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Lists titles of research papers on biochemistry. "Redox control of catalysis in ATP-citrate lyase from rat liver"; "Construction and molecular dynamics of a soluble, enzymatically active form of the Escherichia coli penicillin-binding protein 5 and purification by dye chromatography"; "Studies on the variants of the protein toxins ricin and abrin".

Presents several forthcoming papers published on the December 1, 1983 issue of the "European Journal of Biochemistry." "Purification and Properties of a Thiol Protease From Rat-Liver Nuclei"; Changes in Proteoglycan Compositiion of F9 Teratocarcinoma Cells Upon Differentiation"; "Supercoiling and the Mechanism of Restriction Endonucleases"; Identification of the Gene for DNA Helicase II of Escherichia Coli."

The article presents a list of forthcoming article in the "European Journal of Biochemistry." Some of the article are: Nucleotide sequence of the promoter and amino-terminal encoding region of the escherichia coli pepN gene, Solubilization of the bovine cardiac sarcolemmal binding sites for calcium channel blockers, Hyperchromic effect of collagen induced by human collagenase, etc.

Presents several articles to be published in the "European Journal of Biochemistry." "An Endonuclease From Yeast Mitochondrial Fractions," by R. Morosoli and C. V. Lusena; "Antigenic Relationships Between Pore Proteins of Escherichia coli K12," by N. Overbeeke, G. van Scharrenburg and B. Lugtenberg; "Respiratory Properties of Cytochrome-c-Deficient Mutants of Azotobacter vinelandii," by P.S. Hoffman, T. V. Morgan and D. V. DerVartanian; "Hydroxyl-Ion-Induced Subunit Dissociation of Yeast Cytoplasmic Pyruvate Decarboxylase. A Circular Dichroism Study," by R. F. W. Hopmann; Others.

This article presents information on forthcoming papers on biochemistry, to be published in the periodical "European Journal of Biochemistry." The papers include "Expression in Escherichia Coli of a Secreted Invertebrate Ferritin," by Matthias von Dart, Pauline M. Harrison and Werner Bottke, "Regulation of Protein Kinase A Subunits During Germination of Mucor Rouxii Sporangiospores," by Silvia Rossi and Silvia Moreno and "Optical Spectrum of Myeloperoxidase — Origin of the Red Shift," by René Plans, Younkyoo Kim, Gerald T. Babcock and Ron Wever.

The article presents information on several forthcoming papers to be published in the "European Journal of Biochemistry." Some of the papers, which are included are as: "The Respiratory Burst of Bovine Neutrophils--Role of ab Type Cytochrome and Coenzyme Specificity,"; "Structure of the Capsular Antigen of Neisseria Meningitidis Serogroup K,"; "NMR Studies of the trp Repressor From Escherichia Coli--Characterization and Assignments of Residue Types,"; "Preliminary Identification of the Secondary Structure of the trp Repressor From Escherichia Coli,"; "Identification of the Secondary Structure of the trp Repressor of Escherichia Coli et al."

This article presents information about some forthcoming research papers related to the "European Journal of Biotechnology." Some of those papers are: "Mutation-Induced Instability of Antibiotic-Resistant Mammalian Ribosomes," by P.J. Wejksnora and J.R. Warner; "Phosphoribosylpyrophosphate Synthetase of Escherichia coli. Identification of a Mutant Enzyme," by B. Hove-Jensen, P. Nygaard; "The Fl-Gene Product of Bacteriophage Lambda. Purification and Properties," by S. Benchimol and A. Becker.

The article presents information on various research papers which will be published in the forthcoming issue of the "European Journal of Biochemistry". Some of the papers discusses are "Enzymatic Synthesis of Neolactotetraosylceramide by the N-Acctyllactosamine Synthase of Human Serum". "Rate of Transplantation and Kinetics of Processing of Newly Synthesized Molecules of Two Major Outer-Membrane Proteins, the OmpA and OmpF Proteins of Escherichia Coli," by the researchers I. Crowlesmith and K. Gamon.

The article presents information on the forthcoming papers to be published in the "European Journal of Biochemistry." Some of them are: "Structural studies of the O-antigenic polysaccharide of Fusobacterium necrophorum by M.M. Garcia and M.B. Perry," "The Structure of the gene encoding chicken ribosomal protein L37 a by M. Machida," "Role of acidic lipids in the translocation and channel activity of colicins A and N in Escherichia coli cells by W. Dowhan" and "Purification and sequence determination of guanylate kinase from pig brain by G.E. Schulz."

Lists papers to be published in the "European Journal of Biochemistry" after January 1987. "Expression of bovine pancreatic ribonuclease A in Escherichia coli," by K.P. Nambiar, et al; "Phenobarbital-mediated modulation of gene expression in rat liver — Analysis of cDNA clones; "Complete amino-acid sequence of the natural ATPase inhibitor from the mitochondria of the yeast Candida utilis.

The article presents information on various forthcoming papers to be published in the March 15, 1984 issue of the "European Journal of Biochemistry." Some of them are "Studies on the Structure and Expression of Escherichia Coli pyrC, pyrD and pyrF Using the Cloned Genes," "Rat-Liver Lysosomal Sialidase. Solubilization, Substrate Specificity and Comparison With the Cytosolic Sialidase," "Diamine-Induced Dissociation of the First Component of Human Complement," "Evidence for the Glycoprotein Nature of Retina Glycogen," and "Mapping of Surface Glycoproteins of Trypanosoma Cruzi by Two-Dimensional Electrophoresis. A Correlation With the Cell Invasion Capacity."

Presents several forthcoming papers to be published in the "European Journal of Biochemistry." "Molecular Properties of Two Mutant Species of the Elongation Factor Tu" by P. H. van der Meide, F. J. Duisterwinkel, J. M. de Graaf, B. Kraal, L. Bosch, J. Douglass and T. Blumenthal; "The Mechanism of Uncoupling by Picrate in Escherichia coli K-12 Membrane System," by M. Michels and E. P. Bakker; "Nucleotide Sequence for a Novel Dunk Alpha-Globin Gene," by G. V. Paddock and J. Gaubatz; "Studies on the Actomyosin ATPase and the Role of the Alkali Light Chains," by B. Pope, P. D. Wagner and A. G. Weeds.

