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PubMed2001

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares.

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.

3-Hydroxysteroid Dehydrogenases↗
PubMed2001

Porcine gonadal and placental isozymes of aromatase cytochrome P450: sub-cellular distribution and support by NADPH-cytochrome P450 reductase.

Functional differences exist between the porcine gonadal and placental aromatase cytochrome P450 (P450arom) isozymes that are encoded by separate genes. The present experiments investigated the sub-cellular location of these isozymes, their dependence on the redox partner protein NADPH-cytochrome P450 reductase (P450 reductase) for catalytic activity and the release of steroid intermediates during the aromatization of androgens to estrogen. After differential centrifugation, similar levels of gonadal and placental porcine P450arom were found along with P450 reductase in the microsomal compartment using activity and immunoblot analyses. Activity was stimulated much more by recombinant P450 reductase addition, and higher levels of 19-hydroxy and 19-oxo intermediates accumulated during androstenedione and testosterone metabolism, in cells expressing the gonadal compared to the placental isozyme. No other steroid products were identified by HPLC. Thus, the porcine gonadal P450arom is more sensitive to P450 reductase deprivation than is the placental P450arom, accounting in part for catalytic differences between these isozymes.

Androgens↗
PubMed2004

Cytochrome c oxidase subunit IV as a marker of protein kinase Cepsilon function in neonatal cardiac myocytes: implications for cytochrome c oxidase activity.

We have previously demonstrated that low concentrations of phorbol esters stimulate the selective translocation of protein kinase C (PKC) alpha and epsilon from the cell soluble to the particulate fraction in NCMs (neonatal rat cardiac myocytes). We therefore determined if the in vitro phosphorylation of substrates in these fractions could be used as assays of PKCalpha or epsilon activation. Intact cell phorbol ester treatment caused a decline in the in vitro (32)P-incorporation into several proteins in the cell-soluble fraction. These declines occurred in the presence or absence of in vitro Ca(2+) and probably reflected the exit of PKC isoenzymes from the soluble fraction. In contrast, an approx. 18 kDa protein incorporated (32)P in particulate fractions isolated from 4beta-PMA-treated cells in a Ca(2+)-independent manner. Proteomic and immunoprecipitation analyses indicated that the protein is subunit IV of the cytochrome c oxidase complex (COIV). In vitro phosphorylation of COIV was attenuated by PKC pseudosubstrate peptides. Introduction of an PKCepsilon-selective translocation inhibitor [Johnson, Gray, Chen and Mochly-Rosen (1996) J. Biol. Chem. 271, 24962-24966] into NCMs before 4beta-PMA treatments also attenuated the in vitro phosphorylation of COIV. In mitochondrial extracts from 4beta-PMA-treated NCMs, the PKCepsilon isoenzyme coimmunoprecipitated with COIV, and cytochrome c oxidase activity was enhanced 2-fold. The in vitro phosphorylation of COIV reflects a novel approach for monitoring PKCepsilon function in NCMs. Furthermore, PKCepsilon probably interacts with COIV in NCM mitochondria to enhance electron-transport chain complex IV activity.

Animals↗
PubMed1977

Oxygen intermediates and mixed valence states of cytochrome oxidase: infrared absorption difference spectra of compounds A, B, and C of cytochrome oxidase and oxygen.

A study of the near-infrared absorption spectra of three oxygen compounds of membrane-bound cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase; EC 1.9.3.1) shows that the formation of compound A (oxycytochrome oxidase) causes no significant infrared absorbance changes at -103 degrees. At -64 degrees, the formation of compound C from the mixed-valence state of the oxidase leads to increased absorption at 740-750 nm. The formation of compound B at -84 degrees from the fully reduced state of the oxidase causes increased absorption at 790-800 nm. Further oxidation of cytochrome oxidase results in increased infrared absorption at 820-830 nm at -60 degrees. The position of the infrared absorption band in compound C thus depends at least upon the oxidation-reduction state of heme a and its associated copper atom. Compound C contains two types of oxidized (cupric) copper; that associated with heme a is initially oxidized, and that associated with heme a3 is oxidized as a second step in the reaction with oxygen. Compound C exhibits a unique intense absorption band at 606-609 nm that is tentatively assigned to a charge transfer interaction between heme a3 in the reduced state and its associated copper in the oxidized state, with heme a and its associated copper in the oxidized state.

