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转导;（2） 了解线粒体二磷酸核苷激酶亚型 （NDPK）调节磷酸基团在线粒体之间的流动 腺嘌呤和鸟嘌呤核苷酸池;和（3）阐明遗传 NDPK的线粒体和“胞质”亚型之间的关系。 虽然NDPK的胞质亚型正在接受一个研究小组的密切审查， 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室化已经很长时间了， 被忽视了 鸽肝线粒体的基质空间含有一个 NDPK活性异常高。 GTP/GDP特异性琥珀酸 硫激酶和磷酸烯醇丙酮酸羧激酶也存在于这些细胞中。 线粒体 NDPK的基质同种型已被纯化至均一，并已被纯化。 部分特征。 本提案的具体目标是：（1） 纯化位于线粒体外的线粒体NDPK的第二同种型， 鸽心线粒体内膜，并确定物理和 两种线粒体同工型的酶特性;和（2） 确定每个线粒体的cDNA和氨基酸序列， NDPK亚型。 线粒体亚型cDNA克隆和测序的初步尝试 结果获得了一个明显的细胞溶质的序列， 同种型 为了获得线粒体同种型的序列，寡核苷酸 在PCR中使用引物从一个或多个DNA片段中扩增所需cDNA的片段。 鸽肝的mRNA组分。 线粒体的N端序列 从鸽肝中纯化出NDPK，并测定了NDPK的亚型 线粒体 寡核苷酸引物将对此的几个部分进行 顺序 其他的引物被制作成针对 已知的NDPK 一旦片段被扩增，将制备引物以 通过RACE（cDNA末端的快速扩增）获得3'和5'末端 procedure. 如果这种方法不起作用，则可以使用通过以下方法获得的一种或多种肽： 将对纯化的NDPK的蛋白水解消化进行测序。 此外， 简并寡核苷酸引物将基于实际的 序列的 在获得NDPK的基质同种型的序列后， 位于内膜外的线粒体同种型的序列将 可以通过使用上述方法来寻求。 足够量的鉴定的NDPK的每种线粒体亚型将被施用。 获得，以允许表征天然分子量、 亚基、底物特异性和动力学常数。 两个线粒体 NDPK的形式可以通过在E.杆菌 或者，位于内膜外的同种型将被 通过常规方法纯化。 针对基质形式的抗体在 这些可以提供一种快速的方法来纯化其他同种型，如果有 有足够的交叉反应性。 实现所提出的具体目标将增加重要的知识， 越来越多的信息的属性，角色和功能， NDPK亚型在细胞的各个部分。 任何一种关联 健康知识目前还不确定。 然而，在2010年内， 过去的五年表明，这种酶不仅仅是一种管家， 除此之外，它还在各种细胞功能中发挥调节作用。

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.