A ³¹P‐NMR study of bovine adrenocortical mitochondrial metabolic activities

Abstract

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 P, 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 'P-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.Adrenal cortex mitochondria contain specific enzymes responsible for major steroidogenic reactions such as the side chain cleavage of cholesterol and p-hydroxylation at C11, which play a pivotal role in the biosynthesis of active steroid hormones [l, 21. The corresponding specific hydroxylases are integral mitochondrial membrane cytochrome P-450s, i.e. cholesterol monooxygenase and steroid 1 lp-monooxygenase [3] which activate oxygen using electrons provided by a specific transport pathway involving a flavoprotein (ferredoxin-NADP' reductase) and an iron sulfur protein (adrenodoxin) [4]. The reducing equivalents are specifically provided by NADPH and may come from NADH through an energydependent transhydrogenase or from malate oxidation by the mitochondrial malate dehydrogenase. Simpson and Estabrook [5] have shown that in bovine adrenal cortex mitochondria the malate dehydrogenase pathway was predominant in supporting steroidogenesis [6]. As malate also enters the citrate cycle, intramitochondrial competition takes place between the Correspondence to E. M. Chambaz, BRCE, INSERM U 244,

Metabolism of malate in bovine adrenocortical mitochondria studied by 13C‐NMR spectroscopy

“…This is in agreement with the role of malic enzyme of providing the reducing equivalents to the hydroxylation chain during steroid hydroxylation. This confirms our previous results obtained by 31P-NMR showing that all the phenomena associated with ATP synthesis and respiration are inhibited during steroid hydroxylation in isolated adrenal mitochondria [15].…”

“…The aim of the present study was to investigate the metabolic activities of bovine adrenocortical mitochondria in coupled conditions using ["C]malate as substrate of the citric acid cycle. The results confirm and extend previous data obtained by "P-NMR spectroscopy [15].…”

“…The suspension was centrifuged at 80000 g for 10 min and the mitochondria] pellet was discarded; the supernatant contains essentially ['Tlmalate and is referenced as crude [I3C]malate. Neither ADP nor ATP were identified by "P-NMR in the medium, in agreement with our previous results [15], showing that the ATP formed by the mitochondria accumulated only inside the organelles.…”

“…Perchloric acid extracts were obtained as previously described [15]. The 13C-NMR spectra were acquired used a 40" pulse at 4-s intervals (spectra of carboxyl groups were observed when necessary with a longer delay of 30 s).…”

“…At the end of the incubation, when all the malate has been consumed, the corresponding "P-NMR spectrum showed a triplet at -22.2 ppm and peaks at -11.2 ppm and -8.3 ppm characteristic of the p, u and y phosphorus signals of ATP [15].…”