Further Studies on the Submitochondrial Localization of Cholesterol Side Chain-cleaving Enzyme System in Hog Adrenal Cortex by Sonic Treatment*

Yago, Nagasumi; Kobayashi, Shigeru; Sekiyama, Shigetaka; Kurokawa, Hiromi; Iwai, Yuko; Suzuki, Ichirô; Ichii, Shogo

doi:10.1093/oxfordjournals.jbchem.a129414

Cited by 51 publications

(15 citation statements)

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“…This is substantially higher than the value of 0.03mol/mol reported by Cheng and Kimura [23] for the inner membrane of the bovine adrenal cortex and is also approximately twice as high as values reported for hog adrenal cortex [25] and rat liver [19] inner Table 3.…”

Section: The Cholesterol Content Of the Inner Mitochondrial Membranecontrasting

confidence: 51%

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P‐450_scc incorporated into vesicles prepared from these phospholipids

Tuckey

Stevenson

1985

European Journal of Biochemistry

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Cytochrome P-45OS,,, which catalyses the conversion of cholesterol to pregnenolone in steroidogenic tissues, can be incorporated into artificial phospholipid vesicles and cholesterol binding to the cytochrome is affected by the composition of the vesicles. We have purified the phospholipids from the inner mitochondrial membrane fraction of the bovine corpus luteum where the cytochrome is located. The composition in mol % was 49% phosphatidylcholine, 34% phosphatidylethanolamine, 8.7% cardiolipin, 6.4% lysophosphatidylethanolamine and 1.5% phosphatidylinositol. The ratio of cholesterol to phospholipid (mol/mol) in the inner membrane fraction was 0.14 to 1. The K,,, for cholesterol of purified luteal cytochrome P-45OS,, incorporated into vesicles prepared from the total inner mitochondrial membrane phospholipids was 0.063 mol of cholesterol per mol of phospholipid. Removal of the cardiolipin component of the inner mitochondrial membrane phospholipids prior to preparation of vesicles caused a four fold increase in the Kd of cytochrome P-450 for cholesterol and a two fold increase in K,,,. The data suggests that in the inner mitochondrial membrane of the bovine corpus luteum the cholesterol concentration is less than saturating for cytochrome P-450,,,.Cytochrome P-450,,, which catalyses the conversion of cholesterol to pregnenolone in steroidogenic tissues is located in the inner mitochondrial membrane, on the matrix side [l, 21 and appears to be identical in bovine adrenal cortex and bovine corpus luteum [3]. The cytochrome has its cholesterol binding site in the hydrophobic phospholipid milieu and has a ferredoxin binding site exposed to the matrix [3 -81. Ferredoxin, a one electron carrier, shuttles between ferredoxin reductase and cytochrome P-450,,, supplying the six electrons required to convert cholesterol to pregnenolone [8]. Ferredoxin and ferredoxin reductase also appear identical between the corpus luteum and adrenal cortex [9]. Cytochrome P-450,,, can easily be removed from the membrane with detergents but incorporation of the purified cytochrome into a membrane or detergent micelle is essential before it can catalyse the conversion of the water insoluble cholesterol to pregnenolone.Incorporation of purified cytochrome P-450,,, into artificial phospholipid vesicles has revealed that cholesterol binding to the cytochrome is dependent on the phospholipid composition of the membrane [3 -81. Cardiolopin in particular appears to be a potent stimulator of cholesterol binding to the cytochrome [3,. It has been reported that in the presence of cardiolipin the K,,, for cholesterol expressed as mol cholesterol per mol of phospholipid is as low as 0.07 [6]. However, these studies were done with vesicles prepared from commercial phospholipids, the compositions of which do not reflect closely the composition of the inner mitochondrial Correspondence to

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Section: The Cholesterol Content Of the Inner Mitochondrial Membranecontrasting

confidence: 51%

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P‐450_scc incorporated into vesicles prepared from these phospholipids

Tuckey

Stevenson

1985

European Journal of Biochemistry

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“…In steroidogenic tissues such as adrenal cortex, testis, ovary, and placenta, the initial and rate-limiting step in the pathway leading from cholesterol to steroid hormones is the cleavage of the side chain of cholesterol to yield pregnenolone (1). This reaction, known as cholesterol side-chain cleavage, is catalyzed by a specific form of cytochrome P-450 (P-450,cc) that is localized in the inner mitochondrial membrane (2,3). This protein is synthesized on cytoplasmic ribosomes as a higher molecular weight precursor (Mr 54,500) that is proteolytically processed to the mature form (Mr 49,000) upon insertion into the mitochondrion (4,5).…”

mentioning

confidence: 99%

“…that is localized in the inner mitochondrial membrane (2,3). This protein is synthesized on cytoplasmic ribosomes as a higher molecular weight precursor (Mr 54,500) that is proteolytically processed to the mature form (Mr 49,000) upon insertion into the mitochondrion (4,5).…”

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confidence: 99%

Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450.

