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A sigmoid-type dependence on the inhibitor concentration was observed in the cytochrome c reductase activity for peptide inhibitors (mastoparan and melittin), calmodulin antagonists (W-7 and tamoxifen) and monobutyltin in a reconstituted system comprised of recombinant rat neuronal nitric-oxide synthase (nNOS) and calmodulin (CaM). The increase in the concentration of CaM in the system induced a decrease in the inhibitory effect, indicating that the inhibitors might interfere with the interaction between nNOS and CaM. The changes in the fluorescence spectra of dansylated CaM caused by the addition of mastoparan, melittin and monobutyltin indicated complex formation between CaM and those compounds, which led to the decrease in the effective concentration of CaM available to nNOS. The sigmoid-type inhibition of mastoparan and melittin fit the theoretical equations quite well, assuming that two CaM molecules bind cooperatively to one nNOS homodimer. Monobutyltin, tamoxifen and W-7 were found to inhibit nNOS activity by binding to the CaM binding site of the nNOS homodimer, in addition to the binding of the inhibitors to calmodulin. These compounds inhibited the l -citrulline formation of nNOS from l -arginine, and the inhibitory effects were abrogated by raising the concentration of calmodulin. It became clear that the binding of calmodulin to nNOS can be interfered with in two ways: (1) via a decrease in the effective concentration of calmodulin caused by complex formation between the inhibitor and calmodulin, and (2) via the inhibition of the binding of calmodulin to nNOS caused by the occupation of the binding site by the inhibitor.

Many static protein structures have been determined by X-ray crystallography and nuclear magnetic resonance (NMR). Protein dynamics has also been studied. The structure and dynamics of proteins provide critical information about their functional mechanisms. Recently, mass spectrometry has become an essential method to analyze protein topological structures and protein dynamics. We performed topological analysis of membrane proteins using mass spectrometry coupled with chemical cross-linking. The combination of chemical cross-linking and mass spectrometry is a powerful method for analyzing the topological organization of protein structures, protein complexes, and the interface between interacting subunits. In this paper, we introduce our strategies for studying the membrane-protein and protein-protein interaction sites of P450 17α with its redox partners, NADPH-cytochrome P450 reductase and cytochrome b5.

Steroidogenic acute regulatory (StAR) protein is an important regulatory protein in steroidogenesis and rapidly undergoes proteolysis after import into the mitochondria. In this study, we determined the proteolytic cleavage sites and investigated the effects on the stimulation of steroidogenic activity of the blockage of these sites by mutation. The cleaved StAR proteins, which were purified using an anti-StAR immobilized column, reacted with antiserum against the StAR C-terminal oligopeptide. The molecular weights of the purified proteins were determined by MALDI-TOF mass spectrometry, and were found to be identical to those of the 40-285 and 55-285 amino-acid-regions of the StAR protein. To confirm the identification of the cleavage sites, we constructed site-directed mutants of bovine StAR cDNA, which contained the amino acids R37A/R38A/L40A and/or R53A/R54A/R55A. These mutant StAR proteins expressed in COS-1 cells were not cleaved at positions 39-40 and 54-55, and were processed at sites different from those in the wild-type StAR protein. These mutant proteins stimulated pregnenolone formation at almost the same rate as the wild-type StAR protein in COS-1 cells, which suggests that the cholesterol transfer activity was not affected by the mutation.

Objective: To study the interaction between human steroid 21-hydroxylase (21-OH) and monoclonal antibodies (MAbs) to 21-OH directed to 3 different epitopes recognised by 21-OH autoantibodies characteristic of autoimmune Addison’s disease. Design: Build comparative structural models of 21-OH, 21-OH MAbs and complexes of 21-OH–21-OH MAbs and study the effects of 21-OH MAbs on 21-OH enzyme activity. Then, analyse the relationship between sites important for binding of 21-OH MAbs and 21-OH autoantibodies and sites important for 21-OH enzyme activity. Methods: Variable (V) regions of 21-OH MAbs (M21-OH1, M21-OH3, M21-OH5) were sequenced and models of the MAbs built using structures of antibodies in the database as templates. A comparative model of 21-OH was built using the crystal structure of rabbit cytochrome p450 2c5/3LVdH as template. 21-OH enzyme activity was measured in terms of conversion of [3H]progesterone to deoxycorticosterone and the effect of purified MAb IgGs on 21-OH enzyme activity was assessed. Results: M21-OH1, M21-OH3 and control MAb had no effect on 21-OH enzyme activity with 88.8% ± 24% (n = 6), 86.7% ± 7.6% (n = 6) and 86.5% ± 10.6% (n = 6) of activity remaining in the presence of the respective IgGs. This was consistent with the epitopes for M21-OH1 and M21-OH3 being located distant from 21-OH enzyme active sites in our 21-OH model. The epitope for M21-OH5 which inhibited 21-OH enzyme activity (48.5 ± 8.3% activity remaining; P < 0.001 compared with control MAb IgG) was found close to the redox protein binding site in our 21-OH model. Conclusions: A comparative model of 21-OH has been produced. Analysis of experimental data in the context of the model suggests that M21-OH5 inhibits 21-OH enzyme activity through interference with redox protein binding.

Tributyltin, an environmental pollutant, affected adrenal steroid hormone biosynthesis by two modes of action. Treatment of bovine adrenal cultured cells with 10-100 nM tributyltin for 48 h suppressed cortisol and androstenedione secretion, but induced the accumulation of 17alpha-hydroxyprogesterone and deoxycortisol, indicating that the P450(C21) and P450(11beta) activities were specifically suppressed. Direct inhibition of the enzymatic activities due to tributyltin was not observed in isolated organelles of untreated cells at concentrations less than 10 microM. Western blotting experiments using specific antibodies against steroidogenic enzymes showed that treatment with 1-100 nM tributyltin caused a decrease in cellular P450(C21) and P450(11beta) protein levels, and real-time PCR experiments showed that the decrease in protein content was attributable to decreases in mRNA of the enzymes. Tributyltin at concentrations higher than 100 nM suppressed all steroid biosynthesis in the adrenal cells. This suppression was closely correlated to the decrease in steroidogenic acute regulatory protein. Since nanomolar concentrations of tributyltin disturbed steroidogenesis in mammalian cells, there is the possibility that steroid hormone synthesis in polluted wild animals is affected by this compound.

Objective: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs)in vitro.Design: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21.Methods: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH.Results: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450 nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21.Conclusions: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.

mRNA of cytochrome P450 21-hydroxylase (P450c21) is expressed in the brain, but little is known about the enzymatic properties of P450c21 in the brain. In the present study, we showed, by using various recombinant cytochrome P450 (CYP)2D enzymes and anti-CYP2D4- or P450c21-specific antibodies, that rat brain microsomal steroid 21-hydroxylation is catalyzed not by P450c21, but by CYP2D isoforms. Rat CYP2D4 and human CYP2D6, which are the predominant CYP2D isoforms in the brain, possess 21-hydroxylation activity for both progesterone and 17α-hydroxyprogesterone. In rat brain microsomes, these activities were not inhibited by anti-P450c21 antibodies, but they were effectively inhibited by the CYP2D-specific chemical inhibitor quinidine and by anti-CYP2D4 antibodies. mRNA and protein of CYP2D4 were expressed throughout the brain, especially in cerebellum, striatum, pons, and medulla oblongata, whereas the mRNA and protein levels of P450c21 were extremely low or undetectable. These results support the idea that CYP2D4, not P450c21, works as steroid 21-hydroxylase in the brain. Allopregnanolone, a representative γ-aminobutyric acid receptor modulator, was also hydroxylated at the C-21 position by recombinant CYP2D4 and CYP2D6. Rat brain microsomal allopregnanolone 21-hydroxylation was inhibited by fluoxetine with an IC50 value of 2 μm, suggesting the possibility that the brain CYP2D isoforms regulate levels of neurosteroids such as allopregnanolone, and that this regulation is modified by central nervous system-active drugs such as fluoxetine.

