bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterisation of cAMP‐dependent protein kinase from Candida albicans
Authors: Zelada Alicia , Passeron Susana , Lopes Gomes Suely , Cantore María L.
Published: March 2003

cAMP‐dependent protein kinase (PKA) from Candida albicans yeast cells was isolated and characterised. Structural parameters of the holoenzyme and those of its subunits suggested that C. albicans PKA is a tetramer of 287 kDa composed of two regulatory (R) subunits of 64 kDa and two catalytic (C) subunits of unusually large molecular mass of 78 kDa. The apparent Km for ATP and Kemptide were 30 μM and 60 μM respectively. The [A]0.5 for cAMP activation was 150 nM with a Hill coefficient of 1.6. The holoenzyme undergoes autophosphorylation on the R subunit, a characteristic of the type‐II R subunits. Photoaffinity labeling with 8‐azido‐[32P]cAMP of crude extracts from yeast and mycelial cells strongly suggests that only one type of R subunit is present in the fungus. The R subunit was purified to apparent homogeneity as a protein of 64 kDa. A highly specific polyclonal antiserum raised against the purified protein immunoprecipitated a 64‐kDa protein from crude extracts, indicating that the purified R subunit very probably represents the native form of the protein. The 78‐kDa form of the C subunit was detected in crude extracts and in Mono Q Sepharose column fractions with heterologous anti‐C Ig. It could be isolated by cAMP treatment of the holoenzyme immunoprecipitated from crude extracts with anti‐R serum, but this form could not be purified further. Instead, a 60‐kDa protein with the main characteristics of C subunit was purified to near homogeneity from soluble extracts of yeast cells. Evidence is presented that this protein very probably derives from the 78‐kDa form by proteolytic degradation.
Fluorescence resonance energy transfer mapping of subunit δ in spinach chloroplast F1 ATPase
Authors: Engelbrecht Siegfried , Giakas Enrico , Marx Olaf , Lill Holger
Published: March 2003

Despite the considerable progress in the field of F0F1‐ATPases caused by solving the 2.8‐Å structure of mitochondrial F1 ATPase [Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621−628], little is known about the position and function of the enzyme's small subunits which were not resolved in the X‐ray analysis. We have previously genetically engineered Cys residues into the δ subunit of chloroplast F1 and used these mutant subunits in cross‐linking studies [Lill, H., Hensel, F., Junge, W. & Engelbrecht, S. (1996) J. Biol. Chem. 271, 32 737−32 742]. In this work, various fluorophores have been introduced into the mutant δ subunits and used in fluorescence‐resonance energy‐transfer measurements. The resulting distances were fitted into the framework of existing data. Subunit δ was found to be located between two α/β couples, stretching from the level of the nucleotide binding sites up to a position close to the N‐termini of subunits α and β. These results corroborate and further refine the previously found location of spinach CF1δ at the periphery and membrane‐distal part of CF1, where it may constitute a part of a stator in the rotatory machinery of F0F1.
Oligomeric structure of the repressor of the bacteriophage Mu early operon
Authors: Alazard Robert , Ebel Christine , Venien‐Bryan Catherine , Mourey Lionel , Samama Jean Pierre , Chandler Michael
Published: March 2003

The regulation of the lytic and lysogenic development in the life cycle of bacteriophage Mu is regulated in part by its repressor, c, which binds to three operator sites, O1, O2 and O3, overlapping two divergent promoters. The oligomeric structure of this repressor protein was investigated by hydrodynamic and biochemical methods. Size‐exclusion chromatography, analytical ultracentrifugation, dynamic light scattering, crosslinking and direct electron microscopy observations suggest that c exists primarily as a hexamer with a molecular mass of 120−140 kDa at low concentrations, i.e. in the 10‐μM range. This molecule undergoes a self‐assembly process leading to dodecamers and higher order species as the concentration is further increased in a manner depending on the nature of the solvent. Our results also suggest that these species have an elongated structure, and a possible arrangement of the subunits within the hexamer is proposed. The implication of this unusual quaternary structure for a repressor in its interaction with the operator sites O1 and O2 remains to be elucidated.
Conferring aldosterone synthesis to human CYP11B1 by replacing key amino acid residues with CYP11B2‐specific ones
Authors: Böttner Benjamin , Denner Karsten , Bernhardt Rita
Published: March 2003

Performing residue‐swapping experiments between the highly conserved human steroidogenic proteins CYP11B1 and CYP11B2 we recently demonstrated that replacement of specific residues at position 301, 302 and 320 in the aldosterone‐producing CYP11B2 protein for such residues that were specific for the highly similar cortisol‐producing CYP11B1 protein elevated the 11β‐hydroxylase activity dramatically. Conversely, aldosterone synthesis in the triple mutant was severely impaired. Here we provide evidence that in a reciprocal experiment, CYP11B2‐specific amino acids at position 320 and 335 endowed CYP11B1 with an 18‐oxidase function amounting to 20 % of the CYP11B2 wild‐type activity, thus changing the specificity of steroid hydroxylation by only one point mutation. Combining substitutions at positions 296, 301, 302, 320, 335 and 339 did, however, not result in further enhancement. Paradoxically, 11β‐hydroxylation was not or only marginally affected in CYP11B1 mutants, indicating an alternative structural basis for this activity in CYP11B1 compared with the engineered CYP11B2 variant. Our results suggest that the sequence spanned by amino acids 301 and 335 constitutes part of the substrate‐binding site in CYP11B1 and CYP11B2 as well. By constructing chimeric proteins we further investigated the effect of the C‐terminal portions of both proteins and found that diverging residues at positions 471, 472, 492, 493 and 494 were insignificant for the stereospecificity and regiospecificity of steroid hydroxylation.
An Escherichia coli hydrogenase‐3‐type hydrogenase in methanogenic archaea
Authors: Künkel Andreas , Vorholt Julia A. , Thauer Rudolf K. , Hedderich Reiner
Published: March 2003

