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A Gambacorta

Publications and source records attributed to A Gambacorta.

At least 55 records · Page 3Linked to original sources

Thermostable beta-galactosidase from the archaebacterium Sulfolobus solfataricus. Purification and properties.

A thermophilic and thermostable beta-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 degrees C with o-nitrophenyl beta-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus beta-galactosidase was a tetramer of 240 +/- 8 kDa composed of similar or identical subunits. Comparison of the amino acid composition of beta-galactosidase from S. solfataricus with that from Escherichia coli revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not cross-react with beta-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for beta-galactosidases from other mesophilic and thermophilic sources.

Amino Acids↗

Monopolar-bipolar lipid interactions in model membrane systems.

1H-NMR, dynamic light scattering and negative staining electron microscopy have been used to study the formation and physico-chemical properties of aqueous dispersions of mixtures of monopolar lipids extracted from Sulfolobus solfataricus. This microorganism is a thermophilic archaeobacterium growing optimally at about 85 degrees C and pH 3. The two hydrolytic fractions of the membrane complex lipids that have been studied are: the symmetric lipid glycerol dialkyl glycerol tetraether (GDGT) and the asymmetric lipid glycerol dialkyl nonitol tetraether (GDNT). Electron micrographs of pure and mixed GDNT and GDGT dispersions show the formation of complex structures. Only above a critical monopolar/bipolar lipid ratio, typical of the bipolar lipid, could closed structures be formed and good agreement was obtained in sizing with NMR, electron microscopy and dynamic light scattering. NMR spectra have been carried out at several temperatures from 25 degrees to 85 degrees C, to obtain information on the temperature-dependent structural, dynamic and permeability properties of the co-dispersed vesicles. The results are discussed in terms of the steric constraints and the chemico-physical interactions occurring among the different parts of the molecules and compared with previous studies performed with different physical techniques.

Glyceryl Ethers↗

S-adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus. Purification and characterization of two isoforms.

Two isoforms of methionine adenosyltransferase (S-adenosylmethionine synthetase), A and B, have been partially purified from Sulfolobus solfataricus, a thermophilic archaebacterium optimally growing at 87 degrees C. The chromatographic procedure, involving hydrophobic chromatography on a phenyl-Sepharose column as a major step, results in 330-fold and 150-fold purification of adenosylmethionine synthetase A and B respectively. The apparent molecular masses, estimated by gel filtration, are 180 kDa for A and 75 kDa for B. The A and B isoforms follow Michaelis-Menten kinetics with apparent Km values of 10 microM and 20 microM for L-methionine and of 50 microM and 150 microM for ATP respectively. Adenosylmethionine, a product of the reaction, acts as a powerful non-competitive inhibitor (Ki = 50 microM) of the A isoform while it inhibits only slightly the B isoform. Both isozymes exhibit tripolyphosphatase activity but only that associated with the form A is stimulated by 5 microM adenosylmethionine concentration. The two enzymes absolutely require a divalent cation for the activity, but are not affected by monovalent ions and reducing agents. The optimum temperature is 90 degrees C and no significant loss of activity is observable after incubation of the two isoforms at 100 degrees C in the presence of ATP. The Arrhenius plots observed for both isozymes are biphasic, indicating different activation energies below and above 75 degrees C. The cytoplasmic levels of ATP, methionine and adenosylmethionine are evaluated.

Adenosine Triphosphate↗

Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus.