The rapid effects of steroid hormones on Ca2+ signals have been examined in bovine adrenocortical cells. Among the steroid molecules tested, only corticosterone rapidly stimulated Ca2+ signals upon addition of ACTH, although corticosterone alone did not induce Ca2+ signals. Corticosterone also enhanced steroidogenesis induced by ACTH. The enhancement of ACTH-induced Ca2+ signals was also observed with membrane-impermeable corticosterone conjugated to BSA and was not inhibited by cycloheximide. In addition, corticosterone did not enhance Ca2+ signals induced by ATP or angiotensin II. These results suggest that corticosterone selectively stimulates ACTH-induced Ca2+ signals in a nongenomic way by acting on a target in the plasma membrane. Furthermore, the supernatants of cells incubated with ACTH or ATP enhanced Ca2+ signals, suggesting that steroids produced by such treatment act in an autocrine fashion. Consistent with this idea, these effects were inhibited by inhibitors of steroidogenesis (aminoglutethimide or metyrapone). These results show that steroid molecules synthesized in adrenocortical cells facilitate ACTH-induced Ca2+ signals. Taken together, corticosterone secreted from adrenocortical cells activates ACTH-induced Ca2+ signals and steroidogenesis by nongenomic means.

The conversion of l -arginine (Arg) to Nω-hydroxy- l -arginine (OH-Arg) and further to l -citrulline (Cit) in one cycle of the reaction of purified rat neuronal nitric oxide synthase (nNOS) was measured with the reaction rapid-quenching method using 3H-Arg as the substrate. The produced 3H-OH-Arg was successively converted into 3H-Cit without leaving enzyme. From analysis of the time courses, it was found that the rate-determining step of the multi-cycle reaction of nNOS in the presence of excess amount of Arg was dissociation of produced 3H-Cit from nNOS. With decrease in NADPH concentration, the ratio of amounts of produced OH-Arg/Cit increased under the multi-cycle reaction condition. Under the single cycle reaction condition, decrease in NADPH concentration caused decrease in the rate of hydroxylation reactions both for Arg and OH-Arg but hardly affected the dissociation rate of OH-Arg from the nNOS. On the other hand, decrease in CaM concentration did not change the hydroxylation rate from Arg to OH-Arg but decreased the amount of active complex (CaM–nNOS) under the single cycle reaction condition. Under multi-cycle reaction condition, decrease in CaM concentration did not affect the ratio of produced OH-Arg/Cit. It became clear that there are two modes of regulation in the nNOS reaction.

The Japanese black bear, Ursus thibetanus japonicus, is a seasonal breeder and shows delayed implantation for several months during pregnancy. The objective of this study was to clarify the steroidogenic capability of the corpus luteum and placenta during pregnancy, including both delayed implantation and fetal development, by immunolocalization of steroidogenic enzymes in these organs of the Japanese black bear. Ovaries and placentae from 15 wild Japanese black bears, which had been killed legally by hunters and were thought to be pregnant, were used in an immunocytochemical study to localize the cholesterol side chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17alpha-hydroxylase cytochrome P450 (P450c17) and aromatase cytochrome P450 (P450arom) by the avidin-biotin-peroxidase complex method using polyclonal antisera raised in mammals against P450scc, 3betaHSD, P450c17 and P450arom. P450scc and 3betaHSD were localized in all luteal cells throughout pregnancy. P450c17 was present in a few luteal cells, especially in the outer area of the corpus luteum throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. Cells positively immunostained for P450c17 were significantly smaller than negatively immunostained cells (P < 0.01). P450arom was present sporadically in a few luteal cells throughout pregnancy, but the number of positively immunostained cells decreased during the post-implantation period. The size of cells positively immunostained for P450arom was not significantly different from that of negatively immunostained cells. The whole placenta was negatively immunostained for P450scc, 3betaHSD and P450c17, but P450arom was present in the syncytiotrophoblasts and endothelial cells of maternal blood vessels. These results indicate that, in the Japanese black bear, corpora lutea are a source of progesterone which may play an important role in the maintenance of delayed implantation and fetal development during pregnancy. Corpora lutea have a minimum capability to synthesize androgen in small luteal cells and oestrogen in normal-sized luteal cells during pregnancy, and placentae have the ability to synthesize oestrogen during late pregnancy.

To clarify whether the amphibian brain synthesizes de novo neurosteroids, we examined pregnenolone, pregnenolone sulfate ester, and cytochrome P450 side-chain cleavage enzyme (cytochrome P450scc), an enzyme converting cholesterol to pregnenolone, using amphibians. Pregnenolone and its sulfate ester in the brain, gonad, and plasma of Xenopus laevis were measured by a specific pregnenolone RIA. The concentrations of these two steroids in the female brain were significantly larger than those in the ovary and plasma. A similar tendency was evident in the male. In both sexes, pregnenolone and its sulfate ester were concentrated more highly in the cerebellum than in the telencephalon, diencephalon, or midbrain. An immunoreactive protein band of electrophoretic mobility in the proximity of bovine adrenal P450scc was detected in the Xenopus brain as well as the testis by Western blot analysis. Immunohistochemical analysis indicated that Purkinje cells in the Xenopus cerebellum were specifically immunostained with the P450scc antibody. P450scc-like immunoreactive cells were further found in several telencephalic and diencephalic regions, such as the pallium mediale and nucleus preopticus, in the Xenopus brain. A similar localization of P450scc-like immunoreactive cells was evident in Rana nigromaculata, a seasonally breeding amphibian. In the present study, seasonal changes in pregnenolone and its sulfate ester were further examined as a possible physiological change using R. nigromaculata. In both sexes, pregnenolone concentrations in the brain were almost constant during the seasonally breeding cycle. In contrast, the pregnenolone sulfate concentration in the brain was significantly lower in the hibernating quiescent phase and higher in the breeding and postbreeding active phases, independent of the plasma steroid level. These results taken together suggest that the amphibian brain possesses steroidogenic enzyme P450scc and produces pregnenolone and its sulfate ester. Pregnenolone sulfate may function well during the breeding and postbreeding active phases of the year in the seasonal breeder.

o-Nitrophenyl sulfenyl-modified ACTH (NPS-ACTH) stimulated steroidogenesis acutely in bovine fasciculata-reticularis cells without increase in cellular cAMP synthesis. Application of NPS-ACTH to the cultured cells induced Ca2+ signals in individual cells as detected by video-enhanced microscopic fluorescence measurements. The percentage of Ca2+ signaling cells corresponded well with the increase of steroidogenesis induced by NPS-ACTH below 1 nM. Treatment of the cells with nicardipine, a Ca2+ channel blocker, suppressed the Ca2+ signals except for the transient increase just after the addition of NPS-ACTH and also blocked completely the stimulative effect on the steroidogenesis of NPS-ACTH below 1 nM. At a dosage of NPS-ACTH higher than 10 nM, the stimulative effect of steroidogenesis was partly suppressed by nicardipine and also by AA-861, a lipoxygenase inhibitor. The action of NPS-ACTH might be mediated by both Ca2+ and lipoxygenase metabolite(s) of arachidonic acid as dual second messengers. The effect of ACTH in pM range on the steroidogenesis was suppressed completely by the treatment with nicardipine and AA-861 at the same time, indicating that the action was mediated by both Ca2+ and the lipoxygenase metabolite(s) but not by cAMP. cAMP plays a significant role as a second messenger for ACTH action only at ACTH concentrations greater than 10 pM.

From brains of the adult male zebra finch, we purified a peptide that has a homologous sequence of the large subunit of human U2AF. U2AF is a non-small-nuclear ribonucleoprotein (snRNP) splicing factor required for pre-spliceosome assembly. U2AF consists of a large and a small subunit, whereas only a large subunit is required for in vitro splicing. To show that U2AF large-subunit-like protein exists in the brain of zebra finches, we conducted immunoblot analysis on brain extract, using antiserum against an isolated peptide with primary structures similar to the U2AF large subunit (ppU2AFls). The immunoblot analysis showed that a protein with a molecular weight of about 60 kd reacted with the anti-ppU2AFls-antibody to a putative peptide of the U2AF large subunit. To examine the localization of this protein in the brain, we also conducted immunohistochemical analysis with the anti-peptide. An intense immunoreaction was restricted within the cellular nucleus throughout the brain, suggesting that this protein may contribute to the pre-spliceosome assembly in almost all of the regions of the brain.

