bluuebunny/crossref_metadata_2025_split huggingface dataset:
Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter
Authors: Brandsch Matthias , Knütter Ilka , Thunecke Frank , Hartrodt Bianka , Born Ilona , Börner Volker , Hirche Frank , Fischer Gunter , Neubert Klaus 
Published: March 2003

To elucidate the decisive structural factors relevant for dipeptide–carrier interaction, the affinity of short amide and imide derivatives for the intestinal H+/peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly‐Sar transport in Caco‐2 cells. Dipeptides with proline or alanine in the C‐terminal position displayed affinity constants (Ki) of 0.15–1.2 mm and 0.08–9.5 mm, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N‐terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa‐Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24–70%) and the affinity constants was observed. After correcting the Ki values for the incompetent cis isomers, the Ki corr values of most dipeptides were in a small range of 0.1–0.16 mm. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa‐Pro and Xaa‐Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys‐Pro, Arg‐Pro and Pro‐Pro indicate that additional molecular factors affect their binding at PEPT1. The Ki values obtained for the corresponding Xaa‐Ala dipeptides support this conclusion.Potential substrates or inhibitors of peptide transport were found among Xaa‐piperidides and Xaa‐thiazolidides. Dipeptides with N‐terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro‐Xaa dipeptides such as Pro‐Leu, Pro‐Tyr and Pro‐Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C‐terminal amino acid and the rigidity of the structure.
A proton‐NMR investigation of the fully reduced cytochrome c7 from Desulfuromonas acetoxidans
Authors: Assfalg Michael , Banci Lucia , Bertini Ivano , Bruschi Mireille , Giudici‐Orticoni Marie T. , Turano Paola 
Published: March 2003

The solution structure via 1H NMR of the fully reduced form of cytochrome c7 has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H2 atmosphere after it had been checked that the presence of the hydrogenase did not affect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 ± 1.5 nm and 12.4 ± 1.3 nm for the backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54–61 is present. If this loop is ignored, the rmsd values are 6.2 ± 1.1 nm and 10.2 ± 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution.The structure was analyzed and compared with the already available structure of the fully oxidized protein. Within the indetermination of the two solution structures, the result for the two redox forms is quite similar, confirming the special structural features of the three‐heme cluster. A useful comparison can be made with the available crystal structures of cytochromes c3, which appear to be highly homologous except for the presence of a further heme. Finally, an analysis of the factors affecting the reduction potentials of the heme irons was performed, revealing the importance of net charges in differentiating the reduction potential when the other parameters are kept constant.
Sphingomyelinase treatment of rat hepatocytes inhibits cell‐swelling‐stimulated glycogen synthesis by causing cell shrinkage
Authors: van Sluijters Daphne A. , van Woerkom George M. , Aerts Johannes M. F. G. , Meijer Alfred J. 
Published: March 2003

Breakdown of plasma‐membrane sphingomyelin caused by TNF‐α is known to inhibit glucose metabolism and insulin signalling in muscle and fat cells. In hepatocytes, conversion of glucose to glycogen is strongly activated by amino acid‐induced cell swelling. In order to find out whether breakdown of plasma‐membrane sphingomyelin also inhibits this insulin‐independent process, the effect of addition of sphingomyelinase was studied in rat hepatocytes.Sphingomyelinase (but not ceramide) inhibited glycogen synthesis, caused cell shrinkage, decreased the activity of glycogen synthase a, but had no effect on phosphorylase a. Cell integrity was not affected by sphingomyelinase addition as gluconeogenesis and the intracellular concentration of ATP were unchanged. As a control, glycogen synthesis was studied in HepG2 cells. In these cells, the basal rate of glycogen production was high, could not be stimulated by amino acids, nor be inhibited by sphingomyelinase.Regarding the mechanism responsible for the inhibition of glycogen synthase a, sphingomyelinase did not affect amino acid‐induced, PtdIns 3‐kinase‐dependent, phosphorylation of p70S6 kinase, but caused an increase in intracellular chloride, which is known to inhibit glycogen synthase phosphatase.It is concluded that the decrease in cell volume, following the breakdown of sphingomyelin in the plasma membrane of the hepatocyte, may contribute to the abnormal metabolism of glucose when TNF‐α levels are high.
Complementation of NADPH oxidase in p67‐phox‐deficient CGD patients
Authors: Vergnaud Sabrina , Paclet Marie‐Hélène , El Benna Jamel , Pocidalo Marie‐Anne , Morel Françoise 
Published: March 2003

