bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple binding sites in fibronectin and the staphylococcal fibronectin receptor
Authors: BOZZINI Silvia , VISAI Livia , PIGNATTI Patrizia , PETERSEN Torben E. , SPEZIALE Pietro
Published: March 2005

The binding of fibronectin to Staphylococci exhibits the properties of a ligand‐receptor interaction and has been proposed to mediate bacterial adherence to host tissues.

To localize staphylococcal‐binding sites in fibronectin, the protein was subjected to limited proteolysis and, of the generated fragments, Staphylococci appeared to preferentially bind to the N‐terminal fragment. Different fibronectin fragments were isolated and tested for their ability to inhibit 125I‐fibronectin binding to Staphylococci. The results indicate that only the N‐terminal region effectively competed for fibronectin binding. However, when isolated fragments were adsorbed to microtiter wells, we found that two distinct domains, corresponding to the N‐terminal fragment and to the heparin‐binding peptide mapping close to the C‐terminal end of fibronectin, promoted the attachment of both Staphylococcus aureus Newman and coagulase‐negative strain of Staphylococcus capitis 651.

These same domains were recognized by purified 125I‐labeled staphylococcal receptor, either when immobilized on microtiter wells or probed after adsorbtion onto nitrocellulose membrane.

The heparin‐binding domain is comprised of type‐III‐homology repeats 14, 15 and 16. To determine which repeats participate in this interaction, we isolated and tested repeats type III14 and type III16. We found that the major staphylococcal‐binding site is located in repeat type III14.

The staphylococcal receptor bound the N‐terminal domain of fibronectin with a KD of 1.8 nM, whereas the dissociation constant of the receptor molecule for the internal heparin‐binding domain was 10 nM.

Since the fusion protein ZZ‐FR, which contains the active sequences of fibronectin receptor (D1‐D3) bound only to the N‐terminus, it is reasonable to assume that the bacterial receptor may have additional binding sites outside the D domains, capable of interacting with the internal heparin‐binding domain of fibronectin.
The catalytic site is located on subunit I of the ATPase from Halobacterium saccharovorum
Authors: BONET Maria Luisa , SCHOBERT Brigitte
Published: March 2005

Nucleotide‐binding sites of the ATPase from the halophilic archaebacterium Halobacterium saccharovorum were labeled by ultraviolet irradiation in the presence of [α‐32P]ATP. A high‐affinity site, located on subunit I (98 kDa), was identified as catalytic by the following criteria: ATP bound to subunit I was hydrolyzed and the cross‐linked nucleotide was ADP; the specificity for ATP or ADP compared to that of other nucleotides was high; the tightly bound radionucleotide was exchangeable in the presence of excess unlabeled ATP and Mg2+; photolabeling of this site and enzyme inhibition due to tightly bound ADP were both dependent on the presence of Mg2+ and showed identical Kd values; treatment that restored the activity of the ADP‐inhibited enzyme also led to the release of the tightly bound nucleotide from subunit I. In addition, a non‐catalytic nucleotide‐binding site was found, located on subunit II (71 kDa). This site did not hydrolyze ATP, its occupation was Mg2+ independent and the affinity for ATP and the nucleotide specificity were much lower than that of subunit I. We suspect that this site is nonspecific. These results indicate that H. saccharovorum ATPase is different from F1‐ATPase which contain the catalytic site on the second largest subunit, but may be similar to other archaebacterial and vacuolar ATPases.
Inhibition of the acidification of endosomes and lysosomes by the antibiotic concanamycin B in macrophage J774
Authors: WOO Je‐Tae , SHINOHARA Chikara , SAKAI Kaoru , HASUMI Keiji , ENDO Akira
Published: March 2005