The reactions for the synthesis of aldosterone from deoxycorticosterone were investigated kinetically in the membrane-reconstituted system with bovine cytochrome P45011 beta at 37 degrees C. Reaction rapid-quenching experiments for the metabolism of deoxycorticosterone by P45011 beta showed that aldosterone was produced via corticosterone, not via 18-hydroxydeoxycorticosterone. The kinetic analysis revealed that aldosterone was formed successively from fractions of intermediate metabolites which did not dissociate from P45011 beta. The rate of each reaction step in the successive reactions was estimated from the combination of results of the rapid-quenching experiments and the metabolism of deoxycorticosterone in the presence of an excess amount of substrate, in which the dissociation of final product, aldosterone, from the enzyme was the slowest step in the synthesis from deoxycorticosterone. Under steady-state reaction conditions, the interaction of P45011 beta with P450scc stimulates the production of corticosterone from deoxycorticosterone by about 10-fold but inhibits further reactions from corticosterone. The rapid-quenching experiments showed, however, that the rate constant for the 11 beta-hydroxylation of deoxycorticosterone for corticosterone production in the presence of P450scc was almost the same as that without P450scc. The interaction of P45011 beta with P450scc in the reaction system for deoxycorticosterone metabolism was found to slow the rate of the subsequent 18-hydroxylation of the produced corticosterone and to accelerate the dissociation of the corticosterone from P45011 beta, which stimulated the corticosterone production and inhibited the further reaction for aldosterone synthesis in the steady-state reaction.

Bovine P450(17alpha,lyase) containing an additional four histidine residues at the COOH terminus was expressed in Escherichia coli and purified by one-step column chromatography using Ni-chelate resin. The membrane enzyme was incorporated into liposome membranes having similar lipid composition to that of the endoplasmic reticulum. In the presence of excess substrate, the P450-proteoliposomes metabolize pregnenolone (Delta5-steroid) to 17alpha-hydroxypregnenolone and further to dehydroepiandrosterone. The enzyme catalyzed 17alpha-hydroxylation of progesterone (Delta4-steroid) but did not form androstenedione from progesterone, although the proteoliposomes could catalyze the conversion of 17alpha-hydroxyprogesterone to androstenedione. The kinetic analysis of rapid quenching experiments showed that about 20% of the pregnenolone consumed was converted successively to dehydroepiandrosterone via a fraction of 17alpha-hydroxypregnenolone that did not dissociate from the enzyme. The rapid quenching experiments for progesterone metabolism by the proteoliposomes revealed that the dissociation rate of 17alpha-hydroxyprogesterone was 10 times faster than that of 17alpha-hydroxypregnenolone. The release of the intermediate metabolite of Delta4-steroid is sufficiently faster than the lyase reaction to prevent further reaction by the P450. It is concluded that the dissociation rates of the first hydroxylation metabolites regulate the successive reactions of P450(17alpha,lyase).

To elucidate the steroid-synthesis in the mammalian brain (i. e., neurosteroid), we immunohistochemically studied various kinds of steroidogenic cytochrome P450 enzymes in the rat brain. The primary antibodies used in this study were rabbit polyclonal antibodies to cholesterol side-chain cleavage (SCC), C21-hydroxylase (C21), 11β-hydroxylase (11β), 17α-hydroxylase/C17-20 lyase (17α) and aromatase (AROM). The immunoreactivities for the microsome enzymes, C21 and 17α were located in the neuronal cell-bodies and proximal parts of their fibers, while mitochondria enzymes, SCC and 11β were located in the cell-bodies and their fibers and terminals. These immunoreactivities were distributed in the limbic structures of prosencephalon including hippocampus and amygdaloid complex, the hypothalamus including preoptic area, the cerebellar cortex, and some of other regions. All the sets of these enzymes did not always coexist in the identical cells, however, in the hypothalamus and cerebellum these enzymes are thought to work one after another in adjacent cells, forming the “steroidogenic cellular circuits”. These findings strongly suggest that the steroidsynthesis occurs in the neurons; and the neurosteroids exist in the mammalian brain.

We have reported the localization on yeast microsomes for a modified P450c27 (mic-P450c27) that contains the microsomal targeting signal of bovine P450c17 in front of the mature form of rat mitochondrial P450c27 (Sakaki, T., Akiyoshi-Shibata, M., Yabusaki, Y., and Ohkawa, H. (1992) J. Biol. Chem. 267, 16497-16502). In this study, we found that mic-P450c27 could be reduced by NADPH in the yeast microsomes without supplement of its physiological redox partners, adrenodoxin and NADPH-adrenodoxin reductase. In order to elucidate the direct electron transfer from NADPH-P450 reductase to mic-P450c27, we carried out simultaneous expression of mic-P450c27 and yeast P450 reductase. The reduction rate of mic-P450c27 was increased by overproduction of yeast P450 reductase, roughly in proportion to the reductase content in the microsomes. In addition, we constructed a fused enzyme between mic-P450c27 and yeast P450 reductase. The reduction rate of heme iron in the fused enzyme was too rapid to be measured. These recombinant yeast microsomes showed a notable 27-hydroxylation activity toward 5beta-cholestane-3alpha,7alpha, 12alpha-triol in the absence of adrenodoxin and adrenodoxin reductase. Finally, we purified mic-P450c27 from the recombinant yeast microsomes and reconstituted the hydroxylation system in liposomal membranes using the purified mic-P450c27 and yeast NADPH-P450 reductase. Mic-P450c27 was reduced by NADPH and showed its monooxygenase activity on the reconstituted system. Therefore, yeast NADPH-P450 reductase alone was found to transfer two electrons from NADPH to mic-P450c27. These results clearly show that mic-P450c27 not only localizes on the microsomes but also functions as a microsomal cytochrome P450 that accepts electrons from NADPH-P450 reductase.

The acute activation of adrenal glucocorticoid synthesis by ACTH has long been believed to be mediated by cAMP as the major second messenger, although increases in cellular cAMP concentration have not been observed at low concentrations of ACTH. We found that steroidogenesis in bovine adrenal fasciculata-reticularis cells was activated by the addition of arachidonic acid or its 15-lipoxygenase metabolite, 15-hydroperoxyeicosatetraenoic acid. The cellular 15-lipoxygenase pathway was significantly activated by 1 pM ACTH, at which concentration no increase in cellular cAMP synthesis was observed. The 1 pM ACTH-induced stimulation of steroidogenesis was completely suppressed by a lipoxygenase inhibitor, AA-861. The stimulation was independent of the increase in cellular cAMP. These results show that the action of 1 pM ACTH on steroidogenesis may be mediated by the 15-lipoxygenase metabolite(s) as a solo second messenger. The addition of ACTH at concentrations higher than 10 pM increased both the 15-lipoxygenase activity and cellular cAMP synthesis. Under these conditions, the 15-lipoxygenase metabolite(s) and cAMP were shown to mediate the activation of steroidogenesis synergistically. The presence of a dual second messenger system could explain the stimulation of steroidogenesis by ACTH at physiological concentrations.

In the adrenal cortex, the potent mineralocorticoid, aldosterone, is produced in the zoba glomerulosa but not in the zona fasciculata/reticularis. In rodents and humans, two distinct species of P-450(C18) (aldosterone synthase) and P-450(11beta) (11beta-hydroxylase) are expressed in the adrenal cortex. The selective expression of cytochrome P-450 species in different zones contributes to zone specificity of aldosterone synthesis. In the cow and pig, only one molecular species of P-450(11beta) having both 11beta-hydroxylase and aldosterone synthase activity is expressed throughout the adrenal cortex. P-450(11beta) in the zona fasciculata/reticularis catalyzes the formation of corticosterone but not that of aldosterone from 11-deoxycorticosterone; the same enzyme in the zona glomerulosa produces aldosterone from the same substrate, indicating that a local factor in mitochondria is likely to be involved in the selective suppression of the aldosterone synthetic activity of P-450(11beta) in the zona fasciculata/reticularis. The zone specificity of aldosterone synthesis catalyzed by P-450(11beta) in the bovine adrenal cortex appears to be due to differences in interactions between P-450(11beta) and P-450(SCC) in mitochondria in different cortical zones. Thus, two modes exist for aldosterone biosynthesis in mammals: rodent-human and bovine-porcine modes.

