Search

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]

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."

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.

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.

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.

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.

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 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.

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]

RNA ligase catalyzed the joining of pC-C-Ap with C-A-A in the synthesis of C-A-A-C-C-Ap, which has the sequence of the Escherichia coli tRNAfMet 3′-end. pC-C-A was also shown to be joined to C-A-A without any undesired self-polymerization. Joining of pC-C-A to various synthetic ribotriplets, such as C-C-A, A-A-A, C-C-C, U-U-U, U-A-G, C-C-G AND U-U-C, was performed as well as joining to the partially substituted trimers with a photolabile o-nitrobenzyl group, C-Anbzl-A and C-C-Anbzl. The yields were C-A-A-C-C-A (69 %), C-C-A-C-C-A (38%), A-A-A-C-C-A (66%), C-C-C-C-C-A (71%), U-U-U-C-C-A (50%), U-A-G-C-C-A (23%), C-C-G-C-C-A (43%) AND U-U-C-C-C-A (46%). C-Anbzl-A was a slightly poorer acceptor than C-A-A and C-C-Anbzl did not serve as an acceptor. Recognition of acceptor molecules by RNA ligase is discussed in terms of affinity of oligonucleotides for the enzyme. [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]

Large oligonucleotide fragments obtained by partial digestion of 32P labelled E. coli tRNAF with pancreatic and T1 ribonuclease have been separated and characterized. The information provided by these analyses has permitted the derivation of the total sequence of tRNAF. [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]

The DNA-stimulated 75 000-Mr ATPase described in the preceding paper is shown to be a further catalytic DNA unwinding principle (DNA unwinding enzyme II) made in Escherichia coli cells (the first being the 180 000-Mr ATPase of the cells: DNA unwinding enzyme I). Unwinding depends, strictly, on the supply of ATP. It occurs only under conditions permitting ATP dephosphorylation and it proceeds as long as enzyme molecules are permitted to enter the enzyme · DNA complex. The enzyme binds specifically to single-stranded DNA yielding a complex of only limited stability. These results are interpreted in terms of a distributive mode of action of the enzyme. It is argued that chain separation starts near a single-stranded DNA region and that, forced by continued adsorption of enzyme molecules to the DNA, it develops along the duplex. This mechanism is different from that deduced previously for DNA unwinding enzyme I. Complicated results were obtained using ATPase prepared from rep3 mutant cells. [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]

Peptide studies on two acidic proteins, A1 and A2, from 50-S ribosomes of Escherichia coli indicate a closely related primary structure. 1. Amino acid compositions of the tryptic peptides of the two proteins are identical, with exception of the amino terminal peptide. 2. The N-terminal amino acid sequence of A2-protein is Ser-Ile-Thr-Lys, while that of A1-protein is N-acetyl-Ser-Ile-Thr-Lys. 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent with the physical molecular weight estimate. 4. In 50% of the polypeptide chains, in both A1- or A2-protein, a specific lysine residue is replaced by ε-N-monomethyl-lysine. [ABSTRACT FROM AUTHOR]

Polynucleotide phosphorylase from Escherichia coli cells catalyzes the transfer of a single nucleotidyl residue from each of the 2'(3')-O-isovalerylesters of ADP, GDP, CDP and UDP to the initiator oligonucleotide, A-A-A. The products of these reactions are identified as the 2'-(3')-O-isovaleryl esters of the corresponding tetranucleotides A-A-A-A, A-A-A-G, A-A-A-C and A-A-A-U. The 2'-(3')-O-isovaleryl residue can be removed from the product by treatment with aqueous methanolic ammonic under conditions that do not significantly damage the oligonucleotide chains. The monoaddition of 2'(3')-O-acyl esters of nucleoside diphosphates to an oligonucleotide acceptor is proposed as a method for the stephwise synthesis of oligoribonucleotides of defined sequence. [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]

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]

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]

