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W Möller

Publications and source records attributed to W Möller.

At least 91 records · Page 5Linked to original sources
PubMed1984

The primary structure of elongation factor EF-1 alpha from the brine shrimp Artemia.

cDNA as well as amino acid sequencing has revealed the complete primary structure of elongation factor EF-1 alpha from the brine shrimp Artemia. A comparison with the published sequences of bacterial EF-Tu, mitochondrial EF-Tu and chloroplastic EF-Tu shows that distinct areas of these polypeptide chains are conserved in evolution. The evolutionary distance between prokaryotic and eukaryotic types of EF-Tu is larger than among bacterial and organellar EF- Tus . A number of regions present in both EF-Tu and EF-G from Escherichia coli are also found in EF-1 alpha from Artemia.

Amino Acid Sequence↗
PubMed1983

Synthesis and application of two reagents for the introduction of sulfhydryl groups into proteins.

Two reagents are described which can be used for the introduction of sulfhydryl groups into proteins. Mercaptopropionylhydrazide modifies specifically periodate-oxidized N termini of proteins, provided that the N-terminal residue is serine or threonine. 3-(Phenyldithio)propionimidate introduces a disulfide bond at lysine residues of proteins. Reduction converts the disulfide into a sulfhydryl group. The imidate compound was found to react with a high specificity with only one lysine residue of ribosomal protein L7/L12.

Amino Acids, Sulfur↗
PubMed1983

Genes for elongation factor EF-1 alpha in the brine shrimp Artemia.

A plasmid carrying a cDNA sequence coding for elongation factor EF-1 alpha from Artemia was used to probe blots of mRNA and chromosomal DNA from Artemia. A messenger length for EF-1 alpha corresponding to 1850 nucleotides was found. Southern blots pointed to a limited number (1-4) of genes, coding for EF-1 alpha. From an Artemia gene library a recombinant phage was isolated, which contains genomic sequences of EF-1 alpha. S1-nuclease mapping indicated the presence of intervening sequences within this cloned gene.

Animals↗
PubMed1983

Sequence homology between EF-1 alpha, the alpha-chain of elongation factor 1 from Artemia salina and elongation factor EF-Tu from Escherichia coli.

In the course of a structural analysis of the alpha-chain of elongation factor 1 from Artemia salina cysts, we present four amino acid sequences comprising together half of the polypeptide chain. A comparison of these sequences with the primary structure of elongation factor EF-Tu from Escherichia coli reveals a clear correspondence between the eukaryotic and prokaryotic protein throughout their polypeptide chains. The results support a basic conservation of the structure of the aminoacyl-tRNA carrying enzyme in evolution. The occurrence, in the eukaryotic factor, of several epsilon-trimethyllysine residues, is remarkable.

Amino Acid Sequence↗
PubMed1983

Ribosomal proteins L7/L12 of Escherichia coli. Localization and possible molecular mechanism in translation.

Experiments were performed in order to determine the minimal requirement for the proteins L7/L12 in polyphenylalanine synthesis and elongation factor EF-G-dependent GTP hydrolysis. Via reconstitution, ribosomal particles were prepared containing variable amounts of L7/L12. The L7/L12 content of these particles was carefully determined by the use of 3H-labelled L7/L12 and by radioimmunoassay. The activity of the particles was determined as a function of the L7/L12 content. Our results show that only one dimer of L7/L12 is required for full activity in EF-G-dependent GTP hydrolysis. On the other hand, two L7/L12 dimers are required for polyphenylalanine synthesis. In addition, we have determined the relation between the number of L7/L12 stalks, as observed by electron microscopy, and the L7/L12 content of the 50 S particles. Our interpretation of these results is that each ribosomal particle possesses two L7/L12 binding sites, each site being involved in binding one dimer. Binding of L7/L12 dimer in one site gives rise to formation of the L7/L12 stalk, whereas binding in the other site has no effect on the number of visible stalks.

Bacterial Proteins↗
PubMed1982

Preparation and characterization of fluorescent 50S ribosomes. Specific labeling of ribosomal proteins L7/L12 and L10 of Escherichia coli.

