Evidence of Allosteric Coupling between Substrate Binding and Adx Recognition in the Vitamin D Carbon-24 Hydroxylase CYP24A1.

Metabolic inactivation of 1,25(OH)2D3 requires molecular recognition between the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1) and its cognate redox partner adrenodoxin (Adx). Recent evidence supports a model of CYP24A1 function in which substrate binding and Adx recognition are structurally linked. However, the details of this allosteric connection are not clear. In this study, we utilize chemical cross-linking coupled to mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and CYP24A1 functional assays to inform a working model of a CYP24A1-Adx complex. We report that differential cross-linking internal to CYP24A1 points toward an Adx-induced conformational change that perturbs the F and G helices, which are required for substrate binding. Moreover, the modeled complex suggests that a semiconserved nonpolar interaction at the interface may influence CYP24A1 regioselectivity. Taken together, these findings contribute to our understanding of Adx recognition in a critical vitamin D-inactivating enzyme and provide broader insight regarding the variability inherent in CYP-Adx interactions.

Electrochemical behaviour of human adrenodoxin on a pyrolytic graphite electrode.

Adrenodoxin (Adx) functions as a redox protein in the delivery of electrons to all mitochondrial cytochromes P450. In order to further characterize the human form of this protein, direct electrochemistry of human adrenodoxin (Hadx) has been observed for the first time on a pyrolytic graphite electrode (PGE) modified with poly-L-lysine. A single well-defined redox wave was observed with a midpoint potential of -448+/-3 mV vs. Ag/AgCl (sat. KCl) at scan rates of 10 mV/s and over the pH range 4.0-8.0. At slow scan rates, the reduction process was close to being electrochemically reversible whereas, at faster scan rates, only quasi-reversibility was observed. A correlation was observed between the peak separation (DeltaE) for the cyclic voltammograms and pH over a wide range of scan rates. The variation of DeltaE with pH was at a minimum (optimum reversibility) at pH 7.0 for all scan rates tested. This correlation may suggest that the direct electrochemistry method could possibly provide a means for determining protein or enzyme activity. The electron transfer rate constant, k(s), was determined to be 0.28 s(-1) at pH 7.0 and a small pH dependence was observed. The results obtained in this study demonstrate the facile nature of direct electron transfer for human adrenodoxin, and provide an estimate of the midpoint reduction potential at a pyrolytic graphite electrode via electrostatic immobilisation.

Luteinization and proteolysis in ovarian follicles of Meishan and Large White gilts during the preovulatory period.

This experiment was conducted to determine why follicles luteinize faster in the Meishan breed than in the Large White breed of pig. Follicles were recovered during the late follicular phase from ovaries of both breeds before and after administration of hCG given to mimic the LH surge. First, the patterns of cholesterol transporters (high and low density lipoproteins: HDL and LDL) were compared. Cholesterol transporters detected in follicular fluid consisted of HDL only. Similar amounts of Apolipoprotein A-I were found in all samples. There was no obvious breed effect on minor lipoproteins found in the HDL-rich fraction, and this pattern was altered similarly by hCG in the two breeds. The LDL-rich samples of serum from both breeds contained similar amounts of protein. Second, three steroidogenic enzymes, adrenodoxin, 17 alpha-hydroxylase-lyase (P450(17) alpha) and 3 beta-hydroxysteroid-dehydrogenase (3 beta-HSD) were detected by immunohistochemistry and quantified by image analysis on sections of the two largest follicles. Before hCG treatment, theca interna cells demonstrated immunoreactivities for adrenodoxin (strong), P450(17) alpha and 3 beta-HSD (very strong), whereas granulosa cells displayed immunoreactivities for adrenodoxin only. After hCG treatment, the localization of the enzymes was unchanged but the staining intensity of adrenodoxin on granulosa cells and 3 beta-HSD on theca cells increased (P < 0.01 and P < 0.05, respectively). Breed effects were detected for the amounts of adrenodoxin in theca cells (Meishan > Large White; P < 0.05) and of 17 alpha-hydroxylase (Large White > Meishan, P < 0.01). Breed x treatment interactions were never detected. Finally, gelatinases, plasminogen activator, plasminogen activator inhibitor, tissue inhibitors of metalloproteases (TIMP-1 and TIMP-2) were visualized by direct or reverse zymography or western blotting. Whatever the stage relative to LH administration, follicular fluid from Large White gilts contained more TIMP-1, and TIMP-2 (P < 0.02 and P < 0.01, respectively). No breed effect was detected for the amounts of gelatinases and plasminogen activator inhibitor 1. However, for these parameters, a significant breed x time interaction was obvious, as the Meishan follicles had a greater response to hCG (P < 0.01). Since proteolysis plays a key role in the bioavailability of growth factors such as insulin-like growth factor 1, fibroblast growth factor and transforming growth factor beta, which have the ability to alter gonadotrophin-induced progesterone production in pigs, the differences observed in its control in the present study may explain, at least in part, the different patterns of luteinization observed in Meishan and Large White follicles.

