bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Properties of a Succinyltransferase from Pseudomonas aeruginosa Specific for both Arginine and Ornithine
Authors: Tricot Catherine , Vander Wauven Corinne , Wattiez Ruddy , Falmagne Paul , Stalon Victor 
Published: March 2005

The arginine and ornithine succinyltransferase from Pseudomonas aeruginosa, a bifunctional enzyme involved in the aerobic utilization of arginine and ornithine, has been purified to homogeneity. The apparent molecular mass of the native enzyme was 150 kDa by gel filtration and 140 kDa by polyacrylamide gel electrophoresis under non‐denaturing conditions. After SDS/PAGE two subunits of 35 kDa and 37 kDa were evident, indicating that the enzyme is a heterotetramer. Microsequence analysis of the electroblotted protein bands gave two different but well‐conserved N‐terminal amino acid sequences.The l‐arginine saturation curve followed Henri‐Michaelis kinetics with an apparent Km value of 0.5 mM. The sigmoidal saturation curve for l‐ornithine indicated allosteric behaviour. d‐Arginine, a competitive inhibitor with respect to l‐arginine, reduced l‐ornithine cooperativity. In the presence of spermidine, the l‐ornithine saturation curve became increasingly sigmoidal, the Hill coefficient shifting from 2.5 in the absence of the inhibitor, to 3.5 in the presence of 20 mM spermidine. The l‐arginine analog, l‐homoarginine, was also a substrate of the succinyltransferase, and the saturation of the enzyme by this substrate was also cooperative. All these data confirmed the allosteric nature of the enzyme. Moreover, a mutant growing faster on l‐ornithine than the parent strain had a modified succinyltransferase with a reduced l‐ornithine cooperativity.The fate of l‐homoarginine was different depending on whether the succinyltransferase was induced or not; excreted succinylhomoarginine was found in cultures induced for the transferase activity whereas guanidinovalerate was excreted in non‐induced cultures. The ‘waste’ of succinyl CoA, which could not be regenerated from the excreted succinylhomoarginine, explained the inhibition exerted by l‐homoarginine on growth when ornithine or arginine was used as the growth medium.
A New Type of Ca2+‐Dependent, Mg2+‐Stimulated ATPase of Rat Liver Plasma Membrane
Authors: Heilmann Claus , Spamer Cornelia , Mössner Wolfgang , Dietz Christine , Reutter Werner , Kreisel Wolfgang 
Published: March 2005

Incubation of a glycoprotein fraction obtained from rat liver plasma membrane which has been previously well characterized using [γ‐32P]ATP results in the phosphorylation of a 230‐kDa glycoprotein (pgp230). It is composed of a 120‐kDa subunit (pgp120) and a 110‐kDa subunit (pgp110) linked by interchain disulfide bonds. Peptide maps of pgp120 and pgp110 suggest extensive similarity in their polypeptide chains. Glycan analysis reveals between four and six hybrid‐type oligosaccharide chains for both phosphoproteins. Immunoblotting using monoclonal antibodies and endoglycosidase digestion exclude an identity of pgp120 or pgp110 with the hepatocyte plasma membrane glycoproteins dipeptidylpeptidase IV or the taurocholate transport protein, which co‐purify and comigrate in SDS/PAGE. Protein phosphorylation is ca2+‐dependent (K0.5(Ca2+)= 0.35 μM, in the absence of Mg2+). In the presence of Mg2+, the glycoprotein undergoes rapid cycles of phosphorylation and dephosphorylation, resulting in ATPase activity. Analysis of phosphorylated amino acids identifies phosphothreonine as the major one. Photoaffinity labeling with 8‐azido‐[α‐32P]ATP demonstrates the presence of one or more ATP binding site(s). Preincubation of pgp230 with various purine or pyrimidine nucleotides (ATP, UTP, TTP, ADP, GDP, AMP, CMP) or known P2‐purinoceptor agonists or antagonists (adenosine 5′‐[α,β‐methylene]triphosphate, 2‐methyl‐thio‐adenosine 5′‐triphosphate, suramin) inhibits its phosphorylation by [γ‐32P]ATP.The biological function of pgp230 is unknown at present. Several findings of the present study are compatible with the idea that pgp230 may be involved in a P2‐purinoceptor function of the hepatocyte. Following this concept, a mechanism is discussed where a cytosolically exposed high‐affinity Ca2+‐binding site of pgp230 would allow for receptor feedback control, via phosphorylation and dephosphorylation, by sensing changes in cytosolic Ca2+ concentration.
The Modulation of Enzyme Reaction Rates within Multi‐Enzyme Complexes
Authors: Ricard Jacques , Giudici‐Orticoni Marie‐Thérèse , Gontero Brigitte 
Published: March 2005