Chemical Phenomena↗
PubMed1999

Redox components of cytochrome bc-type enzymes in acidophilic prokaryotes. I. Characterization of the cytochrome bc1-type complex of the acidophilic ferrous ion-oxidizing bacterium Thiobacillus ferrooxidans.

The redox components of the cytochrome bc1 complex from the acidophilic chemolithotrophic organism Thiobacillus ferrooxidans were investigated by potentiometric and spectroscopic techniques. Optical redox titrations demonstrated the presence of two b-type hemes with differing redox midpoint potentials at pH 7.4 (-169 and + 20 mV for bL and bH, respectively). At pH 3.5, by contrast, both hemes appeared to titrate at about +20 mV. Antimycin A, 2-heptyl-4-hydroxyquinoline N-oxide, and stigmatellin induced distinguishable shifts of the b hemes' alpha-bands, providing evidence for the binding of antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide near heme bH (located on the cytosolic side of the membrane) and of stigmatellin near heme bL (located on the periplasmic side of the membrane). The inhibitors stigmatellin, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, and 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone affected the EPR spectrum of the Rieske iron-sulfur center in a way that differs from what has been observed for cytochrome bc1 or b6f complexes. The results obtained demonstrate that the T. ferrooxidans complex, although showing most of the features characteristic for bc1 complexes, contains unique properties that are most probably related to the chemolithotrophicity and/or acidophilicity of its parent organism. A speculative model for reverse electron transfer through the T. ferrooxidans complex is proposed.

Antimycin A↗
PubMed2003

Liver-specific deletion of the NADPH-cytochrome P450 reductase gene: impact on plasma cholesterol homeostasis and the function and regulation of microsomal cytochrome P450 and heme oxygenase.

A mouse model with liver-specific deletion of the NADPH-cytochrome P450 reductase (Cpr) gene (designated Alb-Cre/Cprlox mice) was generated and characterized in this study. Hepatic microsomal CPR expression was significantly reduced at 3 weeks and was barely detectable at 2 months of age in the Alb-Cre+/-/Cprlox+/+ (homozygous) mice, with corresponding decreases in liver microsomal cytochrome P450 (CYP) and heme oxygenase (HO) activities, in pentobarbital clearance, and in total plasma cholesterol level. Nevertheless, the homozygous mice are fertile and are normal in gross appearance and growth rate. However, at 2 months, although not at 3 weeks, the homozygotes had significant increases in liver weight, accompanied by hepatic lipidosis and other pathologic changes. Intriguingly, total microsomal CYP content was increased in the homozygotes about 2-fold at 3 weeks and about 3-fold at 2 months of age; at 2 months, there were varying degrees of induction in protein (1-5-fold) and mRNA expression (0-67-fold) for all CYPs examined. There was also an induction of HO-1 protein (nearly 9-fold) but no induction of HO-2. These data indicate the absence of significant alternative redox partners for liver microsomal CYP and HO, provide in vivo evidence for the significance of hepatic CPR-dependent enzymes in cholesterol homeostasis and systemic drug clearance, and reveal novel regulatory pathways of CYP expression associated with altered cellular homeostasis. The Alb-Cre/Cprlox mouse represents a unique model for studying the in vivo function of hepatic HO and microsomal CYP-dependent pathways in the biotransformation of endogenous and xenobiotic compounds.

Alleles↗
PubMed1997

Mitochondrial ubiquinol-cytochrome c reductase and cytochrome c oxidase: chemotherapeutic targets in malarial parasites.

In order to demonstrate that the mitochondrial electron transport system may be a target for antimalarial drug design in the human malarial parasite Plasmodium falciparum, ubiquinol-cytochrome c reductase and cytochrome c oxidase were purified from mitochondria of the parasite cultivated in vitro. It was found that the catalytic efficiency of the two enzymes from the malarial parasite were markedly lower than those from mouse liver mitochondria. The classical inhibitors affecting different quinone binding sites of the mammalian reductase, antimycin and myxothiazole, which had little antimalarial activities on P.falciparum growth in vitro, were found to exhibit little inhibitory effect against the parasite reductase. The malarial parasite reductase was more sensitive to inhibition by the antimalarial drug, 2-[trans-4-(4'-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone, than the mammalian enzyme, suggesting both the therapeutic potential of the target and the drug.

Animals↗
PubMed1990

Expression of bovine cytochrome P450c21 and its fused enzymes with yeast NADPH-cytochrome P450 reductase in Saccharomyces cerevisiae.