Methanogenic archaea are known to contain two types of [NiFe] hydrogenases designated F420‐reducing hydrogenase and F420‐non‐reducing hydrogenase. We report here that they additionally contain Escherichia coli hydrogenase‐3‐type [NiFe] hydrogenases. The evidence is based on biochemical studies and analysis of the subunit primary structure of this hydrogenase (designated Ech) purified from membranes of acetate‐grown cells of Methanosarcina barkeri. The subunits EchE and EchC of the EchABCDEF complex showed 34 % and 45 % sequence identity to the nickel‐containing large subunit HycE and to the iron‐sulfur cluster containing small subunit HycG, respectively, of the hydrogenase in the formate hydrogen lyase complex from E. coli. Analysis of the totally sequenced genomes of Methanococcus jannaschii and Methanobacterium thermoautotrophicum strain ΔH revealed that these organisms contain similar open reading frames, indicating the presence of an E. coli hydrogenase‐3‐type hydrogenase also in these methanogenic archaea.
One‐step purification of cathepsin D by affinity chromatography using immobilized propeptide sequences
Authors: Wittlin Sergio , Rösel Johannes , Stover David R.
Published: March 2003

In vivo, active cathepsin D proteinase is generated by removal of a 44‐residue propeptide at its N‐terminus. Here we report that mature cathepsin D and pseudocathepsin D (a partially activated form of cathepsin D with 25 amino acid residues removed from the propeptide) bind to the immobilized propeptide, while procathepsin D does not. The N‐terminal 25 amino acid residues of the propeptide are sufficient for this binding. Based on this observation, a simple one‐step procedure was developed to purify mature cathepsin D from whole cell extracts to near homogeneity. This method has the advantage over existing affinity‐purification systems that active forms of the proteinase can be separated from inactive precursors and other aspartic proteinases. Furthermore, this technique was effective for pepsin as well, suggesting it may have general utility for all activated aspartic proteinases and perhaps other families of proteinases.
Guanylate‐cyclase‐inhibitory protein is a frog retinal Ca2+‐binding protein related to mammalian guanylate‐cyclase‐activating proteins
Authors: Li Ning , Fariss Robert N. , Zhang Kai , Otto‐Bruc Annie , Haeseleer Françoise , Bronson Darin , Qin Ning , Yamazaki Akio , Subbaraya Iswari , Milam Ann H. , Palczewski Krzysztof , Baehr Wolfgang
Published: March 2003

Two guanylate‐cyclase‐activating proteins (GCAP) encoded by a tail‐to‐tail gene array have been characterized in the mammalian retina. Using frog retina as a model, we obtained evidence for the presence of a photoreceptor Ca2+‐binding protein closely related to GCAP. This protein (206 amino acids) does not stimulate guanylate cyclase (GC) in low [Ca2+], but inhibits GC in high [Ca2+], and is therefore termed guanylate‐cyclase‐inhibitory protein (GCIP). Sequence analysis indicates that GCIP and GCAP1 and GCAP2 have diverged substantially, but conserved domains present in all vertebrate GCAP are present in GCIP. Moreover, partial characterization of the GCIP gene showed that the positions of two introns in the GCIP gene are identical to positions of corresponding introns of the mammalian GCAP gene array. As to the major differences between GCIP and GCAP, the fourth EF hand Ca2+‐binding motif of GCIP is disabled for Ca2+ binding, and GCIP does not stimulate GC. Monoclonal and polyclonal antibodies raised against recombinant GCIP identified high levels of GCIP in the inner segments, somata and synaptic terminals of frog cone photoreceptors. The results suggest that GCIP is a Ca2+‐binding protein of the GCAP/recoverin subfamily. Its localization in frog cones closely resembles that of GC in mammalian cones. GCIP inhibits GC at high free [Ca2+], competing with GCAP1 and GCAP2 for GC regulatory sites.
Inactivation and destruction of conserved Trp159 of Fe‐superoxide dismutase from Porphyromonas gingivalis by hydrogen peroxide
Authors: Yamakura Fumiyuki , Rardin R. Lynn , Petsko Gregory A. , Ringe Dagmar , Hiraoka B. Yukihiro , Nakayama Koji , Fujimura Tsutomu , Taka Hikari , Murayama Kimie
Published: March 2003

The superoxide dismutase (SOD) of Porphyromonas gingivalis, an obligate anaerobe, was purified from Escherichia coli (sodA sodB mutant) harboring the P. gingivalis SOD‐encoding gene. The purified protein contained both iron and a small amount of manganese. Iron‐ and manganese‐reconstituted SOD, which contained one of these metals exclusively, showed specific activities of 1000 and 1200 U/mg/mol of metals/subunit, respectively. These values were similar to the specific activity of the native enzyme purified from the recombinant E. coli strain. The Fe‐reconstituted enzyme was inactivated by 10 mM hydrogen peroxide to about 5 % of its original activity after a 15 min incubation at 25 +C at pH 7.8, whereas the Mn‐reconstituted enzyme showed no inactivation after 80 min. A concomitant increase in absorbance at 320 nm was observed with inactivation of the Fe‐reconstituted enzyme. Amino acid analysis of the inactivated Fe‐reconstituted enzyme showed a decrease of about 0.7 residues of tryptophan/subunit, a value similar to the iron content of the iron‐reconstituted enzyme. Three major peptides of the digests of the purified SOD with lysylendopeptidase were separated by a reverse‐phase HPLC monitoring at 280 nm. One of the peptides, corresponding to the residues from Gly149 to Lys176, decreased in the HPLC eluent of the H2O2‐inactivated SOD to 20 % of the amount measured for native SOD. Since this peptide contains only one tryptophan residue, it was concluded that the decomposed tryptophan residue is Trp159, which is located midway between the third and fourth metal ligands, Asp157 and His161, and is conserved in aligned amino acid sequences of all known Fe‐SODs and Mn‐SODs. Based on these results, we propose that the differences in hydrogen peroxide sensitivities observed for the Fe‐SODs and Mn‐SODs may be caused by the difference in the identity of the active site metal in the Fe‐SODs and Mn‐SODs and a tuning of the properies of the iron center in the Fe‐SODs.
Helical and coiled‐coil‐forming properties of peptides derived from and inhibiting human immunodeficiency virus type 1 integrase assessed by 1H‐NMR
Authors: Krebs Daniel , Maroun Richard G. , Sourgen Frédéric , Troalen Frédéric , Davoust Daniel , Fermandjian Serge
Published: March 2003