John¹,

Ashley²,

MacDonald³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

109

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Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts -425 and ':950 base pairs long, respectively, which are specific for bovine cholesterol sidechain cleavage cytochrome P-450 (P-450CC), have been identified by using two differential hybridization screening procedures followed by hybrid-selected RNA translation. By using these cloned cDNAs as hybridization probes, an RNA species was identified that had the properties expected of mRNA specific for P-450, with respect to tissue specificity, corticotropin (ACTH)-mediated regulation of synthesis, and size of the protein product synthesized in vitro. In RNA samples obtained from bovine adrenal cortex, from bovine corpus luteum, and from cultured bovine adrenocortical cells, it was found that P450s is encoded by mRNA species -2000 bases long, a mujority of which are polyadenylylated. P-450s, mRNA was not detected in RNA samples prepared from bovine heart, liver, and kidney. Treatment of cultured bovine adrenocortical cells with ACTH resulted in the appearance of elevated levels of P-450, mRNA within 8 hr. Thus, ACTH promotes the enhancement of P-450scc gene transcription or acts to stabilize the transcripts. When pBSCC-2 cDNA was used to probe high molecular weight bovine DNA following treatment with restriction endonucleases, a simple pattern of hybridization was observed indicating that P-450O may be encoded by a single gene.Cytochromes P-450 are a diverse group of heme-containing mixed-function oxidases that are involved in the biotransformation of a wide variety of exogenous and endogenous substrates. The best-studied examples of those forms that metabolize endogenous substrates are the group of cytochromes P-450 required for steroid hormone biosynthesis. In steroidogenic tissues such as adrenal cortex, testis, ovary, and placenta, the initial and rate-limiting step in the pathway leading from cholesterol to steroid hormones is the cleavage of the side chain of cholesterol to yield pregnenolone (1). This reaction, known as cholesterol side-chain cleavage, is catalyzed by a specific form of cytochrome P-450 (P-450,cc) that is localized in the inner mitochondrial membrane (2,3). This protein is synthesized on cytoplasmic ribosomes as a higher molecular weight precursor (Mr 54,500) that is proteolytically processed to the mature form (Mr 49,000) upon insertion into the mitochondrion (4,5). By utilizing bovine adrenocortical cells in primary monolayer culture, it has been demonstrated in this laboratory that the synthesis of P-450,"c can be modulated by corticotropin (ACTH) and that this action of ACTH is mediated by cyclic AMP (6,7). A similar pattern of regulation of P-450Scc synthesis has been observed upon stimulation of bovine ovarian granulosa cells with follicle-stimulating hormone (8) and rat testicular Leydig cells with human chorionic gonadotropin (9). Thus, it appears that the levels of P-4Sscc are maintained in vivo in response to trophic hormone stimulation and that cyclic AMP plays a key role in the regulation of P-45scc synth...

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“…1). Being more polar, hydroxysterols are likely to traverse mitochondrial membranes with greater ease than cholesterol (Mason et al, 1978b) and so more readily reach the site of side-chain cleavage which is thought to be located in the inner mitochondrial membrane (Yago et al, 1970). Intramitochondrial translocation of cholesterol to the steroidogenic sites in the adrenal is slow and probably rate-limiting, and ACTH may act primarily to facilitate this translocation (Mason et al, 1978a).…”

Section: Discussionmentioning

confidence: 99%

Effect of LH factors regulating ovarian cholesterol metabolism and progesterone synthesis in PMSG-primed immature rats

Henderson¹,

Gorban²,

Boyd³

1981

Reproduction

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Administration of an ovulatory dose of LH (10 micrograms, i.v.) to PMSG (4 i.u., s.c.)-primed immature rats increased ovarian pregnenolone levels 5-fold and ovarian progesterone levels 40-fold within 6 h and the levels, although fluctuating, remained elevated for 72 h. Serum progesterone levels mimicked those of the luteal phase in the normal cycle. The sustained increase in steroidogenesis was accompanied by a decrease in both basal and cAMP-stimulatable ovarian cholesterol ester hydrolase activity and a net increase in ovarian cholesterol ester content. Ovarian free cholesterol levels were essentially unchanged during the 72 h study. LH does not, therefore, chronically stimulate steroidogenesis by providing additional substrate for the steroidogenic enzymes through activating cholesterol ester hydrolase to bring about hydrolysis of cholesterol esters. Moreover, ovarian cholesterol esters are unlikely to be the primary source of cholesterol utilized to support luteal steroidogenesis. Studies with isolated mitochondria suggested that the mechanisms by which LH stimulated steroidogenesis were by (a) stimulating mitochondrial pregnenolone production probably by facilitating the intramitochondrial movement of cholesterol of the site of side-chain cleavage, and (b) promoting the metabolism of pregnenolone to progesterone.

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Further Studies on the Submitochondrial Localization of Cholesterol Side Chain-cleaving Enzyme System in Hog Adrenal Cortex by Sonic Treatment*

Cited by 51 publications

References 0 publications

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P‐450_scc incorporated into vesicles prepared from these phospholipids

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P‐450_scc incorporated into vesicles prepared from these phospholipids

Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450.

Effect of LH factors regulating ovarian cholesterol metabolism and progesterone synthesis in PMSG-primed immature rats

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Further Studies on the Submitochondrial Localization of Cholesterol Side Chain-cleaving Enzyme System in Hog Adrenal Cortex by Sonic Treatment*

Cited by 51 publications

References 0 publications

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P‐450scc incorporated into vesicles prepared from these phospholipids

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P‐450scc incorporated into vesicles prepared from these phospholipids

Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450.

Effect of LH factors regulating ovarian cholesterol metabolism and progesterone synthesis in PMSG-primed immature rats

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Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P‐450_scc incorporated into vesicles prepared from these phospholipids

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P‐450_scc incorporated into vesicles prepared from these phospholipids