Recombinant plasmids for expression of bovine cytochrome P450c21 (pA gamma 2), both P450c21 and yeast NADPH-cytochrome P450 reductase (pAR gamma 1), P450c21/yeast reductase fused enzymes (pAF gamma R1, pAF gamma R2, and pAF gamma R20), and yeast reductase/P450c21 fused enzymes (pAFR gamma 1 and pAFR gamma 2) were constructed by using expression vector pAAH5. The plasmids were each introduced into the yeast Saccharomyces cerevisiae AH22 cells. The recombinant yeast strains AH22/pA gamma 2 (Y21) and AH22/pAR gamma 1 (Y21R) produced 2-3 X 10(3) molecules of P450c21 per cell. The cultures of both strains converted progesterone and 17 alpha-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively. The 21-hydroxylase activity per cell of the strain Y21R was about three times higher than that of the strain Y21, probably due to overproduction of yeast reductase. The recombinant yeast strains AH22/pAF gamma R1 (Y21RF1), AH22/pAF gamma R2 (Y21RF2), and AH22/pAF gamma R20 (Y21RF20) produced about 1.1-2.0 X 10(4) molecules per cell of the corresponding P450c21/yeast reductase fused enzymes. The specific 21-hydroxylase activity toward 17 alpha-hydroxyprogesterone per cell of the strains Y21RF1, Y21RF2, and Y21RF20 was about 21, 28, and 49 times higher than that of the strain Y21, respectively. Thus, the fused enzymes were superior to P450c21 in the specific activity and in the expression level in the yeast. The Km values for 17 alpha-hydroxyprogesterone of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 0.29, 0.30, 0.67, and 0.65 microM, respectively. The Vmax values of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 28, 124, 151, and 222 moles/min.mole P450c21 or fused enzyme, respectively. These results indicated that the fused enzymes showed lower affinity for the substrate, probably due to structural modification and higher reaction rates through efficient intramolecular electron transfer as compared with those of P450c21. While the strain AH22/pAFR gamma 2 (YR21F2) produced about 3 X 10(4) molecules per cell of the reductase/P450c21 fused enzyme, the specific 21-hydroxylase activity of the fused enzyme toward 17 alpha-hydroxyprogesterone was extremely low, suggesting that the structure of the fused enzyme might not be suited for electron transfer in yeast microsomes.

17-alpha-Hydroxyprogesterone↗
PubMed1984

The sequence of the gene for cytochrome c oxidase subunit I, a frameshift containing gene for cytochrome c oxidase subunit II and seven unassigned reading frames in Trypanosoma brucei mitochondrial maxi-circle DNA.

A 9.2 kb segment of the maxi-circle of Trypanosoma brucei mitochondrial DNA contains the genes for cytochrome c oxidase subunits I and II (coxI and coxII) and seven Unassigned Reading Frames ("URFs"). The genes for coxI and coxII display considerable homology at the aminoacid level (38 and 25%, respectively) to the corresponding genes in fungal and mammalian mtDNA, the only striking point of divergence being an unusually high cysteine content (about 4.5%). The reading frame coding for cytochrome c oxidase subunit II is discontinuous: the C-terminal portion of about 40 aminoacids, is present in the DNA-sequence in a -1 reading frame with respect to the N-terminal moiety. URF5, 8 and 10, show a low but distinct homology (about 20%) to mammalian mitochondrial URF-1, 4 and 5, respectively. In URF5, the first AUG is found at codon 145, whereas extensive homology to mammalian URF-1 sequences occurs upstream of this position. The possibility exists that UUG can serve as an initiator codon. URF7 and URF9 have a highly unusual aminoacid composition and do not possess AUG or UUG initiator codons. These URFs probably do not have a protein-coding function. The segment does not contain conventional tRNA genes.

Amino Acid Sequence↗
PubMed2000

Developmental expression of cytochrome P450 side-chain cleavage and cytochrome P450 17 alpha-hydroxylase messenger ribonucleic acid and protein in the neonatal hamster ovary.