The antibiotic concanamycin B was found to inhibit oxidized‐low‐density‐lipoprotein(LDL)‐induced accumulation of lipid droplets in the macrophage J774 at a concentration of 5–10 nM. Concanamycin B inhibited cholesteryl‐ester synthesis from [14C]oleate by 50% at 14 nM without affecting the synthesis of triacylglycerol and polar lipids. Degradation of internalized oxidized 125I‐LDL was inhibited by about 80% in cells treated with 25 nM concanamycin B, while cell‐surface binding of oxidized 125I‐LDL at 4°C, uptake of surface‐bound oxidized 125I‐LDL and microsomal acyl‐CoA: cholesterol acyltransferase activity were not significantly affected by the antibiotic at 25 nM. When J774 cells were treated with 25 nM concanamycin B at 37°C for 60 min, there was a reduction of about 50% in the activity of cell‐surface receptors. This reduction appeared to be due to partial trapping of the receptors within the cells. Concanamycin B significantly inhibited ATP‐dependent acidification of endosomes and lysosomes of the J774 cells at a concentration of 4 nM. Since acidic condition of these organelles is required for receptor recycling and hydrolysis of lipoproteins, the results demonstrate that concanamycin‐B inhibition of oxidized‐LDL‐induced accumulation of lipid droplets and cholesteryl esters in macrophages J774 is associated with reduced ATP‐dependent acidification of these organelles.
Identification and purification of the ornithine/citrulline carrier from rat liver mitochondira
Authors: INDIVERI Cesare , TONAZZI Annamaria , PALMIERI Ferdinando
Published: March 2005

The ornithine/citrulline carrier from rat liver mitochondira, solubilized with Triton X‐100 and partially purified on hydroxyapatite, was identified and completely purified by PD‐10, DEAE‐Sephacel and celite chromatography. On SDS/polyacrylamide gel electrophoresis, the purified ornithine/citrulline carrier consisted of a single protein band with an apparent molecular mass of 33.5 kDa. When reconstituted into liposomes the ornithine carrier protein catalyzed an active mersalyl sensitive ornithine/ornithine exchange. It was purified 438‐fold with a recovery of 38% and a protein yield of 0.09% with respect to the extract derived from mitoplasts. The purified and reconstituted protein did not catalyze a significant unidirectional transport of ornithine. Citrulline was found to be the best countersubstrate for the transport of ornithine, followed by lysine and arginine. The exchange activity was inhibited by several sulphydryl reagents.
An 88‐kDa protein of Plastmidium falciparum is related to the band‐3‐binding domain of human erythrocyte ankyrin
Authors: SUETTERLIN Bernd W. , KAPPES Barbara , JENOE Paul , FRANKLIN Richard M.
Published: March 2005

Three typtic‐peptide sequences of an 88‐kDa pair of phosphoproteins of the malaria parasite Plasmodium falciparum were determined. They exhibit a striking similarity to corresponding sequences of the 89‐kDa domain of human erythrocyte ankyrin. [35S]Methionine labeling of the two proteins demonstrated thier parasitic origin. Using an appropriate oligonucletide probe, Southern‐blot analysis of genomic malaria DNA and Norhtern‐blot analysis of malaria RNA suggest the existence of ankyrin‐related sequences in the parasite genome and the presence of an ankyrin‐related transcirpt of about 3.2 kb. Our studies provide further evidence of malaria‐specific analogues of host‐cell proteins, implying an unusual kind of parasite/host interaction.
Lipoamide dehydrogenase from Azotobacter vinelandii
Authors: BENEN Jacques , van BERKEL Willem , VEEGER Cees , de KOK Arie
Published: March 2005

The 10 C‐terminual residues are not visible in the crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii, but can be observed in the crystal structures of the lipoamide dehydrogenases from Pseudomonas putida and Pseudomonas fluorescens. In these structures, the C‐terminus folds back towards the active site and is involved in interactions with the other subunit.

The function of the C‐terminus of lipoamide dehydrogenase from A. vinelandii was studied by deletion of 5, 9 and 14 residues, respectively. Deletion of the last 5 residues does not influence the catalytic properties and conformational stability (thermoinactivation and unfolding by guanidinium hydrochloride). Removal of 9 residues results in an enzyme (enzyme 9) showing decreased conformational stability and high sensitivity toward inhibition by NADH. These features are even more pronounced after deletion of 14 residues (enzyme 14). In addition Tyr16, conserved in all lipoamide dehydrogenases sequenced thus far, and shown from the other structures to be likely to be involved in subunit interaction, was replaced by Phe and Ser. Mutation of Tyr16 also results in a strongly increased sensitivity toward inhibition by NADH. The conformational stability of both Tyr16‐mutated enzymes is comparable to enzyme 9. The results strongly indicate that a hydrogen bridge between tyrosine of one subunit (Tyr16 in the A. vinelandii sequence) and histidine of the other subunit (His470 in the A. vinelandii sequence), exists in the A. Vinelandii enzyme. In the 9 and 14 enzymes this interaction is abolished. It is concluded that this interaction mediates the redox properties of the FAD via the conformation of the C‐terminus containing residues 450–470.
Cyclic nucleotides and intracellular‐calcium homeostasis in human platelets
Authors: BRÜNE Bernhard , ULLRICH Volker
Published: March 2005

