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G Schäfer

Publications and source records attributed to G Schäfer.

At least 19 recordsLinked to original sources
PubMed1995

A secY homologous gene in the crenarchaeon Sulfolobus acidocaldarius.

The nucleotide sequence of an open reading frame, located upstream of the gene for adenylate kinase, was determined in the thermoacidophile crenarchaeon Sulfolobus acidocaldarius. Data bank searches identified the sequence as a secY homologous gene. The DNA derived protein sequence of total 463 amino acids contains 10 hydrophobic domains. A sequence alignment with other prokaryotic and eukaryotic secY sequences reveals significant homology, but the secY primary sequence of S. acidocaldarius shows only a low degree of similarity with the secY counterparts of the euryarchaea Methanococcus vannielii and Haloarcula marismortui. A transcription analysis indicates, that the secY gene is cotranscribed with the gene coding for adenylate kinase.

Adenylate Kinase↗
PubMed1995

Identification of the iron-sulfur clusters in a ferredoxin from the archaeon Sulfolobus acidocaldarius. Evidence for a reduced [3Fe-4S] cluster with pH-dependent electronic properties.

A ferredoxin isolated from the archaeon Sulfolobus acidocaldarius strain DSM 639 has been shown to contain one [3Fe-4S]1 + 10 cluster with a reduction potential of -275 mV and one [4Fe-4S]2+/1+ cluster with a reduction potential of -529 mV at pH 6.4, in the temperature range 0-50 degrees C. The monomer molecular mass was confirmed to be 10907.5 +/- 1.0 Da by electrospray mass spectrometry, as calculated from the published amino acid sequence [Minami, Y. Wakabayashi. S., Wada, K., Matsubara, H., Kerscher, L. & Oesterhelt, D. (1985) J. Biochem. (Tokyo) 97, 745-751], while the holoprotein molecular mass was found to be 11,550 +/- 1.0 Da. The reduced [3Fe-4S]0 cluster was also shown by direct electrochemistry and magnetic circular dichroic spectroscopy to undergo a one-proton uptake reaction as first observed for Azotobacter chroococcum ferredoxin I [George, S. J., Richards, A. J. M., Thomson, A. J. & Yates, M. G. (1984) Biochem. J. 224, 247-251]. The pKa of the protonation step has been determined by a novel thin film electrochemical method to be 5.8. This is significantly different from the pKa of 7.7 determined for A. vinelandii ferredoxin I [Shen, B., Martin, L. L., Butt, J. N., Armstrong, F. A., Stout, C. D., Jensen, J. M., Stephens, P. J., LaMar, G. N., Gorst, C. M. & Burgess, B. K. (1993) J. Biol. Chem. 268, 25928-25939] and indicates that the polypeptide chain around the [3Fe-4S] cluster controls this reaction. Although this appears to be only the second reported case of protonation at or near the reduced [3Fe-4S]0 cluster, its observation in S. acidocaldarius ferredoxin raises the question of the generality of this chemistry for 3Fe clusters. The similarity of the pKa to the estimated intracellular pH of S. acidocaldarius strongly suggests a physiological role for this process.

Amino Acid Sequence↗
PubMed1995

SaRD, a new protein isolated from the extremophile archaeon Sulfolobus acidocaldarius, is a thermostable ribonuclease with DNA-binding properties.

We have isolated the thermostable 9 kDa SaRD-protein from Sulfolobus acidocaldarius which exhibit RNase activity as well as DNA-binding properties (SaRD). The amino acid composition and the sequence of the 16 N-terminal amino acids show similarities to different RNases as well as to DNA-binding proteins from thermophilic archea. The RNase activity was demonstrated by 5S rRNA degradation, thin layer chromatography and a zymogram. The temperature optimum for the RNase activity is 65 degrees C. The pH optimum ranges from 6.5-7.0. DNA-binding properties were shown by gel-shift assays on agarose gels. In a similar way SaRD mediated protection of DNA against DNase I digestion and Sau3A I restriction could be demonstrated. The melting point (Tm) of genomic DNA was raised from 68 degrees C to 90 degrees C by addition of the SaRD-protein. CD spectroscopy indicated that SaRD is very stable near neutral pH and can neither be unfolded by temperatures up to 85% C nor by addition of 8 M urea.

