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Biomedical subjects

G von Jagow

Publications and source records attributed to G von Jagow.

At least 19 recordsLinked to original sources
PubMed2006

[New licensing regulations for physicians. Main areas of reform and first results of the implementation process].

Since October 2003 medical education in Germany has been given a new more up-to-date basis, the "New Licensing Regulations for Physicians". They represent a reform of both the structure and the content of medical education making great demands on medical faculties and medical students; e.g. elective courses during the first and second cycles of the curriculum, interdisciplinary courses (so-called 'cross-sectional courses'), practical clinical courses of several weeks in five specialties, and family medicine as an option for choice during the last year are central issues of the reform. The number of state-controlled medical examinations has been reduced from four to two. It is now the responsibility of medical faculties to assess the knowledge, clinical skills and professional attitudes of students with respect to the numerous specialties which are part of medical education. This paper presents the essential innovations and describes the current state of the implementation process. It also points out problems that deserve reconsideration. Although the full implementation of the new licensing regulations is still in progress, it might be said that they have already initiated important changes in medical education in Germany.

Curriculum↗
PubMed1996

Isolation, characterisation and crystallisation of a water-soluble fragment of the Rieske iron-sulfur protein of bovine heart mitochondrial bc1 complex.

A water-soluble fragment of the bc1 complex from bovine heart mitochondria was isolated containing the intact Rieske [2Fe-2S] cluster. The fragment consists of the last 129 amino acid residues of the Rieske iron-sulfur protein and has a molecular mass of 14592 Da including two iron atoms. The absorption, visible CD, and EPR spectra of the fragment are indistinguishable from those of the membrane-bound iron-sulfur protein. The redox potential as determined by EPR-monitored redox titration was + 306 mV. The far-ultraviolet CD spectrum is indicative of a protein with little regular secondary structure, while significant alpha-helix content was detected in the membrane anchor of the complete iron-sulfur protein. The fragment could be crystallized using poly(ethylene glycol) 6000 as precipitant. Needle-shaped single crystals have been grown by the hanging-drop vapor diffusion technique. These crystals belong to the space group P21 and diffract well beyond 0.2 nm resolution. Phase determination using the multiple-wavelength anomalous-scattering technique is underway.

Amino Acid Sequence↗
PubMed1995

Zinc ions inhibit the QP center of bovine heart mitochondrial bc1 complex by blocking a protonatable group.

Bovine heart bc1 complex is reversibly inhibited by zinc ions with an inhibition constant KI of 10(-7) M at pH > or = 7.0. Binding of zinc is at least a factor of 10 tighter than binding of any other metal ion tested. Essentially complete inhibition of ubihydroquinone:cytochrome c oxidoreductase activity is observed at concentrations of [Zn2+] > 5 microM. Zinc does not affect the Km for the substrates, ubihydroquinone or cytochrome c, but zinc inhibits reduction of the cytochromes by ubihydroquinone through the QP center. A radioactive binding assay using 65Zn revealed one high affinity binding site per bc1 complex with KD < or = 10(-7) M at pH = 7.0 and 3-4 additional low affinity binding sites (KD > 2 x 10(-6) M). Zinc binding does not depend on the redox state of the high potential chain (iron-sulfur protein and cytochrome c1). Zinc binds 3 times tighter to Fe-S-depleted bc1 complex indicating that the zinc binding site is not on the "Rieske" iron-sulfur protein in contrast to a recent report by Lorusso et al. (Lorusso, M., Cocco, T., Sardanella, A.M., Minuto, M., Bonomi, F., and Papa, S. (1991) Eur. J. Biochem. 197, 555-561). Zinc binds to a site which has the same affinity for zinc as for protons. We conclude that the zinc binding site is close to a protonatable group of the bc1 complex with pKa = 7.2 which has not been identified previously. We propose that this group is part of the proton channel at the hydroquinone oxidation center of the bc1 complex.

Animals↗
PubMed1995

Cytochrome-c oxidase in developing rat heart. Enzymic properties and amino-terminal sequences suggest identity of the fetal heart and the adult liver isoform.