Ontogeny of immunoreactive steroidogenic proteins (adrenodoxin and cytochrome p-450(21)) in the interrenals of the teleost Lates calcarifer.

Antisera against bovine adrenodoxin and cytochrome P-450(21) (steroid 21-hydroxylase) cross-reacted with the interrenal cells of the adult Asian seabass (Lates calcarifer); the cells were arranged as cords, two cells thick, in the headkidney. During larval development, cells immunoreactive for adrenodoxin were first observed 1 day posthatching (dph); immunoreactivity for cytochrome P-450(21) was first detected at 1.5 dph. Initially, the interrenal cells occurred as a mass in each headkidney, which only became identifiable histologically at 5 dph. The number of interrenal cells increased with age, becoming associated with the cardinal veins at 14 dph. The present study thus indicates that the posthatching rise in cortisol may originate from the nascent interrenal tissue.

Cloning and sequence analysis of a full-length cDNA of rat adrenodoxin.

A cDNA clone which covers the entire coding region for the precursor of adrenodoxin was isolated from a rat adrenal cDNA library. This precursor consists of amino-terminal 64 residues of extrapeptide for transport into mitochondria and the following 124 residues of mature peptide region. The amino acid sequence of rat mature adrenodoxin showed 85-98% homology with mouse, human, chicken, porcine, bovine and sheep counterparts, whereas that of the extrapeptide showed significantly lower values.

Immunocytochemical localization of heterologously expressed adrenodoxin and its electron acceptor cytochrome P45011B1 in Escherichia coli.

Adrenal steroid hydroxylase P45011B1 and its electron donor adrenodoxin were localized in the cortex of bovine adrenals by immunogold-silver staining. In order to test recently developed heterologous expression systems for both enzymes to enable structure-function studies, immunocytochemical marker methods were applied. Adrenodoxin, the ferredoxin of the adrenal gland, was successfully expressed and for the first time localized in Escherichia coli. By use of ultrathin cryosections and the protein A-gold technique, adrenodoxin was detectable in large amounts in the cytoplasm of the bacterial cells, and, following the insertion of the outer membrane protein A leader sequence of E. coli, also in the periplasmic space. A fusion protein between mature adrenodoxin and human P45011B1 was constructed and clearly localized in E. coli by antibodies against both proteins.

A negative contrast stain for ultra-thin frozen sections.

Ultra-thin frozen sections are ideal substrates for immunolabelling in high resolution electron microscopy. However, visualization of subcellular structures is inferior to that obtained with corresponding plastic sections. Although negative staining is generally effective and even superior to positive staining, the accumulated stain is often too heavy, obscuring morphology and markers used for immunocytochemical localization of antigens. This paper describes the development of a modified negative contrast staining technique in which a high concentration of uranyl acetate is mixed with methyl cellulose at a low pH. Application of this stain to cryosections of cells and tissue resulted in improved visualization of morphological structures characterized by negative images of membranes and cell organelles. Use of this stain is advantageous for morphological and immunocytochemical studies involving ultra-thin frozen sections.

Differential capacity for cholesterol transport and processing in large and small rat luteal cells.