The transfer of cytochrome P-450 from bovine adrenocortical submitochondrial particles (smp) to unilamellar liposome membranes was investigated using a table top ultracentrifuge. Submitochondrial particles were incubated with liposome membranes at 25 degrees C and precipitated by ultracentrifugation at 200000g for a few minutes at 25 degrees C. All liposome vesicles were recovered in the supernatant. Almost no proteins were detected in the supernatant when only smp were incubated and centrifuged. SDS-PAGE revealed one main protein band for the supernatant when smp were incubated with liposome vesicles at 25 degrees C. This band was reactive to anti-P-450scc IgG. Inaccuracy in time for kinetic studies of the transfer was less than 0.5 min. Transfer of P-450scc from smp to liposome membranes was further demonstrated by the decrease in side-chain cleavage activity of smp for endogenous cholesterol after incubation. Cytochromes P-450 accounted for about 70% of the transferred proteins in the liposome membranes, the amount of which increased exponentially with the incubation time. The inverse value of the relaxation time of the transfer increased linearly with the smp concentration and decreased hyperbolically with the liposome concentration. These results coincide with a mechanism by which cytochrome P-450 dissociates from smp membranes, diffuses, and binds to the liposome membranes. In the transfer of cytochrome P-450, the dissociation from smp membranes was deduced to be the rate-limiting step.

Previous investigations demonstrated that carbon tetrachloride (CCl 4 ) was activated by adrenal microsomes, resulting in various functional changes and ultimately in necrosis of the zona reticularis of the gland. Experiments were done to identify the adrenal P450 isozyme(s) involved in the bioactivation of CCl 4 . Incubation of microsomes from the zona reticularis (ZR) of the guinea pig adrenal cortex with CCl 4 plus NADPH caused initiation of lipid peroxidation, covalent binding of CCl 4 -derived radioactivity to protein, and degradation of cytochrome(s) P450. Preincubation of the microsomal preparations with inhibitory antibodies to P450 17α or P450 C12 decreased the corresponding enzyme activities (17α-hydroxylation and 21-hydroxylation), but did not affect the activation of CCl 4 . 1-Aminobenzotriazole (ABT), a suicide inhibitor of some P450 isozymes, decreased the enzyme activities catalysed by an adrenal 52 000 Da (52 kDa) isozyme, but had no effect on the function of P450 17α or P450 C21 . However, ABT completely inhibited the CCl 4 -induced LP and covalent binding in adrenal microsomes. The results indicate that adrenal CCl 4 activation is catalysed by the 52 kDa P450 isozyme and not by the steroid hydroxylases. Localization of the 52 kDa isozyme to the ZR probably accounts for the selective necrosis of this region of the gland by CCl 4 .

Studies were done to determine the effects of a P450 suicide inhibitor, 1-aminobenzotriazole (ABT), on adrenal steroid and xenobiotic metabolism. Incubation of guinea pig adrenal microsomes with ABT plus an NADPH-generating system caused a time-dependent decline in total P450 concentrations. The maximal decrease in P450 levels was approximately 35% and was accompanied by an equimolar decrease in heme content. Western blot analyses indicated that ABT had no effect on P450 apoprotein levels. Benzphetamine (BZ) N-demethylase and benzo[a]pyrene (BP) hydroxylase activities were inhibited almost completely by microsomal incubations with ABT. In contrast, neither steroid 17 α-hydroxylase nor 21-hydroxylase activity was affected by ABT. The steroid-induced type I spectral change in adrenal microsomes also was not affected by ABT, whereas that induced by BZ was eliminated. Similar studies with adrenal mitochondria indicated that ABT had no effect on mitochondrial P450 concentrations or on mitochondrial steroid metabolism. The results demonstrate that the in vitro actions of ABT on adrenal cytochromes P450 are highly selective for those isozymes that catalyze xenobiotic metabolism. Therefore, ABT should serve as a useful probe for further characterization of adrenal xenobiotic-metabolizing P450 isozymes.

Three expression plasmids, pAMC1 for rat P4501A1, pAMR2 for P4501A1 and yeast NADPH-P450 reductase, and pAFCR1 for a fused enzyme between P4501A1 and the reductase, were constructed, and each was introduced into Saccharomyces cerevisiae AH22 cells. The microsomal fraction prepared from the recombinant yeast cells was subjected to kinetic studies of zoxazolamine 6-hydroxylation at 10 degrees C. The apparent Km and Vmax values for hydroxylation by the fused enzyme in AH22/pAFCR1 microsomes were 0.38 mM and 0.42 s-1, respectively. The rate constant for reduction of the fused enzyme with NADPH in the presence of 1 mM zoxazolamine was larger than 50 s-1 using a dual-wavelength stopped-flow spectrometer, indicating that electrons are rapidly transferred from NADPH through FAD and FMN to the heme iron of the fused enzyme. The rate constant kon for substrate binding to the fused enzyme was 25 mM-1.s-1, which is not much different from that of nonfused P4501A1. These results together with spectral data measured during the hydroxylation reaction in the steady state suggest that the rate-limiting step of the reaction by the fused enzyme might be the release of product. On the other hand, the apparent Km and Vmax values for the hydroxylation of P4501A1 in AH22/pAMC1 and AH22/pAMR2 microsomes were 0.32 and 0.33 mM, and 0.015 and 0.29 s-1, respectively. The rate constants for the reduction of P4501A1 were 0.025 and 0.40 s-1, respectively, for AH22/pAMC1 and AH22/pAMR2 microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Purified adrenocortical microsomal P-450C21 was incorporated into vesicle membranes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a molar ratio of 5:3:1. Trypsinolysis of the incorporated P-450C21 resulted in the formation of 30-, 25-, and 20-kDa fragments. Similar fragment formation was observed by trypsinolysis of bovine adrenocortical microsomes with Western blotting using anti-P-450C21 IgG. In the detergent-solubilized state, trypsin cleaved P-450C21 into very small peptides. Washing of the trypsin-treated vesicles with 500 mM Na2CO3 failed to cause these fragments to separate from membranes. N-Terminal amino acid sequencing of these fragments showed that trypsin cleaved the 267 Arg-268Val and 332Arg-333Val bonds of P-450C21. The time course of fragment formation indicated that trypsin cleaved the 267Arg-268Val bond first to produce 30- and 25-kDa fragments and subsequently the 332Arg-333Val bond in the 25-kDa fragment to produce the 20-kDa fragment. Neither 21-hydroxylase activity, the reduced CO difference spectrum, nor the EPR spectrum of digested P-450C21 differed from those of undigested P-450C21. Heat treatment at 50 degrees C for 20 min did not cause any decrease in activity of digested P-450C21, when the substrate progesterone was present. This high stability toward heat treatment was not observed in the solubilized state. Rotational diffusion experiments on P-450C21 showed that the size of the molecule holding the heme was not changed significantly after digestion. On the basis of these results, P-450C21 is concluded to be deeply embedded in the vesicle membranes.

