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R Bernhardt

Publications and source records attributed to R Bernhardt.

At least 19 recordsLinked to original sources
PubMed1997

Pro108 is important for folding and stabilization of adrenal ferredoxin, but does not influence the functional properties of the protein.

The truncated mutant Met-adrenodoxin-(4-107)-peptide of bovine adrenal ferredoxin was expressed as apoprotein in Escherichia coli BL21 and could be reconstituted to the holoform by chemical or enzymatic methods. The reconstituted protein had spectroscopic, functional and redox properties similar to the Met-adrenodoxin-(4-108)-peptide of adrenal ferredoxin, into which the cluster was inserted upon expression in the same Escherichia coli strain. Rate of in vitro cluster insertion into the Met-adrenodoxin-(4-107) apoprotein was much lower than for the Met-adrenodoxin-(4-108) apoprotein under identical conditions. Comparative thermodynamic studies with the Met-adrenodoxin-(4-108)-peptide indicated that removal of Pro108 resulted in an extensive decrease of the overall stability of the protein in either oxidation state. The Met-adrenodoxin-(4-107)-peptide showed a higher sensitivity to urea denaturation and had a sensibly lower denaturation temperature, 44.8 degrees C, compared with 51.7 degrees C for mutant Met-adrenodoxin-(4-108). The stability of the reduced state of both mutants is slightly lower than that of the oxidized state indicating that this protein region does not undergo major structural changes upon reduction.

Adrenal Glands↗
PubMed1997

Specific aspects of electron transfer from adrenodoxin to cytochromes p450scc and p45011beta.

An analysis of the electron transfer kinetics from the reduced [2Fe-2S] center of bovine adrenodoxin and its mutants to the natural electron acceptors, cytochromes P450scc and P45011beta, is the primary focus of this paper. A series of mutant proteins with distinctive structural parameters such as redox potential, microenvironment of the iron-sulfur cluster, electrostatic properties, and conformational stability was used to provide more detailed insight into the contribution of the electronic and conformational states of adrenodoxin to the driving forces of the complex formation of reduced adrenodoxin with cytochromes P450scc and P45011beta and electron transfer. The apparent rate constants of P450scc reduction were generally proportional to the adrenodoxin redox potential under conditions in which the protein-protein interactions were not affected. However, the effect of redox potential differences was shown to be masked by structural and electrostatic effects. In contrast, no correlation of the reduction rates of P45011beta with the redox potential of adrenodoxin mutants was found. Compared with the interaction with P450scc, however, the hydrophobic protein region between the iron-sulfur cluster and the acidic site on the surface of adrenodoxin seems to play an important role for precise complementarity in the tightly associated complex with P45011beta.

Adrenodoxin↗
PubMed1997

Superoxide-mediated reduction of the nitroxide group can prevent detection of nitric oxide by nitronyl nitroxides.

Nitronyl nitroxides (NN), a class of compounds which react with nitric oxide forming imino nitroxides, were applied in different systems for the detection of nitric oxide. Addition of a NN to planar monolayers of bovine aortic endothelial cells (BAEC) activated by Ca2+ ionophore A23187 immediately resulted in a strong decrease of the ozone-mediated .NO chemiluminescence. Simultaneously, a rapid diminution of the electron spin resonance (ESR) signal intensity of the NN (without detectable formation of the corresponding imino nitroxide) was observed; superoxide dismutase partially inhibited this decrease in the NN concentration. Model experiments using hypoxanthine/xanthine oxidase in aqueous solution and KO2 in dimethylsulfoxide as sources of O2.- revealed that there is a rapid reduction of nitronyl nitroxides by superoxide. The second order rate constant for the reaction of the water soluble NN with O2.- was determined to be 8.8 x 10(5) M-1s-1, which is more than two orders of magnitude higher than the value reported previously for reaction with .NO (Woldman et al., BBRC 202, 195-203, 1994). Reduction of the nitronyl nitroxide was also observed in the presence of glutathione, ascorbic acid or rabbit liver microsomes. Incorporation of both nitronyl and imino nitroxides into liposomes strongly decreased reduction by superoxide and other reductants, however, in the presence of microsomes, there was no protective effect by liposomal encapsulation of NN. The results indicate that in biological systems (in addition to other reducing agents) the presence of superoxide can prevent the detection of nitric oxide using nitronyl nitroxides.

