bluuebunny/crossref_metadata_2025_split huggingface dataset:
Coordination of catalytic activities within enzyme complexes
Authors: KELLERSHOHN Nicolas , RICARD Jacques 
Published: March 2005

If two enzymes are physically and permanently associated as a bi‐enzyme complex and if these enzymes catalyze non‐consecutive chemical reactions, either of these reactions may inhibit or activate the other. If these reactions belong to two different metabolic cycles, the functioning of one of these cycles will control the fine tuning of the other.Thus simple kinetic considerations lead to the conclusion that, owing to the spatial organization of enzymes as multimolecular complexes, a fine tuning and a coordination of different metabolic networks, or cycles, may be exerted. It thus appears that channelling of reaction intermediates within a multienzyme complex does not represent the only functional advantage brought about by this type of spatial molecular organization.
Primary sequence, oxidation‐reduction potentials and tertiary‐structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin
Authors: CALDEIRA Jorge , PALMA P. Nuno , REGALLA Manuela , LAMPREIA Jorge , CALVETE Juan , SCHÄFER Wolfram , LEGALL Jean , MOURA Isabel , MOURA José J. G. 
Published: March 2005

Flavodoxin was isolated and purified from Desulfovibrio desulfuricans ATCC 27774, a sulfate‐reducing organism that can also utilize nitrate as an alternative electron acceptor. Mid‐point oxidation‐reduction potentials of this flavodoxin were determined by ultraviolet/visible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E2, for the couple oxidized/semiquinone forms at pH 6.7 and 25°C is –40 mV, while the value for the semiquinone/hydroquinone forms (E1), at the same pH, –387 mV. E2 varies linearly with pH, while E1 is independent of pH at high values. However, at low pH (< 7.0), this value is less negative, compatible with a redox‐linked protonation of the flavodoxin hydroquinone. A comparative study is presented for Desulfovibrio salexigens NCIB 8403 flavodoxin [Moura, I., Moura, J. J. G., Bruschi, M. & LeGall, J. (1980) Biochim. Biophys. Acta 591, 1–8].The complete primary amino acid sequence was obtained by automated Edman degradation from peptides obtained by chemical and enzymic procedures. The amino acid sequence was confirmed by FAB/MS.Using the previously determined tridimensional structure of Desulfovibrio vulgaris flavodoxin as a model [similarity, 48.6%; Watenpaugh, K. D., Sieker, L. C., Jensen, L. H., LeGall, J. & Dubourdieu M. (1972) Proc. Natl Acad. Sci. USA 69, 3185–3188], the tridimensional structure of D. desulfuricans ATCC 27774 flavodoxin was predicted using AMBER force‐field calculations.
The multidrug‐resistance‐reverser verapamil interferes with cellular P‐glycoprotein‐mediated pumping of daunorubicin as a non‐competing substrate
Authors: SPOELSTRA Ellen C. , WESTERHOFF Hans V. , PINEDO Herbert M. , DEKKER Henk , LANKELMA Jan 
Published: March 2005

We examined P‐glycoprotein‐mediated verapamil transport, using two drug‐sensitive and multidrug resistant cell‐line couples, i.e. A2780, 2780AD and SW‐1573, SW‐1573/1R500. The interaction of 3H‐labeled verapamil with cells was measured using a flow‐through system. The verapamil‐containing medium was pumped over the cells and monitored on‐line for radioactivity. In the P‐glycoprotein‐expressing cells, verapamil accumulation was increased by vinblastine and some known multidrug resistant (MDR) modifiers. Subsequent removal of these modifiers caused release of verapamil into the medium against a verapamil concentration gradient. In this manner, we obtained evidence that verapamil is actively transported by the MDR‐related P‐glycoprotein.Using the flow‐through system, we also exposed the cells to flowing culture medium containing daunorubicin, and measured the inhibition of daunorubicin efflux by verapamil. We found that, although the active efflux of daunorubicin was maximally blocked by verapamil short‐term, longer‐term active efflux of daunorubicin resumed. At a daunorubicin concentration in the flowing medium of 5 μM, increasing the verapamil concentration resulted in the same short‐term effects, but in a significantly longer period of a maximal inhibition of daunorubicin efflux from the cells. At a daunorubicin concentration of 20 μM, increasing the verapamil concentration affected neither the short‐term nor the long‐term effects. These and other observations are in agreement with a model in which daunorubicin and verapamil are non‐competing substrates for P‐glycoprotein.In conclusion, we obtained evidence that verapamil is actively transported by the MDR‐related P‐glycoprotein and that verapamil and daunorubicin are non‐competing substrates for P‐glycoprotein. Consequently, the effectiveness of verapamil as an MDR antagonist may be compromised because it is extruded by P‐glycoprotein.
Residues essential for catalytic activity of soybean β‐amylase
Authors: TOTSUKA Atsushi , Van NONG Hai , KADOKAWA Hiroya , KIM Chan‐Shick , ITOH Yoshifumi , FUKAZAWA Chikafusa 
Published: March 2005