In order to clarify periodic changes in the localization of enzymes engaged in estrogen biosynthesis during the estrous cycle, immunohistochemical and fine structural studies were performed using estrous cycling and pregnant hamster ovaries. Results showed that ovulation takes place at midnight between Day 4 and Day 1 in the regular 4 day-cycle hamster. Immunoreactivity for aromatase is localized in the granulosa cells of the secondary follicle and granulosa lutein cells during the morning (10:00am) of Day 1 to the evening (5:00pm) of Day 4; in the night (9:00pm) of Day 4, only the granulosa cells of the Graafian follicle showed a strong immunoreaction.As for 17α-lyase, theca interna cells of the secondary follicle are immunopositive throughout Day 2 to the morning (10:00am) of Day 4. Only a few cells in the theca interna of the Graafian follicles are immunopositive in the evening (5:00pm) of Day 4. No positive cells for this enzyme were detected in the night (9:00pm) of Day 4 or morning (10:00am) of Day 1. The rapid decrease of estrogen biosynthesis occurring just before ovulation is considered to be due to the disappearance of 17α-lyase in the theca interna cells of the ovary.On Day 10 of pregnancy, the granulosa cells of the secondary follicles and both the granulosa and theca lutein cells of the corpora lutea are immunostained with the aromatase antibody, while the theca interna cells of the secondary follicles reacted positively to the 17α-lyase antibody.Only the granulosa and theca interna cells from the large preovulatory Graafian follicle of Day 4 (proestrus) which are positively stained for aromatase as well as 17α-lyase show ultrastructural features typical of steroid secretory cells.

Bovine adrenal P-45011 beta catalyzes the 11 beta- and 18-hydroxylation of corticosteroids as well as aldosterone synthesis. These activities of P-45011 beta were found to be modulated by another mitochondrial cytochrome P-450 species, P-450scc. The presence together of P-45011 beta and P-450scc in liposomal membranes was found to remarkably stimulate the 11 beta-hydroxylase activity of P-45011 beta and also stimulate the cholesterol desmolase activity of P-450scc. The stimulative effect of P-450scc on 11 beta-hydroxylase activity diminished by the addition of protein-free liposomes to proteoliposomes containing P-45011 beta and P-450scc, thus showing P-450scc to interact with P-45011 beta in the same membranes. Kinetic analysis of this effect indicated the formation of an equimolar complex between P-45011 beta and P-450scc on liposomal membranes. P-45011 beta in the complex had not only stimulated activity for 11 beta- and 18-hydroxylation of 11-deoxycorticosterone but also suppressed activity for production of 18-hydroxycorticosterone and aldosterone. When the inner mitochondrial membranes of zona fasciculata-reticularis from bovine adrenal were treated with anti-P-450scc IgG, aldosterone formation was stimulated to a greater extent than that of zona glomerulosa. This indicates the aldosterone synthesizing activity of P-45011 beta in the zona fasciculata-reticularis to be suppressed by interaction with P-450scc. The zone-specific aldosterone synthesis of P-45011 beta in bovine adrenal may possibly be induced by differences in interactions with P-450scc of mitochondrial membranes in each zone.

The hippocampus is essentially involved in learning and memory processes. Its functions are affected by various neuromodulators, including 17β-estradiol, testosterone, and retinoid. Brain-synthesized steroid hormones act as autocrine and paracrine modulators. The regulatory mechanism underlying brain steroidogenesis has not been fully elucidated. Synthesis of sex steroids in the gonads is stimulated by retinoic acids. Therefore, we examined the effects of retinoic acids on estradiol and testosterone biosynthesis in the rat hippocampus. We used cultured hippocampal slices from 10- to 12-d-old male rats to investigate de novo steroidogenesis. The infant rat hippocampus possesses mRNAs for steroidogenic enzymes and retinoid receptors. Slices were used after 24 h of preculture to obtain maximal steroidogenic activity because steroidogenesis in cultured slices decreases with time. The mRNA levels for P45017α, P450 aromatase and estrogen receptor-β in the slices were increased by treatment with 9-cis-retinoic acid but not by all-trans-isomer. The magnitude of stimulation and the shape of the dose-response curve for the mRNA level for P45017α were similar to those for cellular retinoid binding protein type 2, the transcription of which is activated by retinoid X receptor signaling. 9-cis-Retinoic acid also induced a 1.7-fold increase in the protein content of P45017α and a 2-fold increase in de novo synthesis of 17β-estradiol and testosterone. These steroids may be synthesized from a steroid precursor(s), such as pregnenolone or other steroids, or from cholesterol, as so-called neurosteroids. The stimulation of estradiol and testosterone synthesis by 9-cis-retinoic acid might be caused by activation of P45017α transcription via retinoid X receptor signaling.

For clarification of the effects of steroid concentration on steroidogenesis of adrenal microsomes, the kinetic parameters, Km and kcat, were determined in the steady-state for progesterone and 17 alpha-hydroxyprogesterone metabolism catalyzed by P-450C21 and P-450(17 alpha lyase) in guinea pig adrenal microsomes. At a high concentration of progesterone, it was equally metabolized by P-450C21 and P-450(17 alpha lyase), while at a low concentration, it was hydroxylated at 17 alpha-position with twice higher rate than at 21-position. 17 alpha-Hydroxyprogesterone is apparently metabolized preferentially by P-450C21 at any concentration. Although the productions of deoxycortisol and androstenedione from 17 alpha-hydroxyprogesterone were strongly inhibited by progesterone, androstenedione formation from progesterone was not inhibited by a high concentration of progesterone. The addition of liposomal P-450C21 to the reaction medium containing adrenal microsomes caused a decrease in the concentration of 17 alpha-hydroxyprogesterone released into the medium in the steady state reaction, but this had no effect on the activity of androstenedione formation from high concentrations of progesterone. It thus follows that androstenedione is produced by successive monooxygenase reactions without the release of 17 alpha-hydroxyprogesterone from P-450(17 alpha lyase) at a high concentration of progesterone, which is the condition of the adrenal microsomes in vivo.

Rapid modulation of hippocampal synaptic plasticity by estrogen has long been a hot topic, but analysis of molecular mechanisms via synaptic estrogen receptors has been seriously difficult. Here, two types of independent synaptic plasticity, long-term depression (LTD) and spinogenesis, were investigated, in response to 17beta-estradiol and agonists of estrogen receptors using hippocampal slices from adult male rats. Multi-electrode investigations demonstrated that estradiol rapidly enhanced LTD not only in CA1 but also in CA3 and dentate gyrus. Dendritic spine morphology analysis demonstrated that the density of thin type spines was selectively increased in CA1 pyramidal neurons within 2 h after application of 1 nm estradiol. This enhancement of spinogenesis was completely suppressed by mitogen-activated protein (MAP) kinase inhibitor. Only the estrogen receptor (ER) alpha agonist, (propyl-pyrazole-trinyl)tris-phenol (PPT), induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. The ERbeta agonist, (4-hydroxyphenyl)-propionitrile (DPN), suppressed LTD and did not affect spinogenesis. Because the mode of synaptic modulations by estradiol was mostly the same as that by the ERalpha agonist, a search was made for synaptic ERalpha using purified RC-19 antibody qualified using ERalpha knockout (KO) mice. Localization of ERalpha in spines of principal glutamatergic neurons was demonstrated using immunogold electron microscopy and immunohistochemistry. ERalpha was also located in nuclei, cytoplasm and presynapses.

Modulation of hippocampal synaptic plasticity by estrogen has been attracting much attention. Here, we demonstrated the rapid effect of 17b-estradiol on the density and morphology of spines in the stratum oriens (s.o., basal side) and in the stratum lacunosummoleculare (s.l.m., apical side) by imaging Lucifer Yellow-injected CA1 neurons in adult male rat hippocampal slices, because spines in s.o. and s.l.m. have been poorly understood as compared with spines in the stratum radiatum. The application of 1 nM estradiol-induced a rapid increase in the density of spines of pyramidal neurons within 2 h. This increase by estradiol was blocked by Erk MAP kinase inhibitor and estrogen receptor inhibitor in both regions. Effect of blockade by agonists of AMPA receptors and NMDA receptors was different between s.o. and s.l.m. In both regions, ERa agonist PPT induced the same enhancing effect of spinogenesis as that induced by estradiol. � 2006 Published by Elsevier Inc.

