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 - TY - JOUR A1 - Sebald, Walter A1 - Neupert, W. A1 - Weiss, H. T1 - Preparation of Neurospora crassa mitochondria N2 - The fungus Neurospora crassa represents a eukaryotic cell with high biosynthetic activities. Cell mass doubles in 2-4 hr during expone ntial growth , even in simple salt media with sucrose as the sole carbon source. The microorgani sm forms a mycelium of long hyphae durlng vegetative growth . The mitochondria can be isolated under relatively gentle condi tions since a few breaks in the threadlike hyphae are sufficient to cause the outflow of the organelles. This article describes two methods for the physical disruption of the hyphae : (I) The cell s are opened in a grind mill between two rotating corundum di sks. This is a continuous and fast procedure and allows large- and small-scale preparations of mitochondria. (2) Hyphae are ground with sand in a mortar and pestle. This procedure can be applied to microscale preparations of mitochondria starting with minute amounts of cells. Other procedures for the isolation of Neurospora mitochondria after the physical di sruption or the enzymatic degradation of the cell wall have been described elsewhere KW - Biochemie Y1 - 1979 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-82070 ER - TY - JOUR A1 - Sebald, Walter A1 - Wild, G. T1 - Mitochondrial ATPase complex from Neurospora crassa N2 - The A TPase eomplex has been isolated from mitoehondria of N eurospora crassa by immunologieal teehniques. The protein ean be obtained rapidly and qua ntitatively in high purity by miero- or large-seale immunopreeipitation. Immunopreeipitation has been applied to labeled and doubly labeled mitoehondrial proteins in order to investigate the number and moleeular weights of subunit polypeptides , the site of synthesis of subunit polypeptides, and the dieycIohexyIcarbodiimide-binding protein . The A TPase complex obtained by large-seale immunopreeipitation has been used as starting ma terial for the isolation of hydrophobie polypeptides. KW - Biochemie Y1 - 1979 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-82065 ER - TY - JOUR A1 - Sebald, Walter A1 - Werner, S A1 - Weiss, H T1 - Biogenesis of mitochondrial membrane proteins in Neurospora crassa N2 - no abstract available KW - Biochemie KW - Neurospora crassa Y1 - 1979 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-82055 ER - TY - JOUR A1 - Werner, S. A1 - Sebald, Werner T1 - Immunological techniques for studies on the biogenesis of mitochondrial membrane proteins N2 - no abstract available KW - Biochemie Y1 - 1981 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-82044 ER - TY - JOUR A1 - Sebald, Walter A1 - Bücher, T. A1 - Olbrich, B. A1 - Kaudewitz, F. T1 - Electrophoretic pattern of and amino acid incorporation in vitro into the insoluble mitochondrial protein of neurospora crassa wild type and mi-1 mutant N2 - No abstract available KW - Biochemie Y1 - 1968 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62926 ER - TY - JOUR A1 - Sebald, Walter A1 - Hofstötter, T. A1 - Hacker, D. A1 - Bücher, T. T1 - Incorporation of amino acids into mitochondrial protein of the flight muscle of Locusta migratoria in vitro and in vivo in the presence of cycloheximide N2 - No abstract available KW - Biochemie Y1 - 1969 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62919 ER - TY - JOUR A1 - Sebald, Walter A1 - Schwab, A. J. A1 - Bücher, T. T1 - Cycloheximide resistant amino acid incorporation into mitochondrial protein from Neurospora crassa in vivo N2 - No abstract available KW - Biochemie Y1 - 1969 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62900 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 - Neupert, W. A1 - Sebald, Walter A1 - Schwab, A. J. A1 - Massinger, P. A1 - Bücher, T. T1 - Incorporation in vivo of \(^{14}\)C-labelled amino acids into the proteins of mitochondrial ribosomes from Neurospora crassa sensitive to cycloheximide and insensitive to Chloramphenicol N2 - Radioactive amino acids were incorporated in vivo into N eurospora crassa cells, and the mitochondrial ribosomes were isolated. The incorporation of radioactivity into the proteins of these ribosomes was inhibited by cycloheximide, but not by chloramphenicol. It is therefore concluded that these proteins are synthesized on the cycloheximide sensitive and chloramphenicol insensitive cytoplasmic ribosomes. KW - Biochemie Y1 - 1969 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62884 ER - TY - JOUR A1 - Sebald, Walter A1 - Neupert, W. A1 - Birkmayer, G. D. T1 - Internal and external contributions to the biogenesis of mitochondrial proteins N2 - No abstract available KW - Biochemie Y1 - 1970 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62876 