INTRACELLULAR TRANSGLUTAMINASE--GENE ISOLATION, CHARACTERIZATION, AND FUNCTION

该项目的长期目标是确定和描述 细胞内、胞浆形式的血管紧张素转换酶在体内的作用(S) 转谷氨酰胺酶(TGase)。TGase催化胺偶联反应 多胺和其他伯胺来选择内源蛋白质 底物在γ-谷氨酰氨基上的胺交换反应 酸残留物。我们发现植物中的TGase引入了一种 β-(γ-谷氨酰基)-赖氨酸异肽大分子间的交联剂 酶的多肽亚基(L)，核酮糖1，5-二磷酸 羧基酶-加氧酶(Rubisco)。TGase催化L的交联反应 形成二聚体(L2)和四聚体(L4)的单体，其可以是 在伴侣介导的途径中的中间产物导致组装 Rubisco的唯一催化功能形式，L8S8。TGase还拥有 第二种催化活性是喹苷5‘-三磷酸(GTP)- 结合和GTP水解。这些发现启发了两个假设： 第一个假设是，细胞内的TGase，通过它的能力 催化某些蛋白质分子之间的分子间交联，可能 络合四元亚单位组装中的催化剂作用 选择的酶的结构。第二个假设是细胞内 TGase通过其降解GTP的能力，可能作为一种G蛋白发挥作用 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，这四个目标是：(1) 含TGase基因重组噬菌体克隆的鉴定 从从猪瘟病毒中分离出来的mRNA制备的lambdagt11基因表达文库中 紫花苜蓿L幼苗，分离TGase基因，以及 确定其核苷酸序列。(2)将分离的基因转移到 紫花苜蓿TGase转化大肠杆菌宿主，该宿主同时含有 植物大(L)和小(S)多肽的克隆质粒基因拷贝 Rubrisco的亚基。这些重组体将允许对 植物细胞TGase在植物体内的分子伴侣作用 L和S体内组装成具有酶功能的十六聚体L8S8 鲁比斯科。(3)评估TGase可能的G蛋白性质 测定其酶活性及其mRNA的表达 植物幼苗在给药后的合成 信号转导过程。(4)利用 TGase基因的核苷酸序列以开发反义RNA 抑制TGase酶表达的方法学研究 转化的植物。该项目的结果将与 对几乎所有齐聚物所在的生物系统有基本的了解 构建了蛋白质亚单位组件。它还将向前推进 真核细胞系统中G蛋白功能的知识(S)。

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.