The temporal and spatial expression of cytochrome P450 side-chain cleavage (CYP11A1) and cytochrome P450 17 alpha-hydroxylase (CYP17) mRNA and protein during thecal cell differentiation in developing hamster ovaries were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence histochemistry, respectively. Ovaries were collected from 15-day fetal through 20-day-old postnatal hamsters and used either for immunofluorescence detection of enzyme protein or RT-PCR evaluation of enzyme mRNA. Immunoreactivity of CYP11A1 first appeared in the interstitial cells on Day 10 postnatal (PN), and the intensity increased significantly with further ovarian development beyond 11 days of age. In contrast, CYP17 immunostaining was first detected in a few interstitial cells closer to large preantral follicles by Day 12 PN, and their number increased appreciably by Day 14 PN. By age 18-20 days, CYP17-positive cells were localized primarily in the thecal layer of large preantral follicles. A low level of CYP17 and CYP11A1 mRNA was present in fetal ovaries. The CYP17 mRNA levels increased sharply by Day 1 PN but decreased to a low baseline level by Day 2 PN and remained low up to Day 9 PN. Both CYP11A1 and CYP17 mRNA levels increased significantly by Day 10 PN compared to Day 9 PN; however, the increase for CYP11A1 was greater than CYP17. The CYP11A1 mRNA levels decreased noticeably on Day 11 PN and remained relatively stable until Day 14 PN; however, mRNA levels started increasing by Day 15 PN and increased sharply by Day 17 PN onward, corresponding to the increase in CYP11A1 protein in the ovarian interstitium and thecal compartments. On the other hand, CYP17 mRNA expression increased progressively through Day 12 PN. A sharp increase in CYP17 mRNA was noted on Day 13 PN in conjunction with the morphological development of thecal cells; mRNA levels remained steady afterward. The correlation of the increase in enzyme mRNA and protein, especially of CYP17, with the morphological development of thecal layers suggests that the differentiation of interstitial cells into theca may be modulated by multilayered preantral follicles, and the expression of enzyme protein occurs prior to an increase in serum LH.

Animals↗
PubMed1998

A cytochrome cbb3 (cytochrome c) terminal oxidase in Azospirillum brasilense Sp7 supports microaerobic growth.

Spectral analysis indicated the presence of a cytochrome cbb3 oxidase under microaerobic conditions in Azospirillum brasilense Sp7 cells. The corresponding genes (cytNOQP) were isolated by using PCR. These genes are organized in an operon, preceded by a putative anaerobox. The phenotype of an A. brasilense cytN mutant was analyzed. Under aerobic conditions, the specific growth rate during exponential phase (mu(e)) of the A. brasilense cytN mutant was comparable to the wild-type specific growth rate (m(e) of approximately 0.2 h-1). In microaerobic NH4+-supplemented conditions, the low respiration of the A. brasilense cytN mutant affected its specific growth rate (mu(e) of approximately 0.02 h-1) compared to the wild-type specific growth rate (mu(e) of approximately 0.2 h-1). Under nitrogen-fixing conditions, both the growth rates and respiration of the wild type were significantly diminished in comparison to those under NH4+-supplemented conditions. Differences in growth rates and respiration between the wild type and the A. brasilense cytN mutant were less pronounced under these nitrogen-fixing conditions (mu(e) of approximately 0.03 h-1 for the wild type and 0.02 h-1 for the A. brasilense cytN mutant). The nitrogen-fixing capacity of the A. brasilense cytN mutant was still approximately 80% of that determined for the wild-type strain. This leads to the conclusion that the A. brasilense cytochrome cbb3 oxidase is required under microaerobic conditions, when a high respiration rate is needed, but that under nitrogen-fixing conditions the respiration rate does not seem to be a growth-limiting factor.

Aerobiosis↗
PubMed1989

Aromatase cytochrome P450 and cholesterol side-chain cleavage cytochrome P450 in corpora lutea of pregnant rats: diverse regulation by peptide and steroid hormones.