The relationship between agonist‐sensitive calcium compartments and those discharged by the Ca2+‐ATPase inhibitor thapsigargin were studied in human platelets. In this context, calcium mobilization from intracellular pools and manganese in influx was investigated in relation to the effect of altered cyclic‐nucleotide levels. For maximal calcium release from intracellular stores, thapsigargin, compared to a receptor agonist like thrombin, requires the platelet's self‐amplification mechanism, known to generate thromboxane A2. With this lipid mediator formed, thapsigargin released calcium and stimulated manganese influx in a manner similar to thrombin. Blocking the thromboxane receptor by addition of sulotroban (BM13.177) or, alternatively, increasing platelet cAMP or cGMP using prostacyclin or sodium nitroprusside, dramatically reduced the ability of thapsigargin to release calcium from intracellular compartments. The same experimental conditions significantly reduced the rate of manganese influx initiated by thapsigargin compared to thrombin. The experiments indicate that thapsigargin‐sensitive compartments play only a minor role in inducing manganese influx compared to the receptor‐sensitive compartment. Cyclic nucleotides accelerate the redistribution of an agonist‐elevated platelet calcium into the thapsigargin‐sensitive compartment, from which calcium can be released by inhibition of the Ca2+‐ATPase. In human platelets, thapsigargin‐induced calcium increase and influx were responsible for only part the calcium release resulting from inhibition of the corresponding ATPase; another part results from the indirect effect of thapsigargin acting via thromboxane‐A2‐receptor activation. Cyclic nucleotides are therefore an interesting regulatory device which can modify the thapsigargin response by not allowing the self‐amplification mechanism of platelets to operate.
Primary‐structural and evolutionary analyses of the growth‐hormone gene from grass carp (Ctenopharyngodon idellus)
Authors: ZHU Zuoyan , HE Ling , CHEN Thomas T.
Published: March 2005

The growth‐hormone (GH) gene of grass carp, one of the fastest‐growing species of farmed fish, was isolated and the DNA sequenced. Only one GH gene is found in this species. This gene, which is 2.5 kb in length, has five exons and four introns, in common with all of the mammalian and the recently published common‐carp GH genes. In the course of vertebrate evolution, the total lengthsof the intron and the non‐coding region of exon 5 of the GH gene have been shortened by 40–70%, whereas the encoding exons of the gene have been slightly increased. The more closely related species exhibit the closest sequence similarity in their GH genes. For example, the similarity of the exons is 84.1–93.2% between grass carp and common carp (within the same family of Syprinedae), 43.5–82.1% between grass carp and rainbow trout (in different orders of Teleostei) and 45.8–58.6% between glass carp and rat (in different grades of Vertebrata). In addition, similar DNA domains, such as thyroid‐hormone–receptor‐complex‐binding site and cell‐type‐specificciselements involved in regulation of expression of rat and human GH genes, have been localized in the corresponding regions of the grass‐carp GH gene.
O‐Glycosylation and stability
Authors: WILLIAMSON Gary , BELSHAW Nigel J. , NOEL Timothy R. , RING Stephen G. , WILLIAMSON Michael P.
Published: March 2005