ER - TY - JOUR A1 - Weiss, H. A1 - Sebald, Walter A1 - Bücher, T. T1 - Cycloheximide resistant incorporation of amino acids into a polypeptide of the cytochrome oxidase of Neurospora crassa N2 - Radioaetive leueine was ineorporated by N eurospora crassa mitoehondria in vivo in the presence of cyeloheximide. When the membrane protein of these mitochondria was ehromatographieally separated on oleyl polymethaerylie aeid resin, & nurober of fraetions were obtained whieh differ with respeet to their eontents of radioaetivity and eytoehromes. The highest speeifie radioaetivity was found in the fraction eontaining eytoehrome aa3• This fraetion proved to be a pure and enzymatically aetive cytoehrome oxidase. Its ratio of absorbanee at 280 nm (ox)/ 443 nm (red.) was 2.1. By means of sodium dodeeylsulfate gel-electrophoresis, this enzymewas separated into five polypeptides with molecular weights of 30000, 20000, 13000, 10000, and 8000. Only the polypeptide with the molecular weight 20000 displayed a high specific radioaetivity. KW - Biochemie Y1 - 1971 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62866 ER - TY - JOUR A1 - Sebald, Walter A1 - Weiss, H. A1 - Jackl, G. T1 - Inhibition of the assembly of cytochrome oxidase in Neurospora crassa by chloramphenicol N2 - Cytochrome oxidasewas prepared from Neurospora crassa by chromatography on oleyl polymethacrylic acid resin and separated into seven polypeptides by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Incorporation oflabelled amino acids into the single polypeptideswas investigated after a pulse labelling in the absence and presence of chloramphenicol, and afterwashing out the inhibitor. Chloramphenicol (4 mg/ml) inhibited amino acid incorporation into all polypeptides 90-95%• while labeHing of the whole membrane protein was inhibited only 30%• Mter washing out the inhibitor and further growth of the cells. the four smaller polypeptides were highly labelled, whereas the other polypeptides showed only a. small increase in radioactivity. It is concluded that the four small-sized polypeptides of cytochrome oxidase are synthesized but not integrated into the functional enzyme under the action of chloramphenicol. KW - Biochemie Y1 - 1972 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62852 ER - TY - JOUR A1 - Schwab, A. J. A1 - Sebald, Walter A1 - Weiss, H. T1 - Different pool sizes of the precursor polypeptides of cytochrome oxidase from Neurospora crassa. N2 - Pulse-labelling experiments with growing Neurospora crassa revealed that the polypeptides composing the protein moiety of a cytochrome oxidase preparation are derived from at least four independent pools of precursor polypeptides. The pool sizes range from 2 ° f 0 to 25 °/0 of the amount of the corresponding polypeptide present in cytochrome oxidase. The smallest pool is assigned to a polypeptide of mitochondrial origm. Serial pools were found for one of the polypeptides. KW - Biochemie Y1 - 1972 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62841 ER - TY - JOUR A1 - Weiss, H. A1 - Sebald, Walter A1 - Schwab, A. J. A1 - Kleinow, W. A1 - Lorenz, B. T1 - Contribution of mitochondrial and cytoplasmic protein synthesis to the formation of cytochrome b and cytochrome aa\(_3\) N2 - A cytochrome b preparation from Neurospora crassa mitochondria is found to consist of three polypeptides (apparent molecular weight 10 000, 11 000 and 32 000), a cytochrome aa3 preparation of six to seven polypeptides (apparent molecular weight 8 000, 11 000, 13 000, 18 000, 28 000 and 36 000). Selective incorporation of radioactive amino acids by eilher mitochondrial protein synthesis when the cytoplasmic one is blocked or by the cytoplasmic protein synthesis, when the mitochondrial one is blocked, indicates that one cytochrome b polypeptide (mw 32 000) and one to three cytochrome aa3 polypeptides (mw 36 000, 28 000 and 18 000) are mitochondrial translation products, the other cytochrome b and cytochrome aa3 polypeptides cytoplasmic translation products. The delayed appearance of labeling in the cytochrome b and cytochrome aa3 polypeptides compared to the average cell protein after a pulse of <~H leueine revealed that these polypeptides are derived from separate pools of precursor polypeptides. The pool sizes range from 2 p. cent to 25 p. cent of the amount of the corresponding polypeptide present in the cytochromes. The 32 000 molecular weight polypeptide of cytochrome band at least the 18 000 molecular weight polypeptide of cytochrome aa\(_3\) are mitochondrial translation products as well in the fungus Neurospora crassa as in the insect Locusta migratoria. So, despite the fact that the size of mitochondrial DNA and mitochondrial ribosomes is reduced in insects, the