bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determination of flux through different metabolite pathways in Saccharomyces cerevisiae by 1H‐NMR and 13C‐NMR spectroscopy
Authors: TRAN‐DINH Son , HERVE Martine , WIETZERBIN Jaime 
Published: March 2005

We propose an experimental approach combining 1H‐NMR and 13C‐NMR spectroscopy to investigate metabolite flux in cells under physiological conditions and present a mathematical model giving the relationships between the following different parameters: 13C fractional enrichment, fluxes in competing pathways, metabolite concentration and experimental time. This model has been used for determining the absolute and/or relative values of five fluxes involving pyruvate, ethanol, acetyle‐CoA and glutamate via the Krebs cycle in glucose‐grown repressed Saccharomyces cerevisiae cells fed with [1‐13C] glucose and/or unlabeled ethanol. The glucose consumption and the production of various compounds such as ethanol, glycerol, trehalose etc. were studied qualitatively and/or quantitatively as a function of time. The 13C fractional enrichment of [2‐13C]ethanol was determined by observing the proton resonance of the methyl group. Addition of 25 mM unlabeled ethanol shows no significant effect on the glucose consumption or the production of any metabolites. However unlabeled ethanol exerts a strong influence on the enrichment of glutamate C4, but only induces an insignificant change on glutamate C2 and C3. Apart from the fact that ethanol is a potential precursor of acety‐CoA as expected, these results indicate that (a) the probability for citrate and 2‐oxoglutarte to make one turn or more in the Krebs cycle is negligible and (b) the scrambling between C4 and C3 via the glyoxylate shunt is virtually absent.The flux of ethanol formation from pyruvate is about three‐times and nine‐times greater than that of ethanol consumption and acetyl‐CoA formation, respectively, from pyruvate via pyruvate dehydrogenase. Without addition of unlabeled ethanol, the ratio of the integrated resonance of glutamate (C2+C3)/C4 reflecting the activity of pyruvate carboxylase relative to that of citrate synthase, is about 1.1. By comparing the absolute values of the different fluxes, it was found that 88% of the glucose was used to synthetize ethanol but the observed concentration of ethanol in the supernatant represents only 58% of the glucose consumption. The validity of the present model was supported by the data from similar experiments using unlabeled ethanol and non‐NMR techniques.
The solution structures of Escherichia coli trp repressor and trp aporepressor at an intermediate resolution
Authors: ARROWSMITH Cheryl , PACHTER Ruth , ALTMAN Russ , JARDETZKY Oleg 
Published: March 2005

We have determined the solution structures and examined the dynamics of the Escherichia coli trp repressor (a 25‐kDa dimer), with and without the co‐repressor l‐tryptophan, from NMR data. This is the largest protein structure thus far determinéd by NMR. To obtain a set of data sufficient for a structure determination it was essential to resort to isotopic spectral editing. Line broadening observed in this molecular mass range precludes for the most part the measurement of coupling constants and stereospecific assignments, with the inevitable result that the attainable resolution of the final structure will be somewhat lower than the resolution reported for smaller proteins and peptides. Nevertheless the general topology of the protein can be deduced from the subsets of NOEs defining the secondary and tertiary structure, providing a basis for further refinement using the full set of NOEs and energy minimization. We report here (a) an intermediate resolution structure that can be deduced from NMR data, covalent, angular and van‐der‐Waals constraints only, without resort to detailed energy calculations, and (b) the limits of uncertainty within which this structure is valid. An examination of these structures combined with backbone amide exchange data shows that even at this resolution three important conclusions can be drawn: (a) the protein structure changes upon binding tryptophan; (b) the putative DNA binding region is much more flexible than the core of the molecule, with backbone amide proton exchange rates 1000 times faster than in the core; (c) the binding of tryptophan stabilizes the repressor molecule, which is reflected in both the appearance of additional NOEs, and in the slowing of backbone proton exchange rates by factors of 3–10.Sequence‐specific 1H‐NMR assignments and the secondary structure of the holopressor (l‐tryptophan‐bound form) have been reported previously [C. H. Arrowsmith, R. Pachter, R. B. Altman, S. B. Iyer & O. Jardetzky (1990) Biochemistry 29, 6332–6341]. Those for the trp aporepressor (l‐tryptophan‐free form), made using the same methods and conditions as described in the cited paper, are reported here. The secondary structure of the aporepressor was calculated from sequential and medium‐range NOEs and is the same as reported for the holorepressor except that helix E is shorter. The tertiary solution structures for both forms of the repressor were calculated from long‐range NOE data. The two structures are quite similar in overall topology to one another as well as to published crystal structures; however, the DNA binding region appears to be more disordered in solution than in the crystal structures. We propose that the greater flexibility of the DNA binding region observed by us as well as by others [C. L. Lawson, R.‐G. Zhang, R. W. Schevitz, Z. Otwinowski, A. Joachimiak & P. B. Sigler (1988) Proteins 3, 18] plays an important role in the mechanism of DNA binding.
Recombinant aprotinin homologue with new inhibitory specificity for cathepsin G
Authors: BRINKMANN Thomas , SCHNIERER Susanne , TSCHESCHE Harald 
Published: March 2005

