Cowin G J, Willgoss D A, Bartley J, Endre Z H
Department of Medicine, University of Queensland, Royal Brisbane Hospital, Australia.
Biochim Biophys Acta. 1996 Jan 10;1310(1):32-40. doi: 10.1016/0167-4889(95)00142-5.
[2-(13)C]glycine metabolism was studied in freshly isolated rat renal proximal tubules. Mitochondrial coupling of the glycine cleavage complex (GC) and serine hydroxymethyltransferase (SHMT) was confirmed by the formation of three serine isotopomers, [2-(13)C]-, [3-(13)C]- and [2,3-(13)C]serine, detected by 13C-NMR. Incubation with different fractions of 13C-labelled glycine altered the labelling pattern of the serine isotopomers predictably and allowed calculation of the 13C-labelled fractions of total glycine and methylene in N5,N10-methylenetetrahydrofolate (m-THF) available for serine metabolism. Within 20 min there was a fall in labelled glycine (to 42 +/- 3, 68 +/- 3 and 93 +/- 2%, (n = 4, mean +/- S.D.) from 50%, 75% and 100% 13C-labelled added glycine respectively), followed by a slow rate of endogenous glycine formation for up to 80 min incubation. The C2 of glycine was the source of more than 90% of the methylene group of m-THF formed. Gas chromatography-mass spectroscopy (GC-MS) showed that greater than 50% of serine formed was unlabelled. GC and SHMT proceeded in the direction of serine formation. Serine isotopomer analysis by NMR and GC-MS allowed the actions of GC and SHMT and de novo contributions to glycine, serine and m-THF to be monitored in situ in fresh renal proximal tubules.
在新鲜分离的大鼠肾近端小管中研究了[2-(13)C]甘氨酸代谢。通过13C-NMR检测到三种丝氨酸异构体[2-(13)C]-、[3-(13)C]-和[2,3-(13)C]丝氨酸的形成，证实了甘氨酸裂解复合物(GC)和丝氨酸羟甲基转移酶(SHMT)的线粒体偶联。用不同比例的13C标记甘氨酸孵育可预测地改变了丝氨酸异构体的标记模式，并允许计算可用于丝氨酸代谢的N5,N10-亚甲基四氢叶酸(m-THF)中总甘氨酸和亚甲基的13C标记比例。在20分钟内，标记甘氨酸减少（分别从50%、75%和100% 13C标记的添加甘氨酸降至42±3%、68±3%和93±2%，(n = 4，平均值±标准差)），随后在长达80分钟的孵育过程中内源性甘氨酸形成速率缓慢。甘氨酸的C2是形成的m-THF亚甲基的90%以上的来源。气相色谱-质谱(GC-MS)显示，形成的丝氨酸中超过50%未被标记。GC和SHMT朝着丝氨酸形成的方向进行。通过NMR和GC-MS对丝氨酸异构体进行分析，可以在新鲜肾近端小管中原位监测GC和SHMT的作用以及对甘氨酸、丝氨酸和m-THF的从头贡献。