Chronic granulomatous disease (CGD) is due to a functional defect of the O2− generating NADPH oxidase of phagocytes. Epstein–Barr‐virus‐immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein–Barr‐virus‐immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67‐phox‐deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell‐free assay consisting of membrane‐suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic‐activating factors (p67‐phox, p47‐phox, p40‐phox) from healthy or CGD patients. In p67‐phox‐deficient CGD patients, two cytosolic factors are deficient or missing: p67‐phox and p40‐phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67‐phox‐deficient patients with recombinant p67‐phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell‐free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67‐phox and recombinant p40‐phox reversed the previous complementation in a p40‐phox dose‐dependent process.These results suggest that in the reconstitution of oxidase activity, p67‐phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40‐phox from p67‐phox for efficient oxidase activity. In the process, p40‐phox could function as a negative regulatory factor and stabilize the resting state.
Biophysical studies of the development of amyloid fibrils from a peptide fragment of cold shock protein B
Authors: Wilkins Deborah K. , Dobson Christopher M. , Groß Michael 
Published: March 2003

The peptide CspB‐1, which represents residues 1–22 of the cold shock protein CspB from Bacillus subtilis, has been shown to form amyloid fibrils when solutions containing this peptide in aqueous (50%) acetonitrile are diluted in water [M. Großet al. (1999) Protein Science8, 1350–1357] We established conditions in which reproducible kinetic steps associated with the formation of these fibrils can be observed. Studies combining these conditions with a range of biophysical methods reveal that a variety of distinct events occurs during the process that results in amyloid fibrils. A CD spectrum indicative of β structure is observed within 1 min of the solvent shift, and its intensity increases on a longer timescale in at least two kinetic phases. The characteristic wavelength shift of the amyloid‐binding dye Congo Red is established within 30 min of the initiation of the aggregation process and corresponds to one of the phases observed by CD and to changes in the Fourier transform‐infrared spectrum indicative of β structure. Short fibrillar structures begin to be visible under the electron microscope after these events, and longer, well‐defined amyloid fibrils are established on a timescale of hours. NMR spectroscopy shows that there are no significant changes in the concentration of monomeric species in solution during the events leading to fibril formation, but that soluble aggregates too large to be visible in NMR spectra are present throughout the process. A model for amyloid formation by this peptide is presented which is consistent with these kinetic data and with published work on a variety of disease‐related systems. These findings support the concept that the ability to form amyloid fibrils is a generic property of polypeptide chains, and that the mechanism of their formation is similar for different peptides and proteins.
Biosynthesis of the cyanobacterial reserve polymer multi‐L‐arginyl‐poly‐L‐aspartic acid (cyanophycin)
Authors: Berg Holger , Ziegler Karl , Piotukh Kirill , Baier Kerstin , Lockau Wolfgang , Volkmer‐Engert Rudolf 
Published: March 2003

Biosynthesis of the cyanobacterial nitrogen reserve cyanophycin (multi‐l‐arginyl‐poly‐l‐aspartic acid) is catalysed by cyanophycin synthetase, an enzyme that consists of a single kind of polypeptide. Efficient synthesis of the polymer requires ATP, the constituent amino acids aspartic acid and arginine, and a primer like cyanophycin. Using synthetic peptide primers, the course of the biosynthetic reaction was studied. The following results were obtained: (a) sequence analysis suggests that cyanophycin synthetase has two ATP‐binding sites and hence probably two active sites; (b) the enzyme catalyses the formation of cyanophycin‐like polymers of 25–30 kDa apparent molecular mass in vitro; (c) primers are elongated at their C‐terminus; (d) the constituent amino acids are incorporated stepwise, in the order aspartic acid followed by arginine, into the growing polymer. A mechanism for the cyanophycin synthetase reaction is proposed; (e) the specificity of the enzyme for its amino‐acid substrates was also studied. Glutamic acid cannot replace aspartic acid as the acidic amino acid, whereas lysine can replace arginine but is incorporated into cyanophycin at a much lower rate.
Photoinduced intracomplex electron transfer between cytochrome c oxidase and TUPS‐modified cytochrome c
Authors: Kotlyar Alexander , Borovok Natalia , Hazani Miron , Szundi Istvan , Einarsdóttir Ólöf 
Published: March 2003

A novel method for initiating intramolecular electron transfer in cytochrome c oxidase is reported. The method is based upon photoreduction of cytochrome c labeled with thiouredopyrene‐3,6,8‐trisulfonate in complex with cytochrome oxidase. The thiouredopyrene‐3,6,8‐trisulfonate‐labeled cytochrome c was prepared by incubating the thiol reactive form of the dye with yeast iso‐1‐cytochrome c, containing a single cysteine residue. Laser pulse excitation of a stoichiometrical complex between thiouredopyrene‐3,6,8‐trisulfonate‐cytochrome c and bovine heart cytochrome oxidase at low ionic strength resulted in the reduction of cytochrome c by the excited form of thiouredopyrene‐3,6,8‐trisulfonate and subsequent intramolecular electron transfer from the reduced cytochrome c to cytochrome oxidase. The maximum efficiency by a single laser pulse resulted in the reduction of ≈ 17% of cytochrome a, and was achieved only at a 1 : 1 ratio of cytochrome c to cytochrome oxidase. At higher cytochrome c to cytochrome oxidase ratios the heme a reduction was strongly suppressed.
Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site‐directed mutagenesis
Authors: Labrou Nikolas E. , Rigden Danyel J. , Clonis Yannis D. 
Published: March 2003