There is now experimental evidence that association of different enzymes as a multi‐enzyme complex may result in an alteration of the catalytic properties of the enzymes present in this complex. This effect is not related to the channelling of reaction intermediates between different active sites. It appears as a consequence of an information transfer that occurs within the multi‐enzyme complex.A theory, based on statistical thermodynamics, has been developed which provides an understanding, on a physical basis, for how isologous as well as heterologous interactions between identical, or different, enzymes of the complex may modulate the catalytic properties of an oligomeric enzyme of that complex. The theory predicts three possible types of effects: an alteration, through heterologous interactions, of an already existing cooperativity of the oligomeric enzyme within the complex; a co‐operativity, generated by heterologous interactions in the complex that could not occur if the oligomeric enzyme were isolated from the rest of the complex; a Michaelis‐Menten character of the oligomeric enzyme within the complex, but with altered values of Vm and Km relative to what would have been observed with the naked enzyme.All these effects appear as a consequence of a transfer of information between different enzymes of the same multi‐protein complex. The following paper in this journal shows how one can demonstrate and characterize experimentally these effects in a multi‐enzyme complex containing ribulose bisphosphate carboxylase‐oxygenase.
Secondary Structure and Membrane Interaction of PR‐39, a Pro+Arg‐rich Antibacterial Peptide
Authors: Cabiaux Véronique , Agerberths Birgitta , Johansson Jan , Homblé Fabrice , Goormaghtigh Erik , Ruysschaert Jean‐Marie 
Published: March 2005

PR‐39 is a 4719‐Da peptide isolated from pig intestine and belonging to the recently discovered family of Pro + Arg‐rich antibacterial peptides. PR‐39 does not lyse Escherichia coli, instead the lethal action is probably linked to the termination of DNA and protein synthesis [Boman, H. G., Agerberth, B. & Boman, A. (1993) Infect. Immun. 61, 2978–2984].Circular dichroism and Fourier‐transform infrared spectroscopy have been used to investigate the secondary structure of PR‐39 in the absence or presence of lipids. According to the circular dichroic data, this structure is not altered upon incubation of PR‐39 with negatively charged vesicles, although the infrared spectra suggest that the hydrogen bond pattern is modified upon the peptide—lipid interaction. This is detected by a shift in the maximum wavelength of absorption of PR‐39 from 1636 cm−1 in the absence of lipids to 1645 cm−1 in the presence of lipids. We have further addressed the question of the possible mechanism of interaction of PR‐39 with model membranes (liposomes and planar lipid bilayers) whose lipid compositions mimick that of the E. coli inner membrane. PR‐39 induced a calcein release from large unilamellar vesicles, which is dependent upon the peptide concentration and upon the presence of negatively charged lipid (glycerophosphoglycerol) in the membrane. The binding study of PR‐39 to dioleoylglycerophosphoglycerol vesicles suggests that nearly 100% of the added peptide is membrane‐bound. Addition of PR‐39 to a planar lipid bilayer induced a linear increase in the current but no channel formation was observed since no discrete steps of conductance occurred.
Proteolytic Cleavage of the Multienzyme Polypeptide CAD to Release the Mammalian Aspartate Transcarbamoylase
Authors: Hemmens Benjamin , Carrey Elizabeth A. 
Published: March 2005