products have maintained their characteristics. KW - Biochemie Y1 - 1973 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62835 ER - TY - JOUR A1 - Sebald, Walter A1 - Machleidt, W. A1 - Otto, J. T1 - Products of mitochondrial protein synthesis in Neurospora crassa. Determination of equimolar amounts of three products in cytochrome oxidase on the basis of amino-acid analysis N2 - Cytochrome oxidase isolated from N eurospora crassa was resolved into seven protein eomponents by eleetrophoresis in polyaerylamide gels eontaining sodium dodeeylsulfate. The apparent molecular weights were determined tobe 41000, 28500, 21000, 16000, 14000, 11500 and 10000 for the eomponents 1, 2, 3, 4, 5, 6, and 7, respectively. The components 1, 2 and 3 are synthesized on mitochondrial ribosomes as shown by the incorporation of radioactive amino aeids in the presenee of cyeloheximide. Amino-acidanalysis of the isolated components 1, 2 and 3 revealed a high content of apolar amino acids and a low eontent of basic amino aeids compared to an average amino-aeid eomposition of components 4-7. Components 1, 2 and 3 eontribute 27.9°/0, 18°/0 and 14.2°/0 to the whole eytoehrome oxidase protein. This was calculated from the contributions of the single eomponents to the totalleueine eontent of the enzyme and the leueine eontents (nmol leueine per mg protein) of the single eomponents as determined by amino-aeid analysis. Equimolar relations of the components 1, 2 and 3 are found by dividing the amounts of protein by their apparent molecular weights. A stoichiometry of 1:1:1 results assuming a minimal molecular weight of 150000 for the whole cytochrome oxidase protein. On the basis of the heme a content a molecular weight of about 70000 per heme group was determined, using an absorption coeffieient L1e605 (redueed minus oxidized) of 12 mM-1 cm-1• It is concluded that the smallest structural unit of eytochrome oxidase contains two heme groups. KW - Biochemie Y1 - 1973 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62827 ER - TY - JOUR A1 - Jackl, G. A1 - Sebald, Walter T1 - Identification of two products of mitochondrial protein synthesis associated with mitochondrial adenosine triphosphatase from Neurospora crassa N2 - Soluble mitochondrial ATPase (F1) isolated from Neurospora crassa is resolved by dodecylsulfate- gel electrophoresis into five polypeptide bands with apparent molecular weights of 59000, 55000, 36000, 15000 and 12000. At least nine further polypeptides remain associated with ATPase after disintegration of mitochondria with Triton X-100 as shown by the analysis of an immunoprecipitate obtained with antiserum to F 1 A TPase. Two of the associated polypeptides with apparent molecular weights of 19000 and 11000 are translated on mitochondrial ribosomes, as demonstrated by incorporation in vivo of radioactive leueine in the presence of specific inhibitors of mitochondrial (chloramphenicol) and extramitochondrial ( cycloheximide) protein synthesis. The appearance of mitochondrial translation products in the immunoprecipitated A TPase complex is inhibited by' cycloheximide. The same applies for some of the extramitochondrial translation products in the presence of chloramphenicol. This suggests that both types of polypeptides are necessary for the assembly of the A TPase complex. KW - Biochemie Y1 - 1975 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62812 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 - 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 - 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 - Wachter, E. A1 - Tzagoloff, A. T1 - Identification of amino acid substitutions in the dicyclohexylcarbodiimide-binding subunit of the mitochondrial ATPase complex from oligomycin-resistant mutants of Saccharomyces cerevisiae N2 - No abstract available KW - Biochemie Y1 - 1979 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62770 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 - Hoppe, J. A1 - Sebald, Walter T1 - Amino acid sequence of the proteolipid subunit of the proton-translocating ATPase complex from the thermophilic bacterium PS-3 N2 - No abstract available KW - Biochemie Y1 - 1980 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62754 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 - 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 - 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 - 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 - Velours, J. A1 - Esparza, M. A1 - Hoppe, J. A1 - Sebald, Walter A1 - Guerin, B. T1 - Amino acid sequence of a new mitochondrially synthesized proteolipid of the ATP synthase of Saccharomyces cerevisiae N2 - The purification and the amino acid sequence of a proteolipid translated on ribosomes in yeast