Cytochrome P-45011 beta and P-450scc in adrenal cortex: zonal distribution and intramitochondrial localization by the horseradish peroxidase-labeled antibody method.

Mitani, Fumiko; Shimizu, Takamasa; Ueno, R; Ishimura, Yuzuru; Izumi, Shinichi; Komatsú, Noriyuki; Watanabe, Kenji

doi:10.1177/30.10.6813370

Cited by 110 publications

(39 citation statements)

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“…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.…”

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

Perrin

Defaye

Guidicelli

et al. 1990

European Journal of Biochemistry

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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,

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confidence: 99%

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

Perrin

Defaye

Guidicelli

et al. 1990

European Journal of Biochemistry

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“…These hydroxylation reactions are catalysed by specific, integral inner mitochondrial membrane cytochromes P-450 (P-450,,, and P-450 1? @, respectively) which receive reducing equivalents flowing from NADPH through a redox cascade involving adrenodoxin reductase and adrenodoxin, which are mitochondrial matrix components [4]. Adrenodoxin has been proposed to act as a shuttle between adrenodoxin reductase and the cytochromes [5].…”

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confidence: 99%

Phosphorylation of bovine adrenodoxin. Structural study and enzymatic activity

1987

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Adrenodoxin is an iron-sulfur protein which functions as a carrier of reducing equivalents in steroid hydroxylation reactions catalyzed by specific cytochromes P-450 in steroidogenic tissues such as adrenal cortex. Purified bovine adrenocortical adrenodoxin was shown to be selectively phosphorylated upon incubation with purified CAMP-dependent protein kinase, whereas other protein kinases were ineffective. The phosphorylation reaction was completed within 45 min at 30°C and resulted in the optimal incorporation of 1 mol phosphate/mol adrenodoxin. Apoadrenodoxin, lacking the iron-sulfur cluster, was also phosphorylated under similar conditions. An apparent K , of 55 pM with a V,,, of 0.3 pmol 32P incorporated min-l mg adrenodoxin-' was calculated. Phosphorylation resulted in a striking change in several molecular properties of adrenodoxin, such as electrophoretic behavior and hydroxyapatite affinity, thus providing the possibility of clearly separating phosphorylated from unphosphorylated adrenodoxin. In addition, phosphoadrenodoxin became refractory to mild trypsin degradation, whereas this was not the case with apoadrenodoxin. The phosphorylated site of adrenodoxin was identified as a serine residue; study of peptide products resulting from CNBr and proteolytic cleavages of phosphoadrenodoxin suggested that Ser-88 was the target of the phosphorylation reaction. The influence of phosphorylation upon adrenodoxin activity was examined using cholesterol side-chain cleavage and 11 P-hydroxylase (1 1 P) systems, reconstituted from purified components. Phosphorylation of adrenodoxin resulted in an average twofold decrease in its K,,, values for the two specific cytochromes P-450 involved. This effect was paralleled by a positive relationship between the degree of adrenodoxin phosphorylation and its ability to support the overall activity of reconstituted side-chain cleavage and 11 P-hydroxylase systems. Although it remains to be examined whether adrenodoxin is phosphorylated in the intact cell, the present observations suggest that it represents a potential target in the hormonal regulation of the adrenocortical differentiated functions, especially by stimulatory agents acting through a cyclic-AMP-dependent mechanism, such as adrenocorticotropin.Hydroxylation reactions play a pivotal role in the biosynthetic pathways leading to active steroid hormones ultimately secreted by steroidogenic tissues. In adrenal cortex, for example, key steroidogenic steps such as cholesterol sidechain cleavage (scc) and 11 P-hydroxylation (1 1 P) occur in mitochondria [l]. A special interest has been focused on the study of the side-chain cleavage system since it is considered as a limiting step and suggested as the major target in the acute activation of steroidogenesis by hormonal agents such as adrenocorticotropin [2]. In addition, these hydroxylase systems are under long-term hormonal control and may thus be considered as markers of differentiation 131. These hydroxylation reactions are catalysed by specific, integral inner mitochondr...

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“…From our experience with the preembedding method, satisfactory penetration through such mitochondrial walls has been obtained only by using antibodies smaller than Fab'. Indeed, we have recently succeeded in demonstrating the localization of cytochrome P450, adrenodoxin and adrenodoxin reductase in the bovine and rat adrenal cortical cells (3,6,7).…”

Section: Techniques and Proceduresmentioning

confidence: 99%

“…In this context, we have been studying the ultrastructural localization of peptide hormones in the rat and human pituitary glands (21, 22), the enzymes related to adrenocortical steroidogenesis (cytochrome P450, adrenodoxin, adrenodoxin reductase) (3,6,7) and the enzyme related to the adrenocortical lipid metabolism (glutathione peroxidase) (8,9). These immunoelectron microscopic observations can be expected to be extremely useful tools to study the secretory pathways of peptide hormones or functional status of steroidogenic endocrine cells.…”

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confidence: 99%