The 2′,3′‐dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first‐order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mm for the fast phase, 0.45 mm for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min−1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme–oADP product, a process that was reversed after dialysis or after treatment with 2‐mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2‐mercaptoethanol was prevented if the enzyme–oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP‐modified enzyme with trypsin and subsequent separation of peptides by RP‐HPLC gave only one radioactive peak. Amino‐acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the modeller 4 program. The model confirmed that Lys360 is positioned at the NAD+‐binding site. Site‐directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360→Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360→Ala enzyme. The data are discussed in terms of engineering coenzyme specificity.
Chaperone function of mutant versions of αA‐ and αB‐crystallin prepared to pinpoint chaperone binding sites
Authors: Derham Barry K. , van Boekel Martinus A. M. , Muchowski Paul J. , Clark John I. , Horwitz Joseph , Hepburne‐Scott Henry W. , de Jong W. W. , Crabbe M. James C. , Harding John J. 
Published: March 2003

A major stress protein, α‐crystallin, functions as a chaperone. Site‐directed mutagenesis has been used to identify regions of the protein necessary for chaperone function. In this work we have taken some of the previously described mutants produced and assessed their chaperone function by both a traditional heat‐induced aggregation method at elevated temperature and using enzyme methods at 37 °C. In general the different assays gave parallel results indicating that the same property is being measured. Discrepancies were explicable by the heat lability of some mutants. Most mutants had full chaperone function showing the robust nature of α‐crystallin. A mutant corresponding to a minor component of rodent αA‐crystallin, αAins‐crystallin, had decreased chaperone function. Decreased chaperone function was also found for human Ser139→ Arg, Thr144→Arg, Ser59→Ala mutants of αB‐crystallin and double mutants Ser45→Ala/Ser59→Ala, Lys103→ Leu/His104→Ile, and Glu110→His/His111→Glu. A mutant Phe27→Arg that was the subject of previous controversy was shown to be fully active at physiological temperatures.
Novel methylated triterpenoids of the gammacerane series from the nitrogen‐fixing bacterium Bradyrhizobium japonicum USDA 110
Authors: Bravo Jean‐Michel , Perzl Michael , Härtner Thomas , Kannenberg Elmar L. , Rohmer Michel 
Published: March 2003

The nitrogen‐fixing, symbiotic root‐nodule forming bacterium Bradyrhizobium japonicum USDA 110 contained gammacerane derivatives next to triterpenoids of the hopane series. Diploptene, diplopterol, 2β‐methyldiplopterol, aminobacteriohopanetriol and adenosylhopane were accompanied by tetrahymanol and the corresponding novel methylated homologues 2β‐methyltetrahymanol, 20α‐methyltetrahymanol, and 2β,20α‐dimethyltetrahymanol. Incorporation of [2H3]methyl–l‐methionine indicated that the additional methyl groups originated from methionine, probably with S‐adenosylmethionine acting as methyl donor, with retention of the three deuterium atoms. The simultaneous presence of hopane and gammacerane derivatives seems a characteristic feature of the genus Bradyrhizobium and the phylogenetically closely related Rhodopseudomonas palustris.
Exonucleolytic proofreading by p53 protein
Authors: Bakhanashvili Mary 
Published: March 2003