The substitution of amino acids in the reactive site of aprotinin, a bovine serine proteinase inhibitor with potent activity against trypsin, plasmin and tissue kallikrein, led to a change in specificity of the inhibitor.Twelve new aprotinin variants prepared by recombinant DNA technology and expressed in Escherichia coli clearly demonstrated that the neighbouring groups of the P1 residue, in particular P′2, contribute to the specificity of the inhibitor, while earlier investigations on semisynthetically prepared variants revealed the importance of the P1 residue in dominating the inhibitory specificity.Recombinant aprotinin variants which act specifically against chymotrypsin‐like proteinases, were obtained by substitution of the amino acids in position P1 and P'2 by hydrophobic amino acids like phenylalanine, tyrosine and leucine. Some of these variants, particularly those with phenylalanine or leucine substitutions, were also found to exhibit inhibitory activity against cathepsin G with an equilibrium constant of dissociation Ki of 10−8 M. Inhibitory specificity against cathepsin G was not found in any semisynthetic variant prepared earlier.
Tracking of proton flow during transition from anaerobiosis to steady state
Authors: LUVISETTO Siro , SCHMEHL Ibolya , COLA Carmen , AZZONE Giovanni Felice 
Published: March 2005

1. The kinetics of acidification and realkalinization of the matrix after addition of nigericin to respiring and non‐respiring mitochondria, recorded by intramitochondrial pH indicators such as neutral red and 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF), is complementary to that recorded by extramitochondrial pH indicators. The extent of acidification decreases with the logarithm of the KCl concentration and is inhibited by Pi and ammonium ions.2. Proton translocation during respiration has been compared with proton extraction from matrix bulk water. During oxygen pulses to EGTA‐untreated mitochondria, BCECF records an extraction of protons from matrix bulk water of about 2–3 nmol H+/mg, reduced to 1–2 nmol H+/mg in EGTA‐treated mitochondria. Since the amount of proton translocation required to achieve steady state is of the order of 6–7 nmol H+/mg, it appears that 75–90% of the protons are not extracted from matrix bulk water. Only a slight response is recorded by neutral red.3. The effect of permeant cations and of uncouplers on the distribution of proton extraction between membrane and matrix bulk water has been studied in presteady state. During Sr2+ uptake, proton extrusion into cytosolic bulk water, as well as proton extraction from matrix bulk water, corresponds almost to 100% of the protons translocated by the redox proton pumps. In the absence of Sr2+, parallel to the disappearance of the proton extrusion in cytosolic bulk water, the proton extraction from matrix bulk water diminishes to about 20% of the proton translocation.4. The mechanism by which divalent cation uptake and protonophoric uncouplers affect the distribution of proton extraction between matrix bulk water and membrane domains and the nature of the membrane domains are discussed.
The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense
Authors: VANONI Maria A. , NUZZI Lucia , RESCIGNO Maria , ZANETTI Giuliana , CURTI Bruno 
Published: March 2005