We have demonstrated biochemically that the conformation of the proteolytic fragment (mammalian aspartate transcarbamoylase) from the C‐terminus of the 240‐kDa multienzyme polypeptide carrying the activities carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydroorotase (CAD) is similar to that of the catalytic subunits from Escherichia coli aspartate transcarbamoylase. We have measured the extent of unfolding of the mammalian aspartate transcarbamoylase in guanidinium chloride solutions, and have also demonstrated that the protein cross‐reacts with antibodies raised against the E. coli enzyme.CAD is digested by low concentrations of trypsin in the presence of 0.2 mM UTP to release an active aspartate transcarbamoylase domain and a 195‐kDa ‘nicked CAD’ molecule containing active carbamoyl phosphate synthetase. These two products are easily separated by ion‐exchange chromatography. Similar proteolytic cleavage and trimming by elastase releases a family of aspartate transcarbamoylase fragments. Direct N‐terminal sequencing of the aspartate transcarbamoylase fragments confirms predictions of the most accessible residues in the region linking the aspartate transcarbamoylase and dihydroorotase domains. Only the largest of the four fragments generated by elastase retains phosphorylation site 2. When this largest fragment is phosphorylated, the family of aspartate transcarbamoylase fragments is eluted together from ion‐exchange columns in a different fraction from the completely unphosphorylated preparation, demonstrating the affinity of the domains for each other.
Chemical Structure of the Core Region of Escherichia coli J‐5 Lipopolysaccharide
Authors: Müller‐Loennies Sven , Holst Otto , Brade Helmut 
Published: March 2005

The lipopolysaccharide of Escherichia coli J‐5 was sequentially de‐O‐acylated, dephosphorylated, reduced, de‐N‐acylated, and N ‐acetylated. The products were separated by high‐performance anion‐exchange chromatography into a nonasaccharide (1), two octasaccharides (2,3), and a heptasaccharide (4). Compositional analysis, methylation analysis, and NMR spectroscopy revealed the structures of the products as:

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in which 1R is l‐α‐d‐Hepp‐(1–5)‐[α‐Kdop‐(2–4)‐]‐α‐Kdop‐(2–6)‐β‐d‐GlcpNAc‐(1–6)‐d‐GlcN‐Acol, and 2R is l‐α‐d‐Hepp‐(1–5)‐α‐Kdop‐(2–6)‐β‐d‐Glcp NAc‐(1–6)‐d‐GlcNAcol (LD‐Hep, l‐glycero‐d‐manno‐heptose; Kdo, 3‐deoxy‐d‐manno‐octulopyranosonic acid; GlcNAcol, 2‐acet‐amido‐2‐deoxy‐glucitol).Fast‐atom‐bombardment mass spectrometry of de‐O‐acylated and dephosphorylated lipopolysaccharide showed that the isolated oligosaccharides represented the complete carbohydrate moiety of the lipopolysaccharide, and indicated that the non‐reducing terminal d‐GlcN residue in lipopolysaccharide was present as the free base.
Effects of protons on the thrombin‐fibrinogen interaction
Authors: PICOZZI Matilde , LANDOLFI Raffaele , DE CRISTOFARO Raimondo 
Published: March 2005