We have examined the stabilities of the catalytic and binding domains of glucoamylase 1 from Aspergillus niger and how these stabilities are affected by the O‐glycosylated linker glycopeptide which separates the domains. On heating, the catalytic domain unfolds irreversibly, whereas the binding domain unfolds reversibly as shows by differntial scanning calorimetry and by 1H NMR. The stability of three functional peptides, derived from glucoamylase 1, containing the binding domain alone and with 10 or 38 residues of the linker glycopeptide [Williamson, G., Belshaw, N. J. and Williamson, M. (1992) Biochem. J. 282, 423–428] was examined. Refolding in each case was reversible after thermal or chemical denaturation. β‐Cyclodextrin stabilised the binding domain by the same amount when it was part of glucoamylase 1 or an isolated domain. The thermal stability of the catalytic domain was not affected by the binding domain; however, the catalytic domain increased the melting temperature of the binding domain. Furthermore, the linker glycopeptide stabilised the binding domain against reversible thermal and chemical denaturaion by about 10 kJ/mol, but only a portion of the O‐glycosylated residues were required for stabilisation. On a simple molecular mass basis, the linker glycopeptide does not contribute as much as expected to the denaturational enthalpy of glycoamylase 1 and, in addition, shows only a small conformational change on chemical or thermal denaturation; this supports an extended structure for the linker. The results demonstrate that the unfolding pathway of glucomylase 1 depends on the concentration of β‐cyclodextrin and that the presence of the catalytic domain and/or the linker glycopeptide stabilises the binding domain.
Reaction of myeloperoxidase with its product HOCI
Authors: FLORIS René , WEVER Ron
Published: March 2005

The reaction of human myeloperoxidase with its product, hypochlorous acid was investigated using both rapid‐scan spectrophotometry and the stopped‐flow technique. In the reaction of myeloperoxidase with hypochlorous acid a primary compound is found with propersties similar to that of compound I and which is converted into compound II. The primary reaction is strongly pH‐dependent. At pH 7.2 the reaction is too fast to be measured but at higher pH values it is possible to determine the apparent second‐order rate constant. Its value decreases to about 2 × 107 M−1· s−1 at pH 8.3 and to 2.3 (±0.4) × 106 M−1· s−1 at pH 9.2, respectively. The dissociation constant for the formation of the primary compound is 25.7 (± 15.3) μM at pH9.2 and about 2.5μM at pH 8.3. The apparent second‐order rate constant for the formation of compound II is hardly affected by pH and varies between 2 to 5 × 104 M−1· s−1 at pH 10.2 and pH 8.3 respectively.

Reaction of myeloperoxidase with hypochlorous acid also resulted in irreversible partial bleaching of the chromophore. Chloride, which is a substrate of the enzyme not only protects myeloperoxidase against bleaching by hypochlorous acid but also competitively inhibits the binding of hypochlorous acid to myeloperoxidase, a process which also has been observed in the reaction with hydrogen peroxide. It is concluded that hypochlorous acid binds at the heme iron to form compound I.
The GppNHp‐activated adenylyl cyclase complex from turkey erythrocyte membranes can be isolated with its βγ subunits
Authors: BAR‐SINA Allan , MARBACH Irit , SHORR Robert G. L. , LEVITZKI Alexander
Published: March 2005

The adenylyl cyclase complex, derived from turkey erythrocyte membranes, was activated using guanosine 5′‐[β,γ‐imido]triphosphate (Gpp[NH]p) and separated under low‐detergent and low‐salt conditions using conventional molecular‐sieve chromatography followed by high‐pressure ion‐exchange and molecular‐sieve chromatography. Although the complex remains activated with Gpp[NH]p throughout the isolation, the βγ subunits copurify with the cyclase. The stoichiometry of the cyclase to the α subunit of the stimulatory guanosine‐nucleotide‐binding regulatory protein (αs) to the β subunit is close to unity, demonstrating that the βγ subunits do not dissociate from the Gs· cyclase complex (Gs, guanosine‐nucleotide‐binding regulatory protein) upon activation of the enzyme. If the final purification step was performed at high‐salt concentrations, the βi subunits could be separated from the αs· cyclase complex. Previously reported results on bovine brain cyclase also showed that the Gs· cyclase complex remains intact subsequent to activation by hormone and Gpp[NH]p [Marbach, I., Bar‐Sinai, A., Minich, M. and Levitzki, A. (1990) J. Biol. Chem. 265, 9999–10004]. These results using adenylyl cyclase from two different sources, support our previous kinetic experiments which first suggested that βγ subunits are not released from Gs upon cyclase activation. We, therefore, argue that the mode of adenylyl cyclase inhibition by the inhibitory guanosine‐nucleotide‐binding regulatory protein cannot be via shifting the αs to βγ equilibrium as is commonly believed, and an alternate hypothesis is proposed.
Novel aerobic 2‐aminobenzoate metabolism
Authors: ALTENSCHMIDT Uwe , BOKRANZ Martin , FUCHS Georg
Published: March 2005