The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady‐state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both l‐glutamine‐dependent and ammonia‐dependent reactions of the glutamate synthase from A. brasilense follow an identical two‐site uni‐uni bi‐bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2‐oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron‐sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both l‐glutamine‐dependent and ammonia‐dependent reactions, this hydrogen atom equilibrates with the solvent.The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia‐dependent reaction of other glutamate synthases. The ammonia‐dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological l‐glutamine‐dependent activity and requiring the enzyme flavins and iron‐sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. & Meister, A. (1977) J. Biol. Chem. 252, 3501–3508] that the small subunit of glutamate synthase is endowed with a glutamate‐dehydrogenase‐like activity.
NMR study of the structural characteristics of variants of yeast iso‐1‐cytochrome c in which unvaried aromatic residues have been substituted
Authors: THURGOOD Andrew G. P. , DAVIES Anne M. , GREENWOOD Colin , MAUK A. Grant , SMITH Michael , GUILLEMETTE J. Guy , MOORE Geoffrey R. 
Published: March 2005

The structures of variants of yeast iso‐1‐cytochrome c, in which the previously unchanged Tyr48 and Tyr48 + Trp59 have been replaced by Phe, have been characterised by NMR. The NMR data indicated that the structures of the variant cytochromes c are very similar to the wild‐type protein. In particular, the heme environment and interactions of the heme macrocycle were shown to be preserved. The observation of chemical shift differences have allowed for the assessment of conformational changes. The substitution of Trp59 by Phe may have caused a small conformational change, a manifestation of which is the observed chemical shift differences at His39, Val57 and Tyr74.The structural basis for the reduction in redox potential accompanying the amino acid substitutions is discussed and the proposal made that the changes in potential are a direct consequence of the side chain properties and do not result primarily from conformational changes.
Inactivation of pancreatic and gastric lipases by tetrahydrolipstatin and alkyl‐dithio‐5‐(2‐nitrobenzoic acid)
Authors: RANSAC Stéphane , GARGOURI Youssef , MOREAU Hervé , VERGER Robert 
Published: March 2005

We studied the covalent inhibition of lipases by the monolayer technique. We report the inactivation of porcine pancreatic and human and rabbit gastric lipases, acting on mixed monomolecular films of dicaprin containing tetrahydrolipstatin or new hydrophobic disulfide compounds, which can be described as a ‘poisoned‐interface’ system.A kinetic model is presented for depicting the covalent inactivation of lipolytic enzymes at a lipid/water interface. The stoichiometry of the interfacial situation can be described as follows: one lipase molecule embedded among 105 substrate molecules will be inactivated to half its initial velocity by the presence of 10 tetrahydrolipstatin molecules. This inactivation was independent of the surface pressure.When tested in the form of mixed films, all the disulfide compounds investigated specifically reduced the hydrolysis of 1,2‐didecanoyl‐sn‐glycerol films by gastric lipases, but did not affect hydrolysis by pancreatic lipase. With this poisoned‐interface system, tetrahydrolipstatin was found to be the most potent inactivator, whereas disulfide compounds showed a higher degree of selectivity than tetrahydrolipstatin.
Mechanism of action of deoxyribonuclease II from human lymphoblasts
Authors: HAROSH Itzik , BINNINGER David M. , HARRIS Paul V. , MEZZINA Mauro , BOYD James B. 
Published: March 2005

Deoxyribonuclease II has been purified through five fractionation steps from the human lymphoblast cell line K562. Isolation included DEAE‐cellulose and heparin – agarose chromatography followed by fractionation on Mono‐S, Mono‐Q and Superose‐12 FPLC columns. In an extension of previous studies, deoxyribonuclease II was found to introduce a much higher proportion of single‐strand nicks relative to double‐strand breaks into supercoiled DNA than has been reported for linear DNA. Application of DNA sequencing techniques has further revealed a unique resistance of 3′ termini to hydrolysis by this enzyme.Deoxyribonuclease II cleaves at every available site along the duplexed portion of a paired oligonucleotide substrate with the exception of the last four nucleotides. Consistent with previous results, this deoxyribonuclease II is active at low pH in the absence of Mg2+ and is not inhibited by EDTA, but complete inhibition is observed with 100 μM Fe3+. Likewise we confirmed the presence of 3′‐phosphoryl termini on the DNA cleavage products since they failed to function as primers for DNA synthesis catalyzid by Escherichia coli DNA polymerase I.
Geotrichum candidum produces several lipases with markedly different substrate specificities
Authors: SIDEBOTTOM Christopher M. , CHARTON Emmanuelle , DUNN Paul P. J. , MYCOCK Gary , DAVIES Christine , SUTTON Jane L. , MACRAE Alasdair R. , SLABAS Antoni R. 
Published: March 2005