Human immunodeficiency virus type 1 integrase (HIV‐1 IN) which catalyzes viral DNA integration into the host genome of infected cells represents an attractive target for AIDS therapy. We have previously demonstrated the ability of the IN‐(147−175)‐peptide derived from the catalytic core domain of HIV‐1 IN to inhibit the enzyme activity in vitro. IN‐(147−175)‐peptide contains four heptad repeats and displays a high propensity for coiled‐coil formation while its [P159]IN‐(147−175)‐peptide analog (Lys159←Pro in the protein, Lys13←Pro in the peptide) is unable to form a stable coiled‐coil and is devoid of inhibitory activity [Sourgen, F., Maroun, R. G., Frère, V., Bouziane, M., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240, 765−773]. Now, we report results from an NMR study on IN‐(147−175)‐peptide and [P159]IN‐(147−175)‐peptide as well as on an optimized [E156, A163, A167]IN‐(147−175)‐peptide that is a better inhibitor of IN than IN‐(147−175)‐peptide. While in aqueous solution, IN‐(147−175)‐peptide and [P159]IN‐(147−175)‐peptide display only nascent helical features, [E156, A163, A167]IN‐(147−175)‐peptide exhibits 20 % of helical content. In 20 % trifluoroethanol/80 % H2O, the helix content is the highest for [E156, A163, A167]IN‐(147−175)‐peptide (≈70 %) and the lowest for [P159]IN‐(147−175)‐peptide (≈40 %), due to a local helix break caused by the Pro residue. The NHs of residues in the two central helical heptads (a−g) of IN‐(147−175)‐peptide and [E156, A163, A167]IN‐(147−175)‐peptide display a regular periodic variation of their temperature coefficients in 20 % trifluoroethanol. The b, c and f residues on the hydrophilic face of the amphipathic helix show high coefficients reflecting hydrogen bonded NHs, while the a and d residues on the hydrophobic face exhibit low coefficients, near random‐coil values. The particular arrangement of the hydrophobic side‐chains of a and d residues at the coiled‐coil interface reduces the access of trifluoroethanol molecules to their amide groups. The inability of trifluoroethanol molecules to create interactions with the amide C=O groups, these being required to strengthen the intrahelical C=O    H‐N hydrogen bonds, is the main cause for observation of heptadic a and d residues with low NH temperature coefficients. Such effects concern mostly the two central helical heptads of IN‐(147−175)‐peptide and [E156, A163, A167]IN‐(147−175)‐peptide implying that these ones are engaged in stable parallel coiled coils. Our results provide a link between the propensity of peptides for helix formation, their coiled‐coil properties and their efficiency to inhibit IN.
Location of N‐cyclohexyl‐N′‐(4‐dimethyl‐amino‐α‐naphthyl)carbodiimide‐binding site in sarcoplasmic reticulum Ca2+‐transporting ATPase
Authors: Velasco‐Guillén Isabel , Corbalán‐García Senena , Gómez‐Fernández Juan C. , Teruel José A.
Published: March 2003

The Ca2+‐transporting ATPase has been labeled with N‐cyclohexyl‐N′‐(4‐dimethyl‐amino‐α‐naphthyl)carbodiimide (NCD‐4), a fluorescent carbodiimide which reacts with carboxyl groups of acidic residues. It has been reported that NCD‐4 labels a transmembrane portion of the protein at the high‐affinity calcium‐binding sites. We have determined the depth of the calcium‐sensitive probe by quenching the fluorescence by nitroxide‐substituted fatty acids with its spin probe located at different carbons of the fatty acid chain (5, 7, 10, 12 and 16‐nitroxide derivatives). We have found that all the calcium‐sensitive fluorescence is quenched and that the efficiency of quenching decreases as the n‐(4,4‐dimethyl‐3‐oxazolinyloxy) (Doxyl) group is deeper in the membrane. We conclude that the NCD‐4 label which is involved in the high‐affinity calcium‐binding site is located near the water/lipid interface.

The fluorescence of the NCD‐4 bound to that site can be quenched by acrylamide and Cu2+ but not by iodide, probably due to its anionic nature which will be repulsed by the abundance of negative charges of Glu and Asp residues of NCD‐4 located at this site. The hydrophobic location of NCD‐4 was confirmed by the fact that its fluorescence could be quenched by the spin label 2,2,6,6‐tetramethyl‐1‐piperidine‐N‐oxyl but not by 4‐hydroxy‐2,2,6,6‐tetramethyl‐1‐piperidine‐N‐oxyl which is much less hydrophobic.
Mitogen‐activated protein kinase is involved in epidermal‐growth‐factor‐regulated protein phosphorylation in nuclear membranes isolated from JEG‐3 human choriocarcinoma cells
Authors: Bian Lijun , Lei Zhenmin , Rao C. Venkateswara
Published: March 2003