Pseudomonas KB 740 degrades 2‐aminobenzoatee aerobically via a chimeric pathway which combines characterstics of anaerobic and aerobic aromatic metabolism. Atypically, 2‐aminobenzoyl‐CoA is an intermediate, and the activated aromatic acid is not only hydroxylated but also reduced to an alicyclic in a single step. The bacterial strain possesses a small plasmid, pKB 740, which carries all essentail information of this new pathway. Its total nucleotide sequences was determined. It consists of 8280 bp and contains the genes for the two initial enzymes of the pathway; 2‐aminobenzoate‐CoA ligase catalyzes the activation of the aromatic acid, and the flavoenzyme 2‐aminobenzoyl‐CoA monooxygenase/reductase catalyzes the hydroxylation (monooxygenase activity) and subsequent reduction (reductase activity) of the aromatic ring of 2‐aminobenzoyl‐CoA. Further more, five open reading frames (ORF) possibly coding for polypeptides are on the plasmid. Putative promoter sequences were found for two of the ORF. A nucleotide sequence able to form a possible termination loop was located downstream of the gene for 2‐aminobenzoyl‐CoA monooxygenase/ reductase. This gene consists of 2190 bases. The deduced amino acid sequence of the protein (730 residues; calculated molecular mass of the native 729‐residue protein 83 559 Da) contains a consensus sequence for an FAD‐binding site at the N‐terminus and a possible NAD(P)H‐binding site approximately 150 amino acid residues apart from the N‐terminus. The monooxygenase/reductase shows low sequence similarity to the flavoprotein salicylate hudroxylase. Functional and evolutionary aspects of this work are discussed.
X‐ray absorption spectroscopy of soybean lipoxygenase‐1
Authors: Van der HEIJDT Lambertus M. , FEITERS Martinus C. , NAVARATNAM Suppiah , NOLTING Hans‐F. , HERMES Christoph , VELDINK Gerrit A. , VLIEGENTHART Johannes F. G.
Published: March 2005

X‐ray absorption spectra at the Fe K‐edge of the non‐heme iron site in Fe(II) as well as Fe(III) soybean lipoxygenase‐1, in frozen solution or lyophilized, are presented; the latter spectra were obtained by incubation of the Fe(II) enzyme with its product hydroperoxide. An edge shift of about 2–3 eV to higher energy occurs upon oxidation of the Fe(II) enzyme to the Fe(III) species, corresponding to the valence change. The extended X‐ray absorption fine structure shows clear differences in active‐site structure as a result of this conversion. Curve‐fitting on the new data of the Fe(II) enzyme, using the EXCURV88 program, leads to a coordination sphere that is in agreement with the active‐site structure proposed earlier (6 ± 1 N/O ligands at 0.205–0.209 nm with a maximum variance of 0.009nm, including 4 ± 1 imidazole ligands) [Navaratnam, S., Feiters, M. C., Al‐Hakim, M., Allen, J. C., Veldink, G. A. and Vliegenthart, J. F. G. (1988) Biochim. Biophys. Acta 956, 70–76], while for the Fe(III) enzyme a shortening in ligand distances occurs (6 ± 1 N/O ligands at 0.200–0.203 nm with maximum variance of 0.008 nm) and one imidazole is replaced by an oxygen ligand of unknown origin. Lyophilization does not lead to any apparent differences in the iron coordination of either species and gives a much better signal/noise ratio, allowing analysis of a larger range of data.
Sequence of the tuf A gene encoding elongation factor EF‐Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli
Authors: VOSS R. Holger , HARTMANN Roland K. , LIPPMANN Corinna , ALEXANDER Christain , JAHN Olaf , ERDMANN Volker A.
Published: March 2005

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA‐ and tufB‐encoded sequences for elongation factor Tu (EF‐Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182–191). The loop, probably involved in nucleotide binding, is absent in EF‐Tu of the mesophile Escherichia coli. Since EF‐Tu from E. coli is quite unstable, the protein is well‐suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF‐Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C‐terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF‐Tu was overproduced in E. coli using the tac expression system. Identity of the recombinant T. aquaticus EF‐Tu was verified by Western blot analysis, N‐terminal sequencing and GDP binding assays.
The oxygen dependence of the mitochondrial respiration rate in ascites tumor cells
Authors: FERREIRA Jorge
Published: March 2005

