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.

草酸盐主要是在肝脏中由乙醇酸酯形成的甘露乙酸盐中合成的。通常，通过丝氨酸催化的甘氨酸的转化：丙酮酸/丙氨酸：乙二基氨基转移酶（SPT/AGT）是甘酰基代谢的主要范围，但是当肝过氧化物中缺失SPT/AGT时，如原发性高氧化甲素型高胶质溶质溶质的情况时，用于草酸盐的产生，导致高黄油尿症，最后是全身草皮菌。 SPT/AGT位于线粒体，过氧化物酶体或两者都取决于动物物种的食物习惯，并且该双细胞器定位是由替代转录启动和翻译起始中的点突变引起的，这表明只有那些动物物种才能幸存下来通过AGT基因的突变获得，SPT/AGT的适当细胞器定位以满足其食物习惯所产生的代谢需求。乙酸的主要来源仍然难以捉摸，但是如果它是饮食中的乙醇酸本身，它就会富含植物作为光孔培训的中间体。进行了本研究，以评估饮食中的乙醇酸盐对草生成的作用，为此，开发了确定乙醇酸的酶方法。在其中一种方法中，在存在过量的cysteamine的情况下，乙醇酸被乙醇酸氧化酶（GO）氧化，并通过PICO TAG色谱法对产生的硫醇2-羧酸盐氧化。另一方面，在Tris缓冲液（pH 8.3）中进行乙醇酸与GO的反应，并通过GPT，GLDH和G6P-DH的合并作用消除了由L-乳酸形成的丙酮酸。-乙氧基化的合并作用。乙氧基化受到GO和GPT的保护。然后将甘油乙酰化加合物转换为1,5-二苯基甲曲唑，并在515-520 nm处确定。两种方法都可靠地检测到样品中的1 nmol乙醇酸，但是后者更简单，并且与常规使用相关，而前者可以用于确认目的。