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the primary structure of dna binding protein ii from the extreme thermophilic bacterium thermus thermophilus.the primary structure of dna binding protein ii (dna bp ii) from the extreme thermophilic bacterium thermus thermophilus has been established by combination of manual and automated techniques. the protein has 95 residues and a molecular mass of 11,843. comparison of the primary structure with the known sequence data of dna bp ii from clostridium pasteurineum, baccillus stearothermophilus, escherichia coli, rhizobium meliloti, anabena, thermoplasma acidophilum, pseudomonas aeruginosa and bacillus ...19902226865
citrate synthase from the thermophilic archaebacterium thermoplasma acidophilium. cloning and sequencing of the gene.the gene encoding the citric acid cycle enzyme, citrate synthase, has been cloned from the thermoacidophilic archaebacterium, thermoplasma acidophilum. we report the sequencing of this gene and its flanking regions, and the derived amino acid sequence of the enzyme is compared by multiple-sequence alignment analysis with those of citrate synthases from eubacterial and eukaryotic organisms. the similarity is less than 30% between the archaebacterial and non-archaebacterial sequences, although the ...19902269303
cloning and sequencing of the gene coding for the elongation factor 1 alpha from the archaebacterium thermoplasma acidophilum.the gene which encodes the elongation factor 1 alpha (ef-1 alpha) of the archaebacterium thermoplasma acidophilum (tuf-gene) has been cloned and sequenced. the gene coding for elongation factor ef-2 was found downstream from the 3' end of the tuf-gene. comparison of the predicted amino acid sequence of thermoplasma ef-1 alpha with ef-1 alpha sequences of other organisms showed that the highest similarity values were found between t. acidophilum and methanococcus vannielii.19902272495
physicochemical characterization of tetraether lipids from thermoplasma acidophilum. v. evidence for the existence of a metastable state in lipids with acyclic hydrocarbon chains.the main glycophospholipid of thermoplasma acidophilum, grown at 39 degrees c, is composed of a di-isopranol-2,3-glycerotetraether. it has been characterized in hydrated systems by calorimetry. unlike its equivalent grown at 59 degrees c, it shows complex phase properties, which include at least three different phases, (1) a liquid-analogue state (c), which is stable above 20 degrees c, (2) a metastable solid-analogue state (a) formed by supercooling of the liquid-analogue state (c) and (3) a st ...19902337621
organization and expression of the 16s, 23s and 5s ribosomal rna genes from the archaebacterium thermoplasma acidophilum.to elucidate the organization of the transcription units encoding the 16s, 23s and 5s rrnas in the archaebacterium thermoplasma acidophilum, the nucleotide sequences flanking the three rrna genes were determined, and the 5' and 3' termini of the rrna transcripts were mapped by primer extension and nuclease s1 protection. the results show that each of the rrnas is transcribed separately, consistent with the lack of physical proximity among them in the t. acidophilum genome. the transcription init ...19901697064
posttranscriptional modification of trna in thermophilic archaea (archaebacteria).nucleoside modification has been studied in unfractionated trna from 11 thermophilic archaea (archaebacteria), including phylogenetically diverse representatives of thermophilic methanogens and sulfur-metabolizing hyperthermophiles which grow optimally in the temperature range of 56 (thermoplasma acidophilum) to 105 degrees c (pyrodictium occultum), and for comparison from the most thermophilic bacterium (eubacterium) known, thermotoga maritima (80 degrees c). nine nucleosides are found to be un ...19911708763
the multicatalytic proteinase (prosome, proteasome): comparison of the eukaryotic and archaebacterial enzyme.proteasomes isolated and purified from rat muscle tissue and from the archaebacterium thermoplasma acidophilum have a very similar size and shape, but the subunit composition is less complex in the archaebacterium as compared to the eukaryotic particle. the archaebacterial enzyme contains a catalytic site with chymotryptic specificity, which is inhibited by serine proteinase inhibitors and clearly differs from the eukaryotic particle which has a minimum of three catalytic sites for peptide bond ...19911801710
one-step purification of bacterial lipid macroamphiphiles by hydrophobic interaction chromatography.bacterial lipid macroamphiphiles extracted with phenol/water can be purified in one step by hydrophobic interaction chromatography. lipids and the major part of protein are separated from macroamphiphiles during phenol/water extraction. coextracted nucleic acids, polysaccharides, and residual protein are effectively removed by column chromatography on octyl-sepharose whereby macroamphiphiles are primarily adsorbed and later eluted with a buffered propanol gradient. the procedure is applicable to ...19911862938
characterization of a quinole-oxidase activity in crude extracts of thermoplasma acidophilum and isolation of an 18-kda cytochrome.a quinol-oxidase activity was detected in crude extracts of the thermoacidophilic archaebacterium thermoplasma acidophilum. the activity was optimal at ph 5.4 and 50 degrees c. the km for ubiquinol-10 was 18 microm. the enzyme was inhibited by 2n-heptyl-4-hydroxyquinoline n-oxide with a ki of 150 nm. ubiquinols with different side-chain lengths were oxidized at similar rates, whereas menaquinols were converted at 6-12-fold higher rates compared to ubiquinols. membranes from t. acidophilum contai ...19911879426
thermotropic properties of dispersions of cholesterol with tetraether lipids from thermoplasma acidophilum.the main glycophospholipid from thermoplasma acidophilum is composed of a diisopranol-2,3-glycerotetraether. the fraction of pentane cyclizations of its hydrocarbon chains increases with the growth temperature of the source organism (39-59 degrees c). hydrated mixtures of these lipids together with cholesterol have been studied by calorimetry. with the reduction of the phase transition temperatures and enthalpy changes of the transitions, cholesterol is readily incorporated into lipid monolayers ...19911898093
