Properties of ferredoxin reductase and ferredoxin from the bovine corpus luteum

Tuckey, Robert C.; Stevenson, Patricia M.

doi:10.1016/0020-711x(84)90165-4

Cited by 39 publications

(26 citation statements)

References 35 publications

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“…The results of our study and those of others (34) show that there is little vesicle fusion over the typical time period used in the current study (34). P-450scc spontaneously incorporates into these vesicles with its adrenodoxin-binding site facing the exterior (28,31,37). Rapid exchange of P-450scc between vesicles has been reported at high P-450scc concentrations (0.25-0.5 M) but is markedly reduced by the presence of acidic phospholipids in the vesicle membrane (34,38).…”

Section: Fig 4 Concentration Dependence Of N-62mentioning

confidence: 51%

“…The ability of wild type, but not mutant StAR to foster this cholesterol transfer provides compelling evidence that only StAR, cholesterol, and a phospholipid membrane and no other proteins are required for the activity of StAR. and adrenodoxin reductase were purified from bovine adrenals (28,29). Human adrenodoxin was expressed in E. coli and purified as previously described (30).…”

mentioning

confidence: 99%

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Transfer of Cholesterol between Phospholipid Vesicles Mediated by the Steroidogenic Acute Regulatory Protein (StAR)

Tuckey

Headlam

Bose³

et al. 2002

Journal of Biological Chemistry

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The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in steroidogenic cells. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P-450scc converts this cholesterol into pregnenolone. We tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P-450scc to acceptor vesicles containing P-450scc but no cholesterol, using P-450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P-450scc activity in acceptor vesicles 5-10-fold following the addition of donor vesicles. Transfer of cholesterol to acceptor vesicles was rapid and sufficient to maintain a linear rate of pregnenolone synthesis for 10 min. The effect of N-62 StAR in stimulating P-450scc activity was specific for cholesterol transfer and was not due to vesicle fusion or P-450scc exchange between vesicles. Maximum stimulation of P-450scc activity in acceptor vesicles required preincubation of N-62 StAR with phospholipid vesicles prior to adding donor vesicles. The amount of N-62 StAR causing half-maximum stimulation of P-450scc activity in acceptor vesicles was 1.9 M. Half-maximum stimulation required more than a 10-fold higher concentration of R182L N-62 StAR, a mutant associated with congenital lipoid adrenal hyperplasia. N-62 StAR-mediated transfer of cholesterol between vesicles showed low dependence on the cholesterol concentration in the donor vesicles. Thus StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria.The steroidogenic acute regulatory protein (StAR) 1 mediates the rapid stimulation of steroidogenesis induced by the action of tropic hormones on the adrenal, testis, and ovary (1). The essential role of StAR in adrenal and gonadal steroidogenesis was demonstrated by its essential role in congenital lipoid adrenal hyperplasia (2, 3) and has been confirmed in knockout mice (4, 5). Early work showed that StAR is a phosphoprotein (6, 7); phosphorylation of serine 195 doubles StAR activity but is not essential for its action (8). StAR is synthesized as a short-lived 37-kDa preprotein that is cleaved to a more stable 30-kDa form upon its entry into mitochondria. However, deletion of the mitochondrial leader, which prohibits mitochondrial entry, does not diminish the activity of StAR, suggesting that StAR acts on the outer mitochondrial membrane (9 -11). Biophysical studies show that StAR undergoes a pH-dependent conformational change to a partially unfolded molten globule structure (12) when it interacts with membranes (13,14).Although the structure of StAR has not been determined, the crystal structure of the StAR-like domain of MLN64 (15), a highly homologous protein with StAR-like activity (16,17), and the crystal structures of the StAR homologue StAR D4 (18,19) and phosphatidylcholine transfer p...

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Section: Fig 4 Concentration Dependence Of N-62mentioning

confidence: 51%

mentioning

confidence: 99%

Transfer of Cholesterol between Phospholipid Vesicles Mediated by the Steroidogenic Acute Regulatory Protein (StAR)

Tuckey

Headlam

Bose³

et al. 2002

Journal of Biological Chemistry

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“…4B) was expected as this tissue contains a P-450scc enzyme complex, and adrenodoxin from this tissue appears to be identical with that from adrenal (31). The appearance of liver and kidney RNA transcripts that hybridize to adrenodoxin cDNA is more interesting.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, we deduce that the additional COOH-terminal peptide was not found in the original study because (i) it was removed proteolytically during adrenodoxin purification, or (ii) it is a COOH-terminal precursor extrapeptide that is removed upon uptake and processing of the adrenodoxin precursor by mitochondria. In this context, two electrophoretically distinct forms of adrenodoxin have been observed in bovine adrenal cortex and corpus luteum (31). If ii were correct, adrenodoxin would provide the first example of COOH-terminal extrapeptide associated with a mitochondrial precursor encoded by nuclear genes.…”

Section: Resultsmentioning

confidence: 99%

Molecular cloning and amino acid sequence of the precursor form of bovine adrenodoxin: evidence for a previously unidentified COOH-terminal peptide.