The paper presents various articles, which are to be published in the future issues of the European Journal of Biochemistry. Some of the articles to be published are — "Mechanisms for Albumin-Mediated Membrane Damage," by D. Dramas, E. Harvey, Al Lawrence and A. Thomas, "Transfer RNA Precursors Are Accumulated in Escherichia Coli in the Absence of RNase E," by B.K. Ray and D. Apirion, "A New Procedure for the Simultaneous Large-Scale Purification of Bacteriophage-T4-Induced Polynucleotide Kinase, DNA Ligase, RNA Ligase and DNA Polymerase," by G.M. Dolganov, A. V. Chestukhin and M.F. Shemyakin.

Presents a list of articles to be published in the "European Journal of Biochemistry." "Cooperative Effects in the Peptidyltransferase Center of Escherichia Coli Ribosomes," by S. B. Bourd, M. K. Kukhanova, B. P. Gottikh; "Distribution of Secondary Structure Along the Fibronectin Molecule," by S. Yu. Venyaminov, M. L. Metsis, M. A. Chernousov and V. E. Koteliansky; "Chromatin Structure in Barley Nuclei," by G. Mithieux and B. Roux.

From the lipopolysaccharide of Escherichia coli U41/14 (serotype 03 :K2ab(L) : H2) an unusual amino sugar was isolated. By sequential oxidation with periodate and hypoiodite and by oxidation with periodate, followed by detection of acetaldehyde formed, the ammo sugar was characterized as a 2-amino- dideoxyaldose. The hydrochloride of the amino sugar, in comparison to those of 2-amino-2, 6-dideoxy-D-glucose, 2-amino-2,6-dideoxy-L-galactose, 2-amino-2, 6-dideoxy-D-mannose, 2-amino-2, 6-di- deoxy-D-allose, and 2-amino-2, 6-dideoxy-D-gulose, was studied with aid of ion exchange column chromatography. The N-acetylated amino sugar was compared with the N-acetylated reference amino sugars with regard to their relative mobihties in paper chromatography. The ammo sugar alcohols and reference amino sugar alcohols were studied using paper electrophoresis in molybdate buffer. On the basis of the results obtained the unknown amino sugar was identified as 2-amino- 2,6-dideoxymannose (rhamnosamine). Its optical rotation showed it to be of the L-configuration. This is the first report on the natural occurrence of L-rhamnosamine. [ABSTRACT FROM AUTHOR]

The gene for a 104-kDa exocellulase, Cel48A, formerly E6, was cloned from Thermobifida fusca into Escherichia coli and Streptomyces lividans. The DNA sequence revealed a type II cellulose-binding domain at the N-terminus, followed by a FNIII-like domain and ending with a glycosyl hydrolase Family 48 catalytic domain. The enzyme and catalytic domain alone were each expressed in and purified from S. lividans and had very low catalytic activity on swollen cellulose, carboxymethyl cellulose, bacterial microcrystalline cellulose and filter paper. However, in synergistic assays on filter paper, the addition of Cel48A to a balanced mixture of T. fusca endocellulase and exocellulase increased the specific activity from 7.9 to 11.7 µmol cellobiose·min-1·mL-1, more than 15-fold higher than any single enzyme alone. Cel48A retained > 50% of its maximum activity from pH 5 to 9 and from 40 to 60 °C. Using swissmodel, the amino-acid sequence of the Cel48Acd was modeled to the known structure of Clostridium cellulolyticum CelF. Family 48 enzymes are remarkably homologous at 35% identity for all their catalytic domains and some of the properties of the 10 members are discussed. [ABSTRACT FROM AUTHOR]

A subset of the spin systems assigned in the ¹H NMR of the trp repressor in the first paper in this series (our penultimate preceding paper in this journal) can be identified as surface or buried residues on the basis of four independent types of measurement: (a) selective spin-lattice relaxation times; (b) the dependence of line widths on temperature and the concentration of manganous ion; (c) fluorescence quenching; and (d) titration behaviour. Criteria are developed for distinguishing surface and buried residues. The significance for the function of DNA biding proteins is discussed. [ABSTRACT FROM AUTHOR]

This paper presents methods developed in order to analyze experimental results concerning the binding of Escherichia coli DNA-dependent RNA polymerase to DNA at high and at low DNA concentrations, using the filter retention assay. The basic hypotheses, under which the mathematical expressions for describing the kinetics of binding are derived, are as follows. (a) At low DNA concentration: equivalence and independence of the specific binding sites; first-order dependence of the binding reaction on both DNA and protein concentration. (b) At high DNA concentration: equivalence and independence of the non-specific binding sites; no direct transfer or one-dimensional sliding of the protein along the DNA. Comparison between theoretical predictions and experimental results at high DNA concentration will alow one to determine the relative value of the rates of binding of RNA polymerase to different promoters (between 1 and 2 in T5 DNA). Binding experiments performed at low DNA concentration are reported in this paper: these results and the analysis which is reported allow one to determine the value of the rate constant of formation of non-filterable complexes for the system fd DNA (replicative form) · RNA-polymerase (ka = 3.3 × 108 M-1 s-1 in 0.1 M NaCl, 0.01 M MgCl2). [ABSTRACT FROM AUTHOR]

In this paper the question was studied whether Escherichia coli ribosomes which have undergone a specific cleavage of their 16-S rRNA by treatment with the bacteriocin cloacin DF13, are able to enter and complete an elongation cycle. As we have shown in the preceding paper in this journal, these defective ribosomes can form 70-S initiation complexes with MS2 RNA as a messenger. Binding of the second aminoacyl-tRNA (alanyl-tRNA) to the latter complexes led to the formation of fMet-Ala-tRNA which was fully puromyein-sensitive. In addition to these aminoacyl-tRNAs also the third aminoacyl-tRNA (seryl-tRNA) can be bound to defective ribosomes. Dual-label experiments showed that puromycin released all radioactivity from the ribosomes. This indicates that fMet-Ala-Ser-tRNA is formed and is translocated from the A site to the P site. Control ribosomes bind the three aminoacyl-tRNAs in a 1:1:1 ratio. The defective particles bind fMet-tRNA, Ala-tRNA and Ser-tRNA in a ratio of 1:0.5:0.25. Kinetic experiments strongly suggest that at each binding of the ternary complex aminoacyt-tRNA · EF-Tu · GTP to the A site of defective ribosomes, about half of these particles undergo irreversible inactivation. Consequently polypeptide synthesis under our conditions will come to an end after incorporation of only four or five amino acids. The particles which temporarily survive ternary complex binding are fully capable of transpeptidation and translocation. [ABSTRACT FROM AUTHOR]