The DNA-binding protein HU from Escherichia coil is a heterodimer constituted of two poly- peptide chains termed HU-1 and HU-2, of 90 residues each. Their primary structures were established from structural data obtained from tryptic peptides of each monomer in addition to the structural data provided by the automated Edman degradation of the dimer and by peptides derived from cleavage of the dimer with trypsin, chymotrypsin, V8 staphylococcal protease and dilute acid. The results presented in this paper confirm the amino-terminal and carboxy-terminal sequences of the dimer HU reported previously [Laine et al. (1978) FEBS Lett. 89, 116–120]. The amino acid sequences of proteins HU-1 and HU-2 are identical to those of proteins NS-1 and NS-2 respectively, determined independently by Mende et al. [FEBS Lett. (1978) 96, 395–398]. The amino acid sequences of proteins HU-1 and HU-2 are closely related but differ by 28 residues. These proteins are characterized by their high content of hydrophobic residues represented mostly by alanine. In both proteins, half of the basic residues are scattered along the polypeptide chain and the remainder is found within two short sequences located in the carboxy-terminal part of the molecule. No sequence homology could be established between the proteins HU-1 and HU-2 and any one of the five histones from different eukaryotes. [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]

Two lipopolysaccharide preparations were obtained from Escherichia coli 058 by extraction with 45% aqueous phenol and fractional precipitation with cetyltrimethyl ammonium bromide (Cetavlon). Chemical analysis and polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that the two preparations differed only in the extent of the O-specific polysaccharide moiety. The O-specific polysaccharide was characterized with proton magnetic resonance and infrared spectroscopy, optical rotation and paper electrophoresis. Using gas-liquid chromatography and ion-exchange chromatography, it was shown to contain D-mannose, 2-acetamido-2-deoxy-D-glucose, 3-O-(R-1'-carboxyethyl)-L-rhamnose (rhamnolactylic acid), and O-acetyl groups in the molar ratios of 2:1:1:1. The polysaccharide and oligosaccharides obtained from it were subjected to methylation and chromic acid oxidation. The results obtained indicated that the polysaccharide consists of tetrasaccharide repeating units in which the trisaccharide β-GlcNAc1-4αMan-1-4(2/3-O-Ac)-Man is substituted at C-3 of the non-acetylated mannose with rhamnolactylic acid. The repeating units are joined through α-mannosyl-1-3-glucosamine bonds. This structure is identical with that of the cell wall polysaccharide of Shigella dysenteriae type 5. [ABSTRACT FROM AUTHOR]

The lytic enzyme from bacteriophage T7 has been purified from T7-infected Escherichia coli B cells and characterized. In the purification procedure advantage was taken of the reversible binding of the enzyme to chitin. Molecular weight determinations of the enzyme by Sephadex G-75 chromatography and sodium dodecyl sulphate gel electrophoresis gave estimates of 21000 and 18000 respectively. Because the activity of the enzyme falls rapidly to 1-3% of that observed immediately after lysis of the cells, various means to stabilize the enzyme have been tried. Of the methods attempted only binding to the substrate analogue chitin was successful. The enzymic activity was found to be markedly dependent on ionic strength and pH, maximum activity was observed using 5 mM phosphate buffer pH 7.5. MgCl2 was found to enhance the lytic activity 2–3-fold, the optimal concentration being about 0.5 mM. The enzyme was not sensitive to penicillin G or spermine, but was specifically inhibited by p-chloromercuribenzoate. Following digestion of E. coli peptidoglycan the cell wall peptides L-Ala-D-Glu(A2pm), L-Ala-D-Glu(A2pm-D-Ala) and the dimer of the latter were formed, together with paper chromatographically immobile glycan strands. The enzyme, therefore, acts as an N-acetylmuramyl-L-alanine amidase by cleaving the bond between N-acetylmuramic acid and L-alanine in the E. coli peptidoglycan. The enzyme has apparently no effect on the peptidoglycan from Micrococcus luteus, and seems to have a preference for high-molecular-weight substrates. Thus the disaccharide-tetrapeptide GlcNAc-MurNAc-L-Ala-D-Glu(A2pm-D-Ala) and its dimer are not substrates for the enzyme. [ABSTRACT FROM AUTHOR]