Animals↗
PubMed1996

Expression of cytochrome P45011B1 mRNA in the brain of normal and hypertensive transgenic rats.

Cytochrome P45011B1 (11 beta-hydroxylase) was detected in the brain of male rats by in situ hybridization methods. Normal Sprague-Dawley rats were compared to the transgenic strain TGR(mRen2)27, characterized by the expression of the murine Ren-2d renin gene and the development of severe hypertension. Specific riboprobes were generated by in the vitro transcription of a 152 base-pair long cDNA template 35S-labeled riboprobes were hybridized to cryostat sections from adrenal glands and from two different levels of the brain using standard protocols and varying washing conditions. After exposure of the radiolabeled sections to X-ray film, the signals were quantified and compared. Following autoradiography and counterstaining, cytochrome P45011B1 mRNA was clearly localized in the zona fasciculata/reticularis of the adrenal cortex and in distinct layers of the cerebral cortex. High signal densities were obtained in the layers II-IV of the neocortex and in the layer II of the piriform cortex, although the concentrations of cytochrome P45011B1 mRNA were remarkably lower in the central nervous system as compared to adrenal glands. As revealed by the semi-quantitative analysis, there was a slight increase in adrenal 11 beta-hydroxylase mRNA in the transgenic rats, whereas the brain seems to express nearly the same amount of this enzyme in both strains. The cytochrome P45011B1 mRNA expression in distinct cells, probably nerve cells, and especially in regions with high densities of glucocorticoid receptors points to a possible function of brain derived corticosterone in receptor activation.

Adrenal Glands↗
PubMed1996

Reversible, non-denaturing metal substitution in bovine adrenodoxin and spinach ferredoxin and the different reactivities of [2Fe-2S]-cluster-containing proteins.

The non-denaturing substitution of cluster iron by other metals was studied in spinach ferredoxin and in bovine adrenodoxin. Only some of several metal species tested (Cd2+, Zn2+, VO2+, Mn2+, Co2+, Ni2+) caused bleaching of the residual visible absorbance and of the EPR signals of the reduced ferredoxins. No formation of mixed-metal cluster was observed. The most reactive metal species were Cd2+ and Zn2+ and Cd2+ was found to react also with oxidized adrenodoxin. Metal-treated proteins were resolved into a mixture of apoprotein, metal-substituted protein and unreacted holoprotein. Their biological activity was proportional to the residual holoprotein concentration. Spinach ferredoxin and adrenodoxin were found to differ substantially with regard to their metal-substitution reactivity under oxidizing and reducing conditions, reaction time, and formation of apoprotein, which was more pronounced for spinach ferredoxin. Exchange of cluster iron with Cd2+ in adrenodoxin generated stable species containing 2 mol sulfide/mol protein and 2 or 5 mol cadmium/mol protein, respectively. The relative amount of the two substitution products depended on the experimental conditions. CD and NMR data on all the cadmium-substituted proteins suggest that iron replacement led to a significant structural rearrangement. Nevertheless, all the metal-substituted proteins could be re-converted into the native iron-containing form upon incubation with iron in the absence of reductants, of denaturing agents, and of an external source of sulfide. The different reactivity of the two proteins is discussed in terms of the cluster environment, along with the possible physiological relevance of these findings.

Adrenodoxin↗
PubMed1996

Differential regulation of 11 beta-hydroxylase and aldosterone synthase in human adrenocortical H295R cells.