Perinatal development of cytochrome-c oxidase (complex IV) and ubiquinol-cytochrome-c reductase (complex III) was investigated in rat heart and liver by analysing catalytic properties, protein amounts, and subunit isoforms during the transition from the fetal to the adult state. The total amounts of complexes from milligram quantities of tissue, and the portions of isoforms of complex IV, were quantified densitometrically after isolation of the native complexes by blue native polyacrylamide gel electrophoresis and separation of the protein subunits by Tricine/SDS/PAGE [Schägger, H. & von Jagow, G. (1991) Anal. Biochem. 199, 223-231]. A parallel increase of protein amounts and catalytic activities during perinatal development was observed in heart and liver for complex III, but only in liver for complex IV. In heart, both a doubling of the turnover number of complex IV and a lowered Km for cytochrome c were observed. The altered enzymic properties correlated with the increase of heart type subunits VIa and VIII. The fetal enzymes from heart and liver seem to be identical to the adult liver isoform, as deduced from their enzymic properties and identical aminoterminal sequences of subunits VIa and VIII.

Amino Acid Sequence↗
PubMed1995

Human diseases with defects in oxidative phosphorylation. 1. Decreased amounts of assembled oxidative phosphorylation complexes in mitochondrial encephalomyopathies.

The amount of oxidative phosphorylation enzymes in mitochondrial encephalomyopathy patients has been studied by two-dimensional electrophoresis (blue native PAGE/Tricine-SDS-PAGE). Only 20 mg muscle was required to identify and analyse complexes I, III, IV, and V after Coomassie staining. In most cases reduced amounts of the involved complex(es) correlated well with decreased enzyme activities. The reliability of the method was reflected by the constant mutual ratio of the complexes found in all controls. Deviations from normal ratios were found to be more sensitive indicators for a defect than the absolute quantities, which varied considerably within the control group both in the enzymic and in the electrophoretic analysis. The effect of the mitochondrial tRNA(Leu(UUR)) mutation in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes on the amount of oxidative phosphorylation complexes was demonstrated for the first time directly on the protein level. In patients without known DNA mutations, specific defects of single complexes were identified. The new technique is a sensitive method for the identification of oxidative phosphorylation defects, complementary to enzymic measurements.

Adult↗
PubMed1994

Effect of ethoxyformic anhydride on the Rieske iron-sulfur protein of bovine heart ubiquinol: cytochrome c oxidoreductase.

Treatment of bovine heart ubiquinol-cytochrome c oxidoreductase (complex III, bc1 complex) with ethoxyformic anhydride (EFA) inhibits electron transfer between cytochromes b and c1 [Yagi et al., Biochemistry 21 (1982) 4777-4782]. This paper shows that EFA alters the EPR lineshape of the Rieske iron-sulfur cluster in complex III and in the isolated Rieske protein without a significant decrease of spin concentration. The effect of EFA on the Rieske iron-sulfur cluster is competitive with that of Qo site inhibitors, such as stigmatellin, and is completely reversed by hydroxylamine. These results are consistent with the possible ethoxyformylation by EFA of histidine ligands of the Rieske iron-sulfur cluster at the non-iron binding imidazole nitrogens.

Animals↗
PubMed1994

Analysis of cytochrome-b amino acid residues forming the contact face with the iron-sulfur subunit of ubiquinol:cytochrome-c reductase in Saccharomyces cerevisiae.

Four mutations in the mitochondrial cytochrome b of Saccharomyces cerevisiae have been characterized with respect to catalytic properties, inhibitor resistance and subunit interaction. The respiratory-deficient mutant [G137E]cytochrome b and the pseudo-wild-type revertant [G137E, N256K]cytochrome b were described previously [di Rago, J.-P., Netter, P. & Slonimski, P. P. (1990) J. Biol. Chem. 265, 3332-3339; di Rago, J.-P., Netter, P. & Slonimski, P. P. (1990) J. Biol. Chem. 265, 15750-15757]. Two new mutants [N256K]cytochrome b and [N256I]cytochrome b were isolated by dissociation of the second-site suppressor from the original target mutation. The mutants [G137E]cytochrome b and [G137E, N256K]cytochrome b exhibited a high resistance against methoxyacrylate inhibitors, whereas the suppressors [N256K]cytochrome b and [N256I]cytochrome b showed only a slight resistance. Remarkably, all mutants exhibited stigmatellin cross-resistance. The electron-transfer activity from the substrate nonylubiquinol to cytochrome c of mitochondrial membranes was diminished in all mutants. The substitution G137-->E decreases Vmax/Km by one order of magnitude, indicating a reduced catalytic efficiency for ubiquinol. The amino acid exchange at position 256 to a positively charged lysine residue or to a hydrophobic isoleucine residue resulted mainly in a diminished specific activity. The iron-sulfur subunit and the 8.5-kDa subunit were detectable in all mutants at normal levels in immunoblots of membrane preparations, indicating proper assembly of the complex. However, after purification, the mutant bc1 complex lacked the iron-sulfur subunit and the 8.5-kDa subunit. In contrast, the iron-sulfur subunit can only be dissociated from the parental bc1 complex by harsh treatment. These data suggest that residues 137 and 256 in cytochrome b are crucial for cytochrome-b/iron-sulfur protein interaction.