The effect of the oxygen concentration on the rate of oxygen consumption by 786 and TA3 ascites tumor cell lines has been determined under steady‐flow conditions with a membraneless fastresponding O2 electrode and using ascorbate and N,N,N′,N′‐tetramethyl‐p‐phenylenediamine as electron donors. The reaction was initiated by rapid injection of O2 into anaerobically incubated test system. The time‐dependence of the intact cell respiration showed three distinct phases; an early very fast but short duration phase, a subsequent slow phase that prevailed for most of the reaction period and a third phase which preceded the reestablishment of anaerobiosis. Kinetic analysis of the reaction indicated a linkage between the catalytic efficiency and the transmembrane electrochemical potential. The rates of O2 uptake, obtained in the presence of both protonophores and ionophores, were monotonic and pseudo‐first order over 90% of the course of O2 consumption. Extrapolation of the observed rates to zero time, at which zero ΔμH+ and thus constant flow prevails, was used to calculate the oxygen concentration for the half‐maximal respiratory rate, which was found to be in the range 1.55–2.10 μM O2. No noticeable variation in the value of this kinetic parameter was found between the two cell lines used. Possible reasons for discrepancies in published reports on the oxygen dependence of the cytochrome c oxidase activity in various mitochondrial and reconstituted systems are discussed.
Intranuclear compartmentalization of transcribed and nontranscribed c‐myc sequences in Namalva‐S cells
Authors: ANDREEVA Mariana , MARKOVA Desislava , LOIDL Peter , DJONDJUROV Lalio
Published: March 2005

This investigation is centered on the intranuclear localization of transcribed and nontranscribed c‐myc sequences in human Namalva‐S cells bearing t(8;14) translocation. Southern hybridization showed that the breakpoint in the truncated allele of c‐myc lies outside the characteristic 12.8‐kbp EcoRI fragment: as Northern analysis indicated, this reorganization induces a high level of c‐myc transcription. Following high‐salt treatment, EcoRI digestion and centrifugation, isolated nuclei from the same cells are separated into two residual fractions: a heavier P fraction including nuclear matrix structures and a light S fraction representing dehistonized chromatin fibres. Comparative hybridization experiments revealed that the above procedure separates the c‐myc sequences between the two fractions To locate the site of intranuclear c‐myc transcription, we performed run‐on experiments with two fractions, topologically analogous to the residual P and S fractions but maintaining the original chromatin organization. These experiments indicated that chromatin P fraction harbours actively transcribed c‐myc sequences while chromatin S harbours nontranscribed ones. Further experiments have clarified that the transcribed c‐myc sequences are firmly bound to the matrix by multiple attachment sites, arranged throughout the entire gene locus. It was found, moreover, that at the site of attachment the interaction between DNA and the matrix components is realized via proteins. Controls with the β‐globin gene, which is constitutively nonexpressed in Namalva‐S cells but upon induction is highly expressed in murine erythrolcukemia cells, completely confirmed the conclusion we had made for the intranuclear localization of c‐myc. Thus the experiment, presented here support the more common idea that the transcribed and nontranscribed sequences are precisely compartmentalized.
Urokinase binding to laminin‐nidogen
Authors: STEPHENS Ross W. , AUMAILLEY Monique , TIMPL Rupert , REISBERG Tuuli , TAPIOVAARA Hannele , MYÖHÄNEN Heli , MURPHY‐ULLRICH Joanne , VAHERI Anti
Published: March 2005