mitochondria is reported. This protein, which is a subunit of the A TP synthase, was purified by extraction with chloroform/methanol (2/1) and subsequent chromatography on phosphocellulose and reverse phase h.p.l.c. A mol. wt. of 5500 was estimated by chromatography on Bio-Gel P-30 in 8011/o fonnie acid. The complete amino acid sequence of this protein was determined by automated solid phase Edman degradation of the whole protein and of fragments obtained after cleavage with cyanogen bromide. The sequence analysis indicates a length of 48 amino acid residues. The calculated mol. wt. of 5870 corresponds to the value found by gel chromatography. This polypeptide contains three basic residues and no negatively charged side chain. The three basic residues are clustered at the C terminus. The primary structure of this protein is in full agreement with the predicted amino acid sequence of the putative polypeptide encoded by the mitochondrial aap1 gene recently discovered in Saccharomyces cerevisiae. Moreover, this protein shows 5011/o homology with the amino acid sequence of a putative polypeptide encoded by an unidentified reading frame also discovered near the mitochondrial ATPase subunit 6 genein Aspergillus nidulans. KW - Biochemie KW - ATP synthase KW - mitochondrially translated KW - proteolipid KW - sequence subunit Y1 - 1984 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62695 ER - TY - JOUR A1 - Arends, H. A1 - Sebald, Walter T1 - Nucleotide sequence of the cloned mRNA and gene of the ADP/ATP carrier from Neurospora crassa N2 - A cDNA complementary to the mRNA of the ADPIATP carrier from Neurospora crassa was identified among ordered cDNA clones by hybridizing total polyadenylated RNA to pools of 96 cDNA recombinant plasmids and subsequent cellfree translation of hybridization-selected mRNA. Further carrier cDNAs were found by colony fdter hybridization at a frequency of 0.2-0.3%. The gene of the carrier was cloned and isolated on a 4.6-kbp EcoRl fragment of total Neurospora DNA, and the start of the mRNA was determined by Sl nuclease mapping. From the nucleotide sequence of the cDNA and the genomic DNA, the primary structure of the gene, of the mRNA and of the ADP I ATP carrier protein could be deduced. The gene occurs in a single copy in the genome and related genes are absent. It contains two short introns, and a pyrimidine-rieb promoter region. The mRNA has a 46-bp 5 1 end and a 219-bp 3 1 end. There is an open reading frame coding for the 313 amino acid residues of the Neurospora carrier protein. The amino acid sequence is homologous in 148 positions with the established primary structure of the beef heart carrier. KW - Biochemie KW - mitochondrial ADP KW - ATP carrier KW - Neurospora crassa KW - mRNA and gene KW - nucleotide sequence KW - hybrid-selected translation Y1 - 1984 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62684 ER - TY - JOUR A1 - Schmidt, B. A1 - Wachter, E. A1 - Sebald, Walter A1 - Neupert, W. T1 - Processing peptidase of Neurospora mitochondria. Two-step cleavage of imported ATPase subunit 9 N2 - Subunit 9 (dicyclohexylcarbodümide binding protein, 'proteolipid') of the mitochondrial F 1F0-ATPase is a nuclearly coded protein in Neurospora crassa. lt is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved ofT after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2 + for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two dcavage sites in the precursor molecule were determined. The data indicate that: {a) the correct NH2-terminus of the mature protein was generated, (b) the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleueine in position -31. The cleavage sites show similarity ofprimary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NHrterminal part of these polypeptides) into the matrix space of mitochondria. KW - Biochemie Y1 - 1984 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62674 ER - TY - JOUR A1 - Lindenmaier, W. A1 - Dittmar, K. E. A1 - Hauser, H. A1 - Necker, A. A1 - Sebald, Walter T1 - Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo N2 - A method has been developed that allows the isolation of genomic clones from a cosmid library by homologaus recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into A. phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologaus plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 genewas restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively. KW - Biochemie KW - Recombinant DNA KW - DNA mediated gene transfer KW - expression plasmid KW - screening KW - packaging KW - bacteriophage lambda Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62662 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 - Gabellini, N. A1 - Harnisch, U. A1 - McCarthy, J. E. A1 - Hauska, G. A1 - Sebald, Walter T1 - Cloning and expression of the fbc operon encoding the FeS protein, cytochrome b and cytochrome c\(_1\) from the Rhodopseudomonas sphaeroides b/c\(_1\) complex N2 - The gene for the FeS protein of the Rhodopseudomonas sphaeroides b/c1 complex was identified by means of crosshybridization with a segment of the gene encoding the corresponding FeS protein of Neurospora crassa. Plasmids (pRSF1-14) containing the cross-hybridizing region, covering in total 13.5 kb of chromosomal DNA, were expressed in vitro in a homologous system. One RSF plasmid directed the synthesis of all three main polypeptides of the R. sphaeroides blc1 complex: the FeS protein, cytochrome b and cytochrome c1• The FeS protein and cytochrome c1 were apparently synthesized as precursor fonns. None of the pRSF plasmids directed the synthesis of the 10-kd polypeptide found in b/c1 complex preparations. Partial sequencing of the cloned region was performed. Several sites of strong homology between R. sphaeroides and eukaryotic polypeptides of the b/c1 complex were identified. The genes encode the three b/c1 polypeptides in the order: (5') FeS protein, cytochrome b, cytochrome c1• The three genes are transcribed to give a polycistronic mRNA of 2.9 kb. This transcriptional unit has been designated the jbc operon; its coding capacity corresponds to the size of the polycistronic mRNA assuming that only the genes for the FeS protein (jbcF), cytochrome b (jbcß) and cytochrome c1 (jbcC) are present. This could indicate that these three subunits constitute the minimal catalytic unit of the b/c1 complex from photosynthetic membranes. KW - Biochemie KW - R. sphaeroidesl KW - b/c1 complex KW - gene KW - cloning KW - in vitro expression KW - polycistronic mRNA Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62642 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 - McCarthy, J. E. A1 - Sebald, Walter A1 - Gross, G. A1 - Lammers, R. T1 - Enhancement of translational efficiency by the Escherichia coli atpE translational initiation region: its fusion with two human genes N2 - No abstract available KW - Biochemie Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62626 ER - TY - JOUR A1 - Gabellini, N. A1 - Sebald, Walter T1 - Nucleotide sequence and transcription of the fbc operon from Rhodopseudomonas sphaeroides. Evaluation of the deduced amino acid sequences of the FeS protein, cytochrome b and cytochrome c\(_1\) N2 - No abstract available KW - Biochemie Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62615 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 - Hoppe, J. A1 - Gatti, D. A1 - Weber, H. A1 - Sebald, Walter T1 - Labeling of individual amino acid residues in the membrane-embedded F\(_0\) part of the F\(_1\) F\(_0\) ATP synthase from Neurospora crassa. Influence of oligomycin and dicyclohexylcarbodiimide N2 - Three F0 subunits and the F\(_1\) subunit P of the ATP synthase from Neurospora crassa were labeled with the lipophilic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[\(^{125}\)I]iodophenyl)diazirine ([\(^{125}\)I]TID). In the proteolipid subunit which was the most heavily labeled polypeptide labeling was confmed to five residues at the NH2-terminus and five residues at the C-terminus ofthe protein. Labeling occurred at similar positions compared with the homologaus protein (subunit c) in the ATP synthase from Escherichia coli, indicating a similar structure of the proteolipid subunits in their respective organisms. The inhibitors oligomycin and dicyclohexylcarbodiimide did not change the pattern of accessible surface residues in the proteolipid, suggesting that neither inhibitor induces gross conformational changes. However, in the presence of oligomycin, the extent oflabeling in some residues was reduced. Apparently, these residues provide part of the binding site for the inhibitor. After reaction with dicyclohexylcarbodiimide an additional labeled amino acid was found at position 65 corresponding to the invariant carbodümide-binding glutamic acid. These results and previous observations indicate that the carboxyl side chain of Glu-65 is located at the protein-lipid interphase. The idea is discussed that proton translocation occurs at the interphase between different types if F\(_0\) subunits. Dicyclohexylcarbodiimide or oligomycin might disturb this essential interaction between the F\(_0\) subunits. KW - Biochemie Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62598 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 - 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 - Kleene, R. A1 - Pfanner, N. A1 - Pfaller, R. A1 - Link, T. A. A1 - Sebald, Walter A1 - Neupert, W. A1 - Tropschug, M. T1 - Mitochondrial porin of Neurospora crassa: cDNA