Amidase activity of human α‐thrombin toward the synthetic substrate Tosyl‐Gly‐Pro‐Arg‐NH‐Ph and fibrinogen has been studied as a function of pH at t= 25°C, under steady‐state conditions. A viscosity‐perturbation method allowed us to compute the equilibrium binding constant along with the rate constants for the acylation and deacylation reactions. The ionization constants for the groups affecting binding and hydrolysis of the synthetic substrate were measured by application of linkage thermodynamics principles. The binding of the synthetic substrate is controlled by two ionizable groups having pKa values of 7.5 and 8.7 in the free enzyme and 6.3 and 9.8 in the Michaelis adduct. These two groups were found to control the acylation process as well.Thrombin‐fibrinogen interaction has been studied by measurements of steady‐state hydrolysis of the synthetic substrate Phe‐pipecolyl‐Arg‐NH‐Ph in the presence of fibrinogen, used as a competitive inhibitor. This method allowed us to measure the Km of thrombin‐fibrinogen interaction. The values of Km computed at different solution viscosities were used in order to calculate the equilibrium dissociation constant and both k2/k3 and k2/k‐1 ratios. The same residues that were found to control binding of Tosyl‐Gly‐Pro‐Arg‐NH‐Ph to α‐thrombin, do modulate binding of fibrinogen as well. These residues shift their pKa values upon the formation of the Michaelis adduct from 7.5 to 5.7 and from 8.7 to 9.7, respectively. Furthermore the ratio kcat/Km as a function of pH has been obtained by HPLC measurements of fibrinopeptides release. The kcat/Km values along with the ratio k2/k‐1, derived from viscometric experiments, allowed us to calculate the forward‐rate constant, k+1, for the thrombin‐fibrinogen interaction. The association process was found to depend on pH, namely in the alkaline region. The results for Tosyl‐Gly‐Pro‐Arg‐NH‐Ph and fibrinogen are compared and discussed on the basis of the structural elements which differentiate the interactions of these substrates with human α‐thrombin.
Increased thermal aggregation of proteins in ATP‐depleted mammalian cells
Authors: Van NGUYEN Trung , BENSAUDE Olivier 
Published: March 2005

In an attempt to understand the influence of the intracellular environment on protein stability, the thermal denaturation of various reporter proteins was examined within cultured mammalian cells. Loss of solubility and of enzymatic activities were taken as indicators of thermal denaturation. Photinus pyralis luciferase, Escherichia coliβ‐galactosidase, the 70‐kDa constitutive heat‐shock proteins and the 68‐kDa dsRNA‐dependent protein kinase are found mostly in the supernatant fractions of centrifuged lysates from control unshocked mammalian cells. However, when cells are lysed after heat shock, a proportion of the reporter molecules is found to be aggregated to the nuclear pellets. This insolubilization does not affect all cellular proteins; many of them remain unaffected by heat shock.The heat‐induced insolubilization of all four reporter proteins is markedly enhanced when the intracellular ATP concentration is drastically decreased after inhibition of both oxidative phosphorylation and glycolysis. Although ATP molecules bind to luciferase and protect it from thermal inactivation in vitro, the consequences of strong ATP depletion on luciferase thermal stability within the cells are found to be much greater than expected from in vitro data. The 70‐kDa constitutive heat‐shock proteins and the 68‐kDa protein kinase are ATP‐binding proteins but ATP depletion also considerably increases the aggregation of β‐galactosidase to the nuclear pellets, although this enzyme is not known to be an ATP‐binding molecule.Insolubilization of all four reporter proteins occurs in ATP‐depleted cells even at normal growing temperatures (37°C). Protein denaturation may be enhanced either by the aggregation and disappearance of the intracellular ‘free’ chaperones or by the trapping of unfolded protein molecules on chaperones; the chaperone/unfolded protein complexes could not dissociate in the absence of ATP.Enhanced protein denaturation due to ATP depletion is proposed to account for the greater heat sensitivity of ATP‐depleted cells and for the ability of mitochondrial uncouplers to trigger a heat‐shock response in some cells.
The oligosaccharides of the Fe(III)‐Zn(II) purple acid phosphatase of the red kidney bean
Authors: STAHL Bernd , KLABUNDE Thomas , WITZEL Herbert , KREBS Bernt , STEUP Martin , KARAS Michael , HILLENKAMP Franz 
Published: March 2005