We have purified and examined the substrate specificity of four lipases from two strains of the mould Geotrichum candidum, ATCC 34614 and CMICC 335426. We have designated the lipases I and II (ATCC 34614), and A and B (CMICC 335426). The enzymes are monomeric and have similar molecular masses and pI. Thus, lipases I and II have native molecular masses of 50.1 kDa and 55.5 kDa, and pI of 4.61 and 4.47, respectively.Lipases A and B are very similar to lipases I and II with native molecular masses of 53.7 kDa and 48.9 kDa, and pI of 4.71 and 4.50, respectively. Treatment with endo‐β‐N‐acetylglucosaminidase caused a reduction in molecular mass of approximately 4.5 kDa for all four lipases, indicating that these enzymes are glycosylated. Western blotting shows that the lipases are related. However, lipase B from CMICC 335426 shows a remarkable specificity for unsaturated substrates with a double bond at position 9 (cis configuration), and this specificity is not exhibited by the other three lipases. No lipase of this unique specificity has previously been purified to homogeneity. Structural studies using these four lipases should allow insight into the molecular basis of this remarkable specificity.
Molecular and cellular aspects of immunologic tolerance
Authors: NOSSAL Gustav J. V. 
Published: March 2005

This review seeks to explain the most exciting recent data concerning the nature of self/non‐self discrimination by the immune system in a manner accessible to a biochemical readership. The nature of recognition in the two great lymphocyte families, B cells and T cells, is described with special emphasis on the nature of the ligands recognized by each. The history of the field of immunologic tolerance is surveyed, as are the key experiments on conventional mice which provided a conceptual framework. This suggested that tolerance was essentially due to ‘holes’ in the recognition repertoires of both the T and B cell populations so that lymphocytes competent to react to self antigens were not part of the immunologic dictionary. There were essentially two ways to achieve this situation. On the one hand, self antigens might ‘catch’ developing lymphocytes early in their ontogeny and delete the cell, a process of clonal abortion. On the other hand, self antigens might signal lymphocytes (particularly immature cells) in a negative manner, reducing or abolishing their capacity for later responses, without causing death. This process is referred to as clonal anergy. Evidence for both processes exists.Special emphasis is placed on a wave of experimentation beginning in 1988 which imaginatively uses transgenic mouse technology to study tolerance. Transgenic manipulations can produce mice which synthesize foreign antigens in a constitutive and/or inducible manner, sometimes only in specific locations; mice which possess T or B lymphocytes almost all expressing a given receptor of known specificity; and mice which are an immunologic time bomb in that the antigen is present and so too are lymphocytes all endowed with receptors for that antigen. These experiments have vindicated the possibility of both clonal abortion and clonal anergy in both T and B cell populations, the choice of which phenomenon occurs depending on a number of operational circumstance. For T cell tolerance, clonal abortion occurs if the self antigenic determinant concerned is present within the thymus; if not, clonal anergy is more likely. For B cell tolerance, the strength of the negative signal and therefore the choice between abortion and anergy depends on the molar concentration of the self antigen, the capacity for multivalent presentation to a B cell, and the affinity of the B cell's receptor for the antigen in question. Some B cells with low affinity for self antigens certainly escape censorship and remain capable of secreting low affinity anti‐self antibodies, which however do no harm. Mechanisms which prevent the emergence of hypermutated, high‐affinity anti‐self B cells are discussed.
Primary structure of protein moiety of Penicillium notatum phospholipase B deduced from the cDNA
Authors: MASUDA Naoko , KITAMURA Naomi , SAITO Kunihiko 
Published: March 2005