The treatment of highly purified nuclear membranes isolated from JEG‐3 human choriocarcinoma cells with epidermal growth factor (EGF) resulted in an increase of the receptor autophosphorylation and the phosphorylation of several other proteins, including 44‐kDa, 34‐kDa and 24‐kDa proteins, and a decrease in the phosphorylation of a 40‐kDa protein. The kinetics of phosphorylation and the use of RG‐13022, a selective inhibitor of EGF‐receptor kinase activity, suggested that receptor activation was necessary for the phosphorylation response of the other proteins. Tyr was exclusively phosphorylated in the EGF receptor and 24‐kDa proteins, Tyr and Thr were phosphorylated in the 44‐kDa protein, and Ser was phosphorylated in the 34‐kDa protein and dephosphorylated in the 40‐kDa protein. The molecular size, Thr/Tyr phosphorylation, immunoprecipitation and enzymatic activity towards myelin basic protein suggested that the 44‐kDa protein was a mitogen‐activated protein (MAP) kinase. The EGF treatment not only increased phosphorylation but also catalytic activity of MAP kinase and these increases were prevented by the addition of RG‐13022. In summary, this report demonstrates that target cell nuclei contain EGF receptors, which use the MAP kinase signaling pathway.
Identification of Manα1‐3Manα1‐2Man and Man‐linked phosphate on O‐mannosylated recombinant leech‐derived tryptase inhibitor produced by Saccharomyces cerevisiae and determination of the solution conformation of the mannosylated polypeptide
Authors: Bergwerff Aldert A. , Stark Wilhelm , Fendrich Gabriele , Knecht René , Blommers Marcel J. J. , Maerki Walter , Kragten Eddy A. , van Oostrum Jan
Published: March 2003

The production of recombinant leech‐derived tryptase inhibitor (rLDTI) by two different strains of Saccharomyces cerevisiae resulted in the secretion of non‐glycosylated and glycosylated rLTDI. Monosaccharide analysis and α‐mannosidase treatment demonstrated that glycosylated rLDTI was exclusively αmannosylated. A trypsin digest of reduced and S‐carboxymethylated glycosylated rLDTI was separated on a reverse‐phase HPLC column. Glycopeptides identified by a combination of matrix‐assisted laser desorption mass spectrometry, amino acid sequence analysis, and monosaccharide analysis revealed the presence of different glycoforms. It was found that Ser24, Ser33 and Ser36 were partially glycosylated with a single mannose residue, whereas Thr42 in glycosylated rLDTI from both strains was fully occupied with manno‐oligosaccharides with a degree of polymerization ranging over 1−3 and 1−13 depending on the yeast strain. In phosphorylated rLDTI a single phosphate group was predominantly located at the innermost Man residue of units of mannobiose, mannotriose, mannotetraose and mannopentaose at Thr42. Oligosaccharides released by alkaline treatment were reduced by sodium borohydride and separated by high‐pH anion‐exchange chromatography on a CarboPac MA1 column, and analyzed by one‐ and two‐dimensional 1H‐NMR spectroscopy. Besides the major oligosaccharide Manα1‐2Man‐ol, the (for yeast protein O‐glycosylation) unusual Manα1‐3Manα1‐2Man‐ol was determined.

The solution conformation of glycosylated rLDTI was investigated by two‐dimensional NMR spectroscopy. Structure calculations by means of distance geometry showed that glycosylated rLDTI is compactly folded and contained small secondary structure elements. Analysis of the chemical shifts showed that amino acids Val32−Ser33, Ser36−Ser39 and Thr42 were affected by the O‐mannosylation. In addition, changes in chemical shift were observed within the β‐hairpin peptide regions Val13−Ser16 and Gly18−Tyr21 attributed to direct interactions of the mannose residue at Ser36. Furthermore, the protein‐linked oligosaccharides were spatially grouped in a position opposite of the canonical binding loop.
Induction of necrosis by zinc in prostate carcinoma cells and identification of proteins increased in association with this induction
Authors: Iguchi Kazuhiro , Hamatake Michiko , Ishida Ryoji , Usami Yoshiko , Adachi Tetsuo , Yamamoto Hajime , Koshida Kiyoshi , Uchibayashi Tadao , Hirano Kazuyuki
Published: March 2003

Zinc exhibits inhibitory effects on apoptosis, and a deficiency in this metal generally causes this type of cell death to occur. In the present study, we found that exposure to zinc results in necrosis of prostate carcinoma cells. When zinc acetate was added to LNCaP or PC‐3 cells in monolayer culture, they began to detach from the culture dishes, and viability was lost after 4−8 h. Most of the cell death was found to be due to necrosis as determined by double staining with fluorescein‐isothiocyanate‐labeled annexin V and ethidium bromide, and by detection of hypodiploid cells. Associated with the induction of necrosis was an increase in low molecular‐mass proteins, identified by HPLC analysis to be thymosin β10, parathymosin and GAGE in LNCaP cells, and thymosin β4, parathymosin and metallothionein in PC‐3. The time course of the increase of thymosin β10 in LNCaP cells and thymosin β4 in PC‐3 cells was consistent with that of appearance of cell detachment and dead cells. These results indicate that zinc can induce necrosis and suggest that production of proteins including β‐thymosins is involved in induction of processes leading to cell detachment.
The defense‐related rice gene Pir7b encodes an α/β hydrolase fold protein exhibiting esterase activity towards naphthol AS‐esters
Authors: Wäspi Urs , Misteli Beatrice , Hasslacher Meinhard , Jandrositz Anita , Kohlwein Sepp D. , Schwab Helmut , Dudler Robert
Published: March 2003

Acquired resistance of rice to Pyricularia oryzae, the causing agent of rice blast, can be induced by inoculation with the non‐host pathogen Pseudomonas syringae pv. syringae. We have previously cloned a cDNA and a corresponding gene (Pir7b) whose transcripts accumulate upon infiltration with the resistance‐inducing bacteria. The putative encoded product Pir7b exhibits significant sequence similarity to two recently cloned hydroxynitrile lyases from Manihot esculenta (cassava) and Hevea brasisliensis, enzymes involved in the release of hydrogen cyanide from cyanogenic glycosides. As rice does not contain cyanogenic glycosides, a similar function of Pir7b appears unplausible. In order to functionally characterize the protein, recombinant Pir7b was produced in Escherichia coli and Saccharomyces cerevisiae. We show that recombinant Pir7b does not have hydroxynitrile lyase activity, but exhibits esterase activity towards naphthol AS‐acetate. Using Pir7b‐specific antibodies, we show that the protein accumulates in rice leaves inoculated with P. syringae pv. syringae. Both the recombinant and the authentic proteins have an apparent molecular mass of 32 kDa (28.8 kDa calculated) and seem to be active as monomers. Pir7b esterase also exhibits sequence similarity to several expressed sequence tags of Arabidopsis thaliana, indicating that it belongs to a family of proteins widely occuring in plants.
The mitochondrial phosphoglyceroyl‐ATP‐containing polymer, purinogen, is unchanged by cardiac ischaemia and reperfusion but may function in the regulation of free intracellular inorganic phosphate concentrations
Authors: Patel Brinda , Berhane Yoel , Petrovic Arsen , Mowbray John
Published: March 2003