Recently we have shown that heparin and related sulfated polyanions are low‐affinity ligands of the kringle domain in the amino‐terminal region (ATF) of human urokinase (u‐PA), and proposed that this may facilitate loading of u‐PA onto its receptor at the focal contacts between adherent cells and their matrix. We have now tested other components of the cell matrix (fibronectin, vitronectin, thrombospondin and laminin‐nidogen) for u‐PA binding, and found that laminin‐nidogen is also a ligand of the u‐PA ATF. Direct binding assays and competition binding assays with defined fragments of laminin‐nidogen showed that there are u‐PA binding sites in fragment E4 of laminin as well as in nidogen. The long‐arm terminal domain of laminin (fragment E3), which contains a heparin‐binding site, competed for binding of u‐PA to immobilised heparin. However nidogen, which does not bind to heparin, also inhibited binding of u‐PA to heparin, and this effect was also observed with recombinant nidogen and with a fragment of nidogen lacking the carboxy‐terminal domain. Direct binding assays confirmed that u‐PA binds to nidogen through a site in the u‐PA ATF. We conclude that u‐PA binds to laminin‐nidogen by interactions involving the ATF region of u‐PA, the E4 domain of laminin and the rod or amino‐terminal regions of nidogen. Since nidogen is suggested to be an important bridging molecule in the maintenance of the supramolecular organization in basement membranes, the presence of a binding site for u‐PA in nidogen indicates a role for plasminogen activation in basement membrane remodelling.
Distinct N‐glycan fucosylation potentials of three lepidopteran cell lines
Authors: STAUDACHER Erika , KUBELKA Viktoria , MÄRZ Leopold
Published: March 2005

The fucosyltransferase activities of three insect cell lines, MB‐0503 (from Mamestra brassicae), BM‐N (from Bombyx mori) and Sf‐9 (from Spodoptera frugiperda), were investigated and compared with that of honeybee venom glands. Cell extracts and venom gland extracts were incubated with GDP‐[14C]fucose and glycopeptides isolated from human IgG and from bovine fibrin. The labeled oligosaccharide products were released by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase A, fluorescence marked with 2‐aminopyridine and analyzed both by reversed‐phase and size‐fractionation HPLC. They were identified by their elution positions before and after exoglycosidase treatment in comparison with standard oligosaccharides. These experiments revealed distinct fucosylation potentials in the three cell lines tested. While MB‐0503 cells, like honeybee venom glands, are able to transfer fucose into α1–3 and α1–6 linkage to the innermost N‐acetylglucosamine, only α1–6‐fucosyl linkages were detected with BM‐N and Sf‐9 cells.
An intracellular ATP‐dependent calcium pump within the yeast Schizosaccharomyces pombe, encoded by the gene cta3
Authors: HALACHMI Dror , GHISLAIN Michel , EILAM Yael
Published: March 2005

We have permeabilized the plasma membranes of Schizosaccharomyces pombe cell with nystatin and measured ATP‐dependent Ca2+ uptake in the presence of KNO3 and a protonophore in order to inhibit Ca2+ uptake into the vacuole. ATP‐dependent Ca2+ accumulation into non‐vacuolar Ca2+‐storing organelles was detected. This Ca2+ uptake activity was maximal at pH 6 and inhibited by vanadate, the inhibitor of P‐type ATPase. The null mutation of cta3, a putative Ca2+ gene, [Ghislain, M., Goffeau, A., Halachmi, D. and Eilam, Y. (1990) J. Biol. Chem. 265, 18400–18407] strongly reduced the level of ATP‐dependent Ca2+ uptake into non‐vacuolar intracellular storing organelles. This result suggests that cta3 encodes an intracellular ATP‐dependent Ca2+ pump. The residual ATP‐dependent Ca2+ uptake in the mutant strain indicated the presence of a second nonvacuolar, intracellular Ca2+‐ATPase encoded by a different gene.
The structure of pneumococcal lipoteichoic acid
Authors: BEHR Thomas , FISCHER Werner , PETER‐KATALINIĆ Jasna , EGGE Heinz
Published: March 2005

Pneumococcal lipoteichoic acid was extracted and purified by a novel, quick and effective procedure. Structural analysis included methylation, periodate oxidation, Smith degradation, oxidation with CrO3, and fast‐atom‐bombardment mass spectrometry. Hydrolysis with 48% (by mass) HF and subsequent phase partition yielded the lipid anchor (I), the dephosphorylated repeating unit of the chain (II) and a cleavage product of the latter (III). The proposed structures are:

image

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where AATGal is 2‐acetamido‐4‐amino‐2,4,6‐trideoxygalactose, and all sugars are in the pyranose form and belong to the d‐series.

