bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phenylalanine ammonia‐lyase in potato (Solanum tuberosum L.)
Authors: JOOS Hans‐Jürgen , HAHLBROCK Klaus 
Published: March 2005

Potato (Solanum tuberosum L. cv. Datura) contains approximately 40 – 50 phenylalanine ammonia‐lyase (PAL) genes/haploid genome. Considerable cDNA heterogeneity indicates that at least about 10, and probably more, of these genes are potentially active. One subfamily, represented by one selected member (PAL‐1), was analyzed with respect to genomic complexity, nucleotide and deduced amino acid sequence, and mode of constitutive or induced expression. For comparison, a second gene (PAL‐2), representing several subfamilies that are easily distinguished from PAL‐1, was included in these studies. Extensive structural similarities were observed both between the TATA‐proximal portions of the PAL‐1 and PAL‐2 promoters, particularly in the areas containing putative cis‐acting elements, and among all presently known PAL proteins from various higher and lower plants.The relative abundance of PAL mRNA varied greatly in several major potato organs. However, the patterns obtained with probes detecting either total PAL mRNA or more specifically, PAL‐1‐related or PAL‐2‐related mRNA species, were the same within experimental error. Mature leaves contained particularly low levels of PAL mRNA. Infection of these leaves with the pathogenic fungus, Phytophthora infestans, resulted in a large, transient induction of PAL mRNA. The relative timing of PAL‐1 and PAL‐2 mRNA expression, however, differed in compatible (fungus virulent, plant susceptible) but not in incompatible interactions (fungus avirulent, plant resistant). Wounding of leaves caused an extremely rapid and transient induction of both PAL mRNA species.
Biosynthesis of vitamin B12 in anaerobic bacteria
Authors: MUNDER Michael , VOGT Joachim R. A. , VOGLER Bernhard , RENZ Paul 
Published: March 2005

Experiments on the incorporation of erythrose and formate into the 5,6‐dimethylbenzimidazole moiety of vitamin B12 are described.In one experiment, a 1:1 mixture of D‐[1‐13C]erythrose and D‐[1‐13C]threose was added to a Eubacterium limosum fermentation. The vitamin B12 formed was methylated at N3 of its 5,6‐dimethylbenzimidazole part and degraded to 1,5,6‐trimethylbenzimidazole. The 13C‐NMR spectrum of this compound exhibited a single prominent signal at 109.5 ppm due to 13C labeling in C7.This shows that C1 of erythrose or threose was originally incorporated exclusively into C4 of the 5,6‐dimethylbenzimidazole moiety of vitamin B12.In another experiment, sodium [13C]formate was added to a culture of E. limosum. The vitamin B12 isolated was transformed into 1,5,6‐trimethylbenzimidazole as before. The 13C‐NMR spectrum also showed one prominent signal at 142.8 ppm, evoked by 13C at C2.These results demonstrate that erythrose is incorporated into the base part of vitamin B12 regiospecifically and that formate is the precursor of the C2.
Molecular characterization of six intermediate proteins in the processing of mouse protamine P2 precursor
Authors: CHAUVIÈRE Muriel , MARTINAGE Arlette , DEBARLE Michel , SAUTIÈRE Pierre , CHEVAILLIER Philippe 
Published: March 2005