The aim of this investigation was to determine whether a specific luteal subpopulation is responsible for the hypertrophic development of the corpus luteum at midpregnancy in the rat and to determine whether there was an underlying cellular basis for the differential production of steroids by the luteal cell subtypes. To examine this, we have dispersed and separated rat luteal steroidogenic cell populations into small (< 20 microns) and large (> 30 microns) cell types by elutriation. Luteal cells were examined at early (day 3) and midpregnancy (day 14) for differences in protein content and for differential expression of proteins required for steroid production. Specific proteins examined include the P450side chain cleavage enzyme (P450scc), adrenodoxin and adrenodoxin reductase, proteins required for cholesterol conversion to progestagens in the corpus luteum, and sterol carrier protein-2 (SCP2), a protein thought to be involved in intracellular cholesterol transport. The cytochrome P450(17)alpha hydroxylase (P450(17)alpha), a key enzyme responsible for androgen biosynthesis was also examined in the isolated luteal cells. The large luteal cell population displayed an increase in total cellular protein content while the small cell type did not change with luteal development. In addition, the large luteal cells expressed proteins unique to or elevated in that cell type. Analysis by two-dimensional polyacrylamide gel electrophoresis revealed that the large cell-specific proteins had molecular masses of 23 K and 32 K and that a 14 kilodalton (kDa) protein was elevated in the large cell type relative to the small cells. The small luteal cell on day 3 of pregnancy expressed a 36 kDa protein which was barely detectable in the large cell. Immunocytochemical and Western analysis indicated that the large luteal cells contain 5.3-fold more SCP2 (P < 0.05) and 5.6-fold more P450scc (P < 0.001) relative to the small cell type. Immunocytochemical staining of adrenodoxin and adrenodoxin reductase indicate these proteins were elevated in the large cell as well. Human CG administration stimulated P450(17)alpha expression mainly in the large luteal cell population. The results of this investigation indicate, for the first time, that the large luteal cell of the rat, in contrast to the small cell type, undergoes a dramatic increase in protein content with luteal development, and that with this increase in cell size there is a concomitant increase in the large cell capacity to produce steroids. This occurs as a direct result of the enhanced expression of SCP2, P450scc, adrenodoxin and adrenodoxin reductase, proteins specifically required to transport and process cholesterol for steroid production in the large luteal cell.

Introduction of a gonadotropin receptor expression plasmid into immortalized granulosa cells leads to reconstitution of hormone-dependent steroidogenesis.

We have recently succeeded in immortalizing rat granulosa cells by co-transfection with SV-40 DNA and the Ha-ras oncogene. These cells lost their response to gonadotropins, but expressed the cytochrome P450scc mitochondrial system enzymes and produced progesterone and 20 alpha-hydroxy-4-pregnan-3-one (20 alpha-OH-P) upon cAMP stimulation (Suh, B. S., and A. Amsterdam. 1990. Endocrinology. 127:2489-2500; Hanukoglu, I., B. S. Suh, S. Himmelhoch, and A. Amsterdam. 1990. J. Cell Biol. 111:1973-1981). In an attempt to restore the steroidogenic response to gonadotropins in immortalized cells, lutropin/choriogonadotropin (LH/CG-R) receptor expression plasmid was prepared by introducing the complete coding region of LH receptor cDNA (McFarland, K. C., R. Sprengel, H. S. Phillips, M. Köhler, N. Rosemblit, K. Nikolics, D. L. Segaloff, and P. H. Seeburg. 1989. Science (Wash. DC). 245:494-499) into a SV-40 early promoter based eucaryotic expression vector. Granulosa cells from preovulatory follicles were transfected with this LH receptor expression plasmid, together with SV-40 DNA and the Ha-ras oncogene. Cell lines obtained after this triple transfection accumulated cAMP in a dose-dependent manner in response to hCG. Moreover, they produced progesterone and 20 alpha-OH-P upon hCG stimulation with an ED50 of 125 pM and 75 pM, respectively, which is within the physiological range. Concomitantly with hCG induced differentiation, inhibition of cell proliferation was evident following stimulation with hormone concentrations as low as 40 pM. The number of hCG receptor sites per cell after numerous passages and several freezing and thawing cycles was 1.9 x 10(4), they showed a Kd of 180 pM. Stimulation with hCG induced pronounced morphological and biochemical changes in these cells including formation of mitochondrial located adrenodoxin, a marker enzyme for enhanced steroidogenesis. These findings make possible the expression in immortalized granulosa cells, of selectively mutated receptor molecules which preserve their steroidogenic potential, thereby opening the way to analysis of structure-function relationships of the receptor molecule.