localization of subunits in proteasomes from thermoplasma acidophilum by immunoelectron microscopy.the subunit topography of the thermoplasma acidophilum proteasome was determined by immunoelectron microscopy using monospecific antibodies directed against the two constituent subunits (alpha,beta). anti-alpha-subunit igg was found to bind to the outer disks of the cylinder- or barrel-shaped molecule, while the binding sites of the anti-beta-subunit igg were mapped on the two inner rings. probably the homologues of the two subunits in the compositionally more complex but isomorphous eukaryotic ...19911915873
purification and characterisation of an archaebacterial succinate dehydrogenase complex from the plasma membrane of the thermoacidophile sulfolobus acidocaldarius.a succinate dehydrogenase complex was isolated in a three-step purification from plasma membranes of the thermoacidophilic archaebacterium sulfolobus acidocaldarius. it consists of four subunits: a, 66 kda; b, 31 kda; c, 28 kda and d, 12.8 kda. in the 141-kda native protein, the four subunits are present in an equimolar stoichiometry. the complex contains acid-non-extractable flavin, iron and acid-labile sulphide. maximal succinate dehydrogenase activities were recorded at ph 6.5, which coincide ...19911935955
crystallization and preliminary crystallographic study of glucose dehydrogenase from the archaebacterium thermoplasma acidophilum.single crystals of glucose dehydrogenase from the archaebacterium thermoplasma acidophilum were obtained using the hanging-drop vapour diffusion method and polyethylene glycol as a precipitant in the presence of nadp+ at ph 5.4. the crystals belong to the hexagonal space group p6122 or p6522, with unit cell dimensions a = b = 121.9 angstrom, c = 229.6 angstrom and with two molecules in the asymmetric unit.19911960718
cloning and sequencing of the gene encoding the large (alpha-) subunit of the proteasome from thermoplasma acidophilum.the gene encoding the alpha-subunit of the proteasome from the archaebacterium thermoplasma acidophilum was cloned and sequenced. the gene encodes for a polypeptide with 233 amino acid residues and a calculated molecular weight of 25870. sequence similarity of the alpha-subunit with the saccharomyces cerevisiae wild-type suppressor gene scll+ encoded polypeptide, which is probably identical with the subunit yc7-alpha of the yeast proteasome, lends support to a putative role of proteasomes in the ...19911991516
expression and purification of plasmid-encoded thermoplasma acidophilum citrate synthase from escherichia coli.the citrate synthase gene from the thermophilic archaebacterium thermoplasma acidophilum was expressed in escherichia coli, yielding an active product of the expected molecular weight. manipulation of the citrate synthase gene in a series of puc19 constructs showed that the presumed thermoplasma ribosome binding site is recognized by the e. coli ribosome. a rapid purification of the expression product to homogeneity was achieved, based on the thermostability of thermoplasma citrate synthase.19912026248
the three-dimensional structure of proteasomes from thermoplasma acidophilum as determined by electron microscopy using random conical tilting.the three-dimensional structure of proteasomes from the archaebacterium thermoplasma acidophilum has been determined to a resolution of approximately 2 nm from electron micrographs of negatively stained preparations using the method of 'random conical tilting'. the particles turn out to be essentially cylinder-shaped barrels, 15 nm long and 11 nm wide, enclosing a tripartite inner compartiment. an account is given of some of the present limitations which prevent to attain a higher resolution and ...19912037064
cloning and sequencing of the fus-gene encoding elongation factor 2 in the archaebacterium thermoplasma acidophilum.we have cloned a 1.6-kb region of chromosomal dna from thermoplasma acidophilum into escherichia coli using as a probe part of the methanococcus vannielii fus-gene. the sequence of the clone was highly homologous to part of the corresponding methanococcus vannielii gene. by chromosome walking, a 4.7-kb ecori fragment containing the complete gene was isolated. nucleotide sequencing revealed an open reading frame of 2196 nucleotides. the deduced amino acid sequence contains the known peptide seque ...19912044939
incorporation of synthetic peptide helices in membranes of tetraether lipids from thermoplasma acidophilum. a calorimetric study.four analogues of the membrane-modifying, alpha-helical polypeptide antibiotic alamethicin were synthesized. the alpha-helical deca-, undeca-, heptadeca-, and icosapeptides were mixed with the main tetraether lipid of the archaebacterium thermoplasma acidophilum (mpl), dipalmitoylphosphatidylcholine (dppc) and dihexadecylmaltosylglycerol (dhmg) in various ratios and the modification of the lipid phase transition was determined by differential thermal analysis (dta). the polypeptides form mixed p ...19912059650
primary structure of the thermoplasma proteasome and its implications for the structure, function, and evolution of the multicatalytic proteinase.the proteasome or multicatalytic proteinase is a high molecular mass multisubunit complex ubiquitous in eukaryotes but also found in the archaebacterial proteasome is made of two different subunits only, and yet the complexes are almost identical in size and shape. cloning and sequencing the gene encoding the small (beta) subunit of the t. acidophilum complex completes the primary structure of the archaebacterial proteasome. the similarity of the derived amino acid sequences of 233 (alpha) and 2 ...19921734972
localization of a sequence motif complementary to the nuclear localization signal in proteasomes from thermoplasma acidophilum by immunoelectron microscopy.a sequence motif complementary to the nuclear localization signal (nls) has been localized in proteasomes from thermoplasma acidophilum by immunoelectron microscopy using sequence-specific antibodies. the antibodies were generated in two different ways: by immunization with a carrier-coupled peptide and by isolation of the sequence-specific antibody from an immune serum against native proteasomes using a peptide-affinity column. the sequence specificity of the isolated antibody was confirmed by ...19921288618
purification and enzymic characterization of the cytoplasmic pyrophosphatase from the thermoacidophilic archaebacterium thermoplasma acidophilum.cytoplasmic pyrophosphatase has been isolated from the thermoacidophilic archaebacterium thermoplasma acidophilum. the enzyme was purified to electrophoretic homogeneity by combining ion-exchange and affinity-chromatographic separations. this soluble pyrophosphatase probably consists of six identical subunits, since sds/page gave an estimate of about 22 kda for a single subunit and size-exclusion chromatography under non-denaturing conditions indicates a molecular mass of 110 +/- 5 kda. the two ...19921327774