From Escherichia coli 0124 two lipopolysaccharide preparations were obtained with phenol/water extraction and cetavlon precipitation. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and chemical analysis showed that the two preparations differed only in the extent of the O-specific polysaccharide moiety. In passive haemagglutination and gel precipitation the two lipopolysaccharide preparations from E. coli 0124 and the corresponding preparations from Shigella dysenteriae type 3 reacted alike. The O-specific polysaccharide moiety was characterized with proton magnetic resonance spectroscopy, optical rotation and paper electrophoresis. The constituents were determined by gas chromatography and ion-exchange chromatography. The polysaccharide contained glucose (Glc), gatactose (Gal), galactosamine (GalN) and 4-O-(1′-carboxyethyl)-D-glucopyranose (glucolactilic acid, GIcLA) in the molar ratios of 1 : 2 : 1 : 1. Glucolactilic acid, which has a structure similar to muramic acid, was first found in Sh. dysenteriae. The polysaccharide from E. coli 0124 and oligosaccharides obtained from it by partial acid hydrolysis were subjected to methylation analysis using the method of combined gas chromatography—mass spectrometry. The results indicated that the pentasaccharide repeating unit of the polysaccharide is[This symbol cannot be presented in ASCII format] In the polysaccharide the repeating units are joined through galactofuranosidic linkages. This structure is identical with that of the somatic polysaccharide of Sh. dysenterae type 3. [ABSTRACT FROM AUTHOR]

Reduced and carboxymethylated thioredoxin H from Saccharomuces cerevisiae was digested with trypsin and cleaved with cyanogen bromide in Separate experiments. From the amino-acid sequences of isolated peptides after chymotryptic or peptic hydrolysis an alignment of a 17-residue sequence around the redox-active disulfide of thioredoxin II was possible as follows: -Phe-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys-Met-Ile-Ala-Pro-Met-Ile-Glu-Lys- Comparison of this structure with the corresponding amino-acid sequence of the thioredoxin from Escherichia coli B showed identical residues in 10 consecutive positions which included the tryptophan residue, the disulfide ring and the five residues following this on the COOH- terminal side. The COOH-terminal sequence of yeast thioredoxin II was: -Glu-Ala-Ile-Ala-Ser-Asn-Val and the NH2-terminal residue was valine. The sequence results indicated a considerable homology in primary structure between yeast and E. coli thioredoxins and suggested the existence of a common ancestral gene for thioredoxins. The sequence of a peptide from the active site of yeast thioredoxin I showed that the structure around the disulfide bridge was: -Tyr-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys-. This indicated that thioredoxin I and II from yeast have different primary structures. [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]

The experiments reported in this paper lead to the following conclusions: 1. An endonucleolytic activity is associated with the "replication site" in Escherichia coli cells; this endonucleolytic activity attacks the complementary strand of the ψx replicative form DNA only. 2. the viral strand of the ψx-replicative form DNA remains circularly closed during the replication cycle; 3. In the immediate replication product both composing strands are linear; in circularization no strand is preferred; 4. A replication product with both strands closed is transiently found in a structural state which can be, under our experimental conditions, only partially denatured by alkali. Subsequently it is transformed to a typical supercoiled RFI structure. [ABSTRACT FROM AUTHOR]

Valyl-transfer ribonucleic acid was degraded by polynucleotide phosphorylase from Escherichia coli in the presence of phosphate or arsenate. A compound that was identified as 2′&(3′)-O-valyl-adenosine 5′-pyrophosphate was isolated from the phosphorolytic digest of valyl-tRNA while 2′(3′)-O-valydenosine 5′-monophosphate was isolated from the arsenolytic digest. It was concluded that valyl-tRNA can be phosphorolyzed by polynucleotide phosphorylase. The rate of phosphorolysis of aminoacylated tRNA was lower than that of uncharged tRNA. N-blocked derivatives of valyl-tRNA and phenylalanyl-tRNA were also phosphorolyzed by polynucleotide phosphorylase. Some possible uses of nucleoside diphosphates substituted in their suger moiety are suggested. [ABSTRACT FROM AUTHOR]