The lysine residues of guinea pig P450 17alpha were acetylated by acetic anhydride in the absence and presence of NADPH cytochrome P450 reductase (CPR). Eight acetylated peptides were identified in the MALDI-TOF mass spectra of the tryptic fragments from the P450 acetylated without CPR in the limited reaction time of 15 min at ice temperature. The presence of CPR during the acetylation of P450 17alpha prevented double acetylations at K326 and K327 in the J-helix. The activity of P450 17alpha was decreased to 35% by the acetylation, but almost no inactivation was detected in the P450 after acetylation in the presence of CPR. This protection from inactivation shows the importance of K326 and/or K327 in the J-helix of P450 17alpha in the interaction between the two enzymes. Our results provided the first experimental evidence for the importance of the J-helix of P450 in the interaction with CPR. The interaction of P450 17alpha with CPR on the membrane is discussed based on the results of this study, which used molecular modeling.

Modulation of hippocampal synaptic plasticity by estrogen has been attracting much attention. Thorns of thorny excrescences of CA3 hippocampal neurons are post-synaptic regions whose presynaptic partners are mossy fiber terminals. Here we demonstrated the rapid effect of estradiol on the density of thorns of thorny excrescences, by imaging Lucifer Yellow-injected CA3 neurons in adult male rat hippocampal slices. The application of 1nM estradiol induced rapid decrease in the density of thorns on pyramidal neurons within 2h. The estradiol-mediated decrease in the density of thorns was blocked by CNQX (AMPA receptor antagonist) and PD98059 (MAP kinase inhibitor), but not by MK-801 (NMDA receptor antagonist). ERalpha agonist PPT induced the same suppressive effect as that induced by estradiol on the density of thorns, but ERbeta agonist DPN did not affect the density of thorns. Note that a 1nM estradiol treatment did not affect the density of spines in the stratum radiatum and stratum oriens. A search for synaptic ERalpha was performed using purified RC-19 antibody. The localization of ERalpha (67kDa) in the CA3 mossy fiber terminals and thorns was demonstrated using immunogold electron microscopy. These results imply that estradiol drives the signaling pathway including ERalpha and MAP kinase.

Adrenal glucocorticoid synthesis is stimulated by ACTH or its nitrophenylsulphenyl derivative, NPS-ACTH. Acute stimulation of steroid hormone biosynthesis is highly dependent on the expression of steroidogenic acute regulatory (StAR) protein. To determine the regulatory mechanism of StAR expression in bovine fasciculata/reticularis cells, we analyzed the second messenger systems involved in StAR protein expression using cultured cells activated by ACTH and NPS-ACTH. We concluded that cAMP is not the essential second messenger for StAR protein expression, since NPS-ACTH activated StAR protein expression more than ACTH without increase in cellular cAMP. A 15-lipoxygenase metabolite(s) of arachidonic acid stimulated steroidogenesis without increase in StAR protein expression, since AA-861, a lipoxygenase inhibitor, inhibited steroidogenesis without affecting StAR protein expression. Stimulation of StAR protein expression and the corresponding increase in the steroidogenesis were inhibited by nicardipine in cells treated with ACTH or NPS-ACTH. These data indicate that the dominant second messenger for the stimulation of StAR protein expression is Ca2+. Calmodulin-dependent kinase II inhibitors KN-93 and KN-62 suppressed steroidogenic activity without affecting StAR expression. The protein kinase C inhibitor Ro 31-8220 did not show any effects on StAR expression and steroidogenesis. Calmodulin-dependent kinase II and protein kinase C can therefore be concluded not to be involved in StAR protein expression in bovine cells.

In adult mammalian brain, occurrence of the synthesis of estradiol from endogenous cholesterol has been doubted because of the inability to detect dehydroepiandrosterone synthase, P45017α. In adult male rat hippocampal formation, significant localization was demonstrated for both cytochromes P45017α and P450 aromatase, in pyramidal neurons in the CA1–CA3 regions, as well as in the granule cells in the dentate gyrus, by means of immunohistochemical staining of slices. Only a weak immunoreaction of these P450s was observed in astrocytes and oligodendrocytes. ImmunoGold electron microscopy revealed that P45017α and P450 aromatase were localized in pre- and postsynaptic compartments as well as in the endoplasmic reticulum in principal neurons. The expression of these cytochromes was further verified by using Western blot analysis and RT-PCR. Stimulation of hippocampal neurons with N -methyl- d -aspartate induced a significant net production of estradiol. Analysis of radioactive metabolites demonstrated the conversion from [ 3 H]pregnenolone to [ 3 H]estradiol through dehydroepiandrosterone and testosterone. This activity was abolished by the application of specific inhibitors of cytochrome P450s. Interestingly, estradiol was not significantly converted to other steroid metabolites. Taken together with our previous finding of a P450scc-containing neuronal system for pregnenolone synthesis, these results imply that 17β-estradiol is synthesized by P45017α and P450 aromatase localized in hippocampal neurons from endogenous cholesterol. This synthesis may be regulated by a glutamate-mediated synaptic communication that evokes Ca 2+ signals.

Cytochrome P450s in endoplasmic reticulum membranes function in the hydroxylation of exogenous and endogenous hydrophobic substrates concentrated in the membranes. The reactions require electron supplies from NADPH-cytochrome P450 reductase in the same membranes. The membranes play important roles in the reaction of cytochrome P450. The membrane topology of guinea pig P450 17alpha was investigated on the basis of the differences in reactivity to hydrophilic chemical modification reagents between those in the detergent-solubilized state and proteoliposomes. Recombinant guinea pig cytochrome P450 17alpha was purified from Escherichia coli and incorporated into liposome membranes. Lysine residues in the detergent-solubilized P450 17alpha and in the proteoliposomes were acetylated with acetic anhydride at pH 9.0, and the acidic amino acid residues were conjugated with glycinamide at pH 5.0 by the aid of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The modifications were performed under conditions where the denatured form, P420, was not induced. The modified P450 17alpha's were digested by trypsin, and the molecular weights of the peptide fragments were determined by MALDI-TOF mass spectrometry. From the increase in the molecular weights of the peptides, the positions of modifications could be deduced. In the detergent-solubilized state, 11 lysine residues and 7 acidic amino acid residues were modified, among which lysine residues at positions 29, 59, 490, and 492 and acidic residues at 211, 212, and/or 216 were not modified in the proteoliposomes. Both the N- and C-terminal domains and the putative F-G loop were concluded to be in or near the membrane-binding domains of P450 17alpha.

Although cytochrome P45017alpha catalyzes the formation of androgen from both pregnenolone and progesterone, the production of androstenedione from progesterone is a major pathway in the guinea pig, rat, mouse, and hamster. In contrast, human, bovine and sheep P45017alpha produce dehydroepiandrosterone from pregnenolone. Cytochrome P45017alphas from all of these animals have high homology in the amino acid sequence around the 340-370 region. To investigate the substrate preferences for androgen production, we replaced a few amino acids in the 340-370 region of guinea pig P45017alpha with those found in the other animals. The recombinant P45017alphas were expressed in E. coli DH5alpha, purified by column chromatography and incorporated into liposome membranes. The (His)(4) tag in the recombinant P45017alphas had little effect on the interaction with NADPH-P450 reductase in the membranes. The recombinant P45017alphas with a single-position mutation of F344I, H349R or M352L and with double-position mutations of F344I and H349R and triple-position mutations showed decreases in the production of 17alpha-hydroxypregnenolone, androstenedione and dehydroepiandrosterone. The activity for 17alpha-hydroxyprogeterone production was increased significantly by the F344I mutation. The addition of cytochrome b5 did not have much of an effect on the 17alpha-hydroxylation but had a significant effect on androgen production in both the nonmutated and mutated P45017alphas.