Amino Acids↗
PubMed1995

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 at 53 degrees C was 0.32 mM. The membrane-bound enzyme was able to reduce the artificial electron acceptors phenazine methosulfate, N,N,N',N'-tetramethyl-p-phenylenediamine, and 2,6-dichloroindophenol. Succinate oxidation was coupled to oxygen consumption in a completely 2-n-heptyl-4-hydroxyquinoline-N-oxide-sensitive manner. In the oxidized state, T. acidophilum membranes exhibited an almost isotropic EPR spectrum with g-values at gz = 2.017, gy = 2.000, and gx = 1.968 that were assigned to a [3Fe-4S]1+ cluster (S3). Upon reduction with succinate, the membranes displayed a spectrum characteristic of 2Fe-2S clusters (S1), with g-values at gz = 2.029, gy = 1.935, and gx = 1.915. In the dithionite-reduced state, additional resonances can be observed. An axial component, with g-values at gz = 2.057, gy = 1.917, and gx = 1.917 was assigned to a [4Fe-4S]1+ cluster. The saturation behaviour of the S1 cluster was strongly altered in the dithionite-reduced form, thus indicating spin-spin interaction between the S1 center and another paramagnetic center, possibly cluster S2. In both the succinate and the dithionite-reduced membranes, parallel-mode EPR spectra displayed a resonance at g = 14, which may be due to a transition of the S = 2 multiplet of the reduced 3Fe-4S cluster. Spin quantitation yielded a relative stoichiometry of cluster S1 to cluster S3 of 1:1. The results obtained by EPR spectroscopy indicated that the characteristic iron-sulfur cluster S1 [2Fe-2S], S2 [4Fe-4S], and S3 [3Fe-4S], were also present in this archaeal succinate dehydrogenase. EPR redox titrations of T. acidophilum membranes at pH 5.5 yielded a reduction potential of +60 +/- 20 mV for cluster S3 and of +68 +/- 20 mV for cluster S1. The axial [4Fe-4S]2+/1+ center had a reduction potential of -210 +/- 20 mV.

Dithionite↗
PubMed1995

Purification, cloning, and sequencing of archaebacterial pyrophosphatase from the extreme thermoacidophile Sulfolobus acidocaldarius.

Cytoplasmic pyrophosphatases are indispensible for the function of cellular bioenergetics. From the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius, situated at one of the lowest branches of the phylogenetic tree, a cytosolic pyrophosphatase has been isolated and purified 200-fold to electrophoretic homogeneity by combining ion-exchange and gel-exclusion chromatography. The native enzyme consists of a homotetramer of 71 kDa apparent molecular mass; the subunit displays an apparent molecular mass of 17 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an absolute requirement for divalent cations (Mg2+) and a temperature optimum of 75 degrees C coinciding with the growth optimum of the organism; the apparent estimated activation energy is 79.5 kJ/mol. A large variety of cytosolic extracts from other archaebacteria has been probed with a polyclonal antiserum raised against the purified protein; surprisingly, except for an extremely weak signal with S. solfataricus none of the other organisms showed any cross-reactivity. Also, Escherichia coli PPase does not cross-react. Based on N-terminal sequencing the gene has been cloned and sequenced. It codes for a 173-amino-acid protein with a calculated molecular mass of 19,365 kDa. Alignment with known eucaryotic and procaryotic PPases reveals invariant conservation of all residues presently assumed to be involved in metal and substrate binding. Unexpectedly, the highest similarity is found with the enzyme from the phylogenetically extremely distant eubacterium E. coli, but immunological cross-reactivity is absent. Similarity to the only known other archaebacterial PPase is much weaker. Using the 3D structure of the Thermus thermophilus enzyme as a scaffold an energy-minimized structural model is presented, deviating only minimally from the former. The structural features are discussed. The enzyme provides an excellent model for studies of thermostability and folding dynamics since heterologous overexpression has been achieved and genetically mutated forms become accessible.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence of a gene cluster encoding ribosomal proteins in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

A 1.6 kb genomic DNA fragment derived from the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius (DSM 639) comprises four open reading frames. The sequence contains three genes encoding crenarchaeal ribosomal proteins with apparent molecular masses of 6.3 kDa, 15.2 kDa and 9.9 kDa, which all represent strongly basic properties. These were identified by sequence comparison as RL46, RL31 and RL33. One open reading frame encodes a new polypeptide (22.1 kDa, pI = 7.3) with no homology to known proteins. The latter is transcribed as a common mRNA with RL46 and RL31. This gene cluster immediately precedes another cluster including genes encoding the putative SRP receptor alpha subunit as well as the putative secEp.