`The O9-specific mannan of Escherichia coil was synthesized in vitro from GDP-[14C]mannose by membranes which were obtained from a phosphomannose isomerase-less mutant of E. coli O9:K29- :H-, Subsequent treatment of the membranes with dilute acid liberated a neutral product, whereas with aqueous phenol a charged product was obtained. Chromatography on DEAE-cellulose, incubation with alkaline phosphatase and microdetermination showed that the charged mannan was substituted with one phosphate per chain. The neutral 14C-labelled product of the incubation hr vitro was reduced with sodium boro[³H]hydride. After total acid hydrolysis, the radioactive material was chromatographed on paper in the presence of borate. It was found that [³H]glucitol, but no [³H, 14C]mannitol was present. When the neutral product, which was obtained after incubation of 14C-prelabelled membranes with nonradioactive GDP-mannose, was hydrolyzed with and without prior reduction with non-radioactive sodium borohydride, in subsequent paper chromatography, [14C]glucitol or [14C]glucose was found. The glucose was also converted enzymatically to gluconic acid, which was identified by paper electrophoresis. These results show that in the neutral O9-specific mannan glucose is at the reducing end and they indicate that the mannan chain grows at the non-reducing end. This is discussed with respect to the overall mechanism of the biosynthesis of the 09 antigen. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides from a number of mutants of Escherichia coil K-12 were investigated by means of chemical and serological methods. Inhibition of passive hemagglutination and inhibition of precipitation show that L-rhamnose is the immunodominant sugar in the lipopolysaccharide from wild-type E. coli K-12. The disaccharide rhamnosyl-KDO (where KDO is 3-deoxy-D-manno-octulosonic acid) was isolated and characterized after mild acid hydrolysis of the lipopolysaccharide. It is concluded that rhamnose is present in the innermost part of the core as a side-chain substituent on KDO. From crosses between an E. coli K-12 donor and E. coil O8, hybrids were obtained which contained either one or both of the donor rfa and rfb clusters. Serum absorption studies with lipopolysaccharides from these hybrids indicated that the histidine-linked rfb cluster is responsible for the presence of rhamnose in the K-12 core oligosaccharide. Using paper chromatography of 32p-labelled lipopolysaccharides we have found heterogeneous lipopolysaccharide in two strains as well as some differences between two wild-type strains. The latter difference is believed to be due to varying contents of KDO-linked ethanolamine phosphate. The overall results presented together with those described in the companion paper clearly show that the core oligosaccharide in E. coil K-12 has a structure different from the types previously described for other strains of E. coli (designed coli R1 to coil R4). [ABSTRACT FROM AUTHOR]

The polysaccharide moiety of the lipopolysaccharide (polysaccharide I) from Escherichia coli F492 (08:K27-:H-) and the polysaccharide (polysaccharide II) from the rfa mutant F612 (derived from F492) were isolated by extraction with 450% aqueous phenol at 65 °C. Polysaccharide I was obtained in 1–2% yield (based on dry bacteria). It contained mannose (83.5%), glucose (5.7%), galactose (3.4%), heptose (4.6%) and 2-keto-3-deoxy-mannosulonic acid (0.8%). Polysaccharide II was obtained in 0.8–1% yield (based on dry bacteria). It contained 99.2% mannose. With the method of Yphantis it was found that the molecular weights of polysaccharides I and II were 12400 and 10400, respectively. The difference accounted for the core oligosaccharide which was present in polysaccharide I and absent in polysaccharide II. The specific optical rotation was 43 °C for polysaccharide I and 42 °C for polysaccharide II. Both polysaccharides were permethylated. Subsequent hydrolysis, reduction and acetylation, followed by gas-liquid chromatography and mass spectrometry indicated that in both polysaccharides two-thirds of the mannose units were substituted at C-2 and one-third at C-3. These results were confirmed by periodate oxidation. From polysaccharide II nine oligosaccharides were obtained after partial acid hydrolysis by chromatography on Biogel P2, paper chromatography and paper electrophoresis of the borate complexes. The oligosaccharides were reduced with sodium borodeuterate (which labelled the reducing mannose units) and methylated. After hydrolysis, reduction and acetylation the products were analyzed by gas chromatography and mass spectrometry. It is concluded that the poIysaccharides I and II contain about 20 repeating units of α-mannosyl-1,2-α-mannosyl-1,2-α-mannose which are joined through &alph;-1,3 linkages. [ABSTRACT FROM AUTHOR]

Two isogenic derivatives of Escherichia coli K12: strains EA 1 (Met-, Biot-, RCstr and EA 2 (Mei-, Biot-, RCrel) when grown in the presence of ethionine in place of methionine, synthesize ribosomes whose RNA is submethylated and whose proteins contain ethionine. Net ribosome synthesis in the presence of ethionine is greater in the RCrel strain than in the RCstr strain. The precedinig paper describes results obtained with the latter strain. Here we present further studies carried out with the isogenic RCrel strain EA 2 As in the case of the RCstr strain (previous paper) ribosomes synthesized by the RCrel strain grown in the presence of ethionine display defective association properties. A large fraction of these "ethionine" ribosomes is found as free subunits in crude extracts containing 10 mM Mg+ prepared from strain EA 2 grown in an ethionine-containing medium. These free "ethionine" subunits have sedimentation coefficients of 28 S and 45 S approximately while the "ethionine" subunits found associated as 70 S particles in crude bacterial extracts sediment at 30 S and 50 S respectively. Both classes of "ethionine" subunits (free, and associated) can be methylated in vitro in the presence of S-adenosyl methionine with or without the addition of an external source of methylases (100000 x g supernatant of a homologous crude bacterial extract) The latter observation shows that the "ethionine" subunits contain bound methylases. These enzymes do not seem to be normal ribosomal proteins nor to be bound specifically to "ethionine" ribosomes since (a) they are removed by washing the "ethionine" ribosomes with 1 M NH4Cl, and (b) they are found bound to normal methionine ribosomes from which they are also removed by washing with 1 M NH4Cl. [ABSTRACT FROM AUTHOR]