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus, a thermoacidophilic organism isolated from an acidic hot spring (optimal growth conditions: 87 degrees C, pH 3.5) was purified to homogeneity. The enzyme is a dimer (Mr subunit = 53,000) showing microheterogeneity when submitted to chromatofocusing and/or isoelectric focusing analysis (two main bands having pI = 6.8 and 6.3 were observed). The N-terminal sequence (22 residues) does not show any homology with any stretch of known sequence of aspartate aminotransferases from animal and bacterial sources. The apoenzyme can be reconstituted with pyridoxamine 5'-phosphate and/or pyridoxal 5'-phosphate, each subunit binding 1 mol of coenzyme. The absorption maxima of the pyridoxamine and pyridoxal form are centered at 325 and 335 nm, respectively; the shape of the pyridoxal form band does not change with pH. The enzyme has an optimum temperature higher than 95 degrees C, and at 100 degrees C shows a half-inactivation time of 2 h. The above properties seem to be unique even for enzymes from extreme thermophiles (Daniel, R. M. (1986) in Protein Structure, Folding, and Design (Oxender, D. L., ed) pp. 291-296, Alan R. Liss, Inc., New York) and lead to the conclusion that aspartate aminotransferase from S. solfataricus is one of the most thermophilic and thermostable enzymes so far known.

Amino Acid Sequence↗

Tetraether lipid components from a thermoacidophilic archaebacterium. Chemical structure and physical polymorphism.

As a continuation of an X-ray scattering study of the tetraether lipids extracted from the thermophilic archaebacterium Sulfolobus solfataricus, the phase behaviour of four fractions of the complex polar lipid extract (PLE) is described. Each molecule of two of these fractions (P1 and GL) carries an unsubstituted glycerol headgroup, those of another (P2) no such group; the fourth fraction (WPLE) is obtained by water-washing PLE, thus reducing its P2 content from approximately 48% to approximately 24% and increasing the average number of molecules bearing an unsubstituted glycerol headgroup from approximately 0.4 to approximately 0.6. The main result is a striking correlation between the phase behaviour and the average ratio of unsubstituted glycerol headgroups to the total number of headgroups: the fractions P1, GL and WPLE, in which that number is respectively 0.5, 0.5 and 0.3, form rod-containing phases; the fraction P2, in which that number is zero, yields a lamellar phase throughout the phase diagram. An analysis of the dimensions of the structure elements confirms our previous conclusion that, in the presence of a sufficient amount of water, the unsubstituted glycerol headgroups partition preferentially in the hydrocarbon regions rather than at the polar/apolar interfaces. These results, moreover, corroborate our previous conjectures regarding the correlations between the structure of the plasma membrane, the phase behaviour of the lipid extract and life at high temperature.

Archaea↗

A simple chromatographic procedure for the detection of cyclized archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids.

Archaebacterial glycerol-bisdiphytanyl-glycerol tetraether core lipids containing from one to eight cyclopentane rings could be resolved from each other and from the parent uncyclized C40, C40 lipid by TLC. The core lipids of examples from the genera Methanobacterium, Methanobrevibacter, Methanogenium and Methanoplanus did not contain cyclized forms of glycerol-bisdiphytanyl-glycerol tetraethers, whereas the core lipids of Methanosarcina barkeri contained glycerol-bisdiphytanyl-glycerol tetraethers with from one to three cyclopentane rings in each C40 isopranoid chain.

Archaea↗

A novel archaebacterial NAD+-dependent alcohol dehydrogenase. Purification and properties.

An NAD+-dependent alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) was detected in cellular extracts of the extreme thermophilic archaebacterium Sulfolobus solfataricus. The enzyme was purified to homogeneity and shown to be a dimer with a native molecular mass of 71 kDa by sucrose gradient centrifugation and SDS electrophoresis. The enzyme has a broad substrate specificity that includes linear and branched primary alcohols, linear and cyclic secondary alcohols, linear and cyclic ketones and anisaldehyde. The enzyme has an extraordinary thermophilicity and a remarkable thermostability, and appears to have some properties and a structure different from those previously described for thermophilic alcohol dehydrogenases.

Alcohol Dehydrogenase↗

Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties.

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.

Amino Acids↗

Purification and characterization of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium.