Amino Acid Sequence↗
PubMed1995

Purification and characterization of the Rieske iron-sulfur protein from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

The previously detected Rieske iron-sulfur protein from the membranes of the thermoacidophile Sulfolobus acidocaldarius [Anemüller, S., et al. (1993) FEBS Lett. 318, 61-64] was purified to electrophoretic homogeneity and the N-terminal amino acids determined. The apparent molecular weight was estimated to be 32 kDa. The reduced protein displays a rhombic EPR spectrum with gxyz = 1.768, 1.895, 2.035. The average g-value of 1.902 is typical for nitrogen ligand-containing clusters. EPR spin quantification and the iron content indicate the presence of one [2Fe-2S] cluster. The purified protein displays ubiquinol cytochrome c reductase activity. The pH optimum of this reaction is temperature dependent and was determined to be pH 7 at 56 degrees C. The results presented in this study clearly prove that the Sulfolobus Rieske protein belongs to the family of the true Rieske iron-sulfur proteins.

Amino Acid Sequence↗
PubMed1995

Comparison of serine and hippurate as precursor equivalents during infusion of [15N]glycine for measurement of fractional synthetic rates of apolipoprotein B of very-low-density lipoprotein.

Enrichment in hippurate has been measured to indicate precursor enrichment during glycine tracer infusion studies to estimate fractional synthetic rates of individual hepatic export proteins. However, hippurate tends to overestimate precursor enrichment. Since glycine is rapidly converted to serine by liver cells, we compared tracer enrichment in hippurate and serine with that of glycine incorporated into apolipoprotein (apo) B-100. Ten healthy control subjects were studied in the postabsorptive state during an 8-hour primed-constant infusion of [15N]glycine (10 mumol.kg-1.h-1). Apo B of very-low-density lipoprotein (VLDL) was isolated by standard ultracentrifugation and isopropanol precipitation. Glycine and serine were isolated from plasma and hydrolyzed apo B, hippurate was isolated from plasma, and [15N]enrichment was determined by gas chromatography-mass spectrometry. Enrichment in serine and glycine isolated from apo B was identical at all time points, and their enrichment in apo B increased asymptotically, approaching an apparent plateau (mean +/- SD: 91% +/- 10% of calculated plateau at 8 hours) that was taken to represent hepatic protein precursor enrichment. Enrichment in both plasma serine and hippurate followed a biphasic pattern and continued to increase until the end of the study, raising the possibility that precursor enrichment had not reached a steady state during the study. The apo B plateau was lower (factor 0.76 +/- 0.27) than the final enrichment in hippurate and higher (factor 1.38 +/- 0.36) than that in plasma serine; however, predictions of protein precursor enrichment based on either metabolite were flawed by a large coefficient of variation (35% v 26%).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Sulfolobus acidocaldarius terminal oxidase. A kinetic investigation and its structural interpretation.

The thermoacidophilic archaebacterium Sulfolobus acidocaldarius possesses a very unusual terminal oxidase. We report original kinetic experiments on membranes of this microorganism carried out by stopped flow, using time-resolved optical spectroscopy combined with singular value decomposition analysis. The reduced-oxidized kinetic difference spectrum of the Sulfolobus membranes is characterized by three significant peaks in the visible region at 605, 586, and 560 nm. The 605-nm peak and part of the 586-nm peak (cytochrome aa3-type quinol oxidase) are reduced synchronously by both ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) and dithionite, and they are very rapidly oxidized by molecular oxygen. A second pool of cytochromes seems to contribute to the 586-nm peak which is not reduced by ascorbate plus TMPD and reacts very slowly with dithionite. The b-type cytochromes (560 nm peak) are reduced by both reductants and are essentially "non-autoxidizable" at room temperature. Only one CO binding site with spectral features, kinetic properties, and ligand affinity not very dissimilar from those of mammalian cytochrome oxidase can be detected in the ascorbate-reduced membranes. On the contrary, a second CO binding site having unusual properties for aa3 terminal oxidases can be detected in the dithionite-reduced membranes.

Ascorbic Acid↗
PubMed1994

Generation of proton-motive force by an archaeal terminal quinol oxidase from Sulfolobus acidocaldarius.

The terminal quinol oxidase of the cytochrome aa3 type was isolated from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius. In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol. The enzyme was functionally reconstituted into monolayer liposomes composed of archaeal tetraether lipids also derived from S. acidocaldarius. With the electron donor system ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine, the reconstituted enzyme was more active in the archaeal lipids as compared to lipids derived from Escherichia coli at temperatures above 50 degrees C. Due to the low proton permeability of the tetraether lipids, it was possible to generate a steady-state transmembrane electrical potential (delta psi, interior negative), and transmembrane pH gradient (delta pH, interior alkaline) at temperatures up to 70 degrees C. The successful functional reconstitution of the cytochrome aa3-type quinol oxidase from Sulfolobus identifies it as the key energy converter in the respiratory system of this hyperthermophilic archaeon.