Amino Acid Sequence↗
PubMed1994

Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.

Blue native Electrophoresis is a "charge shift" method developed for isolation of native membrane protein complexes from biological membranes that also separates both acidic and basic water-soluble proteins at a fixed pH of 7.5. In combination with a second dimension sodium dodecylsulfate electrophoresis it provides an analytical method for the determination of molecular mass and oligomeric state of nondissociated complexes, of subunit composition, and of degree of purity and for the detection of subcomplexes. The method was applied to analysis of cytochrome bc/bf complexes. By combination of a novel colorless native polyacrylamide gel electrophoresis (CN-PAGE) with blue native BN-PAGE, a two-dimensional native technique was developed that is suitable for preparation of highly pure membrane protein complexes.

Animals↗
PubMed1993

What information do inhibitors provide about the structure of the hydroquinone oxidation site of ubihydroquinone: cytochrome c oxidoreductase?

The Q cycle mechanism of the bc1 complex requires two quinone reaction centers, the hydroquinone oxidation (QP) and the quinone reduction (QN) center. These sites can be distinguished by the specific binding of inhibitors to either of them. A substantial body of information about the hydroquinone oxidation site has been provided by the analysis of the binding of QP site inhibitors to the bc1 complex in different redox states and to preparations depleted of lipid or protein components as well as by functional studies with mutant bc1 complexes selected for resistance toward the inhibitors. The reaction site is formed by at least five protein segments of cytochrome b and parts of the iron-sulfur protein. At least two different binding sites for QP site inhibitors could be detected, one for the methoxyacrylate-type inhibitors binding predominantly to cytochrome b, the other for the chromone-type inhibitors and hydroxyquinones binding predominantly to the iron-sulfur protein. The interactions with the protein segments, between different protein segments, and between protein and ligands (substrate, inhibitors) are discussed in detail and a working model of the QP pocket is proposed.

Amino Acid Sequence↗
PubMed1992

Interactions of phospholipids with the mitochondrial cytochrome-c reductase studied by spin-label ESR and NMR spectroscopy.

Protein/phospholipid interactions in the solubilized mitochondrial ubihydroquinone:cytochrome-c oxidoreductase (bc1 complex) were studied by spin-label electron-spin resonance and by 31P-NMR spectroscopy. Spin-labelled phospholipids were employed to probe the relative binding affinities of a number of phospholipids with regard to the significance of phospholipids for the activity and stability of this multisubunit complex. The protein was titrated with spin-labelled cardiolipin (1,3-bisphosphatidyl-sn-glycerol) and with the spin-labelled analogues of PtdCho and PtdEtn, both of which have been shown recently to elicit a substantial increase in electron-transport activity [Schägger, H., Hagen, T., Roth, B., Brandt, U., Link, T. A. & von Jagow, G. (1990) Eur. J. Biochem. 190, 123-130]. A simplified distribution model showed that neutral phospholipids have much lower protein affinity than cardiolipin. In contrast to the transient weak lipid binding detected by spin-label electron-spin resonance, 31P NMR revealed a tightly bound cardiolipin portion, even after careful delipidation of the complex. Considerable line narrowing was observed after phospholipase A2 digestion of the bound cardiolipin, whereas addition of SDS resulted in complete release. Relative proportions and line widths of mobile and immobilized lipids were obtained by deconvoluting the partially overlapping signals. The current results are discussed with reference to similar findings with other mitochondrial membrane proteins. It is assumed that activation by neutral phospholipids reflects a generalized effect on the protein conformation. Cardiolipin binding is believed to be important for the structural integrity of the mitochondrial protein complexes.

Animals↗
PubMed1992

Determination of the redox properties of the Rieske [2Fe-2S] cluster of bovine heart bc1 complex by direct electrochemistry of a water-soluble fragment.

The redox potential of the Rieske [2Fe-2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25 degrees C and I of 0.01 M was Em = +312 +/- 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with square root of I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k degrees, was calculated to be approximately 2 x 10(-3) cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as delta S degrees = -155 J.K-1.mol-1. The pH dependence of the midpoint potential was followed over pH 5.5-10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex.

Animals↗
PubMed1992

Uncoupling activity and physicochemical properties of derivatives of fluazinam.