The enzyme propylamine transferase, catalyzing the transfer of the propylamine moiety from S-adenosyl(5')-3-methylthiopropylamine to several amine acceptors, has been purified 643-fold in 20% yield from Sulfolobus solfataricus, an extreme thermophilic archaebacterium optimally growing at 87 degrees C. The purified enzyme (specific activity 2.05 units/mg protein), is homogeneous by criteria of gel electrophoresis, gel filtration, isoelectric focusing and ultracentrifugation analysis. The molecular mass of the native enzyme was estimated to be about 110 kDa by gel permeation and ultracentrifugation analysis. The protein migrates on SDS/polyacrylamide gel electrophoresis as a single band of 35 kDa, suggesting that the enzyme is a trimer composed by identical subunits. An optimum pH of 7.5 and an acidic isoelectric point of 5.3 have been calculated. The optimum temperature was 90 degrees C and no loss of activity is observable even after exposure of the purified enzyme to 100 degrees C for 1 h. No reducing agents are required for enzymatic activity. Substrate specificity towards the amine acceptors is rather broad in that 1,3-diaminopropane (Km = 1675 microM), putrescine (Km = 3850 microM), sym-norspermidine (Km = 954 microM) and spermidine (Km = 1539 microM) are recognized as substrates. Conversely S-adenosyl(5')-3-methylthiopropylamine is the only propylamine donor (Km = 7.9 microM) and the deamination of the sulfonium compound prevents the recognition by the enzyme. The reaction is irreversible and initial-rate kinetic studies indicate that the propylamine transfer is operated through a sequential mechanism. 5'-Methylthioadenosine, a product of the reaction, acts as a powerful competitive inhibitor with a Ki of 3.7 microM. Enzyme-substrate binding sites have been investigated with the aid of several substrate analogs and products. Among the compounds assayed, 5'-methylthiotubercidin, S-adenosyl(5')-3-thiopropylamine and S-adenosyl-3-thio-1,8-diaminooctane are the most active inhibitors.

Archaea↗

Glucose dehydrogenase from the thermoacidophilic archaebacterium Sulfolobus solfataricus.

Glucose dehydrogenase has been purified to homogeneity from cell extracts of the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. The enzyme utilizes both NAD+ and NADP+ as coenzyme and catalyses the oxidation of several monosaccharides to the corresponding glyconic acid. Substrate specificity and oxidation rate depend on the coenzyme present; when NAD+ is used, the enzyme binds and oxidizes specifically sugars presenting equatorial orientation of hydroxy groups at C-2, C-3 and C-4. The Mr of the native enzyme is 124,000 and decreases to about 60,000 in the presence of 6 M-guanidinium chloride and to about 30,000 in the presence of 5% (w/v) SDS. The enzyme shows maximal activity at pH 9, 77 degrees C and 20 mM-Mg2+, -Mn2+ or -Ca2+ and is fairly stable in the presence of chaotropic agents and water-miscible organic solvents such as methanol or acetone.

Amino Acids↗

1H and 13C NMR assignment of benzothiophenquinones from the sulfur-oxidizing archaebacterium Sulfolobus solfataricus.

From Sulfolobus solfataricus, a sulfur-oxidizing thermophilic member of archaebacteria, three unusual benzothiophenquinones were isolated. Detailed NMR studies on these quinones, including multipulse mono-dimensional and two-dimensional techniques, were performed to obtain carbon and proton assignments, one-bond, geminal and vicinal coupling constants and T1 relaxation times. This report extends the known quinone composition of thermophilic archaebacteria and further supports the concept that these biomolecules can serve as a useful chemotaxonomic tool.

Archaea↗

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria.

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 degrees C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C40 omega-omega' biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to "physiological" conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type alpha) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type beta'). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type alpha. (3) In all the phases with chains in the alpha conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 A. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the alpha conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Archaea↗

A comparison of the translational diffusion of a normal and a membrane-spanning lipid in L alpha phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers.

We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L alpha phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE). The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus. The translational diffusion was examined between about 15 degrees and 45 degrees C. It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is approximately 2/3 that for NBD-POPE which spans only one monolayer of the bilayer. The result is interpreted in terms of existing models for translational diffusion in lipid membranes.

Archaea↗