In humans the last steps in the synthesis of aldosterone and cortisol rely on the activity of two cytochrome P450 genes termed CYP11B2 (aldosterone synthase; P450aldo) and CYP11B1 (11 beta hydroxylase; P450cl1). The mechanisms which lead to differential expression of these two genes within the adrenal cortex are not well-defined. The human adrenocortical cell line. H295R, was utilized in this study to examine the intracellular second messenger pathways regulating expression of P450aldo and P450c11. using specific ribonuclease protection assays. Treatment of H295R cells with angiotensin II or potassium (K+) caused a time-dependent induction in the level of P450aldo transcripts. While K+ treatment was more specific for the induction of P450aldo mRNA, treatment with angiotensin II increased levels of both P450aldo and P450c11 transcripts. To define the second messenger systems which influence transcript levels for these enzymes, the effects of agonists of the protein kinase A, protein kinase C, and calcium pathways were tested on the expression of P450aldo and P450c11. Activation of the protein kinase A pathway by the agonists, dibutyryl cAMP or forskolin, preferentially increased the P450c11 transcript to a greater degree than P450aldo. Interestingly, activation of the protein kinase C pathway by tetradecanoylphorbol acetate (TPA) did not alter transcripts for either P450aldo or P450c11. The calcium channel agonist BAYK 8644 mimicked the effects of K+ by increasing the transcript for P450aldo. However, the calcium channel blocker nifedipine attenuated the stimulatory effects of angiotensin II and K+ on the levels of P450aldo. However, the calcium channel blocker nifedipine attenuated the stimulatory effects of angiotensin II and K+ on the levels of P450aldo transcripts without affecting the stimulatory effect of dbcAMP. This study demonstrates that the protein kinase A pathway preferentially induces P450c11 mRNA over that of P450aldo. In addition, pharmacologic agents that affect calcium levels provide evidence for an additional regulatory mechanism in modulating the expression of P450aldo. This is of importance since the major physiologic regulators of aldosterone secretion, angiotensin II and K+ are able to increase intracellular calcium but have little effect on intracellular cAMP levels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Engineering a mineralocorticoid- to a glucocorticoid-synthesizing cytochrome P450.

Site-directed mutagenesis of a domain (amino acids 299-338) aligning to the I-helix region of P450cam, P450BM3 and P450terp was used to investigate the different regioselectivities displayed in the hydroxylation reactions performed by human aldosterone synthase (P450aldo) and 11beta-hydroxylase (P45011beta). The two enzymes are 93% identical and are essential for the synthesis of mineralocorticoids and glucocorticoids in the human adrenal gland. Single replacement of P450aldo residues for P45011 beta-specific residues at positions 296, 301, 302, 320, and 335 only gave rise to slightly increased 11beta-hydroxylase activities. However, a L301P/A320V double substitution increased 11beta-hydroxylase activity to 60% as compared with that of P45011 beta. Additionally substituting Ala-320 for Val-320 of P45011 beta further enhanced this activity to 85%. The aldosterone synthase activities of the mutant P450aldo proteins were suppressed to a varying degree, with triple replacement mutant L301P/E302D/A320V retaining only 10% and double replacement mutant L301P/A320V retaining only 13% of the P450aldo wild type activity. These results demonstrate a switch in regio- and stereoselectivities of the engineered P450aldo enzyme due to manipulation of residues at three critical positions, and we attribute the determination of these features in P450aldo to the structure of a region analogous to the I-helix in P450cam.

Amino Acid Sequence↗
PubMed1996

Conformational stability of adrenodoxin mutant proteins.

Adrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4-114 and 4-108, were studied by high-sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 degrees C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change delta Cp = 7.28 +/- 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (delta Cp = 4.29 +/- 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4-108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found.

Adrenodoxin↗
PubMed1996

Changed ratios of glucocorticoids/mineralocorticoids caused by point mutations in the putative I-helix regions of CYP11B1 and CYP11B2.