Three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. No clear function has been assigned to the smallest of these three factors, IF1. Therefore, to investigate the role of this protein in the initiation process in Escherichia coli we have mutated the corresponding gene infA. Because IF1 is essential for cell viability and no mutant selection has so far been described, the infA gene in a plasmid was mutated by site-directed mutagenesis in a strain with a chromosomal infA+ gene, followed by deletion of this infA + gene. Using this approach, the six arginine residues of IF1 were altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. All mutants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Differences in growth phenotypes of the mutants were observed in both minimal and rich media. Some of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type infA+ allele. Several of these recombinants showed reduced growth rate and a partial cold-sensitive phenotype. This paper presents a collection of IF1 mutants designed for in vivo and in vitro studies on the function of IF1. [ABSTRACT FROM AUTHOR]

In the accompanying paper [Adams, H., Scotti, P.A., de Cock, H., Luirink, J. & Tommassen, J. (2002) Eur. J. Biochem. 269 , 5564–5571], we showed that the precursor of outer-membrane protein PhoE of Escherichia coli with a Gly to Leu substitution at position -10 in the signal sequence (G-10L) is targeted to the SecYEG translocon via the signal-recognition particle (SRP) route, instead of via the SecB pathway. Here, we studied the fate of the mutant precursor in a prlA4 mutant strain. prlA mutations, located in the secY gene, have been isolated as suppressors that restore the export of precursors with defective signal sequences. Remarkably, the G-10L mutant precursor, which is normally exported in a wild-type strain, accumulated strongly in a prlA4 mutant strain. In vitro cross-linking experiments revealed that the precursor is correctly targeted to the prlA4 mutant translocon. However, translocation across the cytoplasmic membrane was defective, as appeared from proteinase K-accessibility experiments in pulse-labeled cells. Furthermore, the mutant precursor was found to accumulate when expressed in a secY40 mutant, which is defective in the insertion of integral-membrane proteins but not in protein translocation. Together, these data suggest that SecB and SRP substrates are differently processed at the SecYEG translocon. [ABSTRACT FROM AUTHOR]

The molecular chaperone function of HSP90 is activated under heat-stress conditions. In the present study, we investigated the role of the interactions in the heat-induced activation of HSP90 molecular chaperone. The preceding paper demonstrated two domain–domain interactions of HtpG, an Escherichia coli homologue of mammalian HSP90, i.e. an intra-molecular interaction between the N-terminal and middle domains and an intermolecular one between the middle and C-terminal domains. A bacterial two-hybrid system revealed that the two interactions also existed in human HSP90α. Partners of the interaction between the N-terminal and middle domains of human HSP90α could, but those between the middle and C-terminal domains could not, be replaced by the domains of HtpG. Thus, the interface between the N-terminal and middle domains is essentially unvaried from bacterial to human members of the HSP90-family proteins. The citrate synthase-binding activity of HtpG at an elevated temperature was solely localized in the N-terminal domain, but HSP90α possessed two sites in the N-terminal and other domains. The citrate-synthase-binding activity of the N-terminal domain was suppressed by the association of the middle domain. The complex between the N-terminal and middle domains is labile at elevated temperatures, but the other is stable even at 70 °C. Taken together, we propose the liberation of the N-terminal client-binding domain from the middle suppressor domain is involved in the temperature-dependent activation mechanism of HSP90 molecular chaperone. [ABSTRACT FROM AUTHOR]

Excess of Mg2+ ions is known to inhibit the soluble inorganic pyrophosphatases (PPases). In contrast, the mutant Escherichia coli inorganic pyrophosphatase Asp42→Asn is three times more active than native and retains its activity at high Mg2+ concentration. In this paper, another two mutant variants with Asp42 replaced by Ala or Glu were investigated to characterize the role of Asp42 in catalysis. pH-independent kinetic parameters of MgPPi hydrolysis and the dissociation constants for the activating and inhibitory Mg2+ ions were calculated. It was shown that Mg2+ inhibition of MgPPi hydrolysis by native PPase exhibited uncompetitive kinetics under the saturating substrate concentration. All three substitutions of Asp42 lead to a sharp decrease of inhibitory Mg2+ affinity to the enzyme. These findings allow determination of the sites of inhibitory and substrate Mg2+ ions binding to PPase. Common features of these mutants allow the conclusion that the function of Asp42 is to accurately coordinate the residues implicated in the substrate and the inhibitory Mg2+ ion binding to PPase active site. Structural analysis of PPase complexed with Mg2+ compared with PPase complexed with Mn2+ and reaction products confirms this supposition. [ABSTRACT FROM AUTHOR]

The mechanism of disulfide reduction by thioredoxin in the cell is thought to occur through the formation and subsequent destruction of a mixed-disulfide intermediate between thioredoxin and the substrate. In order to model the interaction, we have prepared a mutant of Escherichia coli thioredoxin where the second cysteine residue of the active site has been replaced by an alanine residue. A specific covalent complex has been prepared between the remaining cysteine residue and a short cysteine-containing peptide. This paper describes the preparation and characterization of the mutant protein both free and in the peptide complex. [ABSTRACT FROM AUTHOR]

An Escherichia coli coupled in vitro transcription-translation system has been modified to allow efficient expression of genes under the control of a T7 promoter. We describe both the characterization and use of two S30 crude extracts prepared from E. coli, namely S30 BL21 (DE3) (containing endogenous T7 RNA polymerase) and S30 BL21 (supplemented with exogenous T7 RNA polymerase). Since transcription by the highly active T7 RNA polymerase is known to overload the translational machinery of E. coli, the ratio betweent mRNA and ribosomes has to be regulated in the coupled in vitro systems. For this purpose, the level of mRNA is controlled by varying the amount of DNA template (S30 extract with endogenous T7 RNA polymerase) or by limited amounts of exogenously added T7 RNA polymerase. The couple in vitro system described in this paper provides two especially useful applications. First, it is most suitable for studying the regulation of gene expression in vitro, second, it can be used to express DNA templates carrying up to 10 genes. We show that genes which are not well expressed in E. coli in vivo because of unfavourable codon usage or plasmid instability are synthesized efficiently in the couple in vitro system. [ABSTRACT FROM AUTHOR]