Catalysis↗
PubMed1994

Evidence for a two proton dependent redox equilibrium in an archaeal Rieske iron-sulfur cluster.

The recently detected Rieske iron-sulfur center in the membrane of the thermoacidophilic archaeon Sulfolobus acidocaldarius (Anemüller et al., 1993, FEBS Lett. 318, 61-64) was further characterized by EPR spectroscopy, coupled to redox-potentiometry and functional studies. The reduction potential is pH-dependent above pH 6, revealing the influence of two ionization equilibria in the oxidized form, with pKaox-values of 6.2 and 8.5. Above pH 9, the slope of the curve is--120 mV/pH-unit. A partially purified fraction exerted a ubiquinol-cytochrome c oxidoreductase activity. To our knowledge, for the first time, in a membrane bound Rieske iron-sulfur protein, unequivocal evidence for a two proton dependent redox equilibrium is presented.

Animals↗
PubMed1994

Cytokine production and DNA synthesis by human peripheral lymphocytes in response to palmitic, stearic, oleic, and linoleic acid.

Effects of palmitic, stearic, oleic, and linoleic acid on mitogen-induced DNA synthesis, on production of IL-1 beta, IL-2, IFN-gamma, and TNF-alpha, and on IL-2R expression were determined in human peripheral lymphocytes. Free fatty acids (FFA) were added over a wide range of concentrations to cells cultured under serum free conditions with fatty acid free albumin. DNA synthesis was stimulated by low and inhibited by high FFA concentrations. Physiological concentrations were stimulatory, except for linoleic acid. Cytokine production became affected by all FFA tested. Palmitic acid enhanced the release of IFN-gamma at concentrations that diminished TNF-alpha production. Saturated fatty acids were significantly more potent than unsaturated fatty acids in affecting cytokine production. IFN-gamma secretion was significantly more stimulated or inhibited by the various FFA compared with the other cytokines. IL-2R expression correlated with the production of IL-2. When tested in combination, stimulatory as well as inhibitory effects of the individual FFA became attenuated. It is suggested that palmitic, stearic, oleic, and linoleic acid are physiological regulators of DNA synthesis and cytokine release in human peripheral lymphocytes. Modulation of FFA ratios may be an effective means for the fine tuning of the immune system. As secretory mechanisms of cytokines appear to exhibit substrate specificity for FFA, the release of individual cytokines may be selectively influenced by FFA.

Animals↗
PubMed1993

Unusual heme structure in cytochrome aa3 from Sulfolobus acidocaldarius: a resonance Raman investigation.

Well-resolved Soret-excited resonance Raman spectra have been obtained in the heme marker band region (1440-1700 cm-1) for the fully oxidized and fully reduced forms of the terminal oxidase aa3 from Sulfolobus acidocaldarius. The results demonstrate that in both heme groups the structural properties depart from those of other aa3 oxidases. In the fully oxidized form, the formyl stretching vibration of heme a is observed at 1656 cm-1, approximately 7 cm-1 higher in frequency than found for beef heart cytochrome c oxidase. The frequency of this vibration shows a slight upshift to 1657 cm-1 in the fully reduced form. Thus, the formyl group of the heme a seems not to be involved in a significant hydrogen bond, in sharp contrast to both beef heart cytochrome c oxidase and other quinol-oxidizing enzymes. For heme a3, two conformers were detected in the fully oxidized state. The observation of two v3 modes at 1482 and 1490 cm-1 indicate the coexistence of both the normal six-coordinated high spin and a new five-coordinated high spin configuration. Both heme a3 species exhibit different formyl stretching vibrations at 1673 and 1666 cm-1, respectively. In the fully reduced enzyme, the identification of two heme a3 conformers is not unambiguous.

Animals↗
PubMed1993

Evidence for a Rieske-type FeS center in the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

A high-potential iron-sulfur cluster with characteristics similar to a Rieske-type center was detected in the plasma membrane of Sulfolobus acidocaldarius by EPR spectroscopy. In the reduced form this center has g-values of gz = 2.031, gy = 1.890 and gx = 1.725 (gav = 1.88, rhombicity = 0.37) and its reduction potential at pH 7.4 was determined to be +325 +/- 10 mV. The The archaebacterial cluster exhibits some unique properties, in comparison to eubacterial and eukaryotic Rieske-type centers. First, the reduction potential is pH-dependent in the range from pH 6.7 to 8.2. Second, the typical inhibitor of Rieske FeS centers, DBMIB, had no effect on the g-values of this cluster. The center is reducible by both NADH and succinate in the presence of cyanide, an inhibitor of the terminal oxidases. The possible role of a Rieske-type center in an organism lacking any c-type cytochromes is discussed.

