Purification and cDNA cloning of indoleacetaldehyde oxidase from maize coleopriles.

乙醛氧化酶（AO）在动物和微生物中已得到广泛的研究。然而，关于这种酶的信息有限。对这种酶的极大兴趣在于其可能参与IAA合成，但其在IAA生物合成中的功能目前尚不清楚。我们认为玉米胚芽鞘中的IAA主要是由色氨酸合成的，并建立了一个由胚芽鞘提取物中的色氨酸合成IAA的体外体系。本研究从玉米胚芽鞘中分离纯化了AO基因，并试图获得其cDNA克隆。结果如下：（1）从3日龄玉米幼苗胚芽鞘中提取的醛氧化酶（AO）纯化倍数约为2,000倍。该酶是由两个150 kDa亚基组成的约300 kDa的同源二聚体。它含有FAD，钼和铁作为辅基;首次证明了植物AO中FAD和金属的存在。(2)除IAA 1d外，其它芳香醛如吲哚-3-醛和苯甲醛也是很好的底物。用小鼠抗玉米AO多克隆抗体进行免疫印迹分析，结果表明玉米AO在胚芽鞘的顶端区域相对富集。(3)从纯化的酶中，测定了部分氨基酸序列。接着，使用PCR方法获得一些cDNA片段。目前，我们已经获得了2个物种的4kb cDNA克隆（可能是全长），并正在继续测定序列。这两个克隆与牛醛氧化酶和动物黄嘌呤氧化酶高度同源。在此获得的序列首次在植物中报道。

Aldchyde oxidase (AO) has been extensively in vestigated in animals and microorganisms. Inplants, however, there has been only a limited information on this enzyme. A great interest in this enzyme has been in its possible involvement in IAA synthesis, but its function in IAA biosynthesis is not presently known. We have suggested that major portion of IAA was synthesized from tryptophan in maize coleoptile, and have also found an in vitro system for formation of IAA from tryptophan in extracts of coleoptiles. In the present study, we purified AO from maize coleoptiles and tried to get cDNA clone (s). The summary of the results are as follows :(1) From coleoptiles of 3-day-old maize seedlings, aldehyde oxidase (AO) was purified around 2,000-fold. The enzyme was an approximately 300 kDa homodimer composed of two 150 kDa subunits. It contained FAD,molybdenum and iron as prosthetic groups ; the presence of FAD and the metals in plant AO was demonstrated for the first time.(2) Besides IAA1d, other aromatic aldehydes such as indole-3-aldehyde and benzaldehyde served as good substrates. Immunoblotting analysis with mouse polyclonal antibodies against the purified maize AO showed that the enzyme was relatively rich in the apical region of maize coleoptile.(3) From the purified enzymes, partial sequences of amino acid were determined. Next, using PCR methods some cDNA segments were obtained. Now, we have 2 species of 4kb cDNA clones (may be full length) and are continuing to determine the sequences. Both clones are highly homologue to bovine aldehyde oxidase and animal xanthine dehydrogenases. The sequences obtained here are reported first in plants.