Expression studies of Propionigenium modestum ATPase genes in various combinations with Escherichia coli ATPase genes were performed in the unc deletion mutant strain E. coli DK8. Plasmids containing the whole unc operon from P. modestum were unable to complement the E. coli unc deletion mutant. Although all ATPase subunits were expressed from the plasmids, there was no detectable ATP hydrolysing activity, indicating that the F1 part was not functional. Transformants expressing an E. coli F1-P. modestum F0 hybrid exhibited considerable ATPase activities. Binding of the F1 part to the membrane was very weak, however, and the coupling between ATP hydrolysis and Na+ transport was impaired. After combining the genes for E. coli ATPase subunits α, β, γ, δ and epsilon and the hydrophilic part of subunit b with P. modestum ATPase subunits a and c and the hydrophobic part of subunit b on a plasmid, a non-functional hybrid ATPase was expressed in E. coli. The ATPase was only loosely bound to the membrane, from which it was solubilized with Triton X-100 and purified. Subunit b and a proteolytic degradation product were the only F0 subunits detectable in the purified enzyme. A stable F0 complex is thus not formed with the hybrid b subunit. The absence of a functional F0 complex was in accord with proton-conduction measurements with bacterial vesicles. The only functional Na+-translocating ATPase expressed in E. coli thus far consists of E. coli subunits α, β, γ and epsilon, and P. modestum subunits δ, a, b and c [Kaim, G. & Dimroth, P. (1993) Eur. J. Biochem. 218, 937-944]. During the cloning conducted in our present study, errors in the sequence entry into the EMBL data bank (accession no. X58461) for the P. modestum ATPase α and β subunits became evident, which are corrected in this paper. [ABSTRACT FROM AUTHOR]

Escherichia coli RNA polymerase has two sites, the i and i + 1, for the binding of the first two substrates. The i site is template- and Mg2+-independent and purine-nucleotide-specific, whereas the i + 1 site is template- and Mg2+-dependent and shows no nucleotide preference. The specificity of the i site for purine nucleotides is well in accord with the fact that most promoters initiate with a purine nucleotide. But there are a few promoters that initiate with a pyrimidine nucleotide. Dinucleotide synthesis at these promoters is completely inhibited by rifampicin. Earlier studies have failed to identify an i site for pyrimidine nucleotides. In this paper, using a fluorescent analog of UTP, namely uridine 5'-[γ-(5-sulfonic acid)naphthylamidate]-triphosphate, abbreviated as UTP[AmNS], we are able to show its binding to RNA polymerase, with a Kd of 0.8 μM, in the absence of Mg2+ and template. This suggests the presence of an i pyrimidine nucleotide site. The fact that UTP-[AmNS] is capable of initiating RNA synthesis from the i site is further evidenced by the abortive transcription analyses at the lac promoter. Fluorescence titration studies performed in the presence and absence of purine initiator molecules indicate that this site is different from the i purine site. Scatchard analysis of the above data indicates the presence of a single binding site for UTP[AmNS] in the absence of Mg2+. Moreover UTP[AmNS] binds to the core enzyme with a Kd of 3.0 μM implying that, unlike the i purine nucleotide site, the sigma protein confers a tighter binding of UTP-[AmNS] to the low-Kd site. Forster's energy transfer measurements using UTP[AmNS] as the donor and rifampicin as the acceptor have been used for estimation of the distance of the i pyrimidine nucleotide site from the rifampicin site. From these measurements, we infer that there is no direct interference of rifampicin with the first [ABSTRACT FROM AUTHOR]

In this paper, the successful expression of trypanosomul triosephosphate isomerase (TIM) from Trypanosoma brucei brucei to high yield in Estherichia coli, using a T7-polymerase-based expression system, is described. Overexpressed trypanosomal TIM is fully active. The measured physicochemical properties of this recombinant TIM and TIM purified from trypanosomes are indistinguishable. Crystals of recombinant TIM have been grown in the presence of 2.4 M ammonium sulphate under the same conditions as for trypanosomally expressed TIM. The recombinant TIM crystal structure has been refined at 0.23 nm resolution; no differences were detected between this structure and the original crystal structure. A TIM mutant was made in which a unique dimer-interface histidine residue (His47) was changed into an asparagine. This variant ([H47N]TIM) could he expressed and purified to homogeneity by a procedure which was somewhat different from the purification of recombinant wild-type TIM. It is shown that the [H47N]TIM dimer is considerably less stable than wild-type trypanosomal TIM. The catalytic activity of [H47N]TIM is concentration dependent. The dilution-dependent inactivation is reversible. His47 is involved in a water-mediated hydrogen bond with Asp385 of the other subunit. The lower stability of the [H47N]TIM dimer implies that this water-mediated hydrogen bond is important for the stability of the TIM dimer. [ABSTRACT FROM AUTHOR]

This paper reports the purification and the properties of a thioredoxin from the fungus Aspergillus nidulans. This thioredoxin is an acidic protein which exhibits an unusual fluorescence emission spectrum, characterized by a high contribution of tyrosine residues. Thioredoxin from A. nidulans cannot serve as a substrate for Escherichia coli thioredoxin reductase. Corn NADP-malate dehydrogenase is activated by this thioredoxin in the presence of dithiothreitol, while fructose-1,6-bisphosphatase is not. The amino acid sequence of Aspergillus thioredoxin was determined by automated Edman degradation after cleavage with trypsin, SV8 protease, chymotrypsin and cyanogen bromide. The masses of tryptic peptides were verified by plasma-desorption mass spectrometry. The mass of the protein was determined by electrospray mass spectrometry and shown to be in agreement with the calculated mass derived from the sequence (Mr = 11 564). Compared to thioredoxins from other sources, the protein from A. nidulans displays a maximal sequence similarity with that from yeast (45%). [ABSTRACT FROM AUTHOR]