Purple acid phosphatase of the common bean Phaseolus vulgaris (KBPase), a dimeric 110‐kDa glycoprotein related to the mammalian purple acid phosphatases with a two‐metal cluster at the active site contains five oligosaccharide side chains/monomer. The N‐linked glycan structures were characterized by selective enzymic degradation in combination with matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS). The purified protein was cleaved by cyanogen bromide. One 30‐kDa large methionine‐free fragment required a further tryptic digest. The peptides were separated by HPLC and the glycosylated species were identified both by their heterogeneous mass spectra and by an immunoassay. None of the glycopeptides proved to have more than one glycosylation site. The composition of the carbohydrate moieties were calculated by comparing the mass spectra of the glycopeptides before and after enzymic deglycosylation. These results were complemented by data from a carbohydrate composition analysis. In four of the five peptides an α1–3 fucose attached to the asparagine‐linked N‐acetylglucosamine prevented removal of the glycan by peptide N‐glycosidase F; peptide N‐glycosidase A removed all carbohydrates from the peptides.To reveal the sequence of the carbohydrate moiety including the linkage positions between the different saccharides, one of the glycopeptides was degraded by specific exoglycosidases. The enzymic degradations by these hydrolases were monitored by mass spectrometry of small aliquots taken at intervals during the reaction. The detailed structure of this one glycan in conjunction with the respective mass spectra and the composition analysis were used to infer the structure of the other four glycans. All glycans of the KBPase have a complex‐type xylose‐containing structure with four of the five having an additional fucose.
Origin of the carbon atoms of biotin
Authors: IFUKU Ohji , MIYAOKA Hiroaki , KOGA Nobuyoshi , KISHIMOTO Jiro , HAZE Shin‐ichirou , WACHI Youji , KAJIWARA Masahiro 
Published: March 2005

The origin of the carbon atoms of pimeloyl‐CoA, the earliest known precursor in the pathway of de novo biotin biosynthesis in Escherichia coli, was investigated by 13C‐NMR spectroscopy.In fermentation of the biotin‐overproducing DRK332/pXBA312 strain of Escherichia coli (a repressor mutant carrying a biotin operon fragment in the plasmid), a high dose of L‐alanine (8 g/l) stimulated dethiobiotin and biotin accumulation. Although L‐alanine is a known precursor of 7‐keto‐8‐aminopelargonic acid in biotin biosynthesis, the 13C‐NMR spectrum of dethiobiotin showed that the C‐3 of L‐[3‐13C]alanine was incorporated into not only the methyl carbon (C‐9) but also alternate carbons (C‐2, C‐4, C‐6) of the side chain, and these latter positions are the same as those labeled with D‐[1‐13C]glucose. These data indicate that L‐alanine can act as an alternative carbon source, suggesting that acetyl‐CoA is a possible precursor for pimeloyl‐CoA synthesis. In accordance with this hypothesis, the C‐1 of sodium (1‐13C)acetate and the C‐2 of sodium (2‐13C)acetate were incorporated into alternate carbons in the side chain of dethiobiotin, i.e., (C‐1, C‐3, C‐5, C‐7) and (C‐1, C‐2, C‐4, C‐6), respectively. These results suggested firstly that in E. coli pimeloyl‐CoA is biosynthesized from L‐alanine and/or acetate via acetyl‐CoA, but not via pimelic acid, which has been suggested as a biotin precursor in other species, and secondly that the carboxyl group of biotin originates from carbon dioxide produced through the tricarboxylic acid cycle.
Threonyl‐tRNA synthetase from yeast Discrimination of 19 amino acids in aminoacylation of tRNAThr‐C‐C‐A and tRNAThr‐C‐C‐A(2′NH2).
Authors: FREIST Wolfgang , STERNBACH Hans , CRAMER Friedrich 
Published: March 2005