cloning and sequencing of the gene for the cytoplasmic inorganic pyrophosphatase from the thermoacidophilic archaebacterium thermoplasma acidophilum.the gene (ppa) from the thermoacidophilic archaebacterium thermoplasma acidophilum, encoding the cytoplasmic pyrophosphatase, has been cloned. two degenerate oligonucleotide probes, synthesized according to the n-terminal amino acid sequence of the isolated protein, were used to screen subgenomic libraries. the dna-derived amino acid sequence of the archaebacterial enzyme allows, for the first time, comparative studies of cytoplasmic pyrophosphatases to be extended to all three urkingdoms. the a ...19921327775
subunit stoichiometry and three-dimensional arrangement in proteasomes from thermoplasma acidophilum.the proteasome or multicatalytic proteinase from the archaebacterium thermoplasma acidophilum is a 700 kda multisubunit protein complex. unlike proteasomes from eukaryotic cells which are composed of 10-20 different subunits, the thermoplasma proteasome is made of only two types of subunit, alpha and beta, which have molecular weights of 25.8 and 22.3 kda, respectively. in this communication we present a three-dimensional stoichiometric model of the archaebacterial proteasome deduced from electr ...19921373380
biochemical properties of the proteasome from thermoplasma acidophilum.we have purified proteasomes to apparent homogeneity from the archaebacterium thermoplasma acidophilum. this proteinase has a molecular mass of about 650 kda and an isoelectric point of 5.6. the proteasome hydrolyses peptide substrates containing an aromatic residue adjacent to the reporter group, as well as [14c]methylated casein optimally at ph 8.5 and 90 degrees c. the enzyme activity is enhanced severalfold by mg2+ and ca2+ at 25-500 mm. this increase in activity results primarily from a cha ...19921396684
nucleotide sequence of the genes encoding the subunits h, b, a' and a'' of the dna-dependent rna polymerase and the initiator trna from thermoplasma acidophilum. 19921408839
black lipid membranes of tetraether lipids from thermoplasma acidophilum.black lipid membranes were formed of tetraether lipids from thermoplasma acidophilum and compared to the bilayer forming lipids diphytanoylphosphatidylcholine and diphythanylglucosylglycerol. bilayer-forming lipids varied in thickness of black lipid membranes due to the organic solvent used. measurements of the specific membrane capacitance (cm = 0.744 microf/cm2) showed that the membrane-spanning tetraether lipids from thermoplasma acidophilum form a monolayer of a constant thickness of 2.5-3.0 ...19921420295
production of an antiserum specific to the adp-ribosylated form of elongation factor 2 from archaebacteria and eukaryotes.an antiserum to adp-ribosylated elongation factor 2 (adpr-ef-2) from s. acidocaldarius was raised in rabbits using stained, homogenized, adpr-ef-2-containing slices from sds-gels as a source of antigen. elongation factor 2 (ef-2) from s. acidocaldarius was cloned in e. coli and the expressed gene product was used in order to adsorb all anti-ef-2 antibodies which do not contain the adp-ribosyl group within their epitopes, as e. coli is unable to synthesize the adp-ribosyl acceptor diphthamide. th ...19921426243
expression of functional thermoplasma acidophilum proteasomes in escherichia coli.the two genes encoding the constituent subunits of the thermoplasma acidophilum proteasome were expressed in escherichia coli yielding fully assembled molecules as shown by electron microscopy. the recombinant proteasomes were purified to homogeneity and were shown to have proteolytic activity indistinguishable from proteasomes isolated from t. acidophilum.19921426246
purification and properties of pyruvate kinase from thermoplasma acidophilum.thermoplasma acidophilum is a thermoacidophilic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). to better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. the enzyme has been purified and has a native m(r) of 250,000. it consists of four, apparently identical subunits each of m(r) 60,000. no remarkable kinetic differen ...19921426985
nucleotide sequence of the gene coding for ribosomal protein s10 from the archaeum thermoplasma acidophilum. 19921508693
conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-coa hydrolase.1. limited proteolysis of citrate synthase from sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-coa + oxaloacetate + h2o----citrate + coash) to 4% but did not affect the hydrolysis of citryl-coa. experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synth ...19921521537
calorimetry of tetraether lipids from thermoplasma acidophilum: incorporation of alamethicin, melittin, valinomycin, and nonactin.the development and application of model membrane systems on the basis of tetraether lipids from thermoplasma acidophilum has been proposed. in this respect incorporation of membrane proteins and ionophores is indispensable and is demonstrated in the case of alamethicin, melittin, nonactin, and valinomycin by calorimetry. dipalmitoylphosphatidylcholine (dppc) and dihexadecylmaltosylglycerol (dhmg) were chosen for comparison. melittin and alamethicin prove to broaden the lipid phase transition an ...19921567197
transcription in vivo and in vitro of the histone-encoding gene hmfb from the hyperthermophilic archaeon methanothermus fervidus.immediately upstream of the hmfb gene, in a dna fragment cloned from methanothermus fervidus, are two identical tandemly repeated copies of a 73-bp sequence that contain the sequence 5'tttatata, which conforms precisely to the consensus tata box element proposed for methanogen promoters. by using this duplicated region as the template dna and a cell-free transcription system derived from methanococcus thermolithotrophicus, transcription in vitro was found to initiate at two identical sites 73 bp ...19921592806
early evolutionary relationships among known life forms inferred from elongation factor ef-2/ef-g sequences: phylogenetic coherence and structure of the archaeal domain.phylogenies were inferred from both the gene and the protein sequences of the translational elongation factor termed ef-2 (for archaea and eukarya) and ef-g (for bacteria). all treeing methods used (distance-matrix, maximum likelihood, and parsimony), including evolutionary parsimony, support the archaeal tree and disprove the "eocyte tree" (i.e., the polyphyly and paraphyly of the archaea). distance-matrix trees derived from both the amino acid and the dna sequence alignments (first and second ...19921602493