We have determined the solution structures and examined thc dynamics of the Escherichia coli trp repressor (a 25-kDa dimer), with and without the co-repressor L-tryptophan, from NMR data. This is the largest protein structure thus far determined by NMR. To obtain a set of data sufficient for a structure determination it was essential to resort to isotopic spectral editing. Line broadening observed in this molecular mass range precludes for the most part the measurement of coupling constants and stereospecific assignments, with the inevitable result that the attainable resolution of the final structure will be somewhat lower than the resolution reported for smaller proteins and peptides. Nevertheless the general topology of the protein can be deduced from the subsets of NOEs defining the secondary and tertiary structure, providing a basis for further refinement using the full set of NOEs and energy minimization. We report here (a) an intermediate resolution structure that can be deduced from NMR data, covalent, angular and van-der-Waals constraints only, without resort to detailed energy calculations, and (b) the limits of uncertainty within which this structure is valid. An examination of trinse structures combined with backbone amide exchange data shows that even at this resolution three important conclusions can be drawn: (a) the protein structure changes upon binding tryptophan; (b) the putative DNA binding region is much more flexible than the core of the molecule, with backbone amide proton exchange rates 1000 times faster than in the core; (c) the binding of tryptophan stabilizes the repressor molecule, which is reflected in both the appearance of additional NOEs, and in the slowing of backbone proton exchange rates by factors of 3-10.Sequence-specific ¹H-NMR assignments and the secondary structure of the holopressor (L-tryptophan-bound form) have been reported previously [C. H. Arrowsmith, R. Pachter, R. B. Altman, S. B. Iyer & O. Jardetzky (1990) Biochemistry 29, 6332-6341]. Those for the trp aporepressor (L-tryptophan-free form), made using the same methods and conditions as described in the cited paper, are reported here. The secondary structure of the aporepressor was calculated from sequential and medium-range NOEs and is the same as reported for the holorepressor except that helix E is shorter. The tertiary solution structures for both forms of the repressor were calculated from long-range NOE data. The two structures are quite similar in overall topology to one another as well as to published crystal structures; however, the DNA binding region appears to be more disordered in solution than in the crystal structures. We propose that the greater flexibility of the DNA binding region observed by us as well as by others [C. L. Lawson, R.-G. Zhang, R. W. Schevitz, Z. Otwinowski, A. Joachimiak & P. B. Sigler (1988) Proteins 3, 18] plays an important role in the mechanism of DNA binding. [ABSTRACT FROM AUTHOR]

Prourokinase is a plasminogen activator of 411 amino acids which displays a clot-lysis activity through a fibrin-dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction. The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain. In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (Δ125-prourokinase) in which 125 amino acids of the N-terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first-order rate constant of renaturation of 0.57 h-1 for Δ125-prourokinase and 0.25 h-1 for prourokinase). Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure. non-glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and Δ125-prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria. After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160000 and 250000 IU/mg protein, respectively, for prourokinase and Δ125-prourokinase. As with prourokinase, the deletion mutant 41 25-prourokinase displays a zymogenic nature, being activated by plasmin to the active two-chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile. [ABSTRACT FROM AUTHOR]

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174-9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA-) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114-12119]. In this paper we present the purification and characterization of one of these thioredoxins. The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin. [ABSTRACT FROM AUTHOR]

UTP-modulated attenuation of transcription is involved in regulating the synthesis of pyrimidine nucleotides in Escherichia coli. Thus, expression of two genes, pyrBI and pyrE, was shown to be under this type of control. The genes encode the two subunits of aspartate transcarbamylase and orotate phosphoribosyltransferase respectively. The levels of these enzymes are inversely correlated with the intracellular concentration of UTP. Modulation of attenuation seems to be a consequence of the effect of UTP concentration on the mRNA chain growth rate. Reducing the UTP pool retards RNA polymerase movement. Mechanistically this will couple the ribosomes translating a leader peptide gene more tightly to the elongating RNA polymerase. The ribosomes will then be more prone to prevent the folding of the mRNA chains into terminating hairpin structures when RNA polymerase is at the attenuator and has to decide whether transcription should terminate or continue into the structural genes. This paper described a study of pyrBI and pyrE gene regulation in cells where the ribosomes move slowly as a result of mutation in rpsL. It appears that expression of the two genes is hyper-regulated by the UTP pool in this type of cells. Furthermore, the attenuator model can only account for the results if it is assumed that UTP-concentration dependent pausing of transcription occurs in vivo in the two pry gene leaders such that RNA polymerase waits for the coupled ribosomes before transcribing into the attenuator regions. [ABSTRACT FROM AUTHOR]

This paper reports the purification and characterization of a thioredoxin system (thioredoxin, thioredoxin reductase, NADPH) from the facultative phototroph Rhodobacter sphaeroides Y. Rhodobacter sph. Y thioredoxin was purified to homogeneity with an assay based on the reduction of 5,5′-dithiobis(2-nitrobenzoic acid) by NADPH and Escherichia coli thioredoxin reductase. Rhodobacter sph. Y thioredoxin reductase was purified with the same assay using NADPH and E. coli thioredoxin. Rhodobacter sph. Y thioredoxin contained 102 amino acid residues and had a single disulfide bond. The two half-cystine residues are part of the active site made up of the sequence -Ala-Glu-Trp-Cys-Gly-Pro-Cys-Arg- which is identical to that of E. coli thioredoxin except for the presence of an Arg instead of a Lys. Rhodobacter sph. Y thioredoxin contains two tryptophan residues. The fluorescence intensity of the tryptophan residues is quenched in oxidized thioredoxin; on reduction, a much smaller increase is observed with Rhodobacter sph. Y thioredoxin than with the E. coli protein. However, the presence of 5 M guanidine · HCl results in the complete exposure of the two tryptophan residues. Rhodobacter sph. Y thioredoxin reductase has structural and functional similarities to E. coli thioredoxin reductase; it has a molecular mass of 68 kDa, and consists of two, probably identical, subunits. Each subunit has one bound FAD molecule. The enzyme is highly specific for NADPH; it is also highly specific for Rhodobacter sph. Y thioredoxin with a Km value of 3.3 ± 0.6 µM. A kinetic study of the two thioredoxin systems shows that they have a high degree of cross-reactivity. [ABSTRACT FROM AUTHOR]

In this paper we show, that the in vivo methylation of the elongation factor Tu from Escherichia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys-56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine. We have undertaken an extensive comparison between the properties of the highly methylated EF-Tu and unmodified EF-Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF-Tu (site I) also appears not to be affected by the methylation of the protein, tRNA binding at site II ts. Although the apparent affinity of tRNA for site U remains unaltered upon methylation of EF-Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys-81 are significantly less stimulated by the tRNA when EF-Tu is methylated. A possible physiological implication of this phenomenon is discussed. [ABSTRACT FROM AUTHOR]

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Escherichia coli and Proteus mirabilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-O-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN II) of the backbone carries, in S. minnesota and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible. It was found that: (a) in all three lipids A GlcN II is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. minnesota and P. mirabilis. In lipids A which were obtained from the P. mirabilis Re mutant grown at low temperature (12°C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetradecanoic acid at GlcN II is positionally replaced by Δ9-hexadecenoic acid (palmitoleic acid). It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to Glecn II are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to Glen I are free or only partially substituted. [ABSTRACT FROM AUTHOR]