For discrimination between threonine and 18 other naturally occuring non‐cognate amino acids by the class II aminoacyl‐tRNA synthetase specific for threonine, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Cys, Met, Val (D= 70–280), indicating that threonine is only 70–280‐times more often esterified to tRNAThr‐C‐C‐A than are these non‐cognate compounds at the same amino acid concentrations. The highest D values have been observed for Gly, Pro, Gln, Leu, Phe, and Lys (D= 1000–2000), for the other non‐cognate amino acids D values are in the medium range 300–1000. Generally, threonyl‐tRNA synthetase is less specific than the class I enzymes specific for Ile, Val, Tyr, Arg, but more specific than the only investigated class II enzyme specific for Lys.In aminoacylation of tRNAThr‐C‐C‐A(2′NH2) discrimination factors D1, are in the range 2–170. From D1, values and AMP‐formation stoichiometry, pre‐transfer proof‐reading factors II1, were determined; post‐transfer proof‐reading factors II2, were determined from D values and AMP‐formation stoichiometry in acylation of tRNAThr‐C‐C‐A. II1, values are in the range 1.8–33, II2, values in the range 1.4–22, thus threonyl‐tRNA synthetase shows the highest post‐transfer proof‐reading activity of six investigated synthetases (specific for Ile, Val, Tyr, Arg, Lys).Initial discrimination factors caused by differences in Gibbs free energies of binding between threonine and non‐cognate amino acids have been calculated from discrimination and proof‐reading factors. Assuming a two‐step binding process, two factors (I1, and I2) have been determined which can be related to hydrophobic interaction forces depending on accessible surface areas of the amino acids. The threonine side chain must be bound by hydrophobic forces and two hydrogen bonds. In contrast to proof‐reading factors obtained with the synthetases specific for Ile, Val, Tyr, Arg, and Lys, proof‐reading factors II1, and II2, obtained with threonyl‐tRNA synthetase are also related to hydrophobic interaction of the amino acid side chains and the enzyme. Threonyl‐tRNA synthetase examines side chain structures of amino acids in the four postulated recognition steps, for each step the enzyme uses special distinct structures or conformations of the binding cleft.
Structural analysis of the functional gene and pseudogene encoding the murine granulocyte colony‐stimulating‐factor receptor
Authors: ITO Yasuhiko , SETO Yoshiyuki , BRANNAN Camilynn I. , COPELAND Neal G. , JENKINS Nancy A. , FUKUNAGA Rikiro , NAGATA Shigekazu 
Published: March 2005

Granulocyte colony‐stimulating factor is a cytokine which specifically regulates the production of neutrophilic granulocytes. The granulocyte colony‐stimulating‐factor receptor (GCSFR) is mainly expressed in neutrophils and their precursor cells. In this study, we isolated the chromosomal gene for murine GCSFR and determined its structure. Like the human GCSFR gene homolog, it consists of 17 exons. The exon‐intron organization of the murine and human GCSFR‐encoding genes are very similar, except that exon 14 and exon 15 in the murine gene are interrupted by a larger intron (greater than 10 kbp) than that found in the human gene (128 bp). This GCSFR‐encoding functional gene (Csfgr) was localized to the distal region of murine chromosome 4 by interspecific backcross mapping.A comparison of the 5′ flanking sequence of murine and human Csfgr revealed that a sequence of approximately 300 bp upstream from the cap site is highly conserved. Within this region, an 18‐nucleotide element conserved in the promoter of the genes for neutrophil‐specific enzymes, was found approximately 140 bp upstream from the cap site, suggesting an involvement of this element in the specific expression of GCSFR in neutrophilic granulocytes.In addition to the functional GCSFR‐encoding gene, we isolated a pseudogene for GCSFR, which is flanked by a 15‐bp direct repeat at the 5′ and 3′ ends, and lacks all introns, exons 1–3 and exons 7–8 of the functional gene. The processed pseudogene has, in its most 5′ region, a sequence of approximately 200 bp that is highly related to the DNA sequence approximately 1.2 kbp upstream of the cap site of the functional gene.