So that the topographic and dynamic properties of the L7/L12--L10 complex in the 50S ribosome of Escherichia coli could be studied, methods and reagents were developed in order to introduce fluorescent groups at specific positions of these proteins. In the case of L7/L12, this was done by attaching an aldehyde group to Lys-51 of the protein by using 4-(4-formylphenoxy)butyrimidate or by converting the amino terminus of L12 into an aldehyde group by periodate oxidation. Subsequent reaction of the aldehyde groups with newly developed hydrazine derivatives of fluorescein and coumarin resulted in specifically labeled L7/L12 derivatives. L10 was modified at the single cysteine residue with N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide. The fluorescent proteins L10 and L7/L12 could be reconstituted into 50S ribosomes. The resulting specifically labeled 50S ribosomes show 25--100% activity in elongation factor G dependent GTPase as well as in polyphenylalanine synthesis. The fluorescent properties of the labeled 50S ribosomes show that these fluorescent derivatives are suitable for energy transfer studies.

Escherichia coli↗
PubMed1982

Fluorescence studies on the location of L7/L12 relative to L10 in the 50S ribosome of Escherichia coli.

The localization of the protein L7/L12 relative to protein L10 in the Escherichia coli ribosome was studied by fluorescence energy transfer. N-[7-(Dimethylamino)-4-methylcoumarinyl]maleimide, coupled to Cys-70 of L10, served as a donor for fluorescein which was attached to Lys-51 or to the N terminus of L7/L12. The binding of the fluorescein-L7/L12 dimers to a strong and a weak binding site in 50S ribosomes could be distinguished. Therefore, it was possible to measure the distances between Cys-70 of L10 and Lys-51 and the N terminus of each L7/L12 dimer. For L7/L12 in the strong binding site, these two distances are both about 43 A, and for L7/L12 in the weak binding site, both are about 56 A.

Energy Transfer↗
PubMed1982

Ribosomal protein S6 from Xenopus laevis ovaries. Isolation, phosphorylation in vivo and cross-reaction with heterologous anti-S6 antibodies.

Ribosomal protein S6 from Xenopus laevis ovaries was prepared by ion-exchange chromatography on phosphocellulose and gel filtration on Sephadex G-75. The protein was identified as S6 from its position on two-dimensional polyacrylamide gels and from its immunological cross-reaction with monoclonal antibody raised against chicken liver S6, and from the fact that it is the major phosphorylated protein of the small subunit. When oocytes were incubated with [32P]orthophosphate in the presence of progesterone, 32P incorporation of 40-S ribosomal proteins was stimulated about 10-fold over controls without hormone. The bulk of the 32P radioactivity was incorporated into protein S6.

Animals↗
PubMed1982

Affinity labels for membrane components involved in the uptake of bile acids and of phallotoxins by hepatocytes. Development of covalently binding derivatives of bile acids and of compounds related to cholecystographic agents.

A series of covalently binding derivatives of bile acids, fusidic acid and of compounds similar to cholecystographic agents were synthesized. Nearly all of them inhibited the development of protrusions on the surface of isolated hepatocytes regularly seen after treatment with phalloidin. The same compounds inhibited the uptake of demethylphalloin and of cholate in a concentration dependent manner. Two kinds of effects could be distinguished: The irreversible part of the inhibition depended on the incubation period and could not be removed by washing procedures. The reversible one was independent on the duration of the preincubation. Final results indicated that the tested derivatives inhibited either both transports, and the phalloidin response of liver cells to the same degree and in the same manner, or were found to be ineffective in all tests. The above parallelism supports the hypothesis that phallotoxins may be translocated by a carrier system normally responsible for the uptake of bile acids from the portal blood.

Affinity Labels↗
PubMed1982

Chemical modification of membrane proteins by brominated taurodehydrocholate in isolated hepatocytes; relationship to the uptake of cholate and of phalloidin and to the sensitivity of hepatocytes to phalloidin.

In vitro treatment of isolated rat hepatocytes with brominated taurodehydrocholic acid (BTC) reduced their sensitivity against phalloidin and inhibited the uptake of phalloidin as well as of cholate in an irreversible and concentration dependent manner. BTC was taken up itself by liver cells; this process was inhibited by 4,4'-diisothiocyano 2,2'-stilbene disulfonate (DIDS). When hepatocytes were incubated with 35S-BTC their plasma membranes contained five labeled protein species with molecular weights of 67,000, 49,000, 38,000, 32,000 and 24,000 as shown by SDS-electrophoresis. No marked difference was observed when isolated plasma membranes from livers were directly treated with the affinity label. DIDS suppressed covalent binding of 35S-BTC to membrane components drastically. Incubation of phalloidin insensitive AS-30D ascites hepatoma cells with 35S-BTC did not result in a chemical modification of the above five proteins. This agrees with an earlier observation that hepatoma cells are unable to take up phalloidin and bile acids (Petzinger et al. 1979; Rufeger and Grundmann 1977; Kroker et al. 1978).

