Size distribution of native cytosolic proteins of Thermoplasma acidophilum

We used molecular sieve chromatography in combination with LC‐MS/MS to identify protein complexes that can serve as templates in the template matching procedures of visual proteomics approaches. By this method the sample complexity was lowered sufficiently to identify 464 proteins and – on the basis of size distribution and bioinformatics analysis – 189 of them could be assigned as subunits of macromolecular complexes over the size of 300 kDa. From these we purified six stable complexes of Thermoplasma acidophilum whose size and subunit composition – analyzed by electron microscopy and MALDI‐TOF‐MS, respectively – verified the accuracy of our method.     

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 differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.     

Low-angle X-ray scattering analysis of the Thermoplasma acidophilum nucleoprotein subunit

A DNA-protein complex isolated from Thermoplasma acidophilum has been examined using low-angle X-ray scattering measurements. In agreement with the results of electron-microscopic studies a diamter of 5.5 nm is deduced. Finally, a simplified model of the DNA-protein particles is discussed postulating a kinked DNA.     

Localization of subunits in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy

The subunit topography of the Thermoplasma acidophilum proteasome was determined by iminunoelectron microscopy using monospecific antibodies directed against the two constituent subunits (,β). Anti--subunit IgG was found to bind to the outer disks of the cylinder- or barrel-shaped molecule, while the binding sites of the anti-β-subunit IgG were mapped on the two inner rings. Probably the homologues of the two subunits in the compositionally more complex but isomorphous eukaryotic proteasomes occupy equivalent positions.     

Proteomics Analysis of Thermoplasma Quinone Droplets

A novel type of lipid droplet/lipoprotein (LD/LP) particle from Thermoplasma acidophilum has been identified recently, and based on biochemical evidences, it was named Thermoplasma Quinone Droplet (TaQD). The major components of TaQDs are menaquinones, and to some extent polar lipids, and the 153 amino acid long Ta0547 vitellogenin‐N domain protein. In this paper, the aim is to identify TaQD proteome components with 1D‐SDS‐PAGE/LC–MS/MS and cross reference them with Edman degradation. TaQD samples isolated with three different purification methods—column chromatography, immunoprecipitation, and LD ultracentrifugation—are analyzed. Proteins Ta0093, Ta0182, Ta0337, Ta0437, Ta0438, Ta0547, and Ta1223a are identified as constituents of the TaQD proteome. The majority of these proteins is uncharacterized and has low molecular weight, and none of them is predicted to take part in lipid metabolism. Bioinformatics analyses does not predict any interaction between these proteins, however, there are indications of interactions with proteins taking part in lipid metabolism. Whether if TaQDs provide platform for lipid metabolism and the interactions between TaQD proteins and lipid metabolism proteins occur in the reality remain for further studies.     

Structural analysis of the plasmid pTA1 isolated from the thermoacidophilic archaeon Thermoplasma acidophilum

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH1.8 and 56°C and has no cell wall. Plasmid pTA1 was found in some strains of the species. We sequenced plasmid pTA1 and analyzed the open reading frames (ORFs). pTA1 was found to be a circular DNA molecule of 15,723 bp. Eighteen ORFs were found; none of the gene products except ORF1 had sequence similarity to known proteins. ORF1 showed similarity to Cdc6, which is involved in genome-replication initiation in Eukarya and Archaea. T. acidophilum has two Cdc6 homologues in the genome. The homologue found in pTA1 is most similar to Tvo3, one of the three Cdc6 homologues found in the genome of Thermoplasma volcanium, among all of the Cdc6 family proteins. The phylogenetic analysis suggested that plasmid pTA1 is possibly originated from the chromosomal DNA of Thermoplasma.     

The nucleotide sequence of the tRNAMMet from the archaebacterium Thermoplasma acidophilum.

Using in vitro labelling techniques, a tRNAMMet from Thermoplasma acidophilum, a member of the Archaebacteriae, has been shown to have the sequence: pGCCGGG Gs4UGGCUCANCUGGAGGAGC m2(2)GCCGGACmUCAUt6AAUCCGGAGGUCUCGGG psi psi CmGAUCCCCGAUCCCGGCACCAOH. Despite the small genome size of this non-parasitic organism, eight modified nucleosides are present, one of which is typically eubacterial, one of which is typically eukaryotic and some of which appear to be unique to the archaebacteria. There is no close sequence homology between this tRNA and that of any other methionine tRNA so far sequenced (less than 70%) but it has almost 90% homology with the nucleotide sequence proposed by Eigen and Oswatitsch for the ancestral quasi-species.     

Organization of rRNA structural genes in the archaebacterium Thermoplasma acidophilum.

In the archaebacterium Thermoplasma acidophilum, each of the structural genes for 5S, 16S and 23S rRNA occur once per genome. In contrast to those of eubacteria and eukaryotes, they appear unlinked. The distance between the 16S and the 23S rDNA is at least 7.5 Kb, that between 23S and 5S rDNA at least 6 Kb and that between 16S and 5S rDNA at least 1.5 Kb. No linkage between those genes has been found by the analysis of recombinant plasmids carrying Bam HI and Hind III rDNA fragments as by hybridizing those plasmids to fragments of Thermoplasma DNA generated by 6 individual restriction endonucleases, recognizing hexanucleotide sequences.     

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila

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 sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences.     