In mouse spermatozoa, DNA is compacted by two protamines mP1 and mP2. Protamine mP2 (63 residues) is synthesized in spermatid nuclei as a precursor pmP2 (106 residues) which is subsequently processed at the end of spermiogenesis [Yelick, P. C., Balhorn, R., Johnson, P. A., Corzett, M., Mazrimas, J. A., Kleene, K. C. & Hecht, N. B. (1987) Mol. Cell. Biol. 7, 2173–2179].Six proteins, three of which were described earlier [Chauvière, M., Martinage, A., Debarle, M., Alimi, E., Sautière, P. & Chevaillier, Ph. (1991) C. R. Acad. Sci. 313, 107–112], have molecular and electrophoretic properties similar to those of pmP2. They were isolated from purified testis nuclei and characterized by amino acid composition, N‐terminal sequence and peptide mapping. From the amino acid compositions, it appears that all six proteins are rich in arginine, cysteine and histidine and are closely related to pmP2 and mP2. The N‐terminal sequence of each protein overlaps a distinct region of the N‐terminal part of pmP2. The C‐terminal part of protamine mP2 starting at arginine 15 is common to all proteins as assessed by amino acid compositions and peptide maps.All these structural data demonstrate that the six isolated proteins are products of pmP2 precursor processing. The six intermediate proteins pmP2/5, pmP2/11, pmP2/16, pmP2/20, pmP2/26 and pmP2/32 which contain 102, 96, 91, 87, 81 and 75 residues, respectively, are generated from the pmP2 precursor after N‐terminal excision of 4, 10, 15, 19, 25 and 31 residues, respectively. The C‐terminal sequence of protamine mP2 is strictly identical to that of its precursor; therefore, no maturation occurs in this part of the molecule. At the present time, the proteolytic pathway involved in the amino‐terminal processing leading to the mature form of the protamine mP2 (63 residues) has not been elucidated. However, the different representation of six intermediates in the testis suggests that some stages of processing are faster than others or that some cleavage sites are preferred. The proteins described in this paper could result either from stepwise excision of N‐terminal residues or from non‐sequential cleavages.
Characterization of ribulose‐1,5‐bisphosphate carboxylase/oxygenase carrying ribulose 1,5‐bisphosphate at its regulatory sites and the mechanism of interaction of this form of the enzyme with ribulose‐1,5‐bisphosphate‐carboxylase/oxygenase activase
Authors: YOKOTA Akiho , TSUJIMOTO Noritaka 
Published: March 2005

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase [Rbu(1,5)P2CO] from plant sources shows a biphasic reaction course when assayed with more than 2 mM ribulose 1,5‐bisphosphate [Rbu(1,5)P2]. In the burst, Rbu(1,5)P2CO has its substrate‐binding sites occupied with Rbu(1,5)P2 for the initial few minutes, then both substrate‐binding and regulatory sites are occupied by Rbu(1,5)P2 in the subsequent linear phase, at physiological concentrations of Rbu(1,5)P2 [A. Yokota (1991) J. Biochem. (Tokyo) 110, 246–252]. This study attempts the characterization of spinach Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the regulatory sites and the interaction of Rbu(1,5)P2CO activase with Rbu(1,5)P2CO purified with poly(ethylene glycol) 4000 without denaturation.Binding of Rbu(1,5)P2 to the regulatory sites strongly influences the temperature dependence of the carboxylase activity of Rbu(1,5)P2CO. The activation energy of Rbu(1,5)P2CO with Rbu(1,5)P2 at the regulatory sites was 40% larger than that without Rbu(1,5)P2 over 30°C, although the binding did not affect the activation energy below this temperature. This caused the almost linear reaction course of the carboxylase reaction at 50°C. The optimum pH for the activity of Rbu(1,5)P2CO carrying Rbu(1,5)P2 at the sites was 8.0–8.2, and increased by about pH 0.2 from that of Rbu(1,5)P2‐CO without Rbu(1,5)P2. The ratio of the activity of the former form to that of the latter increased with increasing pH with an inflection point at pH 8.1. The increase in the ratio was accompanied by a decrease in the hysteric conformational change of Rbu(1,5)P2CO.The ATP‐hydrolyzing activity inherent to Rbu(1,5)P2CO activase was stimulated about twofold by 3–5 mM Rbu(1,5)P2. Rbu(1,5)P2CO in the inactive complex with Rbu(1,5)P2 experienced hysteresis and bound Rbu(1,5)P2 at the regulatory sites during activation in the presence of Rbu(1,5)P2CO activase. Evidence was obtained that Rbu(1,5)P2CO activase promoted the activation of Rbu(1,5)P2CO through binding to the large subunits of Rbu(1,5)P2CO.
On the interpretation of biochemical data by molecular dynamics computer simulation
Authors: van GUNSTEREN Wilfred F. , MARK Alan E. 
Published: March 2005

The application of computer simulation to molecular systems of biochemical interest is reviewed. It is shown that computer simulation is a tool complementary to experimental methods, which can be used to access atomic details inaccessible to experimental probes. Examples are given in which computer simulation augments the experimental information by providing an atomic picture of high resolution with respect to space, energy or time.The usefulness of a computer simulation largely depends on its quality. The most important factors that limit the accuracy of simulated results are discussed. The accuracy of different simulation studies can differ by orders of magnitude. The accuracy will depend on the type of biomolecular system and process studied. It will also depend on the choice of force field, the simulation set‐up and the protocol that is used. A list of quality‐determining factors is given, which may be useful when interpreting simulation studies appearing in the literature.
Mannitol‐specific enzyme II of the phosphoenolpyruvate‐dependent phosphotransferase system of Staphylococcus carnosus
Authors: FISCHER Roland , HENGSTENBERG Wolfgang 
Published: March 2005

