POLYAMINE OXIDASE ROLE IN APOPTOSIS

多胺氧化酶（PAO）催化多胺精胺（spd）或精胺（spm）的氧化裂解以产生二氨基丙烷（dap）、H2 O2和氨基醛衍生物。在植物中，dap是通过Schiff碱还原酶脱羧酶（SBRD）生物合成去甲精脒（nspd）和去甲精胺（nspm）的前体。近年来，有人提出了用PAO对spd和spm进行催化的方法。几个研究人员认为是一种致病因子，通过产物H2 O2和随后的氧化应激，迫使动物细胞进入程序性细胞死亡（凋亡）。本实验室最近从燕麦幼苗中克隆了PAO基因，并对其进行了测序和鉴定。这种新分离的基因的可用性提供了独特的机会，以获得遗传证据的潜在作用（S）的PAO和多胺-catalysts在植物模型试验系统中的细胞凋亡。本申请的假设是：（i）PAO通过其氧化spd或spm产生的H2 O2在细胞凋亡中起致因作用，和（ii）由dap产生的nspd和nspm通过PAO的反馈抑制和H2 O2合成的减少作为凋亡的抑制剂。本研究的具体目的是：（1）利用Ti质粒转化苜蓿，研究PAO基因缺失对植物细胞凋亡的影响。(2)将催化来自dap的nspd和nspm生物合成的酶SBRD的cDNA基因序列用于转化苜蓿植物，以分析PAO从SBRD衍生自dap的nspd和nspm的生物合成解偶联产生H2 O2的后果。目的（1）和（2）是对假设（i）和（ii）部分的直接检验。（三）、以燕麦PAO cDNA序列为探针，尝试从人肾脏和肝脏cDNA文库中分离人PAO基因。一个特征性的人PAO基因将使分子生物学方法的发展成为可能，以研究PAO在产生H2 O2中的作用及其引起动物凋亡的所谓作用。(4)将PAO基因的信号肽的cDNA基因序列与编码荧光蛋白（GFP）的基因连接。将通过荧光成像技术分析由该构建体产生的融合蛋白在用该构建体转化的苜蓿和外植物的组织中，以鉴定PAO的亚细胞定位。通过免疫金抗体试剂原位标记PAO和电子显微镜分析，将证实通过GFP蛋白技术的定位研究。这些结果将有助于定位启动PAO依赖性细胞凋亡的事件的起源。

Polyamine oxidase (PAO) catalyzes oxidative cleavage of polyamines spermine (spd) or spermine (spm) to produce diaminopropane (dap), H2O2, and an aminoaldehyde derivative. In plants, dap is the precursor for biosynthesis of norspermidine (nspd) and norspermine (nspm) via the enzyme, Schiff base reductase decarboxylase (SBRD). Recently, the catabolism of spd and spm by PAO has been proposed. By several investigators to be a causative agent, through product H2O2 and ensuing oxidative stress, which forces animal cells into programmed cell death (apoptosis). The gene for PAO from oat seedlings was recently cloned, sequenced, and characterized in this laboratory. The availability of this newly isolated gene offers unique opportunities to gain genetic evidence for a potential role(s) of PAO and polyamine-catabolism in apoptosis in a plant model test system. The hypothesis of this application is: (i) PAO has a causative role in apoptosis in cells through H2O2 produced by its oxidation of spd or spm, and (ii) the nspd and nspm produced from dap serve as suppressors of apoptosis through feedback inhibition of PAO and reduction in H2O2 synthesis. The specific aims of this proposal are: (1) The PAO cDNA gene sequence will be used to transform alfalfa plants with Ti-plasmid methods to analyze the consequences of controlled PAO- deficiency on apoptosis in plant tissues. (2) The cDNA gene sequence for the enzyme, SBRD, which catalyzes nspd and nspm biosynthesis from dap, will be used to transform alfalfa plants in order to analyze the consequences of uncoupling generation of H202 by PAO from the biosynthesis of nspd and nspm derived from dap by SBRD. Aims (1) and (2) are direct tests of parts (i) and (ii) of the hypothesis. (3). The PAO cDNA gene sequence from oat will be used as a gene probe to attempt the isolation of the human PAO gene from kidney and liver cDNA libraries. A characterized human PAO gene will make possible the development of a molecular biological approach to investigate the role of PAO in generating H202 and its alleged role to elicit animal apoptosis. (4) The cDNA gene sequence of a signal peptide for the PAO gene will be ligated to a gene coding for a fluorescent protein (GFP). The fusion protein produced from this construction will be analyzed by fluorescent imaging techniques in tissue of alfalfa and out plants transformed with this construction in order to identify the subcellular localization of PAO. In situ labeling of PAO by immunogold antibody reagents and electron microscopic analysis, will corroborate the localization studies by the GFP protein technique. These results will aid in localizing the origin of events that initiate PAO-dependent apoptosis.