Leopold Flohé

University of Padua

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Fulvio Ursini

University of Padua

Matilde Maiorino

University of Padua

Wolfgang A. Günzler

Grünenthal Group (Germany)

Regina Brigelius‐Flohé

German Institute of Human Nutrition

Peter M. Steinert

National Institutes of Health

Antonella Roveri

University of Padua

Henryk M. Kalisz

Nerviano Medical Sciences

Ulrich Menge

Helmholtz Centre for Infection Research

Marisa Montemartini

Nerviano Medical Sciences

Everson Nogoceke

Roche (Switzerland)

Cooperative Institutions

Helmholtz Centre for Infection Research

Grünenthal Group (Germany)

Technische Universität Braunschweig

University of Tübingen

University of Padua

Heidelberg University

Harvard University

Universitat de València

Technical University of Munich

National Institutes of Health

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Tryparedoxin II from Crithidia fasciculata

BioFactors (2000)

Marisa Montemartini Peter M. Steinert Mahavir Singh Leopold Flohé Henryk M. Kalisz

Technical university

10.1002/biof.5520110118

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Citations (4)

The Amino-Acid Sequence of Rabbit Cu-Zn Superoxide Dismutase

Biological Chemistry Hoppe-Seyler (1988)

Kurt REINECKE Bernhard Wolf A.M. Michelson K. Puget Gerd Steffens

The primary structure of Cu-Zn superoxide dismutase from rabbit liver was investigated. The reduced and S-carboxymethylated enzyme was treated with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by high-performance liquid chromatography and sequenced by automated Edman degradation. With the exception of the N- and C-terminus the complete sequence was established by means of overlapping peptides. The N-terminus is blocked and thus not susceptible to Edman degradation. The amino-acid composition of the tryptic N-terminal peptide corresponds to that of the cytoplasmatic Cu-Zn superoxide dismutases of other mammals investigated. The chromatographic behaviour of these N-terminal peptides on a reversed phase C18 column is also identical, thus suggesting also for the rabbit Cu-Zn superoxide dismutase the N-terminal sequence Ac-Ala-Thr-Lys. The C-terminus was demonstrated to have the sequence -Ile-Ala-Pro by enzymatic degradation with carboxypeptidase Y. The complete amino-acid sequence of the rabbit Cu-Zn superoxide dismutase consists of 152 amino-acids and shows the expected homology to other Cu-Zn enzymes published so far. The aspartate and six histidine residues known to complex the metal ions are conserved at homologous positions. This also applies for the arginine residue near the C-terminus which is supposed to direct the anionic superoxide radical towards the active centre of the enzyme. The amino acid sequence of the rabbit Cu-Zn superoxide dismutase corresponds to those of other mammals in more than 80% of its amino-acid residues. From a total of 152 amino-acid residues the rabbit shares with rat 128, with mouse 130, with horse 127, with pig 126/127, with cattle 130 and with man 131 amino acids in homologous positions. However the Cu-Zn superoxide dismutases of closely related mammals like rats and mice differ in only five amino acid residues of their sequence. A phylogenetic closer relatedness between lagomorphs and rodents than between other orders of mammals, could not be derived from the sequence data given. Rather rodents and lagomorphs are to be considered as two evolutionary independent orders of mammals.

Edman degradation

Cyanogen bromide

Dismutase

10.1515/bchm3.1988.369.2.715

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The Catalytic Site of Glutathione Peroxidases

Antioxidants and Redox Signaling (2008)

Silvio C. E. Tosatto Valentina Bosello-Travain Federico Fogolari Pierluigi Mauri Antonella Roveri

In GPxs, the redox-active Se or S, is at hydrogen bonding distance from Gln and Trp residues that contribute to catalysis. From sequence homology of >400 sequences and modeling of the DmGPx as a paradigm, Asn136 emerged as a fourth essential component of the active site. Mutational substitution of Asn136 by His, Ala, or Asp results in a dramatic decline of specific activity. Kinetic analysis indicates that k+1, the rate constant for the oxidation of the enzyme, decreases by two to three orders of magnitude, whereas the reductive steps characterized by k′+2 are less affected. Accordingly, MS/MS analysis shows that in Asn136 mutants, the peroxidatic Cys45 stays largely reduced also in the presence of a hydroperoxide, whereas in the wild-type enzyme, it is oxidized, forming a disulfide with the resolving Cys. Computational calculation of pKa values indicates that the residues facing the catalytic thiol, Asn136, Gln80, and, to a lesser extent Trp135, contribute to the dissociation of the thiol group, Asn136 being most relevant. These data disclose that the catalytic site of GPxs has to be redrawn as a tetrad, including Asn136, and suggest a mechanism accounting for the extraordinary catalytic efficiency of GPxs.