To determine which amino acid residues are essential for the catalytic activity of soybean β‐amylase, deoxyoligonucleotide site‐directed mutagenesis was employed against aspartyl, glutamyl, and cysteinyl residues located in highly conserved regions found in β‐amylase family to date. Both substitution of aspartic acid at position 101 and that of glutamic acid at position 186 of the enzyme by neutral and acidic amino acids, respectively, led to the complete elimination of activity, but did not induce any significant changes in circular dichroic spectra or the binding affinity for cyclomaltohexaose, a substrate analogue.Taking account of the results obtained here, the above two amino acid residues are involved in the catalytic site of soybean β‐amylase. The replacement of glutamic acid at position 345 decreased activity to below 6% of the non‐mutant level, implying that this residue may also play a crucial role in β‐amylase activity, although it may not be involved at the catalytic site itself. In contrast, substitution of cysteinyl residue at position 95 by a serinyl residue led to a drastic reducing of the optimal temperature (from 50°C to 30°C), suggesting that this cysteinyl residue is responsible for the thermal stability of the enzyme.
Conformational analysis of 3′‐fluorinated A(2′‐5′)A(2′‐5′)A fragments
Authors: VAN DEN BOOGAART Jacquelien E. , KALINICHENKO Elena N. , PODKOPAEVA Tatjana L. , MIKHAILOPULO Igor A. , ALTONA Cornelis 
Published: March 2005

A one‐ and two‐dimensional NMR study has been performed on seven A(2′–5′)A(2′–5′)A fragments containing 9‐(3′‐fluoro‐3′‐deoxy‐β‐D‐xylofuranosyl)‐adenine (AF) or 3′‐fluoro‐3′‐deoxyadenosine (AF) residues at different positions, and on the corresponding monomers. A(2′–5′)A(2′–5′)A served as a reference compound. The fluoro substituent governs the conformation of the sugar ring: an AF residue displays mainly N‐type sugar and the ring is considerably flattened (øN∼ 30°) compared to AF residues (øS∼ 40°), which exhibit almost pure S‐type conformation. Moreover, in AF moieties the rotamer distribution around torsion angle γ (O5′‐C5′‐C4′‐C3′) and the base orientation are influenced to a large extent by the preseñce of the fluorine substituent. The sugar rings of nonfluorinated residues in the trimers appear rather flexible.A possible correlation between the conformational characteristics of the fluorinated fragments and their biological activity has been found: the fragments that meet the prerequisites for binding to RNase L indeed show enhanced binding to this endonuclease. Furthermore, substitution of the 3′‐OH group of the second residue by hydrogen or of the 3′‐OH group of the 2′‐terminal residue by fluorine or hydrogen results in increased resistance towards 2′–5′‐phosphodiesterase.
Structural organization of the genes for rat von Ebner's gland proteins 1 and 2 reveals their close relationship to lipocalins
Authors: KOCK Kai , AHLERS Christiane , SCHMALE Hartwig 
Published: March 2005

