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AbstractPresent knowledge on peroxiredoxins is reviewed with special emphasis on catalytic principles, specificities and biological function. Peroxiredoxins are low efficiency peroxidases using thiols as reductants. They appear to be fairly promiscuous with respect to the hydroperoxide substrate; the specificities for the donor substrate vary considerably between the subfamilies, comprising GSH, thioredoxin, tryparedoxin and the analogous CXXC motifs in bacterial AhpF proteins. Peroxiredoxins are definitely responsible for antioxidant defense in bacteria (AhpC), yeast (thioredoxin peroxidase) and trypanosomatids (tryparedoxin peroxidase). They are considered to determine virulence of mycobacteria and trypanosomatids. In higher plants they are involved in balancing hydroperoxide production during photosynthesis. In higher animals peroxiredoxins appear to be involved in the redoxregulation of cellular signaling and differentiation, displaying in part opposite effects.

The synthesis of eukaryotic selenoproteins involves the recoding of an internal UGA codon as a site for selenocysteine incorporation. This recoding event is directed by a selenocysteine insertion sequence in the 3'-untranslated region. Because UGA also functions as a signal for peptidyl-tRNA hydrolysis, we have investigated how the rates of translational termination and selenocysteine incorporation relate to cis-acting elements in the mRNA as well as to trans-acting factors in the cytoplasm. We used cis-elements from the phospholipid glutathione peroxidase gene as the basis for this work because of its relatively high efficiency of selenocysteine incorporation. The last two codons preceding the UGA were found to exert a far greater influence on selenocysteine incorporation than nucleotides downstream of it. The efficiency of selenocysteine incorporation was generally much less than 100% but could be partially enhanced by concomitant overexpression of the tRNA(Sec) gene. The combination of two or three UGA codons in one reading frame led to a dramatic reduction in the yield of full-length protein. It is therefore unlikely that multiple incorporations of selenocysteine are processive with respect to the mode of action of the ribosomal complex binding to the UGA site. These observations are discussed in terms of the mechanism of selenoprotein synthesis and its ability to compete with termination at UGA codons.

The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.

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 fasciculata by aqueous two-phase systems and chromatography. The enzyme showed a specific activity of 38 micromol 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 Km 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 kcat 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 (Ki = 0. 08 mM) and trypanothione exerted a feedback inhibition competitive with GSH (Ki = 0.48 mM).

The sequences of urokinase (UK) and tissue-type plasminogen activator (TPA) were aligned with those of chymotrypsin, trypsin, and elastase according to their ‘structurally conserved regions’. In spite of its trypsin-like specificity UK was model-built on the basis of the chymotrypsin structure because of a corresponding disulfide pattern. The extra disulfide bond falls to cysteines 50 and 111d. Insertions can easily be accommodated at the surface. As they occur similarly in both, UK and TPA, a role in plasminogen recognition may be possible. Of the functional positions known to be involved in substrate or inhibitor binding, Asp 97, Lys 143 and Arg 217 (Leu in TPA) may contribute to plasminogen activating specificity. PTI binding may in part be impaired by structural differences at the edge of the binding pocket.

The sequences of urokinase (UK) and tissue‐type plasminogen activator (TPA) were aligned with those of chymotrypsin, trypsin, and elastase according to their ‘structurally conserved regions’. In spite of its trypsin‐like specificity UK was model‐built on the basis of the chymotrypsin structure because of a corresponding disulfide pattern. The extra disulfide bond falls to cysteines 50 and 111d. Insertions can easily be accommodated at the surface. As they occur similarly in both, UK and TPA, a role in plasminogen recognition may be possible. Of the functional positions known to be involved in substrate or inhibitor binding, Asp 97, Lys 143 and Arg 217 (Leu in TPA) may contribute to plasminogen activating specificity. PTI binding may in part be impaired by structural differences at the edge of the binding pocket.