Thiol

10.1089/ars.2008.2055

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Citations (166)

Redox Regulation of NF-kappa B Activation

Free Radical Biology and Medicine (1997)

Leopold Flohé Regina Brigelius-Flohé Claude Saliou Maret G. Traber Lester Packer

Dephosphorylation

Dual-specificity phosphatase

10.1016/s0891-5849(96)00501-1

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Citations (853)

ChemInform Abstract: DESIGNING PROSTACYCLIN ANALOGS

Chemischer Informationsdienst (1984)

Leopold Flohé H. BOEHLKE E Frankus Sook-Hyang Kim W Lintz

10.1002/chin.198401350

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Convenient Isolation and Kinetic Mechanism of Glutathionylspermidine Synthetase from Crithidia fasciculata

Journal of Biological Chemistry (1997)

Kerstin Koenig Ulrich Menge Michael Kieß Victor Wray Leopold Flohé

Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathionylspermidine synthetase and trypanothione synthetase. Glutathionylspermidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculataby aqueous two-phase systems and chromatography. The enzyme showed a specific activity of 38 μmol of glutathionylspermidine formed per min per mg of protein. Its molecular mass was 78 kDa in SDS-polyacrylamide gel electrophoresis, and it appeared predominantly monomeric in native polyacrylamide gel electrophoresis and gel filtration. The isoelectric point was at pH 4.6, and the pH optimum was near 7.6. Partial amino acid sequencing revealed homology with, but low similarity to, the glutathionylspermidine synthetase/amidase of Escherichia coli, and amidase activity was not detected in glutathionylspermidine synthetase of C. fasciculata. The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting K _m values for Mg²⁺-ATP, GSH, and spermidine of 0.25 ± 0.02, 2.51 ± 0.33, and 0.47 ± 0.09 mm, respectively, and a k _cat of 415 ± 78 min⁻¹. Partial reactions at restricted cosubstrate supply were not detected by ³¹P NMR, supporting the necessity of a quarternary complex formation for catalysis. ADP inhibited competitively with respect to ATP (K _i = 0.08 mm) and trypanothione exerted a feedback inhibition competitive with GSH (K _i = 0.48 mm).

Crithidia fasciculata

Isolation

Crithidia

10.1074/jbc.272.18.11908

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STRUCTURE OF TRYPAREDOXIN II

B. Hofmann H. Budde Karsten Bruns Sergio A. Guerrero Henryk M. Kalisz

10.2210/pdb1fg4/pdb

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Time course of IL-6 and TNFα release during endotoxin-induced endotoxin tolerance in rats

Biochemical Pharmacology (1991)

Sascha Flohé Peter C. Heinrich Johannes Schneider Albrecht Wendel Leopold Flohé

10.1016/0006-2952(91)90161-w

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Citations (52)

Peroxiredoxin Systems

Sub-cellular biochemistry/Subcellular biochemistry (2007)

Leopold Flohé J. Robin Harris

10.1007/978-1-4020-6051-9

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Citations (49)

Enrichment of hydrophobic proteinsvia Triton X-114 phase partitioning and hydroxyapatite column chromatography for mass spectrometry

Electrophoresis (2000)

Josef Wissing Sabina Heim Leopold Flohé Ursula Bilitewski Ronald E. Frank

Membrane proteins are the starting point of several signal transduction pathways. Therefore, the separation and identification of these proteins are of great interest in proteome analysis. However, the specific properties of membrane proteins seriously impede their analysis. We present an effective and highly reproducible method for the two-dimensional separation of extremely hydrophobic proteins and demonstrate the advantages of special preseparation procedures for the identification of proteins which have very similar Mr and p/. Using the example of the integral membrane protein very low density lipoprotein (VLDL) receptor (NCBI Acc. # 1730111) and the soluble heat shock protein (HSP) 90 (NCBI Acc. # 386786) we present the applicability of a phase-separation system with Triton X-114. Using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of the protein spots after 2-D separation of the hydrophilic and the strongly hydrophobic protein fraction of human endothelial cells (ECV cell line), we were able to distinguish both proteins.

10.1002/1522-2683(20000701)21:13<2589::aid-elps2589>3.0.co;2-t

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Citations (40)