The mechanisms of adrenal damage induced by 7,12-dimethylbenz (α) anthrancene (DMBA) in 50-day-old female Sprague-Dawley rats were investigated. A single dose of DMBA, either fed (30 mg) per os or injected (6 mg) in a caudal vein, caused inner cortical cell death (cells of the zonae fasciculata and reticularis) by an apoptotic mechanism. Apoptotic cells were identified by cell morphology, and terminal dUTP nick end labeling (TUNEL)-positive cells were seen at 12 hrs post-DMBA, reached a maximum at 36 h, and were accompanied by blood congestion followed by massive hemorrhage leading to post-apoptotic necrosis at 48 and 72 h. The apoptotic cascade involved the up-regulation of Bax, the down-regulation of Bcl-2, and the activation of caspase-3. At 72 h, regeneration as evidenced by the appearance of 5-bromo-2′-deoxyuridine-positive cells began to occur in the damaged inner cortical zones, with the cells proliferating toward the medulla thereafter. Regenerative cells expressed cytochrome P450 11β hydroxylase. The damage was repaired but calcification appeared at 2 weeks post-DMBA, leaving bow-shaped lesions in some adrenals.

Adrenal cytochrome P450 C21 in a membrane-reconstituted system catalyzed 21-hydroxylation of 17alpha-hydroxyprogesterone at a rate higher than that for progesterone in the steady state at 37 degrees C. The rate of product formation in the steady state increased with the concentration of the complex between P450 C21 and the reductase in the membranes. The complex formation was independent of the volume of the reaction, showing that the effective concentrations of the membrane proteins should be defined with the volume of the lipid phase. The rates of conversion of progesterone and 17alpha-hydroxyprogesterone to the product in a single cycle of the P450 C21 reaction were measured with a reaction rapid quenching device. The first-order rate constant for the conversion of progesterone by P450 C21 was 4.3 +/- 0.7 s(-)1, and that for 17alpha-hydroxyprogesterone was 1.8 +/- 0.5 s(-)1 at 37 degrees C. It was found from the analysis of kinetic data that the rate-determining step in 21-hydroxylation of progesterone in the steady state was the dissociation of product from P450 C21, whereas the conversion to deoxycortisol was the rate-determining step in the reaction of 17alpha-hydroxyprogesterone. The difference in the rate-determining steps in the reactions for the two substrates was clearly demonstrated in the pre-steady-state kinetics.

The rat neuronal nitric oxide synthase (nNOS) catalyzes two monooxygenase reactions successively from L-arginine (L-Arg) to L-citrulline (L-Cit) via N(omega)-hydroxy-L-arginine (OH-Arg) without most of OH-Arg leaving the substrate-binding site. In the steady-state reaction conditions, the amount of OH-Arg produced is about 1/30-1/50 that of L-Cit. We found in this study using nNOS purified from an Escherichia coli expression system that the ratio of the amount of OH-Arg to L-Cit (OH-Arg/L-Cit) increased to about 1 at low concentration of NADPH. In one cycle of the nNOS reaction, the decrease in NADPH concentration was found to reduce the rates of two monooxygenase reactions but had little effect on the rate constant of OH-Arg dissociation from the enzyme. The addition of NADP(+), the competitive inhibitor for NADPH, caused the decrease in the rates of monooxygenase reactions in a single cycle of the reaction and the increase in the ratio of OH-Arg/L-Cit in the steady state. At low CaM concentrations, the ratio of OH-Arg/L-Cit was about the same as that at high CaM. In a single cycle of the nNOS reaction, the rate of monooxygenation was not altered by the CaM concentration but the amount of metabolized L-Arg decreased with the decrease in CaM concentration, showing that the amount of active nNOS was regulated by complex formation between nNOS and CaM. It becomes clear that there are two regulatory mechanisms for the successive reaction of nNOS. One controls the rates of monooxygenations and the other controls the amount of active species of nNOS.

Human hepatic cytochrome P450 3A4 (CYP3A4) was expressed in yeast Saccharomyces cerevisiae. While the expression level was high as compared with other human hepatic cytochrome P450s, CYP3A4 showed almost no catalytic activity toward testosterone. Coexpression of CYP3A4 with yeast NADPH–P450 reductase did not give a full activity. Low monooxygenase activity of CYP3A4 was attributed to the insufficient reduction of heme iron of CYP3A4 by NADPH–P450 reductase. To enhance the efficiency of electron transfer from NADPH–P450 reductase to CYP3A4, a fused enzyme was constructed between CYP3A4 and yeast NADPH–P450 reductase. The rapid reduction of the heme iron of the fused enzyme by NADPH was observed. The fused enzyme showed a high testosterone 6β-hydroxylation activity with a sigmoidal velocity saturation curve. However, the coupling efficiency between NADPH utilization and testosterone 6β-hydroxylation was only 10%. Finally, coexpression of the fused enzyme and human cytochrome b5 was examined. A significant decrease in the Km value and a remarkable increase in the coupling efficiency were observed. Substrate-induced spectra revealed that the dissociation constant of the fused enzyme for testosterone significantly decreased with coexpression of human cytochrome b5. These results strongly suggest that human cytochrome b5 directly interacts with the CYP3A4 domain of the fused enzyme and modifies the tertiary structure of substrate binding pocket, resulting in tight binding of the substrate and high coupling efficiency.

Rat neuronal nitric oxide synthase (nNOS) was expressed in Escherichia coli and purified. Although the nitric oxide (NO) complex of the ferric heme was EPR-silent, photo-illumination at 5 K to the NO complex of the ferric nNOS in the substrate-free form produced a new high spin EPR signal similar to that of the ferric heme of N(omega)-nitro-L-arginine-bound nNOS, suggesting that the photo-dissociated NO might move away from the heme. Low photo-dissociability of NO in this complex indicated less restricted movement of the dissociated NO in the distal region of the heme, which might result in the rapid rebinding of the NO to the ferric heme at 5 K. In the presence of substrate L-arginine, derivatives, or product L-citrulline, the photo-products from the ferric NO complexes exhibited large novel EPR signals with a spin-coupled interaction between the ferric heme (S = 5/2) and the photolyzed NO (S = 1/2), suggesting a stereochemically restricted interaction between the photo-dissociated NO and the guanidino- or the ureido-group of the substrate analogues at the distal heme region of nNOS. The photo-product from the NO complex produced from citrulline-bound nNOS might be the same intermediate species as that formed in the last step of the catalytic cycle.

Several kinds of steroidogenic cytochrome P-450 (P-450) catalyze two- or three-step successive monooxygenase reactions in which the product of the preceding reaction is metabolized by the same enzyme without leaving the catalytic site. Adrenal androgen formation by P-45017α,lyase and aldosterone production by P-45011β consist of two and three successive monooxygenation reactions, respectively. We analyze here kinetic parameters of these reactions using the rapid quenching method. The analysis revealed the reaction pathways and the regulatory steps of the successive reactions. (I) Guinea pig P-45017α,lyase catalyzes androstenedione production from progesterone via 17α-hydroxyprogesterone as the intermediate. (II) Bovine P-45017α,lyase catalyzes a successive reaction for androgen formation from pregnenolone but not from progesterone, because the enzyme releases 17α-hydroxyprogesterone 50 times faster than 17α-hydroxypregnenolone. (III) Bovine P-45011β catalyzes a successive reaction for aldosterone production from 11-deoxycorticosterone via corticosterone and 18-hydroxycorticosterone as the intermediates, but not via 18-hydroxy-1l-deoxycorticosterone. (IV) The protein-protein interaction of P-45011β and P-450scc stimulates the release of the intermediate metabolite, corticosterone, from P-45011β. This quick release of the intermediate prevents aldosterone formation. It is demonstrated that release rate of intermediate metabolite from the enzyme regulates successive reactions of steroidogenic P-450s.