Alkaline phosphatase (Escherichia coli) was found to catalyze the synthesis of monophosphate esters of serine, ethanolamine, propanolamine, butanol, glycerol, L-glucose, and tris(hydroxymethyl)aminomethane by phosphoryl transfer from cysteamine S-phosphate (donor) to the respective alcohols (acceptors). Maximum enzymic synthesis of the above esters occurred at pH 7.8. The newly-formed phosphate esters were measured quantitatively after separation of the products by the aid of paper high voltage electrophoresis techniques or by an amino acid analyzer. The percentage of the enzyme-catalyzed synthesis of the new phosphate esters varied from 15-39 0/0 using different acceptors under otherwise identical experimental conditions. It is pertinent to note that the same compounds were phosphorylated to essentially the same extent when the donor (substrate) compound was serine O-phosphate, aminoethanol O-phosphate or p-nitrophenyl O-phosphate. The rates of enzymic consumption of cysteamine S-phosphate (vh + va) were measured at different pH values and at different concentrations of acceptor (Tris or aminoethanol) in the presence of 1.5 M NaCl using 3 mM substrate (cysteamine S-phosphate). The presence of either Tris or aminoethanol gave the same pH profile for the rate of consumption (vh + va) of cysteamine S-phosphate with a maximum value around pH 7.8. This value differs from that found for the same reaction in barbital buffer (pH 9.0). The rate of enzymic consumption of cysteamine S-phosphate was also measured at various substrate and Tris concentrations at pH 7.8 under constant ionic strength (Γ/2 = 1.0). The same Km value (0.24 mM) was obtained at Tris concentrations varying from 0.02 M to 0.5 M. The Vmax values derived from these experiments were linearly related to the Tris concentrations up to 0.5 M Tris. The ph profile of the rate of consumption of p-nitrophenyl phosphate as well as the Km value (0.26 mM) were similar to the corresponding values obtained for cysteamine S-phosphate. The rate of hydrolysis (vh) could not be measured when cysteamine S-phosphate served as the donor compound due to the instability of cysteamine S-phosphate under the conditions required for inorganic phosphate assay. Therefore, the rate of transfer (va = vRSH - vP1) could not be estimated. The pH dependence of the rate of transfer, va, was calculated from experimental data obtained when p-nitrophenyl phosphate served as the donor compound and Tris served as the acceptor. The pH profile for the rate of transfer, va, obtained from these measurements was very similar to that obtained by direct measurement of Tris O-phosphate formation using cysteamine as the donor. It is suggested that phosphoryl transfer occurs through a phosphorylated enzyme intermediate, and therefore, the types of compounds that could serve as acceptors are independent of the donor compounds and depend only on the bond energy of the particular phosphorylated enzyme intermediate. [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]

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]

In this paper, we report the cDNA sequence and deduced primary sequence for human cytosolic seryl-tRNA synthetase, and its expression in Escherichia coli. Two human brain eDNA clones of different origin, containing overlapping fragments coding for human seryl-tRNA synthetase were sequenced: HFBDN14 (fetal brain clone); and IB48 (infant brain clone). For both clones the 5′ region of the cDNA was missing. This 5′ region was obtained via PCR methods using a human brain 5′ RACE-Ready cDNA library. The complete cDNA sequence allowed us to define primers to isolate and characterize the intron/ exon structure of the serS gene, consisting of 10 introns and 11 exons. The introns' sizes range from 283 bp to more than 3000 bp and the size of the exons from 71 bp to 222 bp. The availability of the gone structure of the human enzyme could help to clarify some aspects of the molecular evolution of class-II aminoacyl-tRNA synthetases. The human seryl-tRNA synthetase has been expressed in E. coli, purified (95% pure as determined by SDS/PAGE) and kinetic parameters have been measured for its substrate tRNA. The human seryl-tRNA synthetase sequence (514 amino acid residues) shows significant sequence identity with seryl-tRNA synthetases from E. coli (25 %), Saccharomyces cerevisiae (40%), Arabidopsis thaliana (41%) and Caenorhabditis elegans (60%). The partial sequences from published mammalian seryl-tRNA synthetases are very similar to the human enzyme (94% and 92% identity for mouse and Chinese hamster seryl-tRNA synthetase, respectively). Human seryl-tRNA synthetase, similar to several other class-I and class-II human aminoacyl-tRNA synthetases, is clearly related to its bacterial counterparts, independent of an additional C-terminal domain and a N-terminal insertion identified in the human enzyme. In functional studies, the enzyme aminoacylates calf liver tRNA and prokaryotic E. coli tRNA. [ABSTRACT FROM AUTHOR]