the pyruvate kinase of thermoplasma acidophilum: purification, kinetic characterisation & use as a phylogenetic marker. 19921633935
structure of the archaeal transfer rna nucleoside g*-15 (2-amino-4,7-dihydro- 4-oxo-7-beta-d-ribofuranosyl-1h-pyrrolo[2,3-d]pyrimidine-5-carboximi dam ide (archaeosine)).a number of post-transcriptional modifications in trna are phylogenetically characteristic of the bacterial, eukaryal, or archaeal domains, both with respect to sequence location and molecular structure at the nucleoside level. one of the most distinct such modifications is nucleoside g*, located in archaeal trna at position 15, which in bacterial and eukaryal trnas is a conserved site involved in maintenance of the dihydrouridine loop-t-loop tertiary interactions. g* occurs widely in nearly eve ...19937683667
structural features of 26s and 20s proteasomes.the 26s proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation and has a highly conserved structure from slime molds to humans. the elongated molecule which has a molecular mass of approximately 2,000 kd is formed by a barrel-shaped 20s core complex and two polar 19s complexes. the 20s complex has c2 symmetry and is built by four seven-membered rings of which the outer rings are rotated by 26 degrees relative to the inner rings while the inner rings are in r ...19937697124
atp-independent dna topoisomerase from fervidobacterium islandicum.thermotogales are thermophilic eubacteria belonging to a very slowly evolving branch in the eubacterial tree. in this report, we describe the purification and characterization of an atp-independent dna topoisomerase from the thermotogale, fervidobacterium islandicum. the enzyme, a monomer of about 75 kda, is a type i dna topoisomerase sharing many properties with the other bacterial topoisomerases i: it absolutely requires mg2+ for activity, relaxes negatively but not positively supercoiled dna ...19938241262
preliminary x-ray crystallographic study of the proteasome from thermoplasma acidophilum.single cystals of proteasomes from the archaebacterium thermoplasma acidophilum were obtained using the hanging-drop vapor diffusion method. the crystals diffract to better than 3.0 a and belong to the orthorhombic space group p2(1)2(1)2(1) with unit cell dimensions a = 308.9 a, b = 208.8 a and c = 116.9 a. there is one molecular complex in the asymmetric unit. two potentially useful heavy-atom derivatives have been obtained. the self-rotation function of the native patterson map shows local sev ...19938254679
crystallization and preliminary crystallographic study of citrate synthase from the thermophilic archaeon thermoplasma acidophilum.single crystals of citrate synthase from the archaeon thermoplasma acidophilum were obtained in two forms using the hanging drop vapour diffusion method and polyethylene glycol 3350 as precipitant. type 1 crystals belong to the orthorhombic space group p222(1), with unit cell dimensions a = 80.9 a, b = 103.8 a, c = 98.3 a and one dimer in the asymmetric unit. type 2 crystals belong to the monoclinic space group p2(1), with unit cell dimensions a = 53.8 a, b = 173.8 a, c = 86.7 a and beta = 97.1 ...19938331668
nucleotide sequence of a putative succinate dehydrogenase operon in thermoplasma acidophilum.28 amino acids from the n-terminal region of a putative terminal oxidase from the archaebacterium thermoplasma acidophilum were determined by edman degradation. on basis of this amino acid sequence a degenerated oligonucleotide was synthesized and used as a radioactive probe for southern blot analysis of ecori digested genomic dna. a 2.3 kb ecori fragment strongly hybridized to the probe and size selected genomic library from genomic dna was constructed. several clones scored positive by screeni ...19938334158
cytoskeleton in the archaebacterium thermoplasma acidophilum? viscosity increase in soluble extracts.thermoplasma acidophilum has no cell wall, and so its irregular shape implies the presence of a cytoskeleton. when soluble extracts of t. acidophilum were incubated in vitro they increased in viscosity, suggestive of a polymerizable component. optimal conditions for the viscosity increase coincided with physiological ionic concentrations. electron micrographs of negatively stained extracts showed a meshlike lattice of elements 10 nm in diameter similar to nuclear lamins. however, immunologically ...19938374067
thermoplasma acidophilum proteasomes degrade partially unfolded and ubiquitin-associated proteins.it is shown that proteasomes from the arachaebacterium thermoplasma acidophilum selectively degrade substrate proteins partially unfolded by phenylhydrazine- or hydrogen peroxide-treatment. surprisingly, the pre-incubation of the substrate proteins with ubiquitin is also sufficient to render them susceptible to proteolytic degradation by proteasomes. we propose that, upon spontaneously associating with the substrate protein, ubiquitin exerts a chaotropic effect on it; this may involve the exposu ...19938391997
ubiquitin found in the archaebacterium thermoplasma acidophilum.systematic n-terminal sequencing of the low molecular weight proteins from thermoplasma acidophilum separated by two-dimensional poly-acrylamide gel electrophoresis led to the discovery of a polypeptide with an apparent m(r) of 4.5 kda identical as its first 18 amino acid residues to human ubiquitin. the occurrence of ubiquitin and proteasomes in an archaebacterium strongly suggests that atp-ubiquitin-dependent proteolysis is a cellular function that developed early in evolution.19938392003
cdna cloning of rat proteasome subunit rc7-i, a homologue of yeast pre1 essential for chymotrypsin-like activity.the nucleotide sequence of a cdna that encodes a new subunit, named rc7-i, of the 20 s proteasome of rat hepatoma cells has been determined. the polypeptide predicted from the open reading frame consists of 201 amino acid residues with a calculated molecular weight of 22,912 and isoelectric point of 7.25. approximately 80% of the partial amino acid sequences of several fragments of rc7-i, determined by protein chemical analyses, were found to be in excellent accordance with those deduced from th ...19938405448
