Nissim I, Yudkoff M, Segal S
J Biol Chem. 1985 Nov 15;260(26):13955-67.
Gas chromatography-mass spectrometry was utilized to study the metabolism of [15N]glutamate, [2-15N]glutamine, and [5-15N]glutamine in isolated renal tubules prepared from control and chronically acidotic rats. The main purpose was to determine the nitrogen sources utilized by the kidney in various acid-base states for ammoniagenesis. Incubations were performed in the presence of 2.5 mM 15N-labeled glutamine or glutamate. Experiments with [5-15N]glutamine showed that in control animals approximately 90% of ammonia nitrogen was derived from 5-N of glutamine versus 60% in renal tubules from acidotic rats. Experiments with [2-15N]glutamine or [15N]glutamate indicated that in chronic acidosis approximately 30% of ammonia nitrogen was derived either from 2-N of glutamine or glutamate-N by the activity of glutamate dehydrogenase. Flux through glutamate dehydrogenase was 6-fold higher in chronic acidosis versus control. No 15NH3 could be detected in renal tubules from control rats when [2-15N]glutamine was the substrate. The rates of 15N transfer to other amino acids and to the 6-amino groups of the adenine nucleotides were significantly higher in normal renal tubules versus those from chronically acidotic rats. In tubules from chronically acidotic rats, 15N abundance in 15NH3 and the rate of 15NH3 appearance were significantly higher than that of either the 6-amino group of adenine nucleotides or the 15N-amino acids studied. The data indicate that glutamate dehydrogenase activity rather than glutamate transamination is primarily responsible for augmented ammoniagenesis in chronic acidosis. The contribution of the purine nucleotide cycle to ammonia formation appears to be unimportant in renal tubules from chronically acidotic rats.
利用气相色谱 - 质谱联用技术研究了从对照大鼠和慢性酸中毒大鼠制备的离体肾小管中[15N]谷氨酸、[2 - 15N]谷氨酰胺和[5 - 15N]谷氨酰胺的代谢情况。主要目的是确定肾脏在各种酸碱状态下用于氨生成的氮源。在2.5 mM 15N标记的谷氨酰胺或谷氨酸存在的情况下进行孵育。用[5 - 15N]谷氨酰胺进行的实验表明，在对照动物中，约90%的氨氮来自谷氨酰胺的5 - N，而在酸中毒大鼠的肾小管中这一比例为60%。用[2 - 15N]谷氨酰胺或[15N]谷氨酸进行的实验表明，在慢性酸中毒时，约30%的氨氮来自谷氨酰胺的2 - N或谷氨酸脱氢酶作用下的谷氨酸 - N。与对照相比，慢性酸中毒时通过谷氨酸脱氢酶的通量高6倍。当以[2 - 15N]谷氨酰胺为底物时，在对照大鼠的肾小管中未检测到15NH3。与慢性酸中毒大鼠的肾小管相比，正常肾小管中15N向其他氨基酸和腺嘌呤核苷酸6 - 氨基的转移速率显著更高。在慢性酸中毒大鼠的肾小管中，15NH3中的15N丰度和15NH3的出现速率显著高于所研究的腺嘌呤核苷酸6 - 氨基或15N - 氨基酸。数据表明，谷氨酸脱氢酶活性而非谷氨酸转氨作用是慢性酸中毒时氨生成增加的主要原因。嘌呤核苷酸循环对氨生成的贡献在慢性酸中毒大鼠的肾小管中似乎并不重要。
