ANALYSIS AND PHYSIOLOGICAL IMPLICATIONS OF RENAL 2-OXOGLUTARAMATE METABOLISM
ANALYSIS AND PHYSIOLOGICAL IMPLICATIONS OF RENAL 2-OXOGLUTARAMATE METABOLISM
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DOI：
10.1042/bj2770033
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发表时间：
1991-07-01
影响因子：
4.1
通讯作者：
YUDKOFF, M
中科院分区：
文献类型：
--
作者：
NISSIM, I;WEHRLI, S;YUDKOFF, M
通过谷氨酰胺氨基转移酶（谷氨酰胺氨基转移酶II）途径的通量对肾氨生成的相对意义知之甚少。一个基本的和未解决的问题是，是否2-酮戊二酸（2-OGM），一个产品的转氨酶II反应，脱酰胺产生2-酮戊二酸和NH3，或是否2-OGM积累作为一个不反应的内酰胺，这取决于环境pH值。以及N-15 n.m.r.。证明2-OGM发生作为内酰胺，即5-羟基焦谷氨酸，无论环境pH值。我们的其他目的是确定是否培养的人肾细胞（HK细胞）可以产生2-OGM，并确定pH值依赖性之间的关系NH3和2-OGM生产谷氨酰胺。因此，我们开发了利用5-羟基[4-C-13，1-N-15]焦谷氨酸盐作为标记物质的2-OGM的同位素稀释测定法。与pH 7.4相比，在补充有1 mM-[2-N-15]谷氨酰胺的最小必需培养基中孵育HK细胞，表明在pH 6.8下2-OGM的产量显著较高，而在pH 7.6下的产量较低。类似地，（NH 3）-N-15和[N-15]丙氨酸形成在急性酸中毒中显著更高（pH 6.8），在急性水肿中更低在pH7.4的孵育培养基中加入1 mM-氨基-氧乙酸盐显著减少了[N-15]丙氨酸和2-OGM的产生，但通过谷氨酸脱氢酶途径的（NH 3）-N-15的产生被显著刺激。目前的观察结果表明，转氨酶II途径起着次要作用，通过谷氨酸脱氢酶的通量是调节人肾脏中氨生成的主要位点。
The relative significance of the flux through the glutamine aminotransferase (glutaminase II) pathway to renal ammoniagenesis is poorly understood. A basic and unresolved question is whether 2-oxoglutaramate (2-OGM), a product of the glutaminase II reaction, is deamidated to yield 2-oxoglutarate and NH3, or whether 2-OGM accumulates as an unreactive lactam, depending on the environmental pH. In the current studies we utilized C-13 n.m.r. as well as N-15 n.m.r. to demonstrate that 2-OGM occurs as a lactam, i.e. 5-hydroxypyroglutamate, regardless of the environmental pH. Our additional aims were to determine whether human kidney cells (HK cells) in culture can produce 2-OGM and to ascertain a pH-dependent relationship between NH3 and 2-OGM production from glutamine. We therefore developed an isotope dilution assay for 2-OGM utilizing 5-hydroxy[4-C-13,1-N-15]pyroglutamate as the labelled species. Incubations of HK cells in minimal essential medium supplemented with 1 mM-[2-N-15]glutamine demonstrated significantly higher production of 2-OGM at pH 6.8 and lower production at pH 7.6 compared with pH 7.4. Similarly both (NH3)-N-15 and [N-15]alanine formation were significantly higher in acute acidosis (pH 6.8) and lower in acute alkalosis (pH 7.6) compared with that at physiological pH. Addition of 1 mM-amino-oxyacetate to the incubation medium at pH 7.4 significantly diminished [N-15]alanine and 2-OGM production, but the production of (NH3)-N-15 via the glutamate dehydrogenase pathway was significantly stimulated. The current observations indicate that the glutaminase II pathway plays a minor role and that flux through glutamate dehydrogenase is the predominant site for regulation of ammoniagenesis in human kidney.