cloning, sequencing and expression of the gene encoding glucose dehydrogenase from the thermophilic archaeon thermoplasma acidophilum.the gene encoding glucose dehydrogenase has been identified by southern analysis of doubly restricted genomic thermoplasma acidophilum dna, using two redundant 17-residue oligonucleotide probes reverse translated from protein n-terminal sequence data. a 1670-bp bamh1-ecor1 restriction fragment was ligated into puc19 and puc18 (constructs ptagdh1 and ptagdh2, respectively) and cloned in escherichia coli. the sequence of the whole fragment was determined, and a 1059-bp open reading frame identifie ...19938436115
purification and partial characterisation of a protein of unknown function from thermoplasma acidophilum. 19938449347
molecular characterization of the "26s" proteasome complex from rat liver.the molecular properties of an atp/ubiquitin-dependent "26s" proteasome complex purified from rat liver were examined by physicochemical, biochemical, and morphological analyses. on ultracentrifugation, the proteasome complex sedimented as almost a single component with a sedimentation coefficient of 30.3s. dynamic light-scattering measurements indicated that it has a diffusion coefficient of 1.38 x 10(-7) cm2/sec and a stokes radius of 15.5 nm. from these two coefficients, the protein complex w ...19938003381
existence of a molecular ruler in proteasomes suggested by analysis of degradation products.analysis of the degradation products from two proteins, the insulin b-chain and human hemoglobin, generated by archaebacterial thermoplasma acidophilum 20 s proteasomes, revealed an unexpectedly broad specificity. in spite of the vast number of different peptides found, they fell into a rather narrow size range. this suggests that a molecular ruler exists which determines the length of the cleavage products.19948050567
dissociation and reconstitution of the thermoplasma proteasome.the proteasome from the thermoacidophilic archaeon thermoplasma acidophilum in its native state represents a 20s particle with significant secondary structure (approximately 35% alpha helix) of its subunits. electron microscopy, ultracentrifugal and spectral analysis demonstrate that at ph of less than 3 dissociation to partially denatured subunits occurs. upon dialysis against near neutral ph buffers, at low protein concentration, reconstitution occurs, leading to the restoration of up to 90% o ...19948055945
novel prenylated hemes as cofactors of cytochrome oxidases. archaea have modified hemes a and o.a series of novel hemes with modifications of the isoprenyl side chain has been detected in archaea. heme a(s) was isolated from cytochrome oxidases of the thermoacidophilic archaeon, sulfolobus acidocaldarius. heme a(s) has the same spectroscopic features as heme a but has a hydroxyethylgeranylgeranyl side chain instead of the hydroxyethylfarnesyl group. this variant is also present in other archaeal oxidases as well as in the cytochrome oxidases of a thermophilic eubacterium. other archaea (th ...19948063781
the crystal structure of glucose dehydrogenase from thermoplasma acidophilum.the archaea are a group of organisms distinct from bacteria and eukaryotes. structures of proteins from archaea are of interest because they function in extreme environments and because structural studies may reveal evolutionary relationships between proteins. the enzyme glucose dehydrogenase from the thermophilic archaeon thermoplasma acidophilum is of additional interest because it is involved in an unusual pathway of sugar metabolism.19948081754
the crystal structure of citrate synthase from the thermophilic archaeon, thermoplasma acidophilum.the archaea constitute a phylogenetically distinct, evolutionary domain and comprise organisms that live under environmental extremes of temperature, salinity and/or anaerobicity. different members of the thermophilic archaea tolerate temperatures in the range 55-110 degrees c, and the comparison of the structures of their enzymes with the structurally homogolous enzymes of mesophilic organisms (optimum growth temperature range 15-45 degrees c) may provide important information on the structural ...19947704526
phylogenetic analysis of 70 kd heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus.the evolutionary relationships between archaebacteria, eubacteria and eukaryotic cells are of central importance in biology. the current view is that each of these three groups of organisms constitutes a monophyletic domain, and that eukaryotic cells have evolved fom an archaebacterial ancestor. recent studies on a number of highly conserved protein sequences do not, however, support this view and raise important questions concerning the evolutionary relationships between all extant organisms, p ...19947704574
the 1.5-nm projection structure of hela cell prosome-mcp (proteasome) provided by two-dimensional crystals.we grew two-dimensional crystals of hela cell prosomes, also called multicatalytic proteinases (mcp) and proteasomes, for a structure determination by electron microscopy. the molecules were arranged in side views in these crystals. the crystals have p21 plane group symmetry with one particle per unit cell. this symmetry confirms previously published evidence indicating that eukaryotic prosome-mcps are dimers of two identical halves. structure factors from six crystals each comprising more than ...19947718362
predicted secondary structure of the 20 s proteasome and model structure of the putative peptide channel.secondary structure prediction has made great progress in recent years due to the incorporation of evolutionary information, and may be close to a point where (in combination with biochemical and low-resolution structural data) it can guide the modelling of tertiary structure in cases where no model building is possible by homology. towards this goal it is important to gather information on the performance of prediction methods in advance of the publication of new structures. in anticipation of ...19947957899
occurrence of tertiary and quaternary branched polyamines in thermophilic archaebacteria.polyamines of thermophilic archaebacteria were analysed by high-performance liquid chromatography and gas chromatography. thermoplasma acidophilum and thermoplasma volcanium ubiquitously contained spermidine and spermine. four species of sulfolobus, s. acidocaldarius, s. solfataricus, s. metallicus and s. shibatae, two species of acidianus, a. brierleyi and a. infernus, and metallosphaera sedula, contained norspermidine and norspermine in addition to spermidine and spermine, but quantitative dis ...19947968661
