描述：申请人实验室的长期目标是： (1)了解GTP如何参与监管和整合 线粒体基质内的代谢和能量传递；(2) 了解线粒体亚型二磷酸核苷激酶是如何 (NDPK)调节线粒体之间的磷酸基团流动 腺嘌呤和鸟嘌呤核苷酸的池；以及(3)阐明遗传 线粒体和胞质NDPK亚型之间的关系。 尽管胞质NDPK的异构体正在接受一种 对调控生物学感兴趣的实验室数量、作用、 NDPK的线粒体亚型的性质和区划有很长的历史 被忽视了。鸽肝线粒体的基质间隙含有一个 NDPK活性异常高。GTP/GDP特异性琥珀酸酯 硫代激酶和磷酸烯醇式丙酮酸羧酸激酶也存在于这些 线粒体。 NDPK的基质异构体已纯化至均一状态，并已 有部分特征的。这项建议的具体目的是：(1) 纯化线粒体NDPK的第二个亚型，它位于 家鸽心脏线粒体内膜并测定其生理和功能 两种线粒体亚型的酶学特性；以及(2) 测定每个线粒体的cdna和氨基酸序列。 NDPK的亚型。 线粒体亚型基因的克隆和测序的初步尝试 导致获得了一个明显的胞浆序列 异构体。为了获得线粒体亚型寡核苷酸的序列 将在聚合酶链式反应中使用引物，以从 鸽肝组织中的mRNA组分。线粒体的N-端序列 从鸽肝中纯化的NDPK的亚型已确定 线粒体。将对这一基因的几个部分进行寡核苷酸扩增 序列。另一种是针对人类基因组中高度保守的区域的 已知NDPKs。一旦一个片段被扩增，就会准备好引物来 通过RACE(快速扩增cDNA端)获得3‘和5’端 程序。如果此方法不起作用，则通过 纯化的NDPK的蛋白水解酶将被测序。另外， 将根据实际情况制备简并寡核苷酸引物 序列。在获得NDPK的基质异构体的序列后， 位于内膜外的线粒体亚型的序列将 通过使用上述方法进行搜索。 将鉴定出足够量的每一种线粒体NDPK亚型 获得以允许表征天然相对分子质量、数量 亚基、底物专一性和动力学常数。两者都是线粒体 NDPK的形式可以通过在大肠杆菌中过度表达cDNA来获得。 或者，位于内膜外部的异构体将是 用常规方法提纯的。针对基质形式的抗体在 手，这些可以提供一种快速的方法来纯化另一种异构体，如果有 就是有足够的交叉反应。 实现所提出的具体目标将为 有关属性、角色和功能的信息越来越多 NDPK亚型存在于细胞的不同部位。任何人之间的关联性 对健康的了解目前还不确定。然而，关于NDPK的工作在 过去的五年表明，这种酶不仅仅是一种看家的东西 酶，它还在各种细胞功能中发挥调节功能。

DESCRIPTION: The long term objectives of the applicant's laboratory are to: (1) understand how GTP is involved in the regulation and integration of metabolism and energy transduction within the mitochondrial matrix; (2) understand how mitochondrial isoforms of nucleoside diphosphate kinase (NDPK) regulate the flow of phosphoryl groups between the mitochondrial pools of adenine and guanine nucleotides; and (3) elucidate the genetic relationships between the mitochondrial and "cytosolic" isoforms of NDPK. Although cytosolic isoforms of NDPK are receiving intense scrutiny by a number of laboratories interested in regulatory biology, the roles, properties, and compartmentation of mitochondrial isoforms of NDPK have long been neglected. The matrix space of pigeon liver mitochondria contains an extraordinary high level of NDPK activity. GTP/GDP-specific succinate thiokinase and phosphoenolpyruvate carboxykinase are also present in these mitochondria. The matrix isoform of NDPK has been purified to homogeneity and has been partially characterized. The specific aims of this proposal are to: (1) purify a second isoform of mitochondrial NDPK that is located outside the inner membrane of pigeon heart mitochondria and determine the physical and enzymatic characteristics of the two mitochondrial isoforms; and (2) determine the cDNA and amino acid sequences of each of the mitochondrial isoforms of NDPK. An initial attempt to clone and sequence a cDNA for a mitochondrial isoform of NDPK resulted in obtaining the sequence for an apparent cytosolic isoform. To obtain the sequence to a mitochondrial isoform, oligonucleotide primers will be used in PCR to amplify segments of the desired cDNA from a mRNA fraction of pigeon liver. The N-terminal sequence of the mitochondrial isoform of NDPK has been determined for the purified NDPK from pigeon liver mitochondria. Oligonucleotide primers will be made to several parts of this sequence. Other primers are being made to highly conserved regions within known NDPKs. Once a segment has been amplified, primers will be prepared to obtain the 3' and 5' ends by the RACE (rapid amplification of cDNA ends) procedure. If this approach does not work, one or more peptides obtained by proteolytic digestion of purified NDPK will be sequenced. Additional, degenerate oligonucleotide primers will be prepared based on the actual sequences. After obtaining the sequence to the matrix isoform of NDPK, the sequence of a mitochondrial isoform located outside the inner membrane will be sought by using the approaches described above. Sufficient amounts of each mitochondrial isoform of NDPK identified will be obtained to allow characterization of native molecular weight, number of subunits, substrate specificity, and kinetic constants. Both mitochondrial forms of NDPK may be obtained by over expressing the cDNAs in E. coli. Alternatively, the isoform located outside the inner membrane will be purified by conventional means. Antibodies against the matrix form are in hand and these may provide a quick way to purify the other isoform if there is sufficient cross-reactivity. Accomplishing the proposed specific aims will add important knowledge to the growing body of information about the properties, roles and functions of NDPK isoforms in various parts of the cell. The relatedness of any knowledge to health is uncertain at this time. However, work on NDPK within the past five years indicate that this enzyme is more than a house-keeping enzyme, it also plays regulatory functions in various cellular functions.