Elimination of non-specific binding in western blots from non-reducing gels.

The reaction of some antibodies with Western blots of protein shows strong non-specific binding especially at a region that corresponds to about 70-90 kDa. This binding is independent of protein concentration. Further analysis indicated that the factor responsible for the non-specific binding is 2-mercaptoethanol in the gel sample buffer. Gel electrophoresis of total tissue homogenates in the absence of this reducing agent resulted in dramatic elimination of the non-specific background binding without affecting the mobility of the two proteins we studied.

Immunoblot analysis of cholesterol side-chain cleavage cytochrome P-450 and adrenodoxin in corpora lutea of cyclic and late-pregnant sheep.

The specific contents of cytochrome P-450scc and adrenodoxin in corpora lutea of late pregnant sheep were, respectively, 1/5 and 1/8 that of corpora lutea of the oestrous cycle, suggesting lower steroidogenic enzyme capacity in the former. The contents of Complex V proteins were also lower in the corpora lutea of late pregnancy. It was observed in the immunoblots of both Complex V and cytochrome P-450scc that immunoreactive bands of molecular weights lower than the native proteins were present in the samples from corpora lutea of late pregnancy, indicative of degradation of the native enzymes. It is concluded that corpora lutea of sheep during late pregnancy have a much lower enzyme capacity for steroidogenesis than do those of the oestrous cycle (mid-luteal phase) due to a reduction in the content of cytochrome P-450scc and adrenodoxin. The reduction in the levels of steroidogenic enzyme proteins appears to be unspecific and probably reflects an overall demise in mitochondrial functions.

Mature bovine adrenodoxin contains a 14-amino-acid COOH-terminal extension originally detected by cDNA sequencing.

Since the nucleotide sequence of bovine adrenodoxin cDNA is at variance with protein sequencing data in that it encodes an additional 14 amino acids at the COOH terminus, we used a specific antibody raised against this 14-amino-acid segment to examine its presence in: nascent precursor polypeptide chains, the processed mature adrenodoxin and mitochondria of both steroidogenic and nonsteroidogenic tissues. These studies reveal the presence of the extra peptide in the precursor form derived from in vitro translation and in the newly synthesized mature form as shown by [35S]methionine labeling of proteins in adrenocortical cells. Both the purified COOH-terminal synthetic peptide and purified mature adrenodoxin competed with radiolabeled adrenodoxin for immunoprecipitation by the anti-peptide antibody. Immunoblots revealed the presence of the extra peptide in purified adrenodoxin and in bovine adrenocortical, corpus luteal, kidney and liver mitochondria while it was not detectable in heart mitochondria. Thus, we conclude that mature adrenodoxin and its homologs in non-steroidogenic tissues contain the C-terminal extension following uptake into mitochondria. These results indicate structural homology between adrenodoxin and the iron-sulfur proteins of the kidney and liver and also suggest the presence of a second iron-sulfur protein in kidney and liver.

Kidney and adrenal mitochondria contain two forms of NADPH-adrenodoxin reductase-dependent iron-sulfur proteins. Isolation of the two porcine renal ferredoxins.

Two NADPH-adrenodoxin reductase-dependent iron-sulfur proteins were detected in both porcine kidney and bovine adrenal mitochondria by using high resolution polyacrylamide electrophoresis. Adrenodoxin (Mr = 12,000) constituted the major ferredoxin activity in adrenal mitochondria and a similarly sized protein (Mr = 11,500) was isolated as the major renal ferredoxin activity. A second, higher molecular weight ferredoxin was observed in both adrenal (Mr = 13,300) and kidney (Mr = 13,000) mitochondria. The two renal ferredoxins were isolated by the use of ion exchange, gel exclusion, and preparative electrophoretic techniques. An absorption spectrum typical of [2Fe-2S] ferredoxins was obtained for each protein; however, the larger renal molecule had an unusually high 276 nm absorbance. Immunologic studies revealed a significant degree of antigenic commonality between the two renal proteins as well as specific cross-reactivity of adrenodoxin with antiserum raised against the renal proteins. A possible precursor-product relationship between the paired renal and adrenal ferredoxins is discussed.