Okamura¹,

John²,

Zuber³

et al. 1985

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

111

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Several recombinant cDNA clones specific for the mitochondrial iron-sulfur protein adrenodoxin have been identified in a bovine adrenocortical cDNA library. One clone (pBAdx4) contains a 900-base-pair insert that includes the entire amino acid coding region of the adrenodoxin precursor protein. The amino acid sequence of mature adrenodoxin deduced from the nucleotide sequence of pBAdx4 is identical with that determined by protein sequencing except for three amide changes. The previously undetermined sequence of the adrenodoxin NH2-terminal precursor segment (58 amino acids) contains several basic residues, a characteristic feature of the precursor segment of proteins destined for mitochondria. In addition, a 14 amino acid extension is present at the COOH terminus of the mature adrenodoxin sequence. Whether this represents a COOH-terminal precursor segment is not clear. Three different adrenodoxin mRNAs are present [1.75, 1.4, and 0.95 kilobase(s) long] in bovine adrenocortical RNA. RNA from bovine corpus luteum, liver, and kidney contains transcripts that hybridize to adrenodoxin cDNA. Accumulation of adrenodoxin mRNA occurs in cultured bovine adrenocortical cells after treatment with ACTH or dibutyryl-cAMP, similar to that observed for the mitochondrial steroid hydroxylases that it services-namely, the cholesterol side-chain-cleavage cytochrome P-450 and the steroid 11f3-hydroxylase cytochrome P-450.In steroidogenic tissues, steroid hydroxylations occur in both the mitochondria and endoplasmic reticulum. In bovine adrenal cortex, for example, the cholesterol side-chaincleavage and steroid 11f3-hydroxylase enzyme complexes are in the mitochondria, while the steroid 17a-hydroxylase and 21-hydroxylase enzyme systems are in the endoplasmic reticulum (1). Mitochondrial steroid hydroxylase complexes contain a flavoprotein (adrenodoxin reductase) and an iron-sulfur protein (adrenodoxin) that act to transfer reducing equivalents from NADPH to a specific form of cytochrome P-450 [cholesterol side-chain-cleavage cytochrome P-450 (P-450Scc) and steroid 11-hydroxylase cytochrome P-450 (P-450119) in the examples cited above].The properties of this iron-sulfur protein (2, 3) and its interaction with P-450Scc (4) have been characterized in detail.

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“…Bovine cytochrome P450scc, adrenodoxin and adrenododoxin reductase were isolated from adrenals [26,27]. Human cytochrome P450scc and adrenodoxin were expressed in Escherichia coli and purified as described before [28].…”

Section: Biological Materialsmentioning

confidence: 99%

A novel pathway for sequential transformation of 7‐dehydrocholesterol and expression of the P450scc system in mammalian skin

Slominski¹,

Zjawiony

Wortsman

et al. 2004

European Journal of Biochemistry

225

343

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Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side-chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7-dehydrocholesterol (7-DHC; precursor to cholesterol and vitamin D3) to 7-dehydropregnenolone (7-DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7-DHC into 7-DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7-DHC can form 7-DHP through P450scc side-chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7-steroidal dienes (7-DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB-induced intramolecular rearrangements to vitamin D3-like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.

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Properties of ferredoxin reductase and ferredoxin from the bovine corpus luteum

Cited by 39 publications

References 35 publications

Transfer of Cholesterol between Phospholipid Vesicles Mediated by the Steroidogenic Acute Regulatory Protein (StAR)

Transfer of Cholesterol between Phospholipid Vesicles Mediated by the Steroidogenic Acute Regulatory Protein (StAR)

Molecular cloning and amino acid sequence of the precursor form of bovine adrenodoxin: evidence for a previously unidentified COOH-terminal peptide.

A novel pathway for sequential transformation of 7‐dehydrocholesterol and expression of the P450scc system in mammalian skin

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