Computer modelling and site-directed mutagenesis were employed to investigate the structural basis for the regioselectivity of steroid hydroxylation and to determine whether point mutations of CYP11B1 and CYP11B2 can result in changes in the ratio of glucocorticoids/mineralocorticoids. Single replacement of CYP11B2 residues for CYP11B1-specific residues at positions 296, 301, 302, 320, and 335, belonging to the putative I-helix, gave rise to slightly increased 11 beta-hydroxylase activities. Replacement of 3 amino acids of CYP11B2 by the corresponding residues in CYP11B1 was sufficient to increase cortisol formation from about 5% to 85% of the CYP11B1 wild type level. The aldosterone synthase activities of the mutants were decreased to varying degrees indicating that point mutations at positions 296, 301, 302, and 335 could potentially cause hypoaldosteronism. Replacement of Val-320 of CYP11B1 by alanine, the corresponding residue found in CYP11B2, led to production of aldosterone by this mutant enzyme. This observation suggests that glucocorticoid-remediable hyperaldosteronism could also be due to point mutations located in the CYP11B1 sequence.

Amino Acid Sequence↗
PubMed1996

Functional expression of the guinea pig 11b-hydroxylase in COS-1 cells.

We expressed a guinea pig 11 beta-hydroxylase cDNA (1) in COS-1 cells. In order to find the optimal expression system we compared three expression plasmids, each driven by a different promoter. Although promoters exhibited different transcriptional activities this did not result in different enzymatic activities. Upon cotransfection with bovine adrenodoxin a 5-fold increase of enzyme activity was achieved. A comparison with the bovine 11 beta-hydroxylase clearly demonstrated that the guinea pig enzyme was not able to produce significant amounts of 18-hydroxylated and 18-oxidized products from deoxycorticosterone under the experimental conditions used.

Adrenodoxin↗
PubMed1995

The role of threonine 54 in adrenodoxin for the properties of its iron-sulfur cluster and its electron transfer function.

The amino acid in position 54 of adrenodoxin is strongly conserved among ferredoxins, consisting of a threonine or serine. Its role was studied by analyzing mutants T54S and T54A of bovine adrenodoxin. Absorption, circular dichroism, fluorescence, and electron paramagnetic resonance spectra of mutant T54S show that this substitution has no influence on the formation and stability of the ferredoxin. The redox potential of this mutant, however, was lowered by 55 mV as compared with native adrenodoxin, indicating a role for this residue in redox potential modulation. Incorporation of the iron-sulfur cluster was not impaired in the T54A mutant, although structural features of the oxidized protein were considerably changed. The decreased stability of the T54A mutant as compared with the wild type and mutant T54S indicates that a hydrogen bond donor at this position stabilizes the protein. Both mutants have been shown to be functionally active. Replacement of threonine 54 by serine or alanine, however, leads to rearrangements at the recognition sites for its redox partners. This is reflected by decreased Km and Kd values of both mutants for the cytochromes P450, whereas only T54A displayed a decreased Km value in cytochrome c reduction. Substrate conversion was accelerated (2.2- and 2.4-fold for mutants T54A and T54S, respectively) in the CYP11B1-, but not in the CYP11A1-dependent reaction.

Adrenodoxin↗
PubMed1995

Rabbit liver cytochrome P-450 2B5: high-level expression of the full-length protein in Escherichia coli, purification, and catalytic activity.

Rabbit liver cytochrome P-450 2B5 (P-450 2B5) was expressed in Escherichia coli using the D(+)-galactose-inducible expression vector pJL-2, containing the full-length cDNA encoding P-450 2B5. Stimulation by galactose of protein synthesis in the presence of the heme precursor 5-aminolevulinic acid peaked 72 h after addition to the inducer to yield 108 nmol membrane-bound P-450 2B5 per liter of culture medium. The recombinant enzyme was purified to near homogeneity by a two-column procedure involving chromatography on DE-52 cellulose and hydroxylapatite. The hemoprotein was isolated mainly in the low-spin iron configuration and exhibited a reduced CO-difference spectrum with a Soret band at 451 nm. Second-derivative spectral analysis in the middle-UV region revealed that type I binding of 4-nitroanisole to ferric P-450 2B5 abolished absorption bands ascribable to tyrosine residues within the polypeptide chain. Pseudo-first-order rates of NADPH-driven reduction of the pigment were lower when reconstituted with NADPH-cytochrome P-450 reductase than with the mitochondrial adrenodoxin/NADPH-adrenodoxin reductase redox couple. The enzyme was catalytically active toward 4-nitroanisole and androstenedione; metabolic rates were enhanced to different extents by the presence of cytochrome b5. The recombinant hemoprotein did not catalyze bioactivation of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, a potent pulmonary carcinogen. The methods described here should facilitate further studies on the biophysical basis of the complex interactions of P-450 2B5 with its redox partners.