Previous work in our laboratory demonstrating large unexplained systematic variations in the heart contents of free adenine nucleotides led us to propose the existence of some unrecognised sequestered form and thence to the purification of very labile acid‐insoluble oligomers which we characterised as oligo[3‐phospho‐glyceroyl‐γ‐triphospho(5′)adenosine(3′)], abbreviated to (PG‐ATP)n. More recently, we provided evidence that these oligomers appear to be the end chains of a complex polymer located in the mitochondrial intermembrane space of a number of rat tissues. We called this polymer purinogen and devised a means of assaying it quantitively [Patel, B., Sarcina, M. & Mowbray, J. (1994) Eur. J. Biochem. 220, 663−669]. Here we report measurements of purinogen in perfused hearts subjected to moderate and severe global ischaemia and reperfusion. Measurements of tissue and perfusate nucleotides, nucleosides and purine degradation products demonstrate that ischaemia led to the augmentation of the free nucleotide content by up to 30 % and its re‐sequestration on reperfusion in reversible but not in irreversible ischaemia. The purinogen content was unchanged by ischaemia or reperfusion implying the existence of some other unidentified storage pool. By contrast, glucose addition to glycolytically deprived hearts or removal of Pi from perfusion medium, conditions which might be expected to alter demand for intracellular Pi, led to the quantitative transfer of nucleotides between phosphate‐rich purinogen and free nucleotides. The possibility that purinogen may act as a rapidly accessible reservoir of intracellular inorganic phosphate is discussed.
Evidence of an overlap between the two half‐sites of UAS1‐B/CYC1
Authors: Naït‐Kaoudjt Rachida , Guiard Bernard , Gervais Michel
Published: March 2003

Cyp1p (Hap1p) is a yeast transcriptional regulator belonging to the zinc‐cluster family. CGGNNNTANCGG was identified by PCR selection as the DNA sequence allowing its optimal binding. Nevertheless, this sequence is not a consensus sequence, the simultaneous presence of the two CGGs and the TA generally not being found in the known natural Cyp1p targets. In fact, our previous studies showed that the mechanism of Cyp1p DNA binding was target dependent. Data concerning the binding of Cyp1p to the UAS1‐B/CYC1 are presented here. This target, containing the CGGGGTTTACGG sequence, was found to present the particular ability of stabilizing the binding of only one molecule of some monomeric Cyp1p fragments. This property was used to investigate the actual contribution of the TT and CGG sequences in the binding of Cyp1p. Our results indicate that each CGG belongs to a different half‐site and, in contrast to a previous hypothesis, that the T nucleotide located four bases downstream from the left CGG is essential for the binding of one monomer to each half‐site. The two half‐sites of the UAS1B/CYC1 thus overlap.
Structural determination of the O‐antigenic polysaccharide from Escherichia coli O141
Authors: Färnbäck Magnus , Weintraub Andrej , Widmalm Göran
Published: March 2003

The structure of the O‐antigenic polysaccharide from Escherichia coli O141 has been determined. NMR spectroscopy and sugar and methylation analyses were the principal methods used. The sequence of the sugar residues could be determined by NOESY and heteronuclear multiple‐bond connectivity (HMBC‐) NMR experiments. The polysaccharide is composed of pentasaccharide repeating units with 1 O‐acetyl group/repeating unit. The following structure, where Rha is 6‐deoxymannose is concluded :

α‐
L‐Rhap
1
↓
3
←3)‐β‐
D‐GlcpNAc‐(1←4)‐α‐
D‐Manp‐(1←3)‐α‐
D‐Manp6Ac‐(1←
2
↑
1
β‐
D‐GlcpA
Regulation of iron metabolism in the sanguivore lamprey Lampetra fluviatilis
Authors: Andersen Øivind , Pantopoulos Kostas , Kao Hung‐Teh , Muckenthaler Martina , Youson John H. , Pieribone Vincent
Published: March 2003

Two ferritin cDNAs were cloned from the liver and spinal cord of the sanguivore lamprey Lampetra fluviatilis, an extant representative of the ancient agnathan (jawless) stage in vertebrate evolution. The deduced proteins of 20.2 kDa (H‐subunit) and 20.1 kDa (M‐subunit) display 73  % sequence identity, and both contain the ferroxidase center characteristic of animal H‐ferritin. A highly conserved iron‐responsive element (IRE) was identified in the 5′ untranslated region of lamprey H‐ferritin. Lamprey ferritin IRE forms a specific complex with crude lamprey and rat liver extracts, and with recombinant human iron‐regulatory protein (IRP‐1) in an electrophoretic mobility shift assay. Furthermore, lamprey ferritin IRE competes with labeled human ferritin IRE for binding to IRP in lamprey and mammalian extracts. Two liver cDNA sequences encoding 323 residues and 101 residues of two genetically distinct lamprey IRP were amplified by PCR. Lamprey IRP‐1 and IRP‐2, which are 72  % identical, display about 74  % sequence identity to their presumed homologues in mammals. Northern blot analysis shows that two IRP transcripts of 3.6 kb and 5.8 kb are expressed in lamprey liver. Given the ancient lineage of lampreys, the results indicate that the IRE/IRP regulatory system has remained highly conserved during the evolution of vertebrates.
Structural studies of the O‐antigenic polysaccharide from Escherichia coli O139
Authors: Marie Christèle , Weintraub Andrej , Widmalm Göran
Published: March 2003

The structure of the O‐antigenic polysaccharide from Escherichia coli O139 has been investigated with the aid of NMR spectroscopy, and sugar and methylation analyses. The sequence of the sugar residues was determined by NOESY and heteronuclear‐mutiple‐bond‐connectivity NMR experiments. The polysaccharide is composed of heptasaccharide repeating units containing 0.6 mol O‐acetyl groups/repeating unit. The following structure is concluded, where Rha represents rhamnose.