The tumour suppressor p53 protein plays an important role in maintaining genetic integrity. Recently, p53 was shown to have an intrinsic 3′→5′ exonuclease activity. The current study has extended the characterization of purified wild‐type recombinant p53‐associated 3′→5′ exonuclease function to demonstrate proofreading activity. p53‐associated 3′→5′ exonuclease shows clear preference for degradation of ssDNA over dsDNA substrate. On partial duplex structures, this exonucleolytic activity displays a marked preference for excision of a mismatched vs. a correctly paired 3′ terminus which enables the p53 protein to act as a proofreader. However, p53 displays variation in excision of mismatched base pairs. The results demonstrate that p53 exhibits mispair excision with a specificity of A:A > A:G > A:C opposite the template adenine residue and with a specificity of G:A > G:G > G:T opposite the template guanine residue. Hence, the observed specificity of mismatch excision shows that p53 exonucleolytic proofreading preferentially repairs transversion mutations. As part of an investigation of the functional interaction between p53 and DNA polymerase, the influence of p53 on the accuracy of DNA synthesis was determined with exonuclease‐deficient murine leukemia virus (MLV) reverse transcriptase (RT), representing a relatively low fidelity enzyme. Using an in vitro biochemical assay with 3′‐terminal mismatch‐containing DNA template primers, it was shown that wild‐type recombinant p53 protein enhanced the DNA replication fidelity of MLV RT. A functional interaction between the exonuclease (p53) and polymerase (MLV RT) activities was observed; excision of mispairs by p53 was followed by further elongation onto correctly base‐paired 3′‐termini by MLV RT. Furthermore, the formation of 3′‐mispair and subsequent mispair extension by the enzyme were decreased substantially in the presence of p53. The fact that the exonuclease‐deficient MLV RT is more accurate in the presence of p53, suggests that p53 protein may function as an external proofreading exonuclease for viral enzyme. The observed decrease in initial nucleotide misincorporation and 3′‐terminal mispair extension by MLV RT in the presence of p53, indicates the mechanism by which p53 affects the DNA replication fidelity of exonuclease‐deficient DNA polymerase.
Poly(A) polymerase from Escherichia coli adenylylates the 3′‐hydroxyl residue of nucleosides, nucleoside 5′‐phosphates and nucleoside(5′)oligophospho(5′)nucleosides (NpnN)
Authors: Sillero María A. Günther , Socorro Sónia , Baptista María J. , del Valle Mercedes , de Diego Anabel , Sillero Antonio 
Published: March 2003

The capacity of Escherichia coli poly(A) polymerase to adenylylate the 3′‐OH residue of a variety of nucleosides, nucleoside 5′‐phosphates and dinucleotides of the type nucleoside(5′)oligophospho(5′)nucleoside is described here for the first time. Using micromolar concentrations of [α‐32P]ATP, the following nucleosides/nucleotides were found to be substrates of the reaction: guanosine, AMP, CMP, GMP, IMP, GDP, CTP, dGTP, GTP, XTP, adenosine(5′)diphospho(5′)adenosine (Ap2A), adenosine (5′)triphospho(5′)adenosine (Ap3A), adenosine(5′)tetraphospho(5′)adenosine (Ap4A), adenosine(5′)pentaphospho(5′)adenosine (Ap5A), guanosine(5′)diphospho(5′) guanosine (Gp2G), guanosine(5′)triphospho(5′)guanosine (Gp3G), guanosine(5′)tetraphospho(5′)guanosine (Gp4G), and guanosine(5′)pentaphospho(5′)guanosine (Gp5G). The synthesized products were analysed by TLC or HPLC and characterized by their UV spectra, and by treatment with alkaline phosphatase and snake venom phosphodiesterase. The presence of 1 mm GMP inhibited competitively the polyadenylylation of tRNA. We hypothesize that the type of methods used to measure polyadenylation of RNA is the reason why this novel property of E. coli poly(A) polymerase has not been observed previously.
Microperoxidase 8 catalyzed nitration of phenol by nitrogen dioxide radicals
Authors: Ricoux Remy , Boucher Jean‐Luc , Mansuy Daniel , Mahy Jean‐Pierre 
Published: March 2003

Microperoxidase 8 (MP8) is a heme octapeptide obtained by hydrolytic digestion of horse heart cytochrome c. At pH below 9, the heme iron is axially coordinated to the imidazole side chain of His18 and to a water molecule. Replacement of this weak ligand by H2O2 allows the formation of high‐valent iron‐oxo species which are responsible for both peroxidase‐like and cytochrome P450‐like activities of MP8. This paper shows that MP8 is able to catalyze the nitration of phenol by nitrite. The reaction requires H2O2 and is inhibited by ligands having a high affinity for the iron, catalase and radical scavengers. This suggests that the nitrating species could be NO2• radicals formed by the oxidation of nitrite by high‐valent iron‐oxo species. This new activity of MP8 opens a new access to nitro‐aromatic compounds under mild conditions and validates the use of this minienzyme to mimick heme peroxidases, especially in the reactions of NO‐derived species with biomolecules under oxidative stress conditions.
Alternate routes to conformational specificity in a Greek key β barrel protein
Authors: Qi Xiao‐Fei , Bagby Stefan , Gombos Zoltan , Ikura Mitsuhiko , Chakrabartty Avijit 
Published: March 2003

The N‐terminal domain of protein S, a Greek key calcium‐binding protein from Myxococcus xanthus, forms an atypical molten globule in the calcium‐free state. The structure of this state is characterized by significant conformational fluctuations, which are localized to a subdomain that is not contiguous along the polypeptide chain. The conformational instability of this subdomain appears to arise from repulsive electrostatic interactions of four acidic side chains that are clustered together but are removed from the calcium‐binding sites. This domain can be induced to form a native‐like state through two different routes, calcium binding or reduction of pH. Acid‐induced folding stabilizes the locally unfolded subdomain by selectively removing repulsive interactions without significantly affecting global stability. In contrast, calcium binding appears to increase local stability indirectly by causing global stabilization.