The rat von Ebner's gland protein 1 (VEGP 1) is a secretory protein, which is abundantly expressed in the small acinar von Ebner's salivary glands of the tongue. Based on the primary structure of this protein we have previously suggested that it is a member of the lipocalin superfamily of lipophilic‐ligand carrier proteins. Although the physiological role of VEGP 1 is not clear, it might be involved in sensory or protective functions in the taste epithelium.Here, we report the purification of VEGP 1 and of a closely related secretory polypeptide, VEGP 2, the isolation of a cDNA clone encoding VEGP 2, and the isolation and structural characterization of the genes for both proteins. Protein purification by gel‐filtration and anion‐exchange chromatography using Mono Q revealed the presence of two different immunoreactive VEGP species. N‐terminal sequence determination of peptide fragments isolated after protease Asp‐N digestion allowed the identification of a new VEGP, named VEGP 2, in addition to the previously characterized VEGP 1. The complete VEGP 2 sequence was deduced from a cDNA clone isolated from a von Ebner's gland cDNA library. The VEGP 2 cDNA encodes a protein of 177 amino acids and is 94% identical to VEGP 1.DNA sequence analysis of the rat VEGP 1 and 2 genes isolated from rat genomic libraries revealed that both span about 4.5 kb and contain seven exons. The VEGP 1 and 2 genes are non‐allelic distinct genes in the rat genome and probably arose by gene duplication. The high degree of nucleotide sequence identity in introns A–C (94–100%) points to a recent gene conversion event that included the 5′ part of the genes.The genomic organization of the rat VEGP genes closely resembles that found in other lipocalins such as β‐lactoglobulin, mouse urinary proteins (MUPs) and prostaglandin D synthase, and therefore provides clear evidence that VEGPs belong to this superfamily of proteins.
Deactivation of F0F1 ATPase in intact plant mitochondria
Authors: VALERIO Marie , DIOLEZ Philippe , HARAUX Francis 
Published: March 2005

By using a method especially adapted to intact (pea leaf) mitochondria, we studied the regulation of the F0F1 ATPase by the electrochemical proton gradient (Δμ˜H+) and by the matricial pH. The kinetics of decay of the ATP hydrolase activity was studied immediately after the collapse of the electrochemical proton gradient by an uncoupler. At pH 7.5, three inhibitors of the ATPase (venturicidin, tri‐n‐butyl tin and aurovertin), used at non‐saturating concentrations, inhibited ATP hydrolysis to the same extent throughout the decay. This showed that the activity was totally controlled by the ATPase during all the decay and rules out any involvement of the phosphate or nucleotide carriers. This interpretation was confirmed by the fact that carboxyatractyloside, an inhibitor of the ATP/ADP antiporter, had a strong effect only on the initial rate of ATP hydrolysis, but not on the rate measured after some tens of seconds of decay. Oligomycin, at variance with the other ATPase inhibitors, interfered with the deactivation process, suggesting that its effect depends on the conformational state of the enzyme. Between pH 6.5 and 7.5, the hydrolase activity rose continuously and was still kinetically controlled by the ATPase. At higher pH value, the activity slightly decreased and appeared limited by at least one of the carriers. The activity of the ATPase itself, free of any transport process, seemed to increase monotonously with pH from 6.5 to 8.The electrochemical proton gradient is required to maintain the ATPase active, whereas no effect can be observed on transport processes. Matricial pH, while modulating the apparent catalytic turnover, has no marked effect on the rate of deactivation. These results, obtained with intact mitochondria, extend previous observations on the isolated enzyme and question the binding of IF1 as a rate‐limiting step for ATPase deactivation.
Purification of rat liver plasma membrane glutathione transferase
Authors: HORBACH Martina E. , SIES Helmut , AKERBOOM Theodorus P. M. 
Published: March 2005

Glutathione transferases purified from plasma membrane and microsomal fractions from rat liver share similar enzymic properties. The activity of both proteins with 1‐chloro‐2,4‐dinitrobenzene can be stimulated about 10–15‐fold by N‐ethylmaleimide. No activation is observed using p‐nitrobenzylchloride as a substrate.The enzymes are immunologically related as indicated by Western‐blot analysis using antibodies against the microsomal glutathione transferase or against a synthetic peptide corresponding to the amino acid positions 55–64 of microsomal glutathione transferase. Isolated plasma membrane and microsomal glutathione transferases possess the same amino‐terminal amino acid sequence and digestion with different proteases results in identical fragment patterns as displayed by SDS/PAGE. These data suggest that plasma membrane and microsomal glutathione transferase are identical proteins.
Cell‐specific expression and function of adenylyl cyclases in rat pituitary tumour cell lines
Authors: PAULSSEN Ruth H. , JOHANSEN Per W. , GORDELADZE Jan O. , NYMOEN Øystein , PAULSSEN Eyvind J. , GAUTVIK Kaare M. 
Published: March 2005