←3)‐α‐
D‐GlcpNAc‐(1←3)‐α‐
L‐Rhap‐(1←4)‐α‐
D‐GalpA‐(1←2)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←2)‐α‐
L‐Rhap‐(1←
3
↑
1
β‐
D‐Glcp
Renal and hepatic nitrogen metabolism during NH4Cl ingestion in protein‐deprived rats
Authors: Lardner Anne L. , O'Donovan Daniel J.
Published: March 2003

Three groups of rats were given either a standard protein diet, a protein‐free diet, or a protein‐free diet with the inclusion of 0.28 M NH4Cl in their drinking water, for 10 days. Body, liver and kidney masses were decreased similarly in the protein‐free and protein‐free NH4Cl groups. Ingestion of protein‐free diet resulted in profound systemic acidosis in both groups, the simultaneous consumption of NH4Cl having no further effect. The activities of the urea‐cycle enzymes carbamoyl‐phosphate synthease, ornithine transcarbamoylase, arginosuccinate lyase and arginase were significantly reduced in the protein‐free group, and the simultaneous ingestion of NH4Cl had no further effect. These results indicate that ammonium ingestion does not prevent the decrease in urea cycle enzyme activities during a period of dietary‐protein deprivation. Renal phosphate‐dependent glutaminase activity was unchanged in the protein‐free group, but was significantly higher with simultaneous NH4Cl consumption, suggesting that the renal adaptation to acid ingestion is not compromised by a lack of dietary protein. Urinary ammonia excretion also increased in rats consuming protein‐free diet and NH4Cl. Urinary urea excretion was greater in rats receiving protein‐free diet and NH4Cl than in rats receiving protein‐free diet only, at all time‐points examined. These data demonstrate that urea synthesis is driven primarily by the need to dispose of protein‐derived ammonia rather than bicarbonate.
The C‐terminal peptide of α‐1‐antitrypsin increases low density lipoprotein binding in HepG2 cells
Authors: Janciauskiene Sabina , Lindgren Stefan , Wright H. Tonie
Published: March 2003

Hydrophobic fragments generated from various proteolytically degraded precursor proteins are known to form amyloid fibrils which have biological effects unrelated to precursor function. We examined the effects on HepG2 cells of the fibrillar and soluble C‐terminal peptide [AAT‐(358−394)] generated during the cleavage of the α‐1‐antitrypsin molecule by target proteinase. Soluble and fibrillar forms of AAT‐(358−394)‐peptide increased low‐density lipoprotein (LDL) binding by 1‐fold and 15‐fold, respectively, an effect which was diminished by incubation or coincubation of cells with LDL, but was not affected by the serpin‐enzyme complex. This effect of AAT‐(358−394)‐peptide appears to be on LDL receptor binding and not on the serpin‐enzyme complex (SEC) or LDL receptor‐related protein binding. The terminal deoxynucleotidyl transferase‐biotin dUTP nick‐end labeling assay for apoptosis showed increased DNA fragmentation in cells incubated with AAT‐(358−394)‐peptide fibrils relative to controls and LDL‐incubated cells. [3H]Thymidine incorporation in cells incubated with fibrils for 4 days decreased by 60 %. We conclude that interaction of AAT‐(358−394)‐peptide fibrils with cell surface receptor(s) disturbs intracellular cholesterol homeostasis and induces cell death.
DNA interactions of antitumor trans‐[PtCl2(NH3)(quinoline)]
Authors: Žákovská Alena , Nováková Olga , Balcarová Zdeňka , Bierbach Ulrich , Farrell Nicholas , Brabec Viktor
Published: March 2003

Recent observations that several trans‐platinum complexes exhibit antitumor activity including activity in cisplatin‐resistant tumor cells, violates the classical structure/activity relationships of platinum(II) complexes. According to these relationships, only bifunctional platinum(II) complexes with cis‐oriented leaving ligands should be therapeutically active. In order to contribute to the understanding of mechanisms underlying the antitumor activity of these new trans‐platinum analogs, various biochemical and biophysical methods as well as molecular modeling techniques were employed to study the modifications of DNA by antitumor trans‐[PtCl2(NH3)(quinoline)]. The results indicated that trans‐[PtCl2(NH3)(quinoline)] coordinated monofunctionally to DNA with a similar rate as transplatin. The overall rate of the rearrangement to bifunctional adducts was also similar to that observed in the case of DNA modification by transplatin, i.e. it was relatively slow (after 48 h ≈34 % adducts remained monofunctional). In contrast to transplatin, however, trans‐[PtCl2(NH3)(quinoline)] formed considerably more interstrand cross‐links after 48 h (≈30 %) with a much shorter half‐time (≈5 h) (≈12 % for transplatin, t1/2 > 11 h). The results also suggested that the quinoline ligand in all or in a significant fraction of DNA adducts of trans‐[PtCl2(NH3)(quinoline)], in which platinum is coordinated to base residues, was well positioned to interact with the duplex.