Amino Acid Sequence↗
PubMed1995

Mutational effects on the spectroscopic properties and biological activities of oxidized bovine adrenodoxin, and their structural implications.

Of the aromatic 1H-NMR signals of oxidized bovine adrenodoxin only those of His56 showed intrinsic chemical shift changes upon replacement of Tyr82 by Ser or Leu, that must arise from a loss of a through-space ring-current effect of the tyrosine ring in these mutants. Thus, of the three His residues contained in adrenodoxin, His56 is closest to Tyr82, and hence to the highly acidic determinant region of adrenodoxin that is the interaction site for adrenodoxin reductase and P-450. The strong dependence of the fluorescence intensity of Tyr82 on the residue in position 56 supported this observation. As a consequence of this, the effects of replacement of His56 by Gln or Thr on cytochrome c reduction and cytochromes P-450(11 beta) (CYP11B1)-dependent and P-450scc (CYP11A1)-dependent substrate conversions were studied. No influence on Vmax values was observed for all reactions mediated by the mutants, implying His56 does not play a decisive role in the intramolecular or intermolecular electron transfer. In contrast, the Km values were increased, as was the Ks value for binding of CYP11A1 to the [H56T]adrenodoxin. The secondary structure deduced from further NMR data of adrenodoxin was compared with that of other ferredoxins. Tyr82 is in a region of the molecule containing no secondary-structure elements. The data for Tyr82 are in keeping with the biological activities and suggests it is in a flexible, solvent-exposed region of the molecule.

Adrenodoxin↗
PubMed1995

Bovine adrenodoxin--a mitochondrial iron-sulphur protein--binds to chaperonin GroEL.

The interaction of bovine adrenodoxin with the chaperonin GroEL was investigated using sucrose density centrifugation and analytical ultracentrifugation. It could be clearly established that denatured mature adrenodoxin comigrated in a sucrose density gradient with the GroEL oligomer, indicating that a complex had been formed. Up to 2 moles of adrenodoxin/mol GroEL can be bound. From the partial concentrations, association constants of 4.3 x 10(5) M-1 for the first adrenodoxin molecule and of 1.08 x 10(5) M-1 for the second molecule to the complex, respectively, were calculated. Upon addition of the cochaperonin GroES and Mg-ATP to the adrenodoxin-GroEL complex, adrenodoxin was released, indicating a specific binding between GroEL and adrenodoxin.

Adrenodoxin↗
PubMed1995

Conformational stability of bovine holo and apo adrenodoxin--a scanning calorimetric study.

Holo and apo adrenodoxin were studied by differential scanning calorimetry, absorption spectroscopy, limited proteolysis, and size-exclusion chromatography. To determine the conformational stability of adrenodoxin, a method was found that prevents the irreversible destruction of the iron-sulfur center. The approach makes use of a buffer solution that contains sodium sulfide and mercaptoethanol. The thermal transition of adrenodoxin takes place at Ttrs = 46-57 degrees C, depending on the Na2S concentration with a denaturation enthalpy of delta H = 300-380 kJ/mol. From delta H versus Ttrs a heat capacity change was determined as delta Cp = 7.5 +/- 1.2 kJ/mol/K. The apo protein is less stable than the holo protein as judged by the lower denaturation enthalpy (delta H = 93 +/- 14 kJ/mol at Ttrs = 37.4 +/- 3.3 degrees C) and the higher proteolytic susceptibility. The importance of the iron-sulfur cluster for the conformational stability of adrenodoxin and some conditions for refolding of the thermally denatured protein are discussed.

Adrenodoxin↗