The present study demonstrates cell‐specific distribution and describes distinct functional regulation of different adenylyl cyclases (AC, types I–VI) in rat pituitary cell tumor cell lines (GH12C1, GH3 and GH4C1 cells) and pituitary tissue.Northern‐blot analysis revealed a distinct pattern of cell‐specific expression of the different AC types; Ca2+/calmodulin (CaM)‐insensitive AC type II was found in all cell lines tested except GH12C1 cells. The Ca2+‐inhibitable AC type VI was found in all cell types tested. We observed a lack of the Ca2+/CaM‐sensitive AC type I in GH3 and GH4C1 cells. GH12C1 cells exclusively contained both Ca2+/CaM‐sensitive AC types I and III, the latter previously believed to be specific for olfactory tissue. An additional transcript of AC type III was found in rat brain and rat liver tissue. AC type IV, which is Ca2+/CaM insensitive, could be detected in the prolactin‐producing GH3 and GH4C1 cells and pituitary tissue but not in growth‐hormone‐producing GH12C1 cells.Basal and vasoactive‐intestinal‐peptide‐(VIP)‐releasing‐hormone, somatostatin (SRIF) and thyrothropin‐releasing‐hormone (TRH)‐modulation of AC activity was measured in the presence of 100 μM EGTA, anti‐CaM serum (dilution 1:2000) or 10 μM trifluoroperazine. Antisera against guanine‐nucleotide‐binding protein (G‐protein) α subunits (Gi‐2α, Gsα) and β subunits (Gβ35/36) and CaM were added for functional studies of the SRIF and VIP‐modulated AC in GH12C1 and GH3 cells. These experiments indicate that the VIP and the SRIF receptors are coupled to a Ca2+/CaM‐sensitive AC in GH12C1 cells, different from the AC involved in the regulation of cAMP levels in GH3 and GH4C1 cells. In addition, the βγ‐complex is possibly able to modulate SRIF‐inhibited AC activity by potentiating the inhibitory effect. The TRH receptor in GH3 and GH4C1 cells is coupled to a Ca2+/CaM‐sensitive AC which is different from the already cloned forms of AC types I and III.We, therefore, conclude that hormone regulation of pituitary tumour cell functions differs between the GH cell lines, due to specific utilisation of AC types.
The sex pheromone system of Enterococcus faecalis
Authors: WIRTH Reinhard 
Published: March 2005

The sex pheromone system of Enterococcus faecalis was discovered by observing a clumping reaction of E. faecalis strains during conjugative transfer of plasmids. It was found that only a special type of E. faecalis plasmids, the so‐called sex pheromone plasmids, are transferred via this mechanism. Various experiments, especially by the group of D. B. Clewell, led to the formulation of a model describing how the sex pheromone system works. Small linear peptides, the so‐called sex pheromones, are excreted by strains not possessing the corresponding sex pheromone plasmid. Donor strains harboring the plasmid do not produce the corresponding sex pheromone; they react to the presence of the peptide by production of a plasmid‐encoded adhesin, the so‐called aggregation substance. This adhesin allows contact between the non‐motile mating partners; after conjugative transfer of the plasmid, the former recipient possesses and replicates the new plasmid. Thereby the population of E. faecalis strains is shifted to a high percentage of donor strains. This is especially true because a donor strain will still excrete sex pheromones corresponding to plasmids it does not harbor; therefore, such a strain can also function as recipient for other sex pheromone plasmids it does not possess.Various aspects of this unique plasmid collection mechanism have been studied during the last few years. The data indicate that, with the exception of pAM373, all sex pheromone plasmids possess one DNA region which is highly similar to and codes for the adhesin. It is also becoming more and more clear that regulatory functions/proteins are not conserved between different sex pheromone plasmids. Induction of adhesin synthesis needs the action of a regulatory cascade composed of unique features; at the moment we are just beginning to understand this cascade. By sequencing the first structural gene for one of those adhesins, we realized that the aggregation substance might act also as an adhesin for eucaryotic cells, probably by interaction with integrins. At least in the case of the in vitro cultured pig kidney tubulus cell line LLC‐PK1 this idea could be verified. An interesting aspect of the sex pheromone system of E. faecalis is its evolution. I will discuss the idea that two different components, both of which well might contribute to virulence of the opportunistic pathogenic bacterium, were combined in the species E. faecalis to result in this unique plasmid collection system.
Homology modelling of the catalytic domain of human furin
Authors: SIEZEN Roland J. , CREEMERS John W. M. , VAN DE VEN Wim J. M. 
Published: March 2005