Alkaline phosphodiester cleavage of lipoteichoic acid, followed by treatment with phosphomonoesterase, resulted in the formation of II and IV, with IV as the prevailing species:
image

The linkage between the repeating units was established as phosphodiester bond between ribitol 5‐phosphate and position 6 of the glucosyl residue of adjacent units. The chain was shown to be linked to the lipid anchor by a phosphodiester between its ribitol 5‐phosphate terminus and position 6 of the non‐reducing glucosyl terminus of I. The lipoteichoic acid is polydisperse: the chain length may vary between 2 and 8 repeating units and variations were also observed for the fatty acid composition of the diacylglycerol moiety. Preliminary results suggest that repeating units II and IV are enriched in separate molecular species. All species were associated with Forssman antigenicity, albeit to a various extent when related to the non‐phosphocholine phosphorus. Owing to its unique structure, the described macroamphiphile may be classified as atypical lipoteichoic acid.
Molecular cloning and characterization of the Saccharomyces cerevisiae CYT2 gene encoding cytochrome‐c1– heme lyase
Authors: ZOLLNER Alfred , RÖDEL Gerhard , HAID Albert
Published: March 2005

Cytochrome c1, a subunit of the mitochondrial ubiquinol–cytochrome‐c reductase, is synthesized on cytosolic ribosomes as a precusor protein of 37 kDa. Maturation to the mature 31‐kDa form involves two proteolytic processing steps of the amino‐terminal presequnce. After removal of the amino‐terminal part by the matrix‐localized processing peptidase, the carboxy‐terminal part of the presequence is cleaved off by an unknown intermembrane space protease. This step depends on covalent linkage of heme to the apoprotein. At least two complementation groups (I and II) can be distinguished among mutants of the yeast Saccharomyces cerevisiae, which are defective in this second proteolytic processing, i.e. they accumulate the intermediate‐sized form of cytochrome c1 instead of for cytochrome c1 [Sadler, I., Suda, K., Schatz, G., Kaudewitz, F. & Haid, A., (1984) EMBO J. 3, 2137–2143]. We report on the molecular cloning and characterization of the CYT2 gene representing complementation group I. It maps on chromosome XI and encodes a mitochondrial protein of about 26 kDa. Extensive similarity to Neurospora crassa and S. cerevisiae cytochrome‐c–heme lyase, as well as the phenotype of cyt2 mutants, strogly suggest that we have identified the gene for cytochrome‐c1–heme lyase.
Acetylcholinesterases of the nematode Steinernema carpocapsae
Authors: ARPAGAUS Martine , RICHIER Patricia , BERGE Jean‐Baptiste , TOUTANT Jean‐Pierre
Published: March 2005

We analyzed the molecular forms of acetylcholinesterase (AChE) in the nematode Steinernema carpocapsae. Two major AChEs are involved in acetylcholine hydrolysis. The first class of AChE is highly sensitive to eserine (IC50= 0.05 μM). The corresponding molecular forms are: an amphiphilic 14S form converted into a hydrophilic 14.5S form by mild proteolysis and two hydrophilic 12S and 7S forms. Reduction of the amphiphilic 14S form with 10 mM dithiothreitol produces hydrophilic 7S and 4S forms, indicating that it is an oligomer of hydrophilic catalytic subunits linked by disulfide bond(s) to a hydrophobic structural element that confers the amphiphilicity to the complex. Sedimentation coefficients suggest that 4S, 7S, 12S forms corresond to hydrophilic monomer, dimer, tetramer and that the 14S form is also a tetramer linked to one structural element. The second class of AChE is less sensitive to eserine (IC50= 0.1 mM). Corresponding molecular forms are hydrophilic and amphiphilic 4S forms (monomers) and a major amphiphilic 7S form converted into a hydrophilic dimer by Bacillus thuringiensis phosphatidlinositol‐specific phospholipase C. This amphiphilic 7S form thus possesses a glycolipid anchor. It appears that Steinernema (a very primitive invertebrate) presents AChEs with two types of membrane association that closely resemble those described for amphiphilic G2 and G4 forms of AChE in more evolved animals.
Role of F0 and F1 subunits in the gating and coupling function of mitochondrial H+‐ATP synthase
Authors: ZANOTTI Franco , GUERRIERI Ferruccio , CAPOZZA Giuseppe , FIERMONTE Maria , BERDEN Jan , PAPA Sergio
Published: March 2005

A