[a new species of thermoplasma].an extremely thermoacidophilic strain, es-23, was isolated from a self-heating coal spoil pile, sichuan, china. the new isolate grows between 50 and 75 degrees c with optimum 70 degrees c and ph 1.0-5.0 with optimum 2.0. strain es-23 is aerobic and facultative autotrophic depending both heterotrophically on organic compounds and autotrophically on elemental sulfur as energy source and co2 as carbon source. g-, spherical or oval with 0.9-1.5 microns in diameter. the autotrophic growth showed peri ...19947801633
proteasome sequences in eubacteria. 19947846763
critical elements in proteasome assembly.coexpression of both subunits of the thermoplasma proteasome in escherichia coli yields fully assembled and proteolytically active proteasomes. post-translational processing of the beta-subunit occurs in e. coli as it does in thermoplasma. coexpression of the alpha-subunit and the beta delta pro-subunit, a mutant beta-subunit lacking the propeptide, also yields fully assembled and active proteasomes. this indicates that the beta-propeptide is not essential for the folding and assembly of thermop ...19947634086
proteasome-associated rnas are non-specific.the rna isolated from rnase-treated proteasome preparations from human erythrocytes, hela cells, the archaeon thermoplasma acidophilum and also from recombinant proteasomes of t. acidophilum expressed in escherichia coli was characterized. the rna associated with structurally similar protein particles, namely with the two molecular chaperones, groel from e. coli and with the thermosome from t. acidophilum, served as controls. electrophoretic analysis on polyacrylamide gels of the radioactively e ...19947525280
life at extremely low ph. 19957541113
epr characterization of an archaeal succinate dehydrogenase in the membrane-bound state.the membrane-bound succinate dehydrogenase from the thermoacidophilic archaeon thermoplasma acidophilum was characterized by epr spectroscopy and its functional properties were determined. the highest turnover values of succinate dehydrogenase activity were observed at ph 7.4, which is somewhat above the internal ph value of t. acidophilum. the temperature optimum of the reaction was determined as 78 degrees c and the km value for succinate using phenazine methosulfate as the electron acceptor a ...19957556208
structural features of archaebacterial and eukaryotic proteasomes.the 26s proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. the molecule has a molecular mass of approximately 2000 kd and has a highly conserved structure in eukaryotes. the 26s proteasome is formed by a barrel-shaped 20s core complex and two polar 19s complexes. the 20s complex has c2 symmetry and is formed by four seven-membered rings of which the outer rings (alpha-type subunits) are rotated by 25.7 degrees relative to the inner rings while the inner ...19957565658
catalytic components of proteasomes and the regulation of proteinase activity.the proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. it plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from thermoplasma acidophilum. proteas ...19957565662
morphological variation of new thermoplasma acidophilum isolates from japanese hot springs.we isolated 12 strains of thermoplasma acidophilum from hot springs in hakone, japan. t. acidophilum strains showed morphological variation in the crystal-like structure in the cell and the fibrous structure on the cell surface. two strains tested were sensitive to novobiocin. however, a novobiocin-resistant mutant was obtained by spontaneous mutation.19957574659
molecular cloning of two types of cdna encoding subunit rc6-i of rat proteasomes.a new subunit, named rc6-i, of the rat 20 s proteasome was purified and the partial amino acid sequences of several peptide fragments obtained by digestion with lysyl-endopeptidase were determined by edman degradation. amplification of cdnas encoding rc6-i by the polymerase chain reaction (pcr) technique revealed two types of cdna, tentatively designated as rc6-il and rc6-is in order of size. the nucleotide sequences of the two cdnas are identical except that rc6-il contains an insertion of 18 n ...19957578256
proteolysis. the proteasome: a protein-degrading organelle?the crystal structure of the proteasome suggests that degradation of ubiquitin-protein conjugates is achieved by unfolding the protein substrate and translocating it through a channel into a peptidase-containing chamber.19957583140
proteasome: a complex protease with a new fold and a distinct mechanism.the structure of the proteasome from thermoplasma acidophilum introduces threonine proteases as a fifth class of proteolytic enzymes, and offers insights into the catalytic activity of this complicated piece of molecular machinery with its 14 active sites.19957663937
sighting the cellular shredder. 19957664100
gene of heat shock protein of sulfur-dependent archaeal hyperthermophile desulfurococcus.to elucidate thermoresistance, a gene of a hyperthermophilic heat shock protein (hhsp) was isolated from the hyperthermophile desulfurococcus strain sy which grows at 95 degrees c. the molecular weight of hhsp deduced from the open reading frame was 59,137 (545 amino acid residues). sequence alignments of peptides reveal similarities (evolutionary distances) to the alpha (0.279) and beta (0.296) subunits of thermosome, tf55 (0.343) and human t-complex polypeptide 1. the structure of a thermophil ...19957677788
an archaebacterial elf-1a: new grist for the mill. 19957494488
a proteasome from the methanogenic archaeon methanosarcina thermophila.a 645-kda proteasome was purified from methanosarcina thermophila which had chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities and contained alpha (24-kda) and beta (22-kda) subunits. processing of both subunits was suggested by comparison of n-terminal sequences with the sequences deduced from the alpha- and beta-encoding genes (psma and psmb). alignment of deduced sequences for the alpha and beta subunits revealed high similarity; however, the n-terminal sequence of the alpha ...19957499378
the thermosome of thermoplasma acidophilum and its relationship to the eukaryotic chaperonin tric.a high molecular-mass protein complex from the archaebacterium thermoplasma acidophilum, referred to here as the 'thermosome', is built from two subunits (m(r) 58 and 60). the thermosome has been purified to homogeneity. the molecular mass of the native complex was determined to be 1061 +/- 30 da by scanning transmission electron microscopy. it shows a weak atpase activity and is able to bind denatured polypeptides. averages obtained from electron micrographs of negatively stained molecules in t ...19957867646