The enzyme IImannitol (EIImtl) of the phosphoenolpyruvate‐dependent phosphotransferase system (PTS) catalyses the uptake and concomitant phosphorylation of mannitol by bacteria; it is specified by the gene mtlA. MtlA is located near the genes mtlF and mtlD in the staphylococcal genome, encoding the enzyme IIImtl and the mannitol‐1‐phosphate dehydrogenase, respectively.We present the cloning of the whole operon by a novel complementation system which is generally suitable for cloning Gram‐positive PTS genes. The nucleotide sequence of a 2.5‐kbp subclone spanning mtlA has been determined. From the deduced amino acid sequence, it is predicted that the membrane‐protein EIImtl consists of 505 amino acid residues (54112Da). The protein has the expected hydropathy profile of an integral‐membrane protein. The NH2‐terminal part of the enzyme resides within the membrane, whereas the COOH‐terminus of the enzyme has the properties of a soluble protein. Comparison with the known amino acid sequence of EIImtl of Escherichia coli [Lee, C. A. & Saier, M. H. (1983) J. Biol. Chem. 258, 10 761–10 767] showed significant similarity. The motif containing the cysteine, which is the putative second phosphorylation site in EIImtl of E. coli [Pas, H. H. & Robillard, G. T. (1988) Biochemistry 27, 5835–5839], is well conserved in EIImtl of Staphylococcus carnosus. Chemical modification of the single active site cysteine residue by Ellman's reagent leads to total inactivation, which can be reversed by treatment with 2‐mercaptoethanol.
Lysyl‐tRNA synthetase from yeast
Authors: FREIST Wolfgang , STERNBACH Hans , CRAMER Friedrich 
Published: March 2005

Discrimination factors (D) which are characteristic for discrimination between lysine and 19 naturally occuring non‐cognate amino acids have been determined from kcat and Km values for native and phosphorylated lysyl‐tRNA synthetases from yeast. Generally, both species of this class II aminoacyl‐tRNA synthetase are considerably less specific than the class I synthetases specific for isoleucine, valine, tyrosine, and arginine. D values of the native enzyme are in the range 90–1700, D values of the phosphorylated species in the range 40–770. The phosphorylated enzyme acts faster and less accurately.In aminoacylation of tRNALys‐C‐C‐A(2′NH2) discrimination factors D1 vary over 30–980 for the native and over 8–300 for the phosphorylated enzyme. From AMP formation stoichiometry and D1 values pretransfer proof‐reading factors (II1) of 1.1–56 were calculated for the native enzyme, factors of 1.0–44 for the phosphorylated species. Post‐transfer proof‐reading factors (II2) were calculated from D values and AMP formation stoichiometry in acylation of tRNALys‐C‐C‐A. Pretransfer proof‐reading is the main correction step, posttransfer proof‐reading is less effective or negligible (II2∼ 1–8).Initial discrimination factors (I), which are due to differences in Gibbs free energies of binding between lysine and noncognate substrates (ΔΔGI), were calculated from discrimination and proof‐reading factors. In contrast to class I synthetases, for lysyl‐tRNA synthetase only one initial discrimination step can be assumed and amino acid recognition is reduced to a three‐step process instead of the four‐step recognition observed for the class I synthetases. Plots of ΔΔGI values against accessible surface areas of amino acids show clearly that phosphorylation of the enzyme changes the structures of the amino acid binding sites. This is illustrated by a hypothetical ‘stopper model’ of these sites.
Salt‐dependent and protein‐concentration‐dependent changes in the solution structure of the DNA‐binding histone‐like protein, HBsu, from Bacillus subtilis
Authors: WELFLE Heinz , MISSELWITZ Rolf , WELFLE Karin , GROCH Nicolas , HEINEMANN Udo 
Published: March 2005

