Sherry A D, Sumegi B, Miller B, Cottam G L, Gavva S, Jones J G, Malloy C R
Department of Chemistry, University of Texas at Dallas, Richardson 75083-0688.
Biochemistry. 1994 May 24;33(20):6268-75. doi: 10.1021/bi00186a029.
Metabolism of [2-13C]-, [3-13C]-, and [1,2,3-13C]propionate in perfused rat livers and [2-13C]-acetate in perfused rat hearts has been examined in tissue extracts by 13C NMR. Label from [2-13C]-propionate was preferentially incorporated into the C2 carbon of lactate, alanine, and aspartate in liver tissue while label from [3-13C]propionate appeared preferentially in the C3 carbon of those same molecules. These data suggest that 13C may not be completely randomized in the symmetric citric acid cycle intermediates succinate and fumarate as is normally assumed but that some fraction of those intermediates may be transferred between enzymes in this span of the cycle with conservation of spatial orientation, consistent with recent results obtained in yeast [Sumegi et al. (1990) Biochemistry 29, 9106-9110]. This was confirmed by performing similar experiments with [1,2,3-13C]propionate. Time-dependent asymmetry was also observed between the intensities of the glutamate C2 and C3 resonances and between the aspartate C2 and C3 resonances in 13C NMR spectra of intact hearts and heart extracts during early perfusion with [2-13C]-acetate. A model is presented which predicts that isotopic asymmetry is observed only during the first 2-3 turns of the cycle pools when isotope enters the cycle via acetyl-CoA even if all symmetric cycle intermediates retain a unique molecular orientation on each pass through the citric acid cycle.
利用(^{13}C)核磁共振技术，在组织提取物中研究了灌注大鼠肝脏中([2-^{13}C])、([3-^{13}C])和([1,2,3-^{13}C])丙酸的代谢以及灌注大鼠心脏中([2-^{13}C])乙酸的代谢。肝脏组织中，([2-^{13}C])丙酸的标记优先掺入乳酸、丙氨酸和天冬氨酸的(C2)碳，而([3-^{13}C])丙酸的标记则优先出现在这些相同分子的(C3)碳。这些数据表明，(^{13}C)可能不像通常所认为的那样在对称的柠檬酸循环中间体琥珀酸和延胡索酸中完全随机化，而是这些中间体的一部分可能在循环的这一阶段在酶之间转移，并保持空间取向，这与最近在酵母中获得的结果一致[苏梅吉等人（1990年）《生物化学》29卷，9106 - 9110页]。用([1,2,3-^{13}C])丙酸进行类似实验证实了这一点。在用([2-^{13}C])乙酸早期灌注期间，完整心脏和心脏提取物的(^{13}C)核磁共振谱中，谷氨酸(C2)和(C3)共振强度之间以及天冬氨酸(C2)和(C3)共振强度之间也观察到了时间依赖性不对称。提出了一个模型，该模型预测，即使所有对称的循环中间体在每次通过柠檬酸循环时都保持独特的分子取向，但当同位素通过乙酰辅酶(A)进入循环时，只有在循环池的前2 - 3轮中才会观察到同位素不对称。