the proteasome from thermoplasma acidophilum is neither a cysteine nor a serine protease.the 20 s proteasome, found in eukaryotes and in the archaebacterium thermoplasma acidophilum, forms the proteolytic core of the 26 s proteasome which is the central protease of the non-lysosomal protein degradation pathway. inhibitor studies have indicated that the 20 s proteasome may be an unusual type of cysteine or serine protease and a recent study of the thermoplasma beta subunit has indicated that it carries the proteolytic activity. we have attempted to obtain information on the nature of ...19957867793
functions of the proteasome: the lysis at the end of the tunnel. 19957725095
from the cradle to the grave: ring complexes in the life of a protein. 19957725096
crystal structure of the 20s proteasome from the archaeon t. acidophilum at 3.4 a resolution.the three-dimensional structure of the proteasome from the archaebacterium thermoplasma acidophilum has been elucidated by x-ray crystallographic analysis by means of isomorphous replacement and cyclic averaging. the atomic model was built and refined to a crystallographic r factor of 22.1 percent. the 673-kilodalton protease complex consists of 14 copies of two different subunits, alpha and beta, forming a barrel-shaped structure of four stacked rings. the two inner rings consist of seven beta ...19957725097
proteasome from thermoplasma acidophilum: a threonine protease.the catalytic mechanism of the 20s proteasome from the archaebacterium thermoplasma acidophilum has been analyzed by site-directed mutagenesis of the beta subunit and by inhibitor studies. deletion of the amino-terminal threonine or its mutation to alanine led to inactivation of the enzyme. mutation of the residue to serine led to a fully active enzyme, which was over ten times more sensitive to the serine protease inhibitor 3,4-dichloroisocoumarin. in combination with the crystal structure of a ...19957725107
the thermoplasma acidophilum rpl15 gene encodes a homologue of eukaryotic ribosomal proteins l15/yl10.a gene has been cloned from the archaebacterium, thermoplasma acidophilum, which, on the basis of the deduced amino acid sequence, encodes a homologue of the eukaryotic large subunit ribosomal proteins, l15/yl10. this gene, rpl15, was identified as an open reading frame (orf) located 2.3 kb upstream of the gene encoding the alpha-subunit of the t. acidophilum proteasome. the putative translation product of rpl15 (rpl15) contains 197 amino acid residues, with a m(r) of 22,928 and a basic pi of 11 ...19957733938
archaebacterial and eukaryotic proteasomes prefer different sites in cleaving gonadotropin-releasing hormone.thermoplasma 20 s proteasomes are composed of only two different types of subunits (designated as alpha and beta) but are nearly indistinguishable in their quaternary structure from eukaryotic 20 s proteasomes consisting of 14 distinct subunits. in this study, we compared both the nature and the rate of the proteolytic activities of thermoplasma and of granulosa cell proteasomes on the neurohormone, gonadotropin-releasing hormone (gnrh), the degradation products of which can be unequivocally ide ...19957744730
conformational constraints in protein degradation by the 20s proteasome.conformationally stabilized peptides and unfolding intermediates of bovine alpha-lactalbumin have been used to define the degree of unfolding required for degradation by 20s proteasomes. it appears that complete unfolding and the absence of disulphide bonds are prerequisites for degradation, suggesting that a relatively narrow opening controls access to the inner proteolytic compartment of the barrel-shaped proteasome. this is corroborated by electron microscopy studies showing that the insulin ...19957773788
primary structure of the thermosome from thermoplasma acidophilum.the thermosome, a chaperonin from the archaebacterium thermoplasma acidophilum, consists of two subunits (m(r) 58,000 and 60,000) which assemble into a cylindrical complex of pseudo eight-fold rotational symmetry. the sequences of the two subunits are approximately 60% identical to each other and to tf55 from sulfolobus shibatae, and are 30-40% identical to the subunits of the tcp1 containing ring complex (tric) from the eukaryotic cytosol. a dendrogram of this family of chaperonins contains eig ...19957794526
nuclear localization signals of human and thermoplasma proteasomal alpha subunits are functional in vitro.proteasomes are located both in the nuclei and in the cytoplasm of eukaryotic cells. active transport of these complexes through the nuclear pores has been proposed to be mediated by nuclear localization signals (nls), which have been found in several of the alpha-type proteasomal subunits. we have tested three different putative nls sequences from human alpha-type proteasomal subunits (hsc iota, hsc9, and hsc3), as well as a putative nls-type sequence from the archaeon thermoplasma acidophilum, ...19958618844
expression of an archaeal chaperonin in e. coli: formation of homo- (alpha, beta) and hetero-oligomeric (alpha+beta) thermosome complexes.co-expression of the two genes encoding the alpha- and beta-subunit of the thermoplasma acidophilum thermosome in escherichia coli yielded fully assembled hetero-oligomeric complexes (alpha+beta). surprisingly, also separate expression of both genes resulted in formation of hexadecameric complexes (alpha, beta) in the bacterial cytoplasm. on electron micrographs these complexes were indistinguishable from each other and from the native thermosome. the recombinant alpha-complex as well as the nat ...19958521970
picrophilus gen. nov., fam. nov.: a novel aerobic, heterotrophic, thermoacidophilic genus and family comprising archaea capable of growth around ph 0.two species belonging to a novel genus of archaea, designated picrophilus oshimae and picrophilus torridus, have been isolated from two different solfataric locations in northern japan. one habitat harboring both organisms was a dry, extremely acidic soil (ph < 0.5) that was heated by solfataric gases to about 55 degrees c. in the laboratory both species grew heterotrophically on yeast extract and poorly on tryptone under aerobic conditions at temperatures between 45 and 65 degrees c; they grew ...19958522509
citrate synthase from the hyperthermophilic archaeon, pyrococcus furiosus.the gene encoding the enzyme citrate synthase has been cloned and sequenced from the hyperthermophilic archaeon pyrococcus furiosus, and the derived amino acid sequence has been phylogenetically compared with citrate synthases from archaeal, bacterial and eukaryal organisms. the gene has been over-expressed in escherichia coli to produce an active enzyme that has then been characterized with respect to its kinetic, oligomeric and hyperthermostable properties. a structurally-based sequence alignm ...19958532683
