Purification and cDNA cloning of indoleacetaldehyde oxidase from maize coleopriles.

Aldchyde氧化酶（AO）在动物和微生物中得到了广泛的研究。然而，在植物体内，关于这种酶的信息有限。人们对这种酶的兴趣很大，因为它可能参与了IAA的合成，但它在IAA生物合成中的功能目前还不清楚。我们认为IAA的大部分是由玉米胚芽鞘中的色氨酸合成的，并且还发现了一种由胚芽鞘提取物中的色氨酸形成IAA的体外体系。在本研究中，我们从玉米胚囊中纯化了AO，并试图获得cDNA克隆。结果表明：(1)从3 d龄玉米胚芽中纯化出2000倍左右的醛氧化酶（AO）。该酶是一个约300 kDa的同型二聚体，由两个150 kDa的亚基组成。它含有FAD、钼和铁作为假体基；首次证实了植物AO中FAD和金属的存在。(2)除IAA1d外，其他芳香醛如吲哚-3醛和苯甲醛都是较好的底物。用小鼠多克隆抗体对纯化的玉米AO进行免疫印迹分析，结果表明该酶在玉米胚芽鞘的顶端区域相对丰富。(3)从纯化酶中确定了部分氨基酸序列。接下来，利用PCR方法获得部分cDNA片段。现在，我们有两个物种的4kb cDNA克隆（可能是全长），并继续确定序列。这两个克隆与牛醛氧化酶和动物黄嘌呤脱氢酶高度同源。该序列在植物中首次报道。

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.