The physico-chemical properties and uncoupling activity of eight derivatives of N-phenyl-2-pyridinamines related to the fungicide fluazinam were analyzed using rat liver mitochondria. The uncoupling activity of these compounds relies on the deprotonable secondary amino group. One of the derivatives tested (B-3) was slightly more efficient than fluazinam. By phase-distribution analysis we could show that the N-phenyl-2-pyridinamines are chemicals with moderate hydrophobicity. Deprotonation of the compound reduces the water/octanol partition coefficient by about one order of magnitude. The pKA value of the deprotonable group is affected equally by electron withdrawing substituents of the phenyl- and the pyridinyl-ring, and could be predicted simply from the sum of the Hammett coefficients. The uncoupling efficiency was not dependent on the hydrophobicity of the compound, but appeared to be governed by the pKA of the deprotonable group. This structure/uncoupling characteristic is different from that of the generally more hydrophobic uncouplers of the salicylanilide-type. The pKA resulting in the most efficient uncoupling was found to lie in the range of the pH of the reaction medium. A model based on a solution complexation mechanism, which describes this behaviour, is presented. We conclude that the N-phenyl-2-pyridinamines uncoupled the mitochondria by a simple protonophoric cycle involving protonation/deprotonation in the bulk phase, and that the kinetics of uncoupling were primarily governed by the total concentration of the limiting uncoupler species.

Amines↗
PubMed1991

Significance of the "Rieske" iron-sulfur protein for formation and function of the ubiquinol-oxidation pocket of mitochondrial cytochrome c reductase (bc1 complex).

The binding of specific inhibitors to the ubiquinol oxidation pocket ("QP center") of cytochrome c reductase was analyzed before and after removal of bound phospholipid and the "Rieske" iron-sulfur protein using optical spectroscopy and fluorescence quench binding assays. The enzyme lacking iron-sulfur protein showed almost unchanged, tight binding of the E-beta-methoxyacrylate inhibitors oudemansin A and MOA-stilbene, whereas binding of the chromone inhibitor stigmatellin was almost completely abolished. The affinity of the weak inhibitor 3-undecyl-2-hydroxy-naphthoquinone was decreased. Oudemansin A binding to the defective pocket of the iron-sulfur protein-depleted enzyme was lowered by added phospholipid. It was deduced from these results that the QP center is a spacious pocket formed by domains of cytochrome b, bearing the E-beta-methoxcyacrylate binding site, and the iron-sulfur protein, bearing the stigmatellin binding site. Moreover, removal of the iron-sulfur protein leaves this pocket defective but essentially unchanged in its remaining binding capability. The affinity of three preparations of cytochrome c reductase, the complete, the delipidated, and the iron-sulfur depleted enzyme for E-beta-methoxyacrylate-stilbene, was analyzed for different redox states of the catalytic centers of cytochrome c reductase. The apparent Kd values for the different redox states were interpreted in terms of two conformational states. It is suggested that these changes reflect the two states of the "catalytic switch" proposed recently for the QP pocket of cytochrome c reductase (Brandt, U., and von Jagow, G. (1991) Eur. J. Biochem. 195, 163-170). According to the refined model presented in this work, changeover to the "b" state is triggered by reduction of the iron-sulfur cluster, and changeover back to the "FeS" state is triggered by electron transfer from the low potential onto the high potential heme b center. Our interpretation implies that the stability of the two states is affected by the redox states of the enzyme, but that additionally changing the redox states of the two centers is required for "switching" on a catalytic time scale.

Acrylates↗
PubMed1991

Reactivity of the Bacillus subtilis succinate dehydrogenase complex with quinones.

The succinate dehydrogenase isolated from Bacillus subtilis was found to catalyze the oxidation of succinate with hydrophilic quinones. Either naphthoquinones or benzoquinones served as acceptors. The enzyme activity increased with the redox potential of the quinone. The highest turnover number was commensurate with that of the bacterial succinate respiration in vivo. The succinate dehydrogenase was similarly active in fumarate reduction with quinols. The highest activity was obtained with the most electronegative quinol. The fumarate reductase isolated from Wolinella succinogenes catalyzed succinate oxidation with quinones and fumarate reduction with the corresponding quinols at activities similar to those of the B. subtilis enzyme. Succinate oxidation by the lipophilic quinones, ubiquinone or vitamin K-1, was monitored as cytochrome c reduction using proteoliposomes containing succinate dehydrogenase together with the cytochrome bc1 complex. The activity with ubiquinone or vitamin K-1 was commensurate with the succinate respiratory activity of bacteria or of the bacterial membrane fraction. The results suggest that menaquinone is involved in the succinate respiration of B. subtilis, although its redox potential is unfavorable.

Bacillus subtilis↗