An eighteen-amino-acid peptide having the linear amino acid sequence of human heat-stable enterotoxin (ST) has been synthesized by solid peptide synthesis. The purified peptide could be obtained in yields approaching 25% after purification by size, charge, and high-performance ligand chromatography, amino acid analysis, paper electrophoresis and thin-layer chromatography. The formation of the disulfide bonds was critical for biological and immunological activity and were tentatively determined to be between cysteines 5 and 14, 6 and 10, and 9 and 17. This synthetic peptide had full immunological and biological activity when compared to native ST by enzyme-linked immunosorbent assay and the sucking mouse assay respectively. [ABSTRACT FROM AUTHOR]

Penicillin-binding protein (pbp) 1b, the main DD-transpeptidase/transglycosylase of Escherichia coli, is normally present in the cell in three molecular forms α, β and γ, differenciated by their mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The three molecular forms are enzymatically active in vitro and their relative amounts are kept fairly constant in most labelling experiments with radioactive β-lactam antibiotics. In this paper, we have analyzed the expression of ponB (mrcB), the structural gone for pbp 1b, and the relation among the three forms of pbp 1b in ponB strains lysogenyzed by λ540 (ponB+) recombinant bacteriophages. Our data indicate that ponB is transcribed anti-clockwise on the E. coli chromosome and suggest that pbp 1bx is the first membrane-bound form of pbp 1b able to bind labelled β-lactams, and is the precursor of pbp 1bβ which is, in turn, the precursor of pbp 1bγ. [ABSTRACT FROM AUTHOR]

Enzyme-IIIgle is part of the glucose phosphotransferase system of Escherichia coli and Salmonella typhimturium and is phosphorylated by phosphoenolpyruvate in a reduction requiring enzyme 1 (phosphoeno/pyruvate-protein phospho- transferase), and the histidine-containing phospho-carrier protein HPr. In this paper we report the isolation of IIIgle from E. coil and the characterization of the activc center. Alkaline hydrolysis of [32P]P-IIIgle and chromatography of the hydrolysate suggested that the phosphoryl group is bound to a histidyl residue in P-IIIgle of S. typhimurium. Here we present 1H-NMR measurements of IIIgle and P-IIIgle from E. coli which further substantiate that the phosphoryl group in P-IIIgle is linked to the N-3 position of a histidyl residue. After phosporylation of IIIgle with [32P]Phosphoenolpyruvate, enzyme I and HPr, the phosphorylated protein was cleaved with either alkaline protease from Streptomyces griseus or subtilisin from Bacillus subtilis. According to amino acid analysis both proteases produced the same peptide carrying the phosphoryl group. The amino acid sequence of this peptide was found to be Val-His-Phe-Gly-Ile-Asp. The lower electrophoretic mobility of P-IIIgle on dodecylsulfute/polyacrylamide gels and its stronger binding to the hydrophobic matrix of a reversed-phase column compared to uuphosphorylated protein may indicate a structural change following phosphoenol/pyruvate-dependent phosphorylation. [ABSTRACT FROM AUTHOR]

1. In Escherichia coli ML308 isocitrate dehydrogenase is partially inactivated during growth on acetate [Bennett, P. M. and Holms, W. H. (1975) J. Gen. Micribiol. 87, 37-51]. 2. The active form of isocitrate dehydrogenase was purified to homogeneity from cells grown on glycerol. The key step in the procedure was chromatography on procion-red-Sepharose, from which the enzyme was specifically eluted with NADP+. 3. Two forms of isocitrate dehydrogenase were purified to homogeneity from cells grown on acetate. One form did not bind to procion-red-Sepharose and was essentially inactive; this form could be resolved from the active form by non-denaturing gel electrophoresis. The other form was specifically eluted from procion-red-Sepharose and was partially active; analysis of this form by non-denaturing gel electrophoresis suggested that it was a mixture of the active and inactive forms. 4. The three forms comigrated on denaturing gel electrophoresis and were identical by the criterion of onedimensional peptide mapping. 5. Analysis of the active and inactive forms by sedimentation equilibrium centrifugation and non-denaturing gel electrophoresis showed that they differed in charge but not in size. Amino acid analysis and two-dimensional peptide mapping showed that both forms were directs of identical subunits. 6. The active form of the enzyme contained no detectable alkali-labile phosphate, the inactive form contained 0.8 molecule subunit and the partially active form contained an intermediate amount. 7. The data suggest that the active and inactive forms of isocitrate dehydrogenase differ only in the presence of one phosphate group per subunit in the latter form; this is consistent with our results from phosphorylation of isocitrate dehydrogenase in vitro (Following paper in this journal). 8. The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed. [ABSTRACT FROM AUTHOR]

Escherichia coli ribosomal proteins S4 and S17 were specifically labelled at their thiol groups with the acetylaminoethyl-dansyland/or bimane fluorophores. Each formed a complex with 16-S RNA and, when the other 30-S ribosomal proteins were added, a complete 30-S subunit with at least partial activity. If the 3′ end of the RNA was also labelled (with fluorescein) then the distance between the two fluorophores could be measured by Förster-type energy transfer. The result for S4 was 6.0 nm (60 Å) in the ribonucleoprotein complex and 5.6 nm (56 Å) in the 30-S subunit, and for S17 6.3 nm (63 Å) in the complex and 6.2 nm (62 Å) in the subunit. There is no evidence for a major change in the relative disposition of the 3′ and 5′ ends of the 16-S RNA during formation of the 30-S subunit. Sources of error are discussed, including the question of multiple labelling. In order to measure more accurately the extent of energy transfer a procedure based upon enzymic digestion was developed and is detailed in this paper. [ABSTRACT FROM AUTHOR]