The adducts of trans‐[PtCl2(NH3)(quinoline)] terminated in vitro RNA synthesis preferentially at guanine residues. Surprisingly, the type and extent of conformational alterations induced in DNA indicates that trans‐[PtCl2(NH3)(quinoline)] behaves in some respects like cisplatin, as indicated by the fact that trans‐[PtCl2(NH3)(quinoline)]‐modified DNA is recognized by cisplatin‐specific antibodies. Models for both monofunctional adducts and bifunctional interstrand cross‐links are proposed. Computer‐generated AMBER models show that the combination of monofunctional covalent binding and a stacking interaction between quinoline and the DNA bases can produce a kink in the duplex which is strongly suggestive of the directed bend produced by the major cisplatin‐DNA adduct (1,2 intrastrand cross‐link). Unique DNA adducts of this type formed by trans‐[PtCl2(NH3)(quinoline)] may contribute to the antitumor efficacy of this agent.
Structure of an α‐2,6‐sialylated lipooligosaccharide from Neisseria meningitidis immunotype L1
Authors: Wakarchuk Warren W. , Gilbert Michel , Martin Adèle , Wu Yuyang , Brisson Jean‐Robert , Thibault Pierre , Richards James C.
Published: March 2003

The recent cloning of the lipooligosaccharide (LOS) α‐2,3‐sialyltransferase from Neisseria meningitidis immunotype L3 permitted us to examine other immunotypes for this structural gene. We identified the gene and measured the enzyme activity in the L1 immunotype strain which had previously been reported to lack sialic acid in its LOS because it contains a terminal α‐linked galactose which was thought not to be an acceptor for the sialyltransferase. This finding prompted us to re‐examine the structure of the LOS from the L1 immunotype, which revealed the presence of sialic acid on the terminal α‐linked galactose. Oligosaccharides derived from the LOS were shown to be sialylated by composition and methylation analysis, mass spectrometry and nuclear magnetic resonance. The detailed structural analysis showed the sialic acid to occur only at O6 of the teminal α‐
D‐galactopyranose residue of the α‐
D‐Gal‐1,4‐β‐
D‐Gal‐1,4‐β‐
D‐glc trisaccharide (Pk epitope) chain of the LOS, in the α‐
D configuration. These data are the first report of a α‐2,6‐linked sialic acid in a bacterial LOS or lipopolysaccharide, and also the first report of a sialylated Pk epitope.
Production and properties of a recombinant soluble form of aminopeptidase A
Authors: Vazeux Gilles , Wilk Sherwin , Wilk Elizabeth , Corvol Pierre , Llorens‐Cortès Catherine
Published: March 2003

Aminopeptidase A is a homodimeric membrane‐bound zinc metallopeptidase anchored at the plasma membrane by a 22‐amino‐acid hydrophobic segment. The anchor segment separates a small N‐terminal cytoplasmic domain from a large ectodomain that contains the active site. Site‐directed mutagenesis was performed to investigate the role of the cytoplasmic domain of aminopeptidase A in membrane anchoring and routing of the enzyme. Expression in COS‐7 cells of a mutant lacking the N‐terminal cytoplasmic domain resulted in the efficient secretion of a catalytically active enzyme in the medium. The soluble mutated aminopeptidase A, purified from the medium of a stable cell line, exhibited similar biochemical features to those of the wild‐type enzyme. Pulse/chase metabolic labeling experiments revealed that the soluble form is generated intracellularly at an early stage of biosynthesis, suggesting that the signal peptide/membrane anchor domain of aminopeptidase A is removed in the endoplasmic reticulum through the action of the signal peptidase.
Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans
Authors: Okada Kazunori , Kainou Tomohiro , Tanaka Katsunori , Nakagawa Tsuyoshi , Matsuda Hideyuki , Kawamukai Makoto
Published: March 2003

Decaprenyl diphosphate (decaprenyl‐PP) synthase catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce decaprenyl‐PP, which is used for the side chain of ubiquinone (Q)‐10. We have cloned the synthase gene, designated ddsA, from Gluconobacter suboxydans and expressed it in Escherichia coli. Sequence analysis revealed the presence of an ORF of 948 bp capable of encoding a 33 898‐Da polypeptide that displays high similarity (30−50 %) to other prenyl diphosphate synthases. Expression of the ddsA gene complemented the lethality resulting from a defect in the octaprenyl diphosphate synthase gene of E. coli and produced Q‐10, indicating that Q‐10 can substitute for the function of Q‐8. The His‐tagged DdsA protein was purified to characterize its enzymatic properties. This enzyme required detergent (0.05 % Triton X‐100) and 10 mM Mg2+, for full activity. The Michaelis constants for geranyl diphosphate, all‐E‐farnesyl diphosphate and all‐E‐geranylgeranyl diphosphate were 7.00, 0.50 and 0.32 μM, respectively. Nine single‐amino‐acid substitutions were introduced upstream of conserved region II or VI. Most of the mutants showed a considerable decrease in catalytic activity or shortening of the ultimate chain length. However, the A70G mutant produced a longer‐chain‐length product than wild‐type decaprenyl‐PP synthase, and the A70Y mutant completely abolished the decaprenyl‐PP synthase function, indicating that Ala70 is important for enzyme activity and the determination of the chain‐length properties of DdsA.
Differential usage of the CXC chemokine receptors 1 and 2 by interleukin‐8, granulocyte chemotactic protein‐2 and epithelial‐cell‐derived neutrophil attractant‐78
Authors: Wuyts Anja , Proost Paul , Lenaerts Jean‐Pierre , Ben‐Baruch Adit , Van Damme Jo , Wang Ji Ming
Published: March 2003