Copper binding selectivity of the N- and C-sites in serum (human)- and ovo-transferrin was investigated through copper binding constants, copper dissociation rate constants, and EPR spectra. At pH 7.4, stepwise copper binding constants of serum (human)-transferrin were K1 = 1.8 (±0.6) × 1012M−1and K2 = 1.2 (±0.5) × 1011M−1, and those of ovo-transferrin were K1 = 1.9 (±0.5) × 1011M−1 and K2 = 2.1 (±0.4) × 1011M−1. Absorbance changes resulting from copper binding to the C- or N-site at various ratios of Cu2+/apo-transferrin were separated by a kinetic method. It was clearly indicated that, in serum (human)-transferrin, the copper binding affinity for the C-site was much larger than that for the N-site, whereas in ovo-transferrin, the C- and N-sites have almost the same affinity for copper ions. In the presence of anions (0.1 M KCl or 0.1 M NaClO4), the stepwise copper binding constants of serum (human)-transferrin were almost 10-times smaller than those in the absence of the anions. The selectivity in binding the copper ions to both sites of serum (human)-transferrin in the presence of 0.1 M NaClO4 is much smaller than that in the presence of 0.1 M KCl or in the absence of anions. The copper dissociation rate constants from the N- and C-sites of dicupric serum-Tf were slightly changed by the addition of the anions (0.1 M KCl and 0.1 M NaClO4). EPR spectra of the copper ions of the N-site in dicupric serum-transferrin are dramatically changed respectively by the addition of 0.1 M KCl, 0.1 M NaCl, and 0.1 M NaClO4. This suggests that the change in the coordination geometry of the copper ions occurs at the N-site.

Immunolocalization for four steroidogenic enzymes was performed on an ovary taken from a Hokkaido brown bear during the mating season. This specimen is considered to be in the follicular phase because of the presence of large follicles. In large follicles, cholesterol side-chain cleavage (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were immunolocalized in theca interna cells and granulosa cells. 17 alpha-hydroxylase/C17-C20 lyase cytochrome P450 (P450c 17) was immunolocalized in theca interna cells but not in granulosa cells. Aromatase cytochrome P450 (P450arom) was immunolocalized only in granulosa cells. In medium follicles, however, P450scc and 3 beta HSD were immunolocalized only in theca interna cells, and the immunoreactivity of P450arom was detected in neither theca interna cells nor granulosa cells. Immunoreactivities of P450scc, 3 beta HSD and P450c 17 but not P450arom were detected in interstitial cells. This study suggests that estrogen biosynthesis takes place through interrelation between theca cells and granulosa cells and is explained by the so-called two-cell mechanism. Furthermore, the granulosa cells in large follicles have the capability for pregnenolone and progesterone biosynthesis, and the interstitial cell in the bear ovary is also a steroidogenic site.

The reaction mechanism of androstenedione formation from progesterone was analyzed in a membrane reconstituted system consisting of P-45017 alpha, lyase and NADPH-cytochrome P-450 reductase using a rapid quenching device at 10 degrees C. In these rapid quenching experiments, only the metabolites of [3H]progesterone bound to P-45017 alpha, lyase at the initial stage were detectable during the limited cycles of the P-45017 alpha, lyase reactions (1-120 s). The level of 17 alpha-hydroxy[3H]progesterone increased rapidly in a short period (1-5 s) and then decreased to about half. That of [3H]androstenedione increased gradually from 2 s, which exactly corresponded to the decrease in 17 alpha-hydroxy[3H]progesterone. 17 alpha-Hydroxyprogesterone was conclusively the actual intermediate steroid which did not dissociate from P-45017 alpha, lyase during the successive hydroxylation reaction into androstenedione. A kinetic model can clearly describe the successive reaction catalyzed by P-450 17 alpha, lyase, in which progesterone is converted successively into androstenedione via 17 alpha-hydroxyprogesterone, some of which dissociates from the active site of P-450 17 alpha, lyase and is never metabolized into androstenedione. We analyzed the effects of pH and the amount of NADPH-cytochrome P-450 reductase on the successive reaction and proved that the reaction was regulated by the rate of electron transfer for the conversion of the bound 17 alpha-hydroxyprogesterone to androstenedione. Furthermore, we found that the product dissociation from P-450 17 alpha, lyase is the rate-limiting process in the steady-state metabolism of progesterone by P-450 17 alpha, lyase.

3 beta-Hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD/I) and cytochrome P-450C21 were co-purified from bovine adrenocortical microsomes by an improved method. The 3 beta-HSD/I was successfully incorporated into liposomal membranes in which the enzyme activity was greatly stabilized. Enzymatic activities and kinetic parameters of the 3 beta-HSD/I proteoliposomes were almost the same as those of the solubilized form.

We have recently demonstrated that the avian brain as well as the peripheral steroidogenic glands produces pregnenolone, the main precursor of steroid hormones, on the basis of biochemical studies. Therefore, the immunohistochemical and Western immunoblotting analyses with a polyclonal antibody directed against the purified bovine adrenal cytochrome P450scc, which is involved in pregnenolone formation, was undertaken to investigate the localization of a cytochrome P450scc-like substance in the brain of adult male Japanese quails. P450scc-like immunoreactive cells were distributed in several telencephalic, diencephalic and mesencephalic regions. An intense immunoreaction was observed in somata of Purkinje cells and in the dendrites extending through the cerebellar molecular layer. Clusters of immunoreactive cell bodies were also detected in the hyperstriatum accessorium (HA), the ventral portions of both the archistriatum and the corticoid area, the preoptic area (POA), the anterior hypothalamus (AHy) and the dorsolateral thalamus (DL). Preadsorbing the antibody with cytochrome P450scc resulted in a complete absence of P450scc-like immunoreactivity in all of positively stained cells. On Western immunoblotting with the P450scc antibody, a P450scc-like substance was present in homogenates of the several brain regions that contain immunohistochemically stained cells. Immunohistochemical experiments using both antibodies against P450scc and the glial fibrillary acidic protein (GFA), a specific marker of glial cells, indicated that many HA cells contained both P450scc-like and GFA-like immunoreactivities. However, no immunoreactivity for GFA was observed in Purkinje cells and the cells localized in the ventral portion of the corticoid area, despite the presence of P450scc-like immunoreactivity. These results confirm our previous findings of pregnenolone biosynthesis and suggest the presence of a P450scc-like substance in both neuronal and glial cells of the quail brain.

Androstenedione formation from progesterone by P-450(17 alpha,lyase) was investigated in ovarian microsomes of immature rats treated with pregnant mare serum gonadotropin. Successive monooxygenase reactions in the formation of androstenedione without the intermediate leaving P-450(17 alpha,lyase) were demonstrated by a double-substrate double-label experiment using [14C]progesterone and 17 alpha-[3H]hydroxyprogesterone as substrates and also by specific reduction in the concentration of intermediate 17 alpha-hydroxyprogesterone in the reaction medium by reaction of liposomal P-450C21. A detailed kinetic study on the reactions of P-450(17 alpha,lyase) in microsomes was conducted in the steady state. Kinetic parameters indicated the C17,C20-lyase reaction for 17 alpha-hydroxyprogesterone (Km = 80 nM) to be strongly inhibited by progesterone (Ki = 8 nM). In the presence of a high concentration of progesterone, as in the case of in vivo rat ovary, most androstenedione is concluded to be formed directly from progesterone by successive monooxygenase reactions catalyzed by P-450(17 alpha,lyase). 20 alpha-Dihydroprogesterone competitively inhibited the C17,C20-lyase reaction for 17 alpha-hydroxyprogesterone with Ki = 23 nM, but had only slight effect on progesterone metabolism to androstenedione. 20 alpha-Dihydroprogesterone, thus, cannot be a regulator for androstenedione formation in rat ovary.

Studies were done to determine the role of the 17 alpha-hydroxylase in the conversion of 7 alpha-thiospironolactone (7 alpha-thio-SL) to a reactive metabolite causing the degradation of testicular cytochrome P-450. Incubation of guinea pig testicular microsomes with 7 alpha-thio-SL plus NADPH resulted in an approx. 70% decline in cytochrome P-450 content and even greater loss of 17 alpha-hydroxylase activity. Addition of the 17 alpha-hydroxylase inhibitor, SU-10'603, to the incubation medium prevented the degradation of P-450 by 7 alpha-thio-SL. Similarly, preincubation of testicular microsomes with anti-P-45017 alpha,lyase IgG to inhibit 17 alpha-hydroxylation, diminished the subsequent loss of P-450 caused by 7 alpha-thio-SL. The results indicate that the 17 alpha-hydroxylase catalyzes the conversion of 7 alpha-thio-SL to the reactive metabolite responsible for P-450 destruction. The accompanying loss of 17 alpha-hydroxylase activity supports the hypothesis that suicide inhibition is the mechanism involved.