The mannan-synthesizing system of Escherichia coli O9 was studied with membranes of the rfe- mutant 1357, which also lacks phosphomannose isomerase. These membranes did not incorporate mannose from GDP-mannose. Their reconstitution with UDP-glucose/magnesium chloride was studied under different conditions. Simultaneous incubation of rfe- membranes with 10 mM UDP-glucose, 20 mM magnesium chloride and 5 µM GDP-[14C]mannose resulted in the formation of a glycolipid, the carbohydrate moiety of which was characterized as mannosyl-(1→3)-mannosyl-glucose. This glycolipid was not a mannose acceptor when used in membrane reconstitution experiments. Sequential incubation of rfe- membranes first with 10 mM UDP-glucose/20 mM magnesium chloride and then with GDP-[14C]mannose gave rise to the radioactive mannan of E. coli O9. After the incubation of rfe- membranes with 10 mM UDP-glucose/20 mM magnesium chloride in the absence of GDP-mannose, a glucolipid was extracted with butanol and purified by ion-exchange chromatography on DEAE-cellulose. In reconstitution experiments this glucose-containing lipid functions as a mannose acceptor. It is characterized in the accompanying paper [Weisgerber, C. and Jann, K. (1982) Eur. J. Biochem. 127, 165-168] as α-glucosyldiphosphoundecaprenol. The formation of this glucolipid could be reversed with [14C]UMP using rfe- membranes as enzyme source. In the reverse reaction [14C]UDP-glucose was formed in about 20% yield. When rfe- membranes were incubated with 2 µM UDP-[14C]glucose and 20 mM magnesium chloride, a lipid containing [14C]glucose was formed, although no stimulation of mannose incorporation could be observed at this UDP-glucose concentration. The glucose is bound to a lipid moiety (presumably undecaprenol) via a phosphodiester bridge. On the basis of these results the mechanism of the biosynthesis of the E. coli O9 antigen is discussed. We propose that in the initial reversible reaction α-glucosyldiphosphoundecaprenol is formed by the transfer of α-glucosyl 1-phosphate from UDP-glucose to undecaprenol phosphate. The product functions as mannose acceptor in the subsequent sequential transfer of mannose from GDP-mannose directly. The mannan, together with the glucose unit, is finally translocated from undecaprenol diphosphate to core lipid A, with the formation of the complete lipopolysaccharide (O9 antigen). [ABSTRACT FROM AUTHOR]

Considering enzymatic activities found in bacteria and in animal cells, there are two possible mechanisms for repair of N-methylated purines produced by methylating agents such as the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine. Both mechanisms involve first an enzymatic removal of the methylated bases by glycosylases. The resulting apurinic sites could then be repaired by (a) direct insertion of the correct bases purine insertases or (b) opening of the polynucleotide chain by apurinic endonuclease followed by repair synthesis. As the methods commonly used to detect lesions induced by methylating agents involve alkali, it was thus far not possible to decide between the above possibilities because apurinic sites are by themselves alkali labile. In this paper I describe two methods which avoid alkali and therefore allow the clarification of some aspects of the repair reaction. One of these methods makes use of 95% formamide at 40 °C in place of alkali to denature DNA with pre-existing single-strand breaks, the other measures the capacity of DNA scissions with free 3′-OH groups to act as primer for Escherichia coli DNA polymerase I. Results obtained with both methods make it unlikely that purine insertases play a major role in the repair of apurinic sites. Kinetics of production and repair of single-strand breaks, produced in 3T6 mouse fibroblasts by incubation with N-methyl-N′-nitro-N-nitrosoguanidine, were also examined using the methods of alkaline elution and alkaline sucrose gradient centrifugation. [ABSTRACT FROM AUTHOR]

Escherichia coli K 12 appears to behave as an enzyme. We show in the present paper that, in fact, phosphofructokinase 2 also presents some regulatory properties in vitro: at high concentrations, ATP is an inhibitor of phosphofructokinase 2 and it provokes the tetramerization of the dimeric native enzyme. The binding of the two substrates to phosphofructokinase 2 is sequential and ordered as for phosphofructokinase 1, but in the former case fructose 6-phosphate is the first substrate to be bound and ADP the first product to be released. Each dimer of phosphofructokinase 2 binds two molecules of fructose 6-phosphate but only one molecule of the product fructose 1,6-bisphosphate. Although both phosphofructokinases of E. coli K 12 present regulatory properties in vitro, the mechanism of regulation of the activity of the two enzymes is strikingly different. It can be asked whether or not these mechanisms operate in vivo. [ABSTRACT FROM AUTHOR]

A highly folded form of the ribosomal protein L25 from Escherichia coli can be obtained from urea-denatured preparations. Proton NMR data show that this form of the molecule must have a compact, globular tertiary structure. Spectroscopically it is indistinguishable from L25 prepared by methods which avoid denaturing solvents. Thus L25 is a protein which can be reversibly denatured. The stability and solubility of the folded form of the protein are discussed and primary assignments made for a number of resonances in its NMR spectrum. The paper shows that this folded form of the protein can be characterised using NMR spectroscopy. High-resolution NMR spectroscopy provides a sensitive and general way for the characterisation of protein folds. [ABSTRACT FROM AUTHOR]

Poly(uridylic acid) [poly(U)] and poly(xanthidylic acid) [poly(X)] strongly stimulate the IF-2-dependent binding of fMet-tRNA to 30-S ribosomal subunits from Escherichia coli [Van der Laken et al. (1979) FEBS Lett. 100, 230–234]. The N-formylmethionine moiety is incorporated into poly(phenylalanine) upon subsequent addition of other components required for protein synthesis when poly(U) is used as template. This paper shows that N-acetylated Phe-tRNAPhe (AcPhe-tRNA), but not Phe-tRNAPhe or tRNAPhe, competes with fMet-tRNA for binding to poly(U)-programmed 30-S ribosomal subunits. The two species of N-blocked aminoacyl-tRNA are bound to poly(U)-programmed and poly(X)-programmed 30-S subunits in a ratio that is linearly dependent on the ratio of the two species added. With poly(U) as template there is no apparent preference for either fMet-tRNA or AcPhe-tRNA, whereas with poly(X) there is a 2–3-fold preference for fMet-tRNA. The initiation factor IF-2, which is strictly required for the binding of N-blocked aminoacyi-tRNAs, has a higher affinity for fMet-tRNA than for AcPhe-tRNA. It is concluded that (a) interaction of the 30-S ribosomal subunit with poly(U) or poly(X) leads to IF-2-dependent binding of N-blocked aminoacyl-tRNA; (b) the initiation factor IF-2 discriminates in favour of fMet-tRNA; (c) the presence of the cognate codon discriminates in favour of the corresponding N-blocked aminoacyl-tRNA. [ABSTRACT FROM AUTHOR]