Purification and biochemical characterization of hepatic ferredoxin (hepatoredoxin) from bovine liver mitochondria.

Hepatic ferredoxin (hepatoredoxin) was purified from bovine liver mitochondria. The monomeric molecular mass of the hepatoredoxin was larger than that of adrenocortical ferredoxin (adrenodoxin) from bovine adrenocortical mitochondria at 14 kDa. We studied the amino acid residues and NH2-terminal sequence of this protein. The hepatoredoxin was organ-specific protein. The optical absorption spectrum of oxidized hepatoredoxin had two peaks, at 414 and 455 nm in the visible region. Hepatoredoxin formed an immunoprecipitin line against anti-adrenodoxin immunoglobulin in Ouchterlony double diffusion, and an immunochemical staining band in Western blotting.

[Various properties of purified hepatoredoxin from bovine liver mitochondria]. / Nekotorye svoistva ochishchennogo gepatoredoksina iz mitokhondrii pecheni byka.

Hepatoredoxin purified to homogeneity from bovine liver mitochondria has been characterized for the first time in terms of its most important physico-chemical properties. The protein was found to contain in its active center a [2Fe-2S] cluster and has in the oxidized state an absorption maxima at 280, 320, 415 and 455 nm. The spectrophotometric index of purity, A415/A280 of the homogeneous native preparation is 0.84; extinction coefficient, epsilon 415, is 9800 M-1cm-1. The Mr of hepatoredoxin as evidenced by data from SDS gel electrophoresis is 12 500 Da; pI is 4.2. Hepatoredoxin is necessary for the reconstitution of the C27-steroid hydroxylase activity and can be substituted for by a related protein, adrenodoxin. All the above parameters as well as the circular dichroism spectra, immunochemical properties and sequence of the initial five N-terminal amino acids of hepatoredoxin and adrenodoxin are either coincident or very close. At the same time, the amino acid composition of these ferredoxins, apart from some common features, has individual peculiarities.

Cross-linking studies of adrenocortical cytochrome P-450scc. Evidence for a covalent complex with adrenodoxin and localization of the adrenodoxin-binding domain.

A cleavable cross-linking reagent, dimethyl-3,3'-dithiobispropionimidate, was used to study the molecular organization of adrenocortical cytochrome P-450scc. Extensive cross-linking was found to occur, resulting in the formation of heterologous oligomers up to octamer. The covalently cross-linked complex of adrenocortical cytochrome P-450scc with adrenodoxin has been obtained by using dimethyl-3,3'-dithiobispropionimidate. In the presence of NADPH and adrenodoxin reductase, electron transfer to cytochrome P-450scc occurs in the complex, and, in the presence of cholesterol, the latter effectively oxidizes to pregnenolone. By using covalently immobilized adrenodoxin and heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate, the adrenodoxin-binding site was shown to be located in the heme-containing, catalytic domain of cytochrome P-450scc. The data obtained indicate the existence of two different sites on the adrenodoxin molecule that are responsible for the interaction with adrenodoxin reductase and cytochrome P-450scc. This is consistent with the model mechanism of electron transfer in the organized complex.

[Localization of the adrenodoxin-binding fragment of cholesterol hydroxylating cytochrome P-450 from adrenal cortex mitochondria]. / Lokalizatsiia adrenodoksinsviazyvaiushchego fragmenta kholesteringidroksiliruiushchego tsitokhroma P-450 iz mitokhondrii kory nadpochechnikov.

The interaction between cytochrome P-450scc and adrenodoxin has been studied using cleavable cross-linking reagents and limited trypsinolysis. The data obtained indicate that the site responsible for adrenodoxin binding is located on the NH2-terminal fragment F1 of cytochrome P-450scc.

[Structural organization of adrenodoxin using limited proteolysis]. / Issledovanie strukturnoi organizatsii adrenodoksina metodom organichennogo proteoliza.

The microheterogeneity of adrenodoxin preparation was established by endogenous proteolysis. The controlled limited trypsinolysis and endogenous proteolysis result in modification of the COOH-terminus of the polypeptide chain with a formation of a protein with Mr = 10 000. The interaction of this protein and of the native protein with cholesterol-specific cytochrome P-450 and adrenodoxin reductase occurs in a similar way.