Phospholipase B has not yet been well defined. The most important points about this enzyme are its relationships with lysophospholipase and phospholipase A1. As reported [Saito, K., Sugatani, J. & Okumura, T. (1991) Methods Enzymol. 197, 446–456], Penicillium notatum phospholipase B is a glycoprotein with a molecular mass of 95 kDa and intrinsic lysophospholipase and phospholipase B activities; however, by endogenous proteolytic modification, its phospholipase B activity is lost almost completely, whereas its lysophospholipase activity remains unchanged.A cDNA library of P. notatum was screened by hybridization with two synthetic oligodeoxyribonucleotide probes, which corresponds to two different pentapeptides of the enzyme. A hybridization‐positive clone, pPLB18, was isolated and its nucleotide sequence was determined. The deduced amino acid sequence was quite different from that found previously. Therefore, we rescreened the cDNA library with a Sau3AI fragment derived from pPLB18 and isolated a new clone, pPLB15. Comparison of the nucleotide sequences of pPLB15 and pPLB18 revealed that pPLB18 contained an insertion sequence of 53 bp. Consequently, the reading frame was open downstream for 603 amino acid residues.From the assigned sequence, it was deduced that the limited proteolysis occurred between Leu175 and Asp176; eight cysteine residues and 16 potential N‐glycosylation sites were also found. No amino acid sequence similarity was found with other proteins, including other phospholipases.
Extremely thermostable glutamate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus furiosus
Authors: CONSALVI Valerio , CHIARALUCE Roberta , POLITI Laura , VACCARO Rosaria , DE ROSA Mario , SCANDURRA Roberto 
Published: March 2005

The hyperthermophilic archaebacterium Pyrococcus furiosus contains high levels of NAD(P)‐dependent glutamate dehydrogenase activity. The enzyme could be involved in the first step of nitrogen metabolism, catalyzing the conversion of 2‐oxoglutarate and ammonia to glutamate. The enzyme, purified to homogeneity, is a hexamer of 290 kDa (subunit mass 48 kDa). Isoelectric‐focusing analysis of the purified enzyme showed a pI of 4.5. The enzyme shows strict specificity for 2‐oxoglutarate and l‐glutamate but utilizes both NADH and NADPH as cofactors. The purified enzyme reveals an outstanding thermal stability (the half‐life for thermal inactivation at 100°C was 12 h), totally independent of enzyme concentration. P. furiosus glutamate dehydrogenase represents 20% of the total protein; this elevated concentration raises questions about the roles of this enzyme in the metabolism of P. furiosus.
Molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of Trypanosoma brucei
Authors: ALLERT Sylvie , ERNEST Isabelle , POLISZCZAK Annick , OPPERDOES Fred R. , MICHELS Paul A. M. 
Published: March 2005

In Trypanosoma brucei (stock 427) genes encoding the glycolytic enzyme pyruvate kinase are present on two homologous chromosomes. We have cloned and characterized one of the alleles. Two large, tandemly arranged open reading frames were found, each coding for a pyruvate kinase polypeptide of 498 amino acids. The gene sequences differ at 15 positions, resulting in five amino acid substitutions. The calculated molecular masses of the polypeptides are 54 378 Da and 54 363 Da. These values are somewhat smaller than those reported for the subunit molecular mass of the purified protein, which is 57–59 kDa. However, in vitro translation of the DNA region corresponding to the open reading frame, and translation of the RNA in a wheat‐germ lysate, yielded a product that comigrated exactly with the native polypeptide in SDS/PAGE.The overall identity between the sequences of the trypanosomal enzyme and the enzymes from other sources is 41–51%. The conserved residues are not equally distributed over the polypeptide. The primary structure of domains A and, to a lesser extent, B, which constitute the active site, are rather well conserved. In contrast, the sequence of domain C, which supposedly is involved in the regulation of the enzyme activity, is much more variable.The cytosolically located pyruvate kinase of T. brucei lacks the specific features found in the majority of the glycolytic enzymes of this organism that are sequestered in a microbody‐like organelle, the glycosome. It has neither a relatively high subunit molecular mass, due to unique insertions or terminal extensions, nor a high excess of positively charged amino acids. The polypeptide is shorter than that of most other pyruvate kinases and the calculated net charge is only + 3.