The solution structure of the histone‐like DNA‐binding protein, HBsu, from Bacillus subtilis in 2 mM sodium cacodylate, pH 7.5, is sensitive to the ionic strength of the buffer. This was shown by circular dichroism measurements at different concentrations of sodium chloride and potassium fluoride. The stability of HBsu is also influenced; at HBsu concentrations of about 0.1 mg · ml−1, melting temperatures of 32°C and 55°C were found in the absence of potassium fluoride and in the presence of 0.5 M potassium fluoride, respectively, exhibiting drastic ionic‐strength‐dependent differences in the temperature‐induced unfolding of HBsu.Furthermore, at low ionic strength, circular dichroism spectra vary markedly depending on the HBsu concentration in the approximate range 0.2–3 mg · ml−1. Such protein‐concentration dependent differences in the spectra were not observed in the presence of 0.5 M potassium fluoride. Very similar circular dichroism spectra of HBsu and the histone‐like DNA‐binding protein of Bacillus stearothermophilus (HBst) at high ionic strength, indicate comparable structures of both proteins under these conditions. Estimation of the secondary structure content from the circular dichroism spectra yields data which are in satisfactory agreement with the values obtained from the crystal structure of HBst.Transition temperatures of 45°C and 61°C were found in differential scanning calorimetric measurements performed with HBsu in potassium‐fluoride‐free buffer and in the presence of 0.5 M potassium fluoride, respectively. The thermodynamic data point to the melting of native HBsu dimers into two denatured monomers.
Selective effect of poly(lysine) on the enhancement of the lyn tyrosine protein kinase activity
Authors: DONELLA‐DEANA Arianna , MARIN Oriano , BRUNATI Anna Maria , PINNA Lorenzo A. 
Published: March 2005

A tyrosine protein kinase (TPK‐I), isolated from rat spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451–457] and recently identified as the product of the lyn oncogene [Brunati, A. M., Donella‐Deana, A., Ralph, S., Marchiori, F., Borin, G., Fischer, S. & Pinna, L. A. (1991) Biochim. Biophys. Acta 1901, 123–126], is stimulated by a variety of effectors, including poly(lysine), heparin and very high NaCl concentrations. The efficacy of these compounds is variably dependent on the nature of the phosphoacceptor peptide substrates. Here we show that poly(lysine), but neither NaCl nor heparin, specifically enhances the phosphorylation efficiency of lyn TPK for the peptide EDNEYTA (src peptide). It reproduces the main autophosphorylation site of pp60c‐src (Tyr416), which is entirely conserved in lyn TPK. The favourable effect of poly(lysine) is accounted for by both a dramatic drop of the Km value (70 μM versus 670 μM) and more than a threefold increase in Vmax. The effect is not so evident with a variety of different peptides, either unrelated to the src peptide (e.g. angiotensin II, AAYAA) or derived from the src peptide by single or multiple substitutions of the residues located on the N‐terminal side of tyrosine. In particular, the responsiveness to poly(lysine) is dramatically reduced whenever alanine is replaced for either asparagine at position – 2 or glutamic acid at position – 1, either in the src heptapeptide or in its shorter derivative, the pentapeptide NEYTA. In sharp contrast, the favourable effect of 2 M NaCl, which is invariably accounted for only by an increased Vmax, is especially evident with peptides like angiotensin II and AAYAA, whose phosphorylation is less sensitive to poly(lysine) stimulation. The phosphorylation of the src peptides are actually inhibited rather than stimulated by 2 M NaCl. Consistent with this, lyn TPK autophosphorylation is also dramatically stimulated by poly(lysine) while being inhibited by 2 M NaCl.These data show that poly(lysine) deeply alters the selectivity of lyn TPK by conferring to it an enhanced activity and an especially high affinity toward peptides that reproduce the conserved autophosphorylation site of the TPK of the src family. It is suggested that endogenous compound, whose effect is mimicked by poly(lysine) in vitro, may play a relevant role in determining the specificity of lyn TPK in vivo and possibly of other TPK of the src family.
Limited proteolysis as a probe of conformational changes in aspartate aminotransferase from Sulfolobus solfataricus
Authors: ARNONE M. I. , BIROLO L. , GIAMBERINI M. , CUBELLIS M. V. , NITTI G. , SANNIA G. , MARINO G. 
Published: March 2005

The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein.Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez‐Carrion, M. (1984) J. Biol. Chem. 259, 723–728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323–326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (>60°C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25–75°C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line.Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non‐linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of kcat is non‐linear as well.
Enzymatic reduction of benzoyl‐CoA to alicyclic compounds, a key reaction in anaerobic aromatic metabolism
Authors: KOCH Jürgen , FUCHS Georg 
Published: March 2005