The inflammatory response is mediated by a family of chemotactic cytokines, designated chemokines. The receptor usage of the CXC chemokine granulocyte chemotactic protein‐2 (GCP‐2) was compared with that of interleukin‐8 (IL‐8) and epithelial‐cell‐derived neutrophil attractant‐78 (ENA‐78). Chemokine activities were evaluated by measurement of intracellular calcium increase and by chemotaxis and binding assays, using CXC chemokine receptor (CXCR)‐transfected cell lines. GCP‐2 was equally potent at inducing a rise in [Ca2+]i in both CXCR1‐transfected and CXCR2‐transfected cells (minimal effective concentration 3 nM). IL‐8 augmented the [Ca2+]i more efficiently in CXCR1‐transfectants than in CXCR2‐transfectants, whereas for ENA‐78, threefold higher concentrations were necessary to obtain a calcium response in CXCR1‐transfected cells than in CXCR2‐transfectants. GCP‐2 desensitized the calcium increase induced by IL‐8 in both CXCR1‐transfected and CXCR2‐transfected cells, but ENA‐78 only affected the IL‐8‐induced calcium response in CXCR2‐transfectants. The half‐maximal effective concentrations for migration of CXCR2‐transfectants in response to GCP‐2 and ENA‐78 were similar (0.1 nM), whereas GCP‐2 was tenfold more potent than ENA‐78 on CXCR1‐transfectants. Half‐maximal migration of CXCR1‐transfected and CXCR2‐transfected cells was obtained with IL‐8 at concentrations of no more than 0.01 nM. Radiolabeled IL‐8 could efficiently be displaced from CXCR2 by IL‐8, GCP‐2 and ENA‐78. In contrast, only IL‐8 and GCP‐2 but not ENA‐78, competed for 125I‐IL‐8 binding to CXCR1. From these data, it can be concluded that, in addition to IL‐8, GCP‐2, but not ENA‐78, efficiently binds to both CXCR1 and CXCR2. The differential receptor usage of the structurally related ELR+CXC chemokines GCP‐2 and ENA‐78 is indicative of a different role in inflammatory reactions.
Dimers of Thermus thermophilus elongation factor Ts are required for its function as a nucleotide exchange factor of elongation factor Tu
Authors: Nesper Martina , Nock Steffen , Sedlák Erik , Antalík Marián , Podhradský Dušan , Sprinzl Mathias
Published: March 2003

Elongation factor Ts (EF‐Ts) promotes the formation of active GTP‐bound elongation factor Tu (EF‐Tu) by accelerating the dissociation of GDP from the EF‐Tu  GDP complex. Thermus thermophilus EF‐Ts forms a dimer in solution, which is stabilised by interaction of a three‐stranded antiparallel β‐sheet from each of the two EF‐Ts molecules. A disulfide bridge and several hydrophobic interactions are the main structural elements which stabilise the dimer [Jiang, Y., Nock, S., Nesper, M., Sprinzl, M. & Sigler, P. B. (1996) Biochemistry 35, 10 269−10 278]. Site‐directed mutagenesis was used to study the dimer formation and the effect of dimerization on the nucleotide exchange activity. The presence of the covalent disulfide bridge between the Cys190 residues has no effect on the activity. However, this disulfide bridge is not a necessary condition for the activity of EF‐Ts. The amino acid residues Leu73, Cys190 and Phe192 form a hydrophobic core on the dimerization interface. Their replacement by Asp, Ala and Asp, respectively, influences to different degrees the stability of EF‐Ts, the ability of EF‐Ts to form dimers, and the ability to interact with EF‐Tu. EF‐Ts variants which were unable to form dimers were also inactive in the nucleotide exchange on EF‐Tu. CD spectroscopy confirmed that this loss of activity is not due to changes in EF‐Ts secondary structure. By calorimetric measurements, it was demonstrated that the dimer formation considerably contributes to the thermostability of T. thermophilus EF‐Ts. Dimerization of T. thermophilus EF‐Ts is required to fulfil its physiological function in protein biosynthesis and probably represents a strategy of the translation system in this thermophile to withstand high temperatures. The biochemical data presented here are supported by the recently solved structure of the T. thermophilus EF‐Tu  EF‐Ts complex [Wang, Y., Jiang, Y., Meyering‐Voss, M., Sprinzl, M. & Sigler, P. B. (1997) Nature Struct. Biol. 4, 650−656] in which each EF‐Tu in the dyad symmetrical heterotetrameric complex interacts with two subunits of EF‐Ts.
Mechanisms of melanogenesis inhibition by tumor necrosis factor‐α in B16/F10 mouse melanoma cells
Authors: Martínez‐Esparza María , Jiménez‐Cervantes Celia , Solano Francisco , Lozano José Antonio , García‐Borrón José Carlos
Published: March 2003

The inflammatory cytokine tumor necrosis factor‐α (TNF‐α) is present in the dermal and epidermal layers of normal skin [Kilgus, O., Payer, E., Schreiber, S., Elbe, A., Strohal, R. & Stingl, G. (1993) J. Invest. Dermatol. 100, 674−680]. Its local concentrations are modified by several stimuli, including wound healing and ultraviolet irradiation. Moreover, TNF‐α inhibits melanogenesis in normal melanocytes [Swope, V., Abdel‐Malek, Z., Kassem, L. & Norlund, J. (1991) J. Invest. Dermatol. 96, 180−185], and is, therefore, a potential autocrine/paracrine regulator of pigmentation. We have analyzed the mechanisms of this effect using B16/F10 melanoma cells as a model.

Nanomolar concentrations of TNF‐α inhibit the tyrosine hydroxylase and dopa oxidase activities of B16/F10 melanocytes, to less than 30 % control levels, without effects on tyrosinase‐related protein 2/dopachrome tautomerase (TRP2/DCT). The 50 % inhibition was obtained at 1 nM TNF‐α and 48 h treatment. The effect of TNF‐α was noticeable after 6 h treatment, and maximal after 24 h. This inhibition is explained by decreased intracellular levels of tyrosinase and tyrosinase‐related protein 1 (TRP1), but not of TRP2/DCT as detected by Western blotting. Northern‐blot experiments showed that the inhibitory effect is partially explained by a reduced accumulation of the corresponding mRNAs, that dropped to about 50 % of control values (48 h treatment, 5 nM TNF‐α). Moreover, the tyrosine hydroxylase and dopa oxidase activities decreased more rapidly in TNF‐α‐treated cells than in control cells, under conditions of inhibition of protein synthesis. This suggests a TNF‐mediated reduction of tyrosinase half‐life. However, the possibility of an inhibitory post‐translational modification of the enzyme induced by TNF cannot be ruled out. Therefore, the inhibitory effect of TNF‐α on tyrosinase and TRP‐1 results from combined effect on mRNA levels and enzymatic activity or protein stability.