Cell Membrane↗
PubMed1993

Archaebacterial adenylate kinase from the thermoacidophile Sulfolobus acidocaldarius: purification, characterization, and partial sequence.

For the first time an adenylate kinase of archaebacterial origin has been purified to homogeneity. The enzyme from the extreme thermoacidophile Sulfolobus acidocaldarius (DSM 639) has been found to consist of a 23- to 24-kDa polypeptide likely to form a dimer under in vitro conditions. Its temperature optimum is about 90 degrees C; the pH optimum is 5.3-6.0, depending on the observed direction of the reaction. The KM values for ATP, AMP, and ADP are almost equal (0.6-0.7 mM). The enzyme is absolutely specific for AMP as phosphate acceptor but has a broad specificity for nucleotide triphosphates as donors. It requires divalent metals for activity with Mg2+ > Mn2+ > Ca2+ > Zn2+ in the order of decreasing potency. Distinct from mammalian enzymes the sensitivity toward the typical inhibitor diadenosine-5,5'-pentaphosphate is extremely low; > 300 microM is required for 50% inhibition, suggesting an altered distance between the AMP-/ATP-binding sites. The thermostability of the protein decreases sharply at room temperatures above 90 degrees C. It exhibits unusual stability, however, toward acidic pH. A 41-residue N-terminal sequence has been determined which allowed us to construct probes for the genetic approach. From residue 8-15 the protein contains the typical glycine rich P-loop as well as another conserved sequence stretch 21 residues further. Isolation of the gene is in progress.

Adenosine Monophosphate↗
PubMed1993

Identification, cloning, and expression of the gene for adenylate kinase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

An adenylate kinase gene from a member of the archaebacterial kingdom, the thermoacidophilic archaebacterium (archaeon) Sulfolobus acidocaldarius, has been cloned and sequenced for the first time. Two degenerate oligonucleotide probes, based on the N-terminal amino acid sequence information, led to the amplification of a gene-specific DNA fragment, used to screen subgenomic libraries. Comparing the DNA-derived amino acid sequence of total 194 residues with those of known procaryotic and eucaryotic adenylate kinases revealed only a low degree of similarity, except for a glycine-rich region close to the N-terminus, the so-called P-loop. Using inducible expression systems catalytically active S. acidocaldarius adenylate kinase was produced in large amounts. Although the total length of the protein and the results of alignment procedures suggest a closer relation to eucaryotic than to procaryotic sequences, the archaebacterial enzyme may represent a novel class of adenylate kinases. This is corroborated by the finding that an antiserum against this protein does not cross-react with Escherichia coli nor yeast or rabbit adenylate kinases for example.

Adenylate Kinase↗
PubMed1993

No evidence for feedback inhibition of hepatic apolipoprotein B (apo B) production after extracorporeal low density lipoprotein precipitation as determined by [1-13C]leucine infusion in normal volunteers.

To determine the impact of an acute reduction of the circulating mass of apolipoprotein B (apo B) on apo B metabolism we studied six healthy male volunteers before (day 0), 1 day after (day 2), and 7 days after (day 8) an LDL apheresis treatment which reduced apo B mass by 59%. Appearance of newly synthesized apo B in plasma VLDL and LDL was studied using a primed-constant infusion of [1-13C]-leucine. VLDL apo B pool size and fractional VLDL apo B production rate calculated using a one-compartment model were similar on all 3 study days. Absolute VLDL apo B production was not statistically different throughout the study (19.7 +/- 12.3, 19.5 +/- 7.5, 29.1 +/- 17.7 mg kg-1 day-1). LDL apo B fractional production rate was increased on day 2 (0.38 +/- 0.17, 0.68 +/- 0.08, 0.37 +/- 0.06 pools day-1 on days 0, 2, and 8; P < 0.01). Absolute LDL apo B production, however, remained constant throughout the study (10.8 +/- 3.3, 11.0 +/- 1.9, 10.8 +/- 3.1 mg kg-1 day-1). We conclude that in healthy male volunteers acute reduction of the circulating apo B mass by LDL apheresis does not affect apo B metabolism significantly.

Adult↗