Different anaerobic bacteria can oxidize a variety of aromatic compounds completely to CO2 via one common aromatic intermediate, benzoyl‐CoA. It has been postulated that anaerobically the aromatic nucleus of benzoyl‐CoA becomes reduced. An oxygen‐sensitive enzyme system is described catalyzing the reduction of benzoyl‐CoA to trans‐2‐hydroxycyclohexanecarboxyl‐CoA in a denitrifying Pseudomonas species grown anaerobically on benzoate plus nitrate. The assay mixture consists of cell extract, [U‐14C]benzoyl‐CoA, a [U‐14C]benzoyl‐CoA‐generating system (consisting of [U‐14C]benzoate, purified benzoate‐CoA ligase, Mg2+‐ATP, coenzyme A), an ATP‐regenerating system (consisting of phosphoenolpyruvate, pyruvate kinase, myokinase), and a low‐potential reductant [titanium(III) citrate]. The optimal pH is about 7, the specific activity 10 nmol benzoyl‐CoA reduced min−1× mg−1 protein. The apparent Km for benzoyl‐CoA is below 50 μM. Five major products were found. One product is cyclohex‐1‐enecarboxyl‐CoA which must have been formed by a benzoyl‐CoA reductase. The other product is probably trans‐2‐hydroxycyclohexanecarboxyl‐CoA rather than the cis‐stereoisomer; this product must have been formed by a cyclohex‐1‐enecarboxyl‐CoA hydratase. Two other products are likely to be intermediates of benzoyl‐CoA reduction to cyclohex‐1‐enecarboxyl‐CoA, suggesting that the reduction reaction is more complex. An early formed fifth product is more polar than cyclohexanecarboxyl‐ or cyclohex‐1‐enecarboxyl‐CoA.The enzyme system is under oxygen control since it was not found in cells grown aerobically on benzoate. It is induced by aromatic compounds since its activity is low in cells grown anaerobically on acetate. The actual inducer is probably benzoyl‐CoA rather than benzoate. This conclusion is drawn from the fact that the system is also present in cells grown anaerobically on phenol, phenylacetate, 4‐hydroxybenzoate, or 2‐aminobenzoate; the anaerobic metabolism of these compounds has been shown in this organism to proceed directly via benzoyl‐CoA rather than via free benzoate.
Structure of the N‐linked oligosaccharides of the human transferrin receptor
Authors: ORBERGER Georg , GEYER Rudolf , STIRM Stephan , TAUBER Rudolf 
Published: March 2005

Human transferrin receptor was isolated from placenta and from the hepatocarcinoma cell line Hep G2. Asparagine‐linked oligosaccharides were released by treatment of tryptic glycopeptides with endo‐β‐N‐acetylglucosaminidase H or peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F. Oligosaccharide alditols were fractionated by anion‐exchange high‐performance liquid chromatography and by high‐pH anion‐exchange chromatography. Glycans from placental transferrin receptor were further characterized, after desialylation, by methylation analysis and, in part, by liquid secondary‐ion mass spectrometry. Sialylation of placental transferrin receptor was examined by lectin affinity blotting with Sambucus nigra agglutinin and Maackia amurensis agglutinin. In order to trace possible inter‐individual differences in N‐glycosylation of the receptor, two preparations of placental transferrin receptor purified from two donors were compared.The results demonstrate that human transferrin receptor from placenta predominantly carries diantennary and triantennary N‐acetyllactosaminic glycans as well as hybrid‐type species, the galactose residues of which being almost completely substituted with (α2–3)‐linked sialic acid residues. Distinct differences were noted in the glycosylation pattern of the receptor from different individuals. Transferrin receptor from donor A carried predominantly diantennary and triantennary complex‐type glycans, in part fucosylated at the innermost N‐acetylglucosamine residue, in addition to small amounts of bisected and of incomplete diantennary species. Placental transferrin receptor from donor B predominantly carried triantennary N‐acetyllactosaminic glycans without fucose and hybrid‐type oligosaccharides with four or five mannose residues. Distinct from placental transferrin receptor, the receptor from Hep G2 cells contained larger amounts of oligomannosidic glycans with six to nine mannose residues and tetrasialylated complex‐type oligosaccharides apart from mono‐, di‐ and trisialylated species.