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A study on the precursor of glyoxylate in plants. Trials to reduce oxalate production in hyperoxalurias.

植物中乙醛酸前体的研究。

基本信息

批准号：
08670168
负责人：
ICHIYAMA Arata
金额：
$ 1.22万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Oxalate is synthesized mainly in the liver from glyoxylate which is formed from glycolate in liver peroxisomes. Usually, the conversion to glycine catalyzed by serine : pyruvate/alanine : glyoxylate aminotransferase (SPT/AGT) is the main rout of glyoxylate metabolism, but when SPT/AGT is missing in liver peroxisomes as in the case of primary hyperoxaluria type 1 more glyoxylate is utilized for oxalate production resulting in hyperoxaluria and finally systemic oxalosis. SPT/AGT locates in either mitochondria, peroxisomes or both depending on the food habit of animal species, and this dual organelle localization is caused by an alternative transcription initiation and point mutations in the translation initiation AUG codon, suggesting that only those animal species survive who gained, by mutations of the AGT gene, the proper organelle localization of SPT/AGT to meet the metabolic needs arisen from their food habit. The major source of glycolate is still elusive, but if it is the dietary glycolate itself it is rich in plant as an intermediate of photorespiration. The present study was undertaken to evaluate the contribution of dietary glycolate to oxalogenesis, and for this purpose enzymic methods for determination of glycolate were developed. In one of the methods, glycolate is oxidized by glycolate oxidase (GO) in the presence of excess cysteamine and resulting thiazolidine 2-carboxylate is quantitated by pico tag chromatography. In the other, the reaction of glycolate with GO is performed in Tris buffer (pH 8.3) and pyruvate formed from L-lactate is eliminated by the combined actions of GPT,GlDH and G6P-DH.Glyoxylate protected from both GO and GPT as Tris-glyoxylate adduct is then converted to 1,5-diphenylformazan and determined at 515-520 nm. Both methods reliably detect 1 nmol of glycolate in the sample, but the latter is simpler and pertinent to routine use while the former may be used for confirmatory purposes.

草酸主要在肝脏中由乙醛酸合成，乙醛酸由肝脏过氧化物酶体中的乙醇酸形成。通常，由丝氨酸：丙酮酸/丙氨酸：乙醛酸氨基转移酶（SPT/AGT）催化的转化为甘氨酸是乙醛酸代谢的主要途径，但是当SPT/AGT在肝脏过氧化物酶体中缺失时，如在原发性高尿酸血症1型的情况下，更多的乙醛酸用于草酸盐产生，导致高尿酸血症和最终的全身性草酸盐中毒。SPT/AGT定位于线粒体、过氧化物酶体或两者中，这取决于动物物种的饮食习惯，并且这种双重细胞器定位是由交替的转录起始和翻译起始AUG密码子的点突变引起的，这表明只有那些通过AGT基因突变获得SPT/AGT的适当细胞器定位以满足其饮食习惯引起的代谢需要的动物物种才能存活。乙醇酸的主要来源仍然是难以捉摸的，但如果它是膳食乙醇酸本身，它是丰富的植物作为光呼吸的中间体。本研究旨在探讨饲粮中乙醇酸对草酸生成的影响，并建立了乙醇酸的酶法测定方法。在一种方法中，在过量半胱胺存在下，通过乙醇酸氧化酶（GO）氧化乙醇酸盐，并通过皮科标签色谱法定量所得噻唑烷2-羧酸盐。另一种方法是在pH8.3的Tris缓冲液中，乙醇酸与GO反应，L-乳酸生成的丙酮酸被GPT、GlDH和G6 P-DH的共同作用消除，而不受GO和GPT保护的乙醛酸作为Tris-乙醛酸加合物，转化为1，5-二苯基甲，并在515-520 nm处测定。这两种方法都能可靠地检测到样品中1 nmol的乙醇酸盐，但后者更简单，适用于常规使用，而前者可用于确证目的。