reconstitution of bacteriorhodopsin and atp synthase from micrococcus luteus into liposomes of the purified main tetraether lipid from thermoplasma acidophilum: proton conductance and light-driven atp synthesis.the archaebacterium thermoplasma acidophilum is cultivated at 59 degrees c in a medium containing sulfuric acid of ph 2. the purified bipolar membrane spanning main phospholipid (mpl) of this organism can be used to produce stable liposomes of 100-500 nm in diameter either using a french pressure cell detergent dialysis or sonication. despite a potassium diffusion potential of 186 mv very low ionic permeability of sonicated mpl liposomes was measured using the potassium binding fluorescent indic ...19958565113
a non-alpha-like dna polymerase from the hyperthermophilic archaeon pyrococcus furiosus.we identified a dna polymerase with properties that differed from those of alpha-like dna polymerase (pol i), in pyrococcus furiosus, a hyperthermophilic archaeon. the novel dna polymerase (pol ii) was partially purified and characterized. the dna polymerizing activity of pol ii was relatively sensitive to dideoxythymidine-triphosphate (ddttp) and it was inhibited by n-ethylmaleimide, but not by aphidicolin. activity staining gel electrophoresis showed that the dna polymerizing activity was deri ...19958787781
catalytic mechanism of the 20s proteasome of thermoplasma acidophilum revealed by x-ray crystallography. 19958824425
tricorn protease--the core of a modular proteolytic system.large macromolecular assemblies have evolved as a means of compartmentalizing reactions in organisms lacking membrane-bounded compartments. a tricorn-shaped protease was isolated from the archaeon thermoplasma and was shown to form a multisubunit proteolytic complex. the 120-kilodalton monomer assembled to form a hexameric toroid that could assemble further into a capsid structure. tricorn protease appeared to act as the core of a proteolytic system; when it interacted with several smaller prote ...19968910281
glucose dehydrogenase from the halophilic archaeon haloferax mediterranei: enzyme purification, characterisation and n-terminal sequence.an nad(p)-glucose dehydrogenase from the extremely halophilic archaeon, haloferax mediterranei, has been purified to electrophoretic homogeneity. the purified enzyme has been characterised with respect to its cofactor specificity, subunit composition and its salt and thermal stability. the n-terminal amino acid sequence has been determined and n-terminus alignment with sequences of other glucose dehydrogenases shows that the halophilic enzyme most closely resembles the nad(p)-linked glucose dehy ...19968925901
tricorn protease (tri) interacting factor 1 from thermoplasma acidophilum is a proline iminopeptidase.tricorn protease (tri), a high molecular mass complex from the archaeon t. acidophilum, forms the core of a modular proteolytic system; upon interacting with low molecular mass factors intrinsic activities are enhanced and novel activities are generated. here we characterize the first factor, f1, which turns out to be homologous with several bacterial proline iminopeptidases (pips). surprisingly, it cleaves not only typical pip substrates such as h-pro-amc, but a wide spectrum of amino acid subs ...19968946961
hats off to the tricorn protease. 19968966603
archaeal-eubacterial mergers in the origin of eukarya: phylogenetic classification of life.a symbiosis-based phylogeny leads to a consistent, useful classification system for all life. "kingdoms" and "domains" are replaced by biological names for the most inclusive taxa: prokarya (bacteria) and eukarya (symbiosis-derived nucleated organisms). the earliest eukarya, anaerobic mastigotes, hypothetically originated from permanent whole-cell fusion between members of archaea (e.g., thermoplasma-like organisms) and of eubacteria (e.g., spirochaeta-like organisms). molecular biology, life-hi ...19968577716
in vitro hydrogen production by glucose dehydrogenase and hydrogenase.a new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. the reaction is based on the oxidation of glucose by thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of nadph by pyrococcus furiosus hydrogenase. stoichiometric yields of hydrogen were produced from glucose with the continuous recycling of cofactor. this simple system may provide a method for the biological production of hydrogen from renewable sources. in a ...19969631013
isolation and characterization of flagella and flagellin proteins from the thermoacidophilic archaea thermoplasma volcanium and sulfolobus shibatae.isolated flagellar filaments of sulfolobus shibatae were 15 nm in diameter, and they were composed of two major flagellins which have m(r)s of 31,000 and 33,000 and which stained positively for glycoprotein. the flagellar filaments of thermoplasma volcanium were 12 nm in diameter and were composed of one major flagellin which has an m(r) of 41,000 and which also stained positively for glycoprotein. n-terminal amino acid sequencing indicated that 18 of the n-terminal 20 amino acid positions of th ...19968550530
structural and sequence comparisons of quinone oxidoreductase, zeta-crystallin, and glucose and alcohol dehydrogenases.quinone oxidoreductase, zeta-crystallin, glucose dehydrogenase, and alcohol dehydrogenase belong to a superfamily of medium-chain dehydrogenase/reductases. the crystal structures of escherichia coli quinone oxidoreductase (qor) and thermoplasma acidophilum glucose dehydrogenase have recently been determined and are compared here with the well-known structure of horse liver alcohol dehydrogenase. a structurally based comparison of these three enzymes confirms that they possess extensive overall s ...19968638928
processing of n3, a mammalian proteasome beta-type subunit.proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from thermoplasma acidophilum. rn3 is the beta-type subunit, n3, of rat proteasomes which has been implicated in the peptidylglutamyl-peptide hydrolase activity of the proteinase complex. we have expressed recombinant rn3 protein in escherichia coli in order to raise subunit-specific polyclonal antibodies ...19968645151
amino acid sequence alignment of a "small' citrate synthase from pseudomonas aeruginosa pac 514, with other citrate synthase sequences. 19968674717
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