Affinity Labels↗
PubMed1981

Protein synthesis in artemia salina. Eucaryotic elongation factor eEF-Ts is a transphosphorylase.

It is thought that eucaryotic elongation factor eEF-Ts catalyzes the replacement of GDP for GTP on eucaryotic elongation factor eEF-Tu. We have found that eEF-Ts displays a strong nucleoside diphosphate phosphotransferase activity. This transferase activity resides in a dimer molecule of a subunit molecular weight close to 30,000. The transfosforylating activity of eEF-Ts results in a stimulatory effect of ATP, GTP, UTP and CTP on protein synthesis provided that GDP is present. The specificity for guanine nucleotides in protein synthesis resides only in eEF-Tu.

Adenosine Triphosphate↗
PubMed1981

Structural analysis of ribosomal protein L7/L12 by the heterobifunctional cross-linker 4-(6-formyl-3-azidophenoxy)butyrimidate.

The structure of the dimeric form of the protein L7/L12 from ribosomes from Escherichia coli was studied by using the heterobifunctional cross-linker 4-(6-formyl-3-azidophenoxy)butyrimidate. The imidate group of the cross-linker reacts very specifically with Lys-51 of L7/L12. Subsequent cross-linking of this modified L7/L12 by reductive alkylation of the aldehyde group of the cross-linker results in the formation of a covalent cross-link between both polypeptide chains of the L7/L12 dimer. This covalently cross-linked dimer is fully active in reconstitution of elongation factor G dependent GTP hydrolysis of 50S cores lacking L7/L12, suggesting a conformation of the cross-linked protein similar to the conformation of native L7/L12. Analysis of the tryptic peptides of cross-linked L7/L12 shows the points of attachment of the cross-linker to be Lys-51 in one polypeptide chain and Lys-29 in the other. On the basis of a combination of this result with published data, a structure for the N-terminal region of L7/L12 dimers is proposed. The important feature of this model is a shifted parallel alignment of both polypeptide chains resulting in one free N-terminal stretch for each L7/L12 dimer which attaches the protein to the ribosome via protein L10.

Amino Acid Sequence↗
PubMed1981

Photochemical cross-linking of elongation factor G to 70-S ribosomes from Escherichia coli by 4-(6-formyl-3-azidophenoxy)butyrimidate.

Ribosomal proteins situated at or near the binding site of elongation factor G (EF-G) on the Escherichia coli ribosome have been identified by use of the heterobifunctional cross-linker 4-(6-formyl-3-azidophenoxy)butyrimidate. Four different preparations of EF-G, in which the number of cross-linker molecules coupled to EF-G ranged from four to seven, all cross-linked to 50-S subunit proteins L1, L3 and L11 as well as to 30-S subunit proteins S3 and S4. Cross-linking of EF-G to ribosomal proteins was tested electrophoretically. In the case of L7/L12 and L11 immunological methods were also used. Cross-linking of EF-G to L1, L3, L11, S3 and S4 is specific as judged from the fact that addition of unmodified EF-G and of thiostrepton results in less cross-linking. The cross-linking data suggests that the binding site for EF-G includes several proteins which are located at the interface between the 30-S and 50-S subunits.

Cross-Linking Reagents↗
PubMed1981

Partial purification of the messenger RNA for eukaryotic elongation factor Tu from Artemia salina.

Polyadenylated RNA from developing Artemia salina cysts was fractionated by centrifugation through a sucrose gradient containing methylmercuric hydroxide (CH3HgOH). Aliquots of each fraction were directly added to a rabbit reticulocyte lysate to program protein synthesis in vitro. The translation products were assayed for eukaryotic elongation factor Tu (eEF-Tu) by immunoprecipitation with an antibody raised in rabbits and purified by affinity chromatography. The immunoprecipitated radioactivity was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Sequences coding for eEF-Tu sediment in the 20-S region of the gradient and form a major component of the poly(A)-containing RNA. The mRNA of the 20-S region, comprising about 10% of the poly(a)-containing RNA fractionated on the gradient, has been translated in vitro and 30% of the translation products represent immunoprecipitable eEF-Tu protein chains with an Mr of 50000.

Animals↗