Purification and cDNA cloning of indoleacetaldehyde oxidase from maize coleopriles.

醛氧化酶（AO）已在动物和微生物中得到广泛研究。然而，在植物体内，有关这种酶的信息非常有限。人们对这种酶的极大兴趣在于它可能参与 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IAA的主要部分是由玉米胚芽鞘中的色氨酸合成的，并且还发现了从胚芽鞘提取物中的色氨酸形成IAA的体外系统。在本研究中，我们从玉米胚芽鞘中纯化了AO并试图获得cDNA克隆。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)纯化了2000倍左右。该酶是由两个 150 kDa 亚基组成的约 300 kDa 同二聚体。以FAD、钼、铁为辅基；首次证明FAD和植物AO中金属的存在。(2)除IAA1d外，其他芳香醛如3-吲哚醛和苯甲醛都是良好的底物。用针对纯化的玉米AO的小鼠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区相对丰富。(3)从纯化的酶中确定了部分氨基酸序列。接下来，使用PCR方法获得了一些cDNA片段。现在，我们有2种4kb cDNA克隆（可能是全长）并且正在继续确定序列。两个克隆与牛醛氧化酶和动物黄嘌呤脱氢酶高度同源。这里获得的序列首先在植物中报道。

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.