cloning, in vitro expression and import into mitochondria N2 - No abstract available KW - Biochemie Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62566 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 - Weigel, U. A1 - Meyer, M. A1 - Sebald, Walter T1 - Mutant proteins of human interleukin 2. Renaturation yield, proliferative activity and receptor binding N2 - No abstract available KW - Biochemie Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62543 ER - TY - JOUR A1 - Merz, H. A1 - Fliedner, A. A1 - Lehrnbecher, T. A1 - Sebald, Walter A1 - Müller-Hermelink, H. K. A1 - Feller, A. C. T1 - Cytokine expression in B-cell non-Hodgkin lymphomas N2 - No abstract available KW - Biochemie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62539 ER - TY - JOUR A1 - Tony, H. P. A1 - Lehrnbecher, T. A1 - Merz, H. A1 - Sebald, Werner A1 - Wilhelm, M. T1 - Regulation of IL-4 responsiveness in lymphoma B cells N2 - The responsiveness to IL-4 with and without costimulation with anti-IgM antibodies or phorbolester was studied in 35 cases of low grade non-Hodgkin Iymphoma by analyzing enhancement of CD23 and HLA dass li expression. The predominant phenotype responds directly to IL-4. Separate differentiation states can be distinguished according to coordinate or differential upregulation of CD23 and HLA dass II molecules by IL-4 alone, and differences in responsiveness to anti-IgM antibodies. A particular subgroup of B-lymphoma cells defines a separate stage of B-eeil differentiation. They fail to express high affinity binding sites for IL-4 and accordingly do not respond to IL-4- mediated signals. Cross-linking membrane lgM receptors or direct activation of protein kinase C via phorbolester induces IL-4 receptor expression and subsequent IL-4 reactivity. KW - Biochemie KW - B lymphocytes KW - CD23 KW - CLL KW - HLA class ll KW - IL-4 KW - IL-4-receptor KW - membrane immunoglobulin Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62520 ER - TY - JOUR A1 - Preiser, J. C. A1 - Schmartz, D. A1 - Van der Linden, P. A1 - Content, J. A1 - Vanden Bussche, P. A1 - Buurman, W. A1 - Sebald, Werner A1 - Dupont, E. A1 - Pinsky, M. R. A1 - Vincent, J. L. T1 - Interleukin-6 administration has no acute hemodynamic or hematologic effect in the dog N2 - To investigate the possible hemodynamic efl'ects of interleukin-6 (IL-6), a single dose of 15 mcg/kg of recombinant IL-6 isolated from Escherichia coli was injected intravenously in six pentobarbital-anesthetized dogs. After 30 min, saline infusion was performed to maintain the - pulmonary artery balloon-occluded pressure at baseline Ievel. The animals were observed for up to 5 hours. No other hemodynamic alteration was observed than a gradual decline in cardiac output attributed to anesthesia. Hematologic variables, blood glucose, and total serum proteins were also constant. IL-6 levels were markedly elevated in the blood, bot no tumor necrosis factor activity was detected. Thus a primary role for IL-6 in the early cardiovascular alterations associated with septic shock seems unlikely. KW - Biochemie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62511 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 - Merz, H. A1 - Fliedner, A. A1 - Orscheschek, K. A1 - Binder, T. A1 - Sebald, Walter A1 - Müller-Hermelink, H. K. A1 - Feller, A. C. T1 - Cytokine expression in T-cell lymphomas and Hodgkin's disease. Its possible implication in autocrine or paracrine production as a potential basis for neoplastic growth N2 - No abstract available KW - Biochemie Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62483 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 - Lehrnbecher, T. A1 - Merz, H. A1 - Sebald, Walter A1 - Poot, M. T1 - Interleukin 4 drives phytohemagglutinin-activated T cells through several cell cycles: no synergism between interleukin 2 and interleukin 4 N2 - Cell kinetic studies of T cells stimulated with the interleukin 2 (11-2), D-4, or both lymphokines were performed with conventional [3H] thymidine incorporation and with the bivariate BrdU/Hoechst technique. 11-2 and 11-4 are able to drive phytohemagglutininactivated T cells through more than one cell cycle. Neither synergistic nor inhibitory efl'ect on T -cell proliferationwas seen for the stimulation with both 11-2 and 11-4 as compared with the effect ofll-2 alone. The quantitative data ofthe cell cycle distribution ofphytohemagglutininactivated T cells suggestthat the population ofll-4-responsive cells is at least an overlapping population, if not a real subset of the ·population of the 11-2-responsive cells. KW - Biochemie KW - BrdU-Hoechst KW - cell cycle KW - flow cytometry KW - interleukin 2 KW - interleukin 4 Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62491 ER -