Tryparedoxin peroxidase from Crithidia fasciculata is an essential component of the trypanothione-dependent hydroperoxide metabolism in the trypanosomatids (Nogoceke, E., Gommel, D. U., Kiebeta, M., Kalisz, H. M., and Flohé, L. (1997) Biol. Chem. 378, 827-836). The tryparedoxin peroxidase gene and its flanking regions have been isolated and sequenced from a C. fasciculata genomic DNA library. It consists of an open reading frame of 564 base pairs encoding a protein of 188 amino acid residues. The gene, modified to encode 6 additional histidine residues, was expressed in Escherichia coli and the recombinant protein was purified to homogeneity by metal chelating chromatography. Recombinant tryparedoxin peroxidase has a subunit molecular mass of 21884 +/- 22 and contains two isoforms of pI 6.2 and 6.3. It exhibits a kinetic pattern identical to that of the authentic tryparedoxin peroxidase and has a similar specific activity of 2.51 units mg-1. The enzyme unequivocally belongs to the peroxiredoxin family of proteins, whose members have been found in all phyla. A phylogenetic tree comprising 47 protein and DNA sequences showed tryparedoxin peroxidase and a homologous Trypanosoma brucei sequence to form a distinct molecular clade. The consensus sequence: xnAx5-6Fx9Gx3Vx2Fx1Px2Fx1FVCPTEx21Sx1Dx7Wx16-19Dx15- 16Gx3Rx2Fx2Dx27Ax 1Qx4-11Cx1-3Wxn was demonstrated by alignment of the sequences of tryparedoxin peroxidase and 8 other peroxiredoxins with established peroxidase function.

Saruplase — a recombinant single-chain urokinase-type plasminogen activator was identified immunohistochemically in normal rat tissue after intravenous administration by means of a polyclonal antibody. For this purpose, rat tissues were fixed in various ways (liquid nitrogen, ethanol, formaldehyd solution). Saruplase could be detected by the PAP method, streptavidinbiotin system and indirect immunofluorescence in the kidney (proximal tubule), liver (hepatocytes, Kupffer cells) and spleen (reticular cells). Saruplase was not localized in the rat endothelium. It is discussed that the ratspecific receptors for urokinase-type plasminogen activator on endothelial cells cannot bind Saruplase due to the extreme species specificity.

Summary The literature on glutathione peroxidase (glutathione: H2O2 oxidoreductase, EC 1.11.1.9) is reviewed.

Zusammenfassung Eine Methode zur Kristallisation von größeren Mengen gereinigtem HbF aus menschlichem Nabelschnurblut wird beschrieben.

THE crystal shape of pure human adult haemoglobin (HbA) has unanimously been described as that of well-developed, symmetrical bipyramids1,2. Investigations of the crystal forms of human foetal haemoglobin (HbF), however, have revealed divergent results: hexagonal rhombic plates3, probably monoclinic forms4and a mixture of dodecahedra and square prisms5have been reported. All these investigations were carried out on cord blood. Our methods allow the investigation of pure crystalline foetal haemoglobin.

The pathophysiologic relevanceof altered eicosanoid metabolism and free-radical production during endotoxemia is reviewed. A direct or complement-mediated release of eicosanoids - depending on the species, the tissue, and the kind of endotoxin - has been found to be associated with endotoxemic shock. Prostacyelin appears to bepartially responsible for the early decrease in blood pressure and clearly induces the sustained hypotension of endotoxemia but is irrelevant to its fatal outcome. In contrast, simultaneous administration of vasodilating prostanoids such as prostaglandin E1 and metabolically stable analogues of prostacyclin prevents endotoxin lethality. Thromboxane A2 may contribute to platelet aggregation and vasoconstriction, but its quantitative importance often is obscured by other mediators. The peptidoleukotrienes, in particular leukotriene D4 , contribute to lung damage and - in galactosamine-sensitized rats, at least - to fatal liver destruction. Little is known about the role of leukotriene B4 in endotoxemia. Endotoxin suppresses rather than stimulates superoxide release from phagocytes in vitro but leads to massive free-radical formation mediated by complement activation in vivo and, thereby, contributes to lung injury.