In previous studies we have shown that aromatase cytochrome P450 (P450arom) mRNA and protein increase markedly in luteal tissue between days 10-19 of gestation, whereas cholesterol side-chain cleavage cytochrome P450 (P450scc) appears to be constitutively maintained regardless of hormonal changes occurring during pregnancy. To identify pituitary and placental hormones that regulate these two P450 enzymes in the rat corpus luteum, serum LH activity and pituitary PRL release were selectively inhibited by administration of LH antiserum (LH-Ab) or CB-154, respectively. Placental hormones were removed by hysterectomy. Hormonal activities were replaced by the administration of hCG, PRL, testosterone (T), or estradiol (E), given individually or in combination. Induction of aromatase mRNA transcripts (3.3, 2.6, and 1.9 kilobases) and protein (54,000 mol wt) between days 10-15 of gestation was blocked by either surgical hysterectomy or LH-Ab treatment. Hysterectomy on day 10 combined with CB-154 abolished not only aromatase mRNA, but also markedly reduced P450scc mRNA (2.0 kilobases) by day 12. Induction of aromatase was partially restored in the day 10-15 hysterectomized rats by treatment with PRL plus E (most effective), PRL plus T, or PRL alone, but not by either T or E alone. Similar results were observed 2 days after hysterectomy (day 12), except that hysterectomy alone caused a transient 3.5-fold increase in P450arom mRNA and protein, most likely due to a transient release of pituitary LH. Aromatase mRNA and protein were also increased in intact pregnant rats treated with hCG between days 10-12. However, no effect of hCG was observed before (days 8-10) or after (days 13-19) midgestation. Likewise, LH-Ab had no effect if given after day 13. Despite hormone-specific regulation of the content of aromatase protein, E biosynthesis in vitro was not strictly related to aromatase enzyme content. We conclude that aromatase mRNA and protein are maintained by PRL at a low level of expression in the first half of pregnancy, can be modulated by LH at midgestation, and are subsequently induced to high levels in the second half of gestation by placental factors (rat placental lactogen-1 and T) and the conversion of T to E in the corpus luteum. P450scc appears to be constitutively maintained. Thus, two P450 genes known to be regulated by LH/cAMP in the rat follicle are controlled by diverse peptide and steroid signal transduction mechanisms in the corpus luteum.

Animals↗
PubMed1987

Noncoordinate regulation of de novo synthesis of cytochrome P-450 cholesterol side-chain cleavage and cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase in mouse Leydig cell cultures: relation to steroid production.

The role of cAMP in the regulation of the amount and synthesis of cytochrome P-450 cholesterol side-chain cleavage (P-450scc) and cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase (P-450(17 alpha) was investigated in mouse Leydig cell cultures. In the absence of cAMP, the amount of immunoreactive P-450(17 alpha) decreased to less than 5% by day 4 and was undetectable between days 7 and 11. In contrast, the amount of immunoreactive P-450scc remained relatively constant throughout the same period. Treatment of Leydig cell cultures for 4 days with 0.05 mM 8-bromo-cAMP initiated on day 7 increased the amount of P-450(17 alpha) with relatively little effect on the amount of P-450scc. The rate of de novo synthesis of each of the P-450 enzymes was studied by determining [35S]methionine incorporation into newly synthesized protein. In the absence of cAMP, de novo synthesis of P-450(17 alpha) ceased while the rate of de novo synthesis of P-450scc increased with time in culture between days 2 and 11. Treatment with cAMP initiated on day 7 of culture caused a time-dependent increase in the rate of de novo synthesis of P-450(17 alpha) on days 9 and 11 equivalent to 40% and 60%, respectively, of that observed in freshly isolated Leydig cells. The rate of de novo synthesis of P-450scc was increased 2-fold relative to untreated cultures on days 9 and 11. De novo synthesis of P-450(17 alpha) ceased when cAMP was removed on day 11 and restored when cAMP was added again on day 13 of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Mitochondrial inner membrane bound Pet1402 protein is rapidly imported into mitochondria and affects the integrity of the cytochrome oxidase and ubiquinol-cytochrome c oxidoreductase complexes.

In vitro synthesized Pet1402 precursor protein is very rapidly and efficiently imported into isolated mitochondria. The import depends on a membrane potential and functional mtHsp70. The mitochondrial targeting sequence of the Pet1402 precursor protein is removed by the matrix processing peptidase MPP and the mature protein is firmly embedded in the inner mitochondrial membrane. The Pet1402 protein is required for the integrity of the cytochrome oxidase and ubiquinol-cytochrome c oxidoreductase complexes.

Biological Transport↗
PubMed1997

Immunolocalization of 3 beta-hydroxysteroid dehydrogenase, cytochrome P450 17 alpha-hydroxylase/17,20-lyase and cytochrome P450 aromatase in the equine corpus luteum of dioestrus and early pregnancy.