Purification and characterization of a DNA polymerase from the archaebacterium Thermoplasma acidophilum

A thermophilic DNA polymerase has been purified to near homogeneity from the archaebacterium Thermoplasma acidophilum. Analysis of the purified enzyme by sodium dodecyl sulfate gel electrophoresis revealed a single polypeptide of 88 kDa which co-sediments with the DNA polymerase activity on sucrose gradients. Combination of sedimentation and gel filtration analyses indicates that this DNA polymerase is an 88-kDa monomeric enzyme in its native form. The DNA polymerase is resistant to aphidicolin, slightly sensitive to 2',3'-dideoxyribosylthymine triphosphate and inhibited by N-ethylmaleimide when preincubation with this reagent is performed at 65 degrees C. We find that a 3'----5' exonuclease activity is associated with the purified DNA polymerase; the two activities of the enzyme are optimal at 65 degrees C but the exonuclease activity is active in a broader range of lower temperatures and is more thermostable than the DNA polymerase activity.     

Purification and properties of malate dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum

Malate dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum is purified 50-fold to electrophoretic homogeneity. The purified enzyme crystallizes readily. Native malate dehydrogenase shows a relative molecular mass of 144 000. It is a tetramer of identical subunits with a relative molecular mass of 36 600. Malate dehydrogenase from Thermoplasma uses both NADH and NADPH as coenzyme to reduce oxaloacetate. The enzyme shows A-side (pro-R) stereospecificity for both coenzymes. The pH optimum for the reduction of oxaloacetate in the presence of NADH is found to be at pH 8.1. At pH 7.4 the Km value for oxaloacetate is found to be 5.6 microM while for NADH a value of 11.7 microM is found. The homogeneous enzyme shows a turnover number of kcat = 108 s-1.     

The nucleotide sequence of the 5S rRNA from the archaebacterium Thermoplasma acidophilum.

The complete nucleotide sequence of the 5S ribosomal RNA isolated from the archaebacterium Thermoplasma acidophilum has been determined. The sequence is: pG GCAACGGUCAUAGCAGCAGGGAAACACCAGAUCCCAUUCCGAACUCGACGGUUAAGCCUGCUGCGUAUUGCGUUGUACU GUAUGCCGCGAGGGUACGGGAAGCGCAAUAUGCUGUUACCAC(U)OH. The homology with the 55 rRNA from another archaebacterial species, Halobacterium cutirubrum, is only 60.6% and other 55 rRNAs are even less homologous. Examination of the potential for forming secondary structure is revealing. T. acidophilum does not conform to the usual models employed for either procaryotic or eucaryotic 5S rRNAs. Instead this 5S rRNA has a mixture of the characteristic features of each. On the whole this 5S rRNA does however appear more eucaryotic than eubacterial. These results give further support to the notion that the archaebacteria represent an extremely early divergence among entities with procaryotic organization.     

Identification,heterologous expression and detection of enzymatic activity of an asparaginase from the archaeon Thermoplasma acidophilum

Abstract

Asparaginases (ASNases) participate in the metabolism of all living organisms in the hydrolysis of free asparagines. Bacterial ASNases are used in cancer chemotherapy as they efficiently deplete amino acids. However, allergic reactions and silent inactivation represent critical limitations to their extended use. The rationale of this study was to identify, express, and characterize a plant-type L-ASNase from the archaeon Thermoplasma acidophilum as an enzyme with potentially improved characteristics. The Ta0338 orf was cloned into the pET28a(+) expression vector and overexpressed as a soluble protein with a molecular weight of 32 kDa. The quantity of recombinant L-ASNase produced in Escherichia coli was estimated as 9.68 mg/l. The purified protein showed evident autocatalytic processing of the zymogen at 4° and 37°C at physiological pH of 7.2 and clearly generated the expected alpha and beta subunits of 18 and 13 kDa, respectively. We propose that Ta-ASNase represents a potential biotechnological product for therapeutic purposes.     

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.     

The Mass Spectra of the Lankacidin Antibiotics

Bundlins A and B, antibiotics elaborated by Streptomyces sp. 6642 GC₁, have the unique structures which possess a seventeen-membered carbon skeleton fused with an unusual β-keto-δ-Mactonic system and a pyruvamide side chain. In the course of the structural studies of the antibiotics, we found that these compounds showed the interesting fragmentations in their mass spectra and in consequence, the investigation about the interpretation of the principal peaks together with their formation mechanism was undertaken by the aid of high resolution mass spectrometry and the measurement of meta stable ions. In addition to the two antibiotics, the mass spectra of related compounds designated T–2636 D and T-2636 F were also investigated.     

Purification and characterization of glucose dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.

Glucose dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme is a tetramer of polypeptide chain Mr 38,000 +/- 3000, it is catalytically active with both NAD+ and NADP+ cofactors, and it is thermostable and remarkably resistant to a variety of organic solvents. The amino acid composition was determined and compared with those of the glucose dehydrogenases from the archaebacterium Sulfolobus solfataricus and the eubacteria Bacillus subtilis and Bacillus megaterium. The N-terminal amino acid sequence of the Thermoplasma acidophilum enzyme was determined to be: (S/T)-E-Q-K-A-I-V-T-D-A-P-K-G-G-V-K-Y-T-T-I-D-M-P-E.     

嗜酸热原体菌的超氧化物歧化酶的结构模建和进化踪迹分析

为考察来源于极端高温酸性环境中的超氧化物歧化酶,建立了一个由嗜酸热原体茵的sod基因编码的蛋白质的可靠三维分子结构,并对这个蛋白质进行进化踪迹分析识别出了11个踪迹残基?其中,残基Asn39,Gly105和Glu162的位置随机遍布整个结构,其余的残基却全部都明显地聚于一个靠近铁原子的子类当中?由此从在三维结构确认sod基因编码含铁的超氧化物歧化酶.此外,那些被识别的靠近铁原子的踪迹残基可能跟铁的绑定和催化功能直接相关?