Perrin A, Defaye G, Guidicelli C, Chambaz E, Albrand J P, Roby C
Laboratoire de Biochimie des Régulations Cellulaires Endocrines, l'Institut National de la Santé et de la Recherche Médicale, Grenoble, France.
Eur J Biochem. 1990 Oct 5;193(1):283-9. doi: 10.1111/j.1432-1033.1990.tb19334.x.
High-field 31P-NMR spectroscopy has been used to study the metabolic activities of coupled bovine adrenocortical mitochondria in vitro. These differentiated organelles use oxygen as a substrate to support both oxidative phosphorylation and specific steroid hydroxylation reactions. The NMR technique allowed the resolution of two inorganic phosphate signals, attributed to the matrix and external medium phosphate pools, at low and high field, respectively. These signals were used to calculate the respective Pi concentrations and to obtain the pH of the two corresponding compartments. In addition, the NMR spectra displayed resonance signals corresponding to ADP added to the medium and to ATP synthesized during oxidative phosphorylation. NMR analysis of the mitochondrial perchloric acid extracts identified the major phosphate-containing metabolites, namely NADP+, NAD+, phosphocholine, phosphoethanolamine, sn-glycero-(3)phosphocholine, AMP, ADP, ATP and Pi. Upon addition of ADP and malate to the oxygenated suspension, the kinetics of mitochondrial external Pi consumption and of ATP synthesis, along with the intra- and extraorganelle pH variations could be monitored over time periods of approximately 30 min, in the absence and presence of different steroid hydroxylation substrates. A major observation was that oxidative phosphorylation, which takes place in the absence of steroid, was markedly inhibited as soon as steroid hydroxylation was operating. These observations show the potential of 31P-NMR spectroscopy in the study of metabolic activities of isolated intact mitochondrial organelles. Such an approach appears promising for further determination of the underlying mechanisms in the balance between vital oxidative phosphorylation and differentiated steroid hydroxylation which are under hormonal control in adrenocortical mitochondria as well as in other steroidogenic cell systems.
高场31P核磁共振波谱已被用于体外研究偶联的牛肾上腺皮质线粒体的代谢活性。这些分化的细胞器利用氧气作为底物来支持氧化磷酸化和特定的类固醇羟化反应。核磁共振技术分别在低场和高场分辨出两个无机磷酸盐信号，分别归因于基质和外部介质中的磷酸盐池。这些信号被用于计算各自的磷酸浓度，并获得两个相应区室的pH值。此外，核磁共振谱显示出与添加到介质中的ADP以及氧化磷酸化过程中合成的ATP相对应的共振信号。线粒体高氯酸提取物的核磁共振分析确定了主要的含磷代谢物，即NADP+、NAD+、磷酸胆碱、磷酸乙醇胺、sn-甘油-(3)磷酸胆碱、AMP、ADP、ATP和Pi。向充氧悬浮液中添加ADP和苹果酸后，在有无不同类固醇羟化底物的情况下，可在约30分钟的时间段内监测线粒体外部磷酸消耗和ATP合成的动力学，以及细胞器内外pH值的变化。一个主要的观察结果是，在没有类固醇的情况下发生的氧化磷酸化，一旦类固醇羟化开始进行就会受到显著抑制。这些观察结果表明31P核磁共振波谱在研究分离的完整线粒体细胞器代谢活性方面的潜力。这种方法对于进一步确定肾上腺皮质线粒体以及其他类固醇生成细胞系统中受激素控制的重要氧化磷酸化和分化的类固醇羟化之间平衡的潜在机制似乎很有前景。