The onset of equine chorionic gonadotrophin (eCG) secretion in pregnant mares is associated with an increase in luteal androgen and oestrogen production. The luteal cell type(s) responsible for the increased production of androgens and oestrogens has not been identified in the equine corpus luteum. In this study, we examined the pattern of expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450(17 alpha)) and cytochrome P450 aromatase (P450arom) by immunohistochemistry in equine luteal tissue collected during dioestrus (days 7-10; n = 4) and early pregnancy, before (days 29-35; n = 4) and after (days 39-45; n = 4) the onset of endogenous eCG secretion. All luteal cells expressed 3 beta-HSD, P450(17 alpha) and P450arom. The distribution of 3 beta-HSD, P450(17 alpha) and P450arom did not differ with stage of the reproductive cycle. The intensity of immunohistochemical staining for 3 beta-HSD did not appear to differ with reproductive stage. In contrast, the intensity of immunostaining for P450(17 alpha) increased after the onset of eCG secretion. The intensity of immunostaining for P450arom increased during pregnancy before the onset of eCG secretion and diminished after the onset of eCG secretion to the intensity seen in dioestrous corpora lutea. This finding suggests that androgen and oestrogen production is not compartmentalized within the equine corpus luteum. Both large and small luteal cells express the steroidogenic enzymes necessary for oestrogen production, and the intensity of immunostaining for P450(17 alpha) and P450arom appears to be stage-specific.

3-Hydroxysteroid Dehydrogenases↗
PubMed2002

Genetically engineered bacterial cells co-expressing human cytochrome P450 with NADPH-cytochrome P450 reductase: prediction of metabolism and toxicity of drugs in humans.

Genetically engineered bacterial cells expressing human cytochrome P450 (CYP) have been developed as new tools to predict the metabolism and toxicity of drugs in humans. There are various host cells for the heterologous expression of a form of CYP. Among them, bacterial cells such as Escherichia coli (E. coli) have advantages with regard to ease of use and high yield of protein. CYP protein could be first expressed by the modification of the N-terminal amino acid sequence in E. coli cells in 1991. Since then, many forms of human CYP have been successfully expressed in E. coli cells. Since the E. coli cells do not possess endogeneous electron transport systems to support the full catalytic activity of CYP, E. coli strains co-expressing both human CYP and NADPH-cytochrome P450 reductase (OR) have been established. Each form of CYP expressed in the E. coli cells efficiently catalyzed the oxidation of a representative substrate at an efficient rate, indicating that the OR was sufficiently expressed to support the catalytic activity of CYP. According to the studies performed so far, the modification of the N-terminal amino acid sequence of CYP did not seem to affect the catalytic properties of CYP. The human CYP expressed in the E. coli cells were applicable for studies to determine a metabolic pathway(s) of drugs and to estimate kinetic parameters of drug metabolism by human CYP. Drug-drug interactions caused by inhibition of the metabolism of drugs by human CYP could also be examined by in vitro inhibition studies with CYP expressed in the E. coli cells. Recently, human CYP was co-expressed with the OR in Salmonella typhimurium (S. typhimurium) cells used for mutation assay (Ames test) by applying the technology for the expression of human CYP and the OR in E. coli cells, to evaluate whether chemicals including drugs are metabolically activated by human CYP and show mutagenicity. These strains of bacteria are considered as useful tools to study the metabolism and the toxicity of drugs in humans.

Journal Article↗
PubMed1992

Relationship between cardiolipin content and cytochrome c oxidase activity of cytochrome aa3 in Paracoccus denitrificans.

Using cultivation at different oxygen tensions, the molar ratio of cardiolipin to haem a in cells of Paracoccus denitrificans was varied systematically from 30 to 300. The molecular activity of cytochrome aa3 (with N, N, N', N'-tetramethyl-p-phenylenediamine as substrate) remained unchanged in this interval, this ruling out any regulatory effect of physiological cardiolipin levels on the terminal oxidase. Titration of anaerobically grown cells with cyanide indicated the presence of cytochrome aa3 which accounted for about 1/4 of the total electron flow from TMPD to oxygen.

Aerobiosis↗
PubMed1987

Reconstitution of monomeric cytochrome c oxidase into phospholipid vesicles yields functionally interacting cytochrome aa3 units.

When the carbon monoxide complex of fully reduced cytochrome c oxidase, reconstituted into liposomes, is mixed with oxygen-containing buffer, complex kinetic progress curves are observed. This pattern is seen irrespective of whether the oxidase used in reconstitution is the dimeric or monomeric (subunit III-depleted) enzyme. These findings are interpreted in the light of similar experiments on the detergent-solubilized enzyme reported by Gibson and Greenwood (Gibson, Q.H., and Greenwood, C. (1963) Biochem. J. 86, 541-554) and confirmed by ourselves. We conclude that reconstitution of monomeric (subunit III-less) enzyme yields, preferentially, vesicles containing more than one functional unit, possibly associated as dimers. This result is of significance to our understanding of the relationships between aggregation state and proton pumping capacity of cytochrome oxidase.

Animals↗