We have incorporated chloroplast ribosomal proteins (R-proteins) L12 and S18 into Escherichia coli ribosomes and examined the hybrid ribosomes for their ability to form polysomes in vivo and perform poly(U)-dependent poly(Phe) synthesis in vitro. The rye chloroplast S18 used for the experiment is a highly divergent protein (170 amino acid residues; E. coli S18, 74 residues), containing a repeating, chloroplast-specific, heptapeptide motif, and has amino acid sequence identity of only 35% to E. coli S18. When expressed in E. coli, chloroplast S18 was assembled in E. coli ribosomes. The latter formed polysomes in vivo at about the same rate as the host ribosomes, indicating that the replacement of E. coli S18 with its chloroplast homologue has only a minor, if any, effect on function. The L12 protein is much more conserved in sequence and chain length, and is known to have a very important function. The Arabidopsis chloroplast L12 used in the experiment was incorporated into E. coli 50S subunits that associated with the 30S subunits to form ribosomes, but the latter were unable to form polysomes. This result indicates functional inactivation of E. coli ribosomes by a chloroplast R-protein. To further confirm this result, we overproduced chloroplast L12 through the use of a secretion, vector and purified the protein to homogeneity. Chloroplast L12 could be efficiently incorporated in vitro into L7/12-1acking E. coli ribosomes, but the hybrid ribosomes were totally inactive in poly(U)-dependent poly(Phe) synthesis. Computer modeling of the spatial structure of all known chloroplast L12 proteins (using E. coli L12 coordinates) indicated a 'chloroplast loop' present only in chloroplast L12. The presence of this loop might have a role in the observed inactivation. Taken together with previously reported results (summarized in this paper), it would appear that the features of chloroplast R-proteins concerned with specific functions are more divergent than their assembly properties. We have previously described methods suitable for overproduction and purification of chloroplast Rproteins that are encoded in organellar DNA (≈20), but that gave poor yield for those encoded in the nuclear DNA (≈45). Here we describe a method that overcomes this problem and allows the purification of nucleus-encoded chloroplast R-proteins in milligram quantities. [ABSTRACT FROM AUTHOR]

This paper describes a biochemical study on the protein composition of crystalline inclusions (cores) from plant peroxisomes. By SDS/PAGE and immunoblotting, a catalase of 59 kDa was identified as the predominant protein component in purified cores from sunflower (Helianthus annuus L.) cotyledons. A 55-kDa catalase was the only additional peptide detected. In contrast to in cores, the 55-kDa catalase was the major catalase protein in matrix fractions obtained from lysed peroxisomes. These findings suggested two peroxisomal populations of catalase differing in molecular structure and subperoxisomal compartmentation in sunflower cotyledons. Evidence for different amino acid sequences of the two catalases was found by peptide mapping with endoproteinase Glu-C, by expressing a eDNA encoding matrix catalase in Escherichia coli, and by partial amino acid sequencing of peptide fragments from 59-kDa core catalase. These results contradict the previous view that the formation of cores occurred via condensation of matrix catalase, and indicate that new concepts on the biogenesis and physiological function of plant peroxisomal cores need to be developed. [ABSTRACT FROM AUTHOR]

This paper reports the biochemical characterization of the flavoprotein L-aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holoenzyme in an unproteolysed form. L-Aspartate oxidase is a monomer of 60 kDa containing 1 mol of non-covalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. L-Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of L-aspartate, binds to the enzyme with a dissociation constant (Kd) equal to 1.4 μM. The enzyme binds FAD by a simple second-order process with Kd 0.67 μM. Site-directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme. [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]