INTRACELLULAR TRANSGLUTAMINASE--GENE ISOLATION, CHARACTERIZATION, AND FUNCTION

该项目的长期目标是确定和描述 细胞内、细胞溶质形式的 酶，转氨酶（TGase）。 TGase催化胺缀合 多胺和其他伯胺的选择内源性蛋白质 通过在γ-异丙胺基氨基上的胺交换反应 酸残留物 我们已经发现，植物中的TGase引入了 β-（γ-氨基）-赖氨酸异肽之间的大交联 该酶的多肽亚基（L），核酮糖1，5-二磷酸 羧化酶加氧酶（Rubisco）。 TGase催化L 单体以形成二聚体（L2）和四聚体（L4），其可以是 分子伴侣介导的途径中的中间体， Rubisco的唯一催化功能形式，L 8 S8。 TGase还拥有 第二催化活性，其为5 ′-三磷酸喹核苷（GTP）- 结合和GTP水解。 这些发现激发了两种假设： 第一个假设是，细胞内的TGase，通过其能力， 催化一些蛋白质分子之间的分子间交联，可以 在复杂的四元亚基的组装中起催化剂的作用 选择酶的结构。 第二个假设是细胞内 TGase，通过其水解GTP的能力，可以作为G蛋白发挥功能 在植物的信号转导过程中。 这些假设将是 通过调查四个具体目标进行测试，这些目标是：（1） 表征含有TGase基因的重组噬菌体克隆， 从从分离的mRNA制备的cDNA表达文库中， 苜蓿幼苗，分离TGase的基因，和 确定其核苷酸序列。 (2)为了转移分离的基因， 苜蓿TGase进入大肠杆菌宿主，其同时含有 植物大（L）和小（S）多肽的克隆质粒基因拷贝 Rubisco的亚基。 这些重组体将允许研究 植物细胞TGase在介导蛋白质合成中的分子伴侣作用 将L和S体内组装成酶促功能性十六聚体，L 8 S8 rubisco。 (3)通过以下方法评价TGase的推定G蛋白性质： 测定其酶活性及其mRNA的表达 在施用干扰植物幼苗的药剂后， 信号转导过程 (4)为了利用知识， TGase基因的核苷酸序列，以便利用反义RNA 抑制TGase酶表达的方法 转化植物 该项目的结果将与 基本了解几乎所有的生物系统， 构建蛋白质亚基组装体。 它还将推进 了解G蛋白在真核细胞系统中的功能。

The long-term objectives of this project are to identify and characterize the in vivo functional role(s) for the intracellular, cytosolic form of the enzyme, transglutaminase (TGase). TGase catalyzes amine-conjugations of polyamines and other primary amines to select endogenous protein substrates through an amine-exchange reaction at gamma-glutaminyl amino acid residues. We have discovered that TGase in plants introduces an epsilon- (gamma-glutaminyl) -lysine isopeptide crosslink between large polypeptide subunits (L) of the enzyme, ribulose 1, 5-bisphosphate carboxylase-oxygenase (rubisco). TGase catalyzes crosslinkage of L monomers to form dimers (L2) and tetrameres (L4), which may be intermediates in a chaperone-mediated pathway leading to the assembly of the only catalytically functional form of rubisco, L8S8. TGase also has a second catalytic activity, that being quanosine 5'-triphosphate (GTP)- binding and GTP hydrolysis. These findings inspire two hypotheses: The first hypothesis is that intracellular TGase, through its capacity to catalyze intermolecular crosslinks between some protein molecules, may function as a catalyst in the assembly of complex quaternary subunit structures of select enzymes. The second hypothesis is that intracellular TGase, through its capacity to hydrolyze GTP, may function as a G-protein in signal transduction processes in plants. These hypotheses will be tested by investigating four specific aims, which are: (1) To characterize recombinant phage clones containing the TGase gene derived from a lambdagt11 cDNA expression library prepared from mRNA isolated from Medicago sativa L (alfalfa) seedlings, isolate the gene for TGase, and determine its nucleotide sequence. (2) To transfer the isolated gene for alfalfa TGase into an Escherichia coli host which simultaneously contains cloned plasmid gene copies of plant large (L) and small (S) polypeptide subunits for rubisco. These recombinants will permit an investigation of the putative chaperone role of plant cellular TGase in mediating the in vivo assembly of L and S into enzymatically functional hexadecameric, L8S8 rubisco. (3) To evaluate putative G-protein properties of TGase by determining the expression of its enzymatic activity and its mRNA synthesis in plant seedlings after administration of agents that perturb signal transduction processes. (4) To utilize knowledge of the nucleotide sequence of the TGase gene in order to exploit anti-sense RNA methodology for suppression of the expression of the TGase enzyme in transformed plants. The results of this project will be relevant to the basic understanding of virtually all biological systems where oligomeric protein subunit assemblies are constructed. It will also advance knowledge of G-protein function(s) in eukaryotic cell systems.