Purification and cDNA cloning of indoleacetaldehyde oxidase from maize coleopriles.

在动物和微生物中已有广泛的研究。然而，在植物中，关于这种酶的信息有限。人们对该酶很感兴趣的是它可能参与了IAA的合成，但它在IAA生物合成中的作用目前还不清楚。我们认为玉米胚芽鞘中的大部分IAA是由色氨酸合成的，并发现了从胚芽鞘提取液中色氨酸合成IAA的体外体系。在本研究中，我们从玉米胚芽鞘中提纯了AO，并试图获得克隆(S)。主要研究结果如下：(1)从3日龄玉米幼苗的胚芽鞘中，乙醛氧化酶(AO)的纯度提高了2000倍左右。该酶为约300 kDa的同源二聚体，由两个150 kDa的亚基组成。它含有FAD、钼和铁作为辅基，首次证明了FAD和金属在植物AO中的存在。(2)除IAA1d外，其他芳香醛如吲哚-3-醛和苯甲醛是很好的底物。用小鼠抗玉米AO的多克隆抗体进行免疫印迹分析表明，该酶在玉米胚芽鞘顶端相对丰富。(3)从纯化的酶中测定了部分氨基酸序列。其次，利用聚合酶链式反应方法获得了部分基因片段。现在，我们有两种4kb的cDNA克隆(可能是全长)，并正在继续测定序列。这两个克隆都与牛醛氧化酶和动物黄嘌呤脱氢酶高度同源。这里获得的序列是首次在植物中报道的。

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.