A model is presented for the three‐dimensional structure of the catalytic domain of the human serine proteinase furin and its interaction with model substrates. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin, and it also applies to other members of the eukaryotic subtilisin‐like proprotein convertases. Predictions are made of the general protein fold, inserted loops, disulfide bonds, Ca2+‐binding sites and salt bridges. A detailed prediction of the substrate‐binding region attempts to explain the basis of specificity for multiple basic residues preceding the cleavage site. Specific acidic residues in the S1, S2 and S4 subsites of the substrate‐binding region of furin are identified which appear to be of particular importance, while residues of the S2′, S3, S5 and S6 subsites may also contribute to substrate binding.Based on this model, protein engineering can be employed not only to test the predicted enzyme‐substrate interactions, as demonstrated for human furin, but, equally importantly, to design proprotein convertases with a desired specificity, or to design novel substrates or inhibitors.
A few amino acid substitutions are responsible for the higher thermostability of a novel NAD+‐dependent bacillar alcohol dehydrogenase
Authors: CANNIO Raffaele , ROSSI Mosè , BARTOLUCCI Simonetta 
Published: March 2005

The gene adh‐hT encoding a thermostable and thermophilic NAD+‐dependent alcohol dehydrogenase (ADH) from the novel and more thermophilic Bacillus stearothermophilus LLD‐R strain was cloned and its nucleotide sequence determined. The deduced protein sequence shows remarkable amino acid substitutions when compared to the sequence of the protein isolated from strain NCA1503 and significant similarity with the highly thermostable ADH from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The alignment of these sequences led to the identification of three amino acid replacements probably responsible for the higher thermostability of the novel bacillar ADH. Adh‐hT gene expression in Escherichia coli, a fast purification procedure and the characterization of the recombinant enzyme are also described.
Mass spectrometric analysis of human soluble catechol O‐methyltransferase expressed in Escherichia coli
Authors: VILBOIS Francis , CASPERS Patrick , DA PRADA Mosé , LANG Gabrielle , KARRER Catherine , LAHM Hans‐Werner , CESURA Andrea M. 
Published: March 2005

Technological advances in the field of mass spectrometry (MS) are providing powerful analytical means for the investigation of proteins and peptides. In the present work we have used pneumatically assisted electrospray (ion‐spray) MS for the biochemical characterization of recombinant human catechol O‐methyltransferase (rhCOMT). hCOMT could be expressed in Escherichia coli in large quantities but in two forms of different size, both enzymically active. Electrospray MS analysis showed that the smaller rhCOMT protein had a molecular mass of 24352±2Da, corresponding to the calculated value for native hCOMT (without the initiating methionine), whereas that mass of the larger protein was of 25775±4Da.To investigate the molecular differences between the two proteins, they were digested with trypsin and the peptides produced analysed by electrospray MS. Neither protein apparently contained disulfide bridges and the observed molecular masses of the tryptic peptides corresponded to the calculated values. It was possible to determine, however, that the larger protein contained an extended C‐terminus with the correct sequence GPGSEAGP plus an additional stretch, EDLR. This C‐terminal extension resulted from ribosomal frameshift at the codon of the last proline (CCC, rare codon in prokaryotes). In fact, rightward frameshifting would produce a hCOMT form with an additional stretch of 11 amino acid (EDLRSHHHHHH) and the calculated molecular mass of this protein (25773.5Da) is in good agreement with our experimental result.The differential reactivity of the cysteine residues of the correct rhCOMT enzyme, in the presence and in the absence of S‐adenosyl‐L‐methionine (AdoMet) and MgCl2, was also studied. 5‐Iodoacetamido fluorescein (5‐IAF) was used as thiol‐modifying reagent. Under the conditions used, 5‐IAF rapidly inactivated rhCOMT but the presence of AdoMet and MgCl2 partially protected it from inactivation. The 5‐IAF‐labeled tryptic peptides were separated by HPLC and then submitted to electrospray MS and tandem MS. Several cysteine residues appeared to be readily available to chemical modification by 5‐IAF. Incorporation of 5‐IAF occurred to a larger extent into Cys32, Cys68, Cys94 and Cys172. AdoMet and MgCl2 markedly reduced the label incorporation into Cys68 and Cys94, therefore suggesting that these residues belong to a region at or near the binding site of AdoMet.