TY - JOUR A1 - Kruse, N. A1 - Shen, B. J. A1 - Arnold, S. A1 - Tony, H. P. A1 - Müller, T. A1 - Sebald, Walter T1 - Two distinct functional sites of human interleukin 4 are identified by variants impaired in either receptor binding or receptor activation N2 - Interleukin 4 (IL-4) exerts a decisive role in the coord.ination of proteelive immune responses against parasites, particularly helminths. A disregulation of ll.r4 function is possibly involved in the genesis of allergic disease states. The search for important amino acid residues in human ll.r4 by mutational analysis of charged invariant amino acid positions identified two distinct functional sites in the 4-helix-bundle protein. Site 1 was marked by amino acid substitutions of the glutamic acid at position 9 in helix A and arginine at position 88 in helix C. Exchanges at both positions led to IL-4 variants deficient in binding to the extracellular domain of the ll.r4 receptor (IL-4ReJ. In parallel, up to 1000-fold increased concentrations of this type of variant were required to induce T -cell proliferation and B-eeil CD23 expression. Site 2 was marked by amino acid exchanges in helix D at positions 121, 124 and 125 (arginine, tyrosine and serine respectively in the wild-type).ß.A variants affected at site 2 exhibited partial agonist activity during T -cell proliferation; however, they still bound with high affinity to IL-4Rex. [The generation of an IL-4 antagonist by replacing tyrosine 124 with aspartic acid has been described before by Kruse et al. (1992) (EMBO }., 11, 3237-3244)]. These findings indicate that IL-4 functions by bind.ing IL-4Rex via site 1 which is constituted by residues on helices A and C. They further suggest that the association of a second, still undetined receptor protein with site 2 in helix D activates the receptor system and generates a transmembrane signal. KW - Biochemie KW - drug design/partial agonists KW - receptor signalling Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62451 ER - TY - JOUR A1 - McCarthy, J. E. A1 - Schairer, H. U. A1 - Sebald, Walter T1 - Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation N2 - The c, b and ö subunit genes of the Escherichia coli atp operon were cloned individually in an expression vector between the tac fusion promoter and the galK gene. The relative rates of subunit synthesis directed by the cloned genes were similar in vitro andin vivo and compared favourably with the subunit stoichiometry of the assembled proton-translocating A TP synthase of E. coli in vivo. The rate of synthesis of subunit c was at least six times that of subunit b and 18 times that of subunit ö. Progressive shortening of the long intercistronic sequence lying upstream of the subunit c gene showed that maximal expression of this gene is dependent upon the presence of a sequence stretching > 20 bp upstream of the Shine-Dalgarno site. This sequence thus acts to enhance the rate of translational initiation. The possibility that similar sequences might perform the same function in other operons of E. coli and bacteriophage A is also discussed. Translation of the subunit b cistron is partially coupled to translation of the preceding subunit c cistron. In conclusion, the expression of all the atp operon genes could be adjusted to accommodate the subunit requirements of A TP synthase assembly primarily by means of mechanisms which control the efficiency of translational initiation and re-initiation at the respective cistron start codons. KW - Biochemie KW - E. coli atp operon KW - subunit stoichiometry KW - in vitro and in vivo expression KW - translational initiation Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62657 ER - TY - JOUR A1 - Hoppe, J. A1 - Sebald, Walter T1 - Topological studies suggest that the pathway of the protons through F\(_0\) is provided by amino acid residues accessible from the lipid phase N2 - The structure of the F0 part of ATP synthases from E. coli and Neurospora crassa was analyzed by hydrophobic surface labeling with [125I]TID. In the E. co/i F0 all three subunits were freely accessible to the reagent, suggesting that these subunits are independently integrated in the membrane. Labeted amino acid residues were identified by Edman degradation of the dicyclohexylcarbodiimide binding (DCCD) proteins from E. coli and Neurospora crassa. The very similar patterns obtained with the two homologaus proteins suggested the existence of tightly packed cx-helices. The oligomeric structure of the DCCD binding protein appeared to be very rigid since little, if any, change in the labeling patternwas observed upon addition of oligomycin or DCCD to membranes from Neurospora crassa. When membrancs were pretrcated with DCCD prior to the reaction with [125I]TID an additionally labeled amino acid appeared at the position of Glu·65 which binds DCCD covalently, indicating the Jocation of this inhibitor on the outside of the oligomer. It is suggested that proton conduction occurs at the surface of the oligomer of the DCCD binding protein. Possibly this oligomer rotates against the subunit a or b and thus enables proton translocation. Conserved residues in subunit a, probably located in the Iipid bilayer, might participate in the pro· ton translocation mechanism. N2 - La structure de la partie F0 de l'ATP synthase a ete analysee au moyen de marquage par /e reactif hydrophobe TJD[125 I]. Les trois sous-unites de E. coli F0 sont accessibles au reactif ce qui semble indiquer que ces sous-unites sont integrees dans Ia membrane defaron independante. Les amino-acides marques ont ete identifies par Ia degradation d'Edman des profeines d'E. coli et de Neurospora associees au dicyclohexylcarbodiimide (DCCD). L 'analogie des courbes obtenues pour /es deux proteines homologues suggere l'existence d'a-helices rangees de faron serree. La structure oligomerique de Ia proreine associee au DCCD semble etre tres rigide puisque pratiquement aucun changement dans /e marquage n 'a ete observe par addition d'oligomycine ou de DCCD aux membranes de Neurospora crassa. Quand /es membranes sont traitees avec /e DCCD avant Ia reaction avec TJD[125 I], un amino-acide additionnellement marque apparait a Ia position Glu·65 et forme avec le DCCD une Iiaison covalente. Ce dernier resu/tat indique Ia localisation de cet inhibiteur a /'exterieur de J'oligo· mere. II semble donc que Ia conduction des protons ait lieu a Ia surface de /'oligomere de Ia proteine associee au DCCD. II serait possib/e que l'o/igomere se retourne contre Ia sous-unite a ou b, permettunt de ce fait Ia translocation des protons. Les residus conserves de Ia sous-unite a, probab/ement Jocalises dans Ia double couche lipidique, pourraient participer au mecanisme de translocation des protons. KW - Biochemie KW - conduction de protons KW - proteines membranaires KW - carbenes KW - proton conduction KW - membrane proteins KW - carbenes Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62602 ER - TY - JOUR A1 - Schairer, H. U. A1 - Hoppe, J. A1 - Sebald, Walter A1 - Friedl, P. T1 - Topological and functional aspects of the proton conductor, F\(_0\), of the Escherichia coli ATP-synthase N2 - The isolated H\(^+\) conductor, F\(_0\) , of the Escherichia co1i ATP-synthase consists of three subunits, a, b, and c. H\(^+\) -permeable liposomes can be reconstit~ted with F\(_0\) and lipids; addition of F\(_1\)-ATPase reconstitutes a functional ATP-synthase. Mutants with altered or misslng F\(_0\) subunits are defective in H\(^+\) conduction. Thus, all three subunits are necessary for the expression of H\(^+\) conduction. The subunits a and b contain binding sites for F\(_1\)• Computer calculations, cross-links, membrane-permeating photo-reactive labels, and proteases were used to develop tentative structural models for the individual F\(_0\) subunits. KW - Biochemie Y1 - 1982 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62721 ER - TY - JOUR A1 - Flügge, U. I. A1 - Fischer, K. A1 - Gross, A. A1 - Sebald, Walter A1 - Lottspeich, F. A1 - Eckerskorn, C. T1 - The triose phosphate-3-phosphoglycerate-phosphate translocator from spinach chloroplasts: nucleotide sequence of a full-length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts N2 - No abstract available KW - Biochemie Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62559 ER - TY - JOUR A1 - Hoppe, J. A1 - Friedl, P. A1 - Schairer, H. U. A1 - Sebald, Walter A1 - Meyenburg, K. von A1 - Jorgensen, B. B. T1 - The topology of the proton translocating F\(_0\) component of the ATP synthase from E. coli K12: studies with proteases N2 - The accessibility of the three F\(_0\) subunits a, b and c from the Escherichia coli Kll A TP synthase to various proteases was studied in F\(_1\)-depleted inverted membrane vesicles. Subunit b was very sensitive to all applied proteases. Chymotrypsin produced a defined fragment of mol. wt. 1S 000 which remained tightly bound to the membrane. The cleavage site was located at the C-terminal region of subunit b. Larger amounts of proteases were necessary to attack subunit a (mol. wt. 30 000). There was no detectable deavage of subunit c. It is suggested that the major hydrophilic part of subunit b extends from the membrane into the cytoplasm and is in contact with the F\(_1\) sector. The F\(_1\) sector was found to afford some protection against proteolysis oftheb subunit in vitro andin vivo. Protease digestion bad no influence on the electro-impelled H\(^+\) conduction via F\(_0\) bot ATP-dependent H\(^+\) translocation could not be reconstituted upon binding of F\(_1\)• A possible role for subunit b as a linker between catalytic events on the F\(_1\) component and the proton pathway across the membrane is discussed. KW - Biochemie KW - protein pathway KW - ATPase mutants Y1 - 1983 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62718 ER - TY - JOUR A1 - Hoppe, J. A1 - Schairer, H. U. A1 - Sebald, Walter T1 - The proteolipid of a mutant ATPase from Escherichia coli defective in H\(^+\)-conduction contains a glycine instead of the carbodiimide-reactive aspartyl residue N2 - No abstract available KW - Biochemie Y1 - 1980 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62769 ER - TY - JOUR A1 - Harnisch, U. A1 - Weiss, H. A1 - Sebald, Walter T1 - The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing N2 - No abstract available KW - Biochemie Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62631 ER - TY - JOUR A1 - Römisch, J. A1 - Tropschug, M. A1 - Sebald, Walter A1 - Weiss, H. T1 - The primary structure of cytochrome c\(_1\) from Neurospora crassa N2 - No abstract available KW - Biochemie Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62578 ER - TY - JOUR A1 - Viebrock, A. A1 - Perz, A. A1 - Sebald, Walter T1 - The imported preprotein of the proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa. Molecular cloning and sequencing of the mRNA N2 - No abstract available KW - Biochemie Y1 - 1982 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62742 ER - TY - JOUR A1 - Weich, H. A. A1 - Sebald, Walter A1 - Schairer, H. U. A1 - Hoppe, J. T1 - The human osteosarcoma cell line U-2 OS expresses a 3.8 kilobase mRNA which codes for the sequence of the PDGF-B chain N2 - A cDNA clone of about 2500 basepairswas prepared from the human osteosarcoma cellline U-2 OS by hybridizing with a v-sis probe. Sequence analysis showed that this cDNA contains the coding region for the PDGF-B chain. Here we report that the mitogen secreted by these osteosarcoma cells contains the PDGF-B chain and is probably a homodimer of two B-chains. KW - Biochemie KW - Platelet-derived growthfactor KW - cDNA KW - Oncogene KW - Tumor cell Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62588 ER - TY - JOUR A1 - Sebald, Walter A1 - Graf, T. A1 - Lukins, H. B. T1 - The dicyclohexylcarbodiimide-binding protein of the mitochondrial ATPase complex from Neurospora crassa and Saccharomyces cerevisiae. Identification and isolation N2 - Incubation of mitochondria from Neuraspara crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low-molecular-weight polypeptide. This dicyclohexylcarbodiimide-binding protein is identical with the smallest subunit (Mr 8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein. The dicyclohexylcarbodiimide-binding protein is extracted from whole mitochondria with neutral chloroformjmethanol both in the free and in the inhibitor-modified form. In Neuraspara and yeast, this extraction is highly selective and the protein is obtained in homogeneaus form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide Iabel is enriched in the purified protein up to 50-fold compared to whole mitochondria. Based on the amino acid analysis, the dicyclohexylcarbodiimide-binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N-terminal ~mino acid is tyrosine in Neuraspara and formylmethionine in yeast. KW - Biochemie Y1 - 1979 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62792 ER - TY - JOUR A1 - Graf, T. A1 - Sebald, Walter T1 - The dicyclohexylcarbodiimide-binding protein of the mitochondrial ATPase complex from beef heart. Isolation and amino acid composition N2 - No abstract available KW - Biochemie Y1 - 1978 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62806 ER - TY - JOUR A1 - Michel, R. A1 - Wachter, E. A1 - Sebald, Walter T1 - Synthesis of a larger precursor for the proteolipid subunit of the mitochondrial ATPase complex of Neurospora crassa in a cell-free wheat germ system N2 - No abstract available KW - Biochemie Y1 - 1979 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62789 ER - TY - JOUR A1 - Sebald, Walter A1 - Friedl, P. A1 - Schairer, H. U. A1 - Hoppe, J. T1 - Structure and genetics of the H\(^+\)-conducting F\(_0\) portion of the ATP synthase N2 - The ATP synthase occurs in remarkably conserved form in procaryotic and eucaryotic cells. Thus, our present knowledge of ATP synthase is derived from sturlies of the enzyme from different organisms, each affering specific experimental possibilities. In recent tim es, research on the H\(^+\) -conducting F0 part of the ATP synthase has been greatly stimulated by two developments in the Escherichio coli system. Firstly, the purification and reconstitution of the whole ATP synthase as weil as the proton conductor Fa from E. coli have been achieved. These functionally active preparations are well defined in terms of subunit composition, similar to the thermophilic enzyme from PS-3 studied by Kagawa's group.u Secondly, the genetics and the molecular cloning of the genes of all the F\(_0\) subunits from E. coli yielded information on the function of subunit polypeptides and essential amino acid residues. Furthermore, the amino acid sequence of hydrophobic F\(_0\) subunits, which are difficult to analyze by protein-chemical techniques, could be derived from the nucleotide sequence of the genes. These achievements, which shall be briefly summarized in the next part of this communication, provide the framework to study specific aspects of the structure and function of the F\(_0\) subunits. KW - Biochemie Y1 - 1982 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62733 ER - TY - JOUR A1 - Dummer, R. A1 - Posseckert, G. A1 - Nestle, F. A1 - Witzgall, R. A1 - Burger, M. A1 - Becker, J. C. A1 - Schäfer, E. A1 - Wiede, J. A1 - Sebald, Walter A1 - Burg, G. T1 - Soluble interleukin-2 receptors inhibit interleukin 2-dependent proliferation and cytotoxicity: explanation for diminished natural killer cell activity in cutaneous T-cell lymphomas in vivo? N2 - No abstract available KW - Biochemie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62473 ER - TY - JOUR A1 - Kruse, N. A1 - Lehrnbecher, T. A1 - Sebald, Walter T1 - Site-directed mutagenesis reveals the importance of disulfide bridges and aromatic residues for structure and proliferative activity of human interleukin-4 N2 - Mutant proteins (muteins) of human lnterleukin-4 (llA) were constructed by means of in vitro mutagenesis. The muteins were expressed in E. co/1, submitted to a renaturation and purification protocol and analysed for biological activity. Exchange of the cysteines at either position 46 or 99 which form one of the three disulfide bridges resulted. in a nearly co•mplete loss · of biological actiyity and an unstable protein. The exchange of tyrosine 124 also inactivated the protein, while a mutation of tyrosine 56 left some residual activity. Exchange of the other four cysteines or of · the single tryptophane had smaller etTects. KW - Biochemie KW - Interleukin 4 (human) KW - Recombinant KW - ln vitro mutagenesis KW - Structure-function Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62505 ER - TY - JOUR A1 - Demchuk, E. A1 - Mueller, T. A1 - Oschkinat, H. A1 - Sebald, Walter A1 - Wade, R. C. T1 - Receptor binding properties of four-helix-bundle growth factors deduced from electrostatic analysis N2 - Hormones of the hematopoietin class mediate signal transduction by binding to specific transmembrane receptors. Structural data show that the human growth hormone (hGH) forms a complex with a homodimeric receptor and that hGH is a member of a class of hematopoietins possessing an antiparallel 4-a-helix bundle fold. Mutagenesis experiments suggest that electrostatic interactions may have an important influence on hormonereceptor recognition. In order to examine the specificity of hormone-receptor complexation, an analysis was made of the electrostatic potentials of hGH, interleukin-2 (IL-2), interleukin-4 (IL-4), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the hGH and IL-4 receptors. The binding surfaces of hGH and its receptor, and of IL-4 and its receptor, show complementary electrostatic potentials. The potentials of the hGH and its receptor display approximately 2-fold rotational symmetry because the receptor subunits are identical. In contrast, the potentials of GM-CSF and IL-2 Iack such symmetry, consistent with their known high affinity for hetero-oligomeric receptors. Analysis of the electrostatic potentials supports a recently proposed hetero-oligomeric model for a high-affinity IL-4 receptor and suggests a possible new receptor binding mode for G-CSF; it also provides valuable information for guiding structural and mutagenesis studies of signal-transducing proteins and their receptors. KW - Biochemie KW - cytokines KW - electrostatic potential KW - hematopoietic receptors KW - human growth factor KW - interleukins KW - molecular recognition Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62424 ER - TY - JOUR A1 - Neupert, W. A1 - Sebald, Walter A1 - Schwab, A. J. A1 - Pfaller, A. A1 - Bücher, T. T1 - Puromycin sensitivity of ribosomal label after incorporation of \(^{14}\)C-labelled amino acids into isolated mitochondria from Neurospora crassa N2 - Radioactive amino acids were incorporated into isolated mitochondria from Neurospora crassa. Then the mitochondrial ribosomes were isolated and submitted to density gradient centrifugation. A preferential labelling of polysomes was observed. However, when the mitochondrial suspension was treated with puromycin after amino acid incorporation, no radioactivity could be detected in either the monosomes or the polysomes. The conclusion is drawn that isolated mitochondria under these conditions do not incorporate significant amounts of amino acids into proteins of their ribosomes. KW - Biochemie Y1 - 1969 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62899 ER - TY - JOUR A1 - Weiss, H. A1 - Sebald, Walter T1 - Purification of cytochrome oxidase from Neurospora crassa and other sources N2 - A chromatographic procedure 1 is described by means of which cytochrome oxidase has been purified from a variety of organisms including the fungus N eurospora crassa,2,3 the unicellular alga Po/ytoma mirum, 4 the insect Locusta migratoria ,5 the frog Xenopus muel/eri,4 and the mammal Rattus norwegicus. 4 This procedure can be used to equal effect for large-scale preparations, starting from grams of mitochondrial protein, or for small-scale preparations starting from milligrams. The cytochrome oxidase preparations from the different organisms are enzymically active. They show similar subunit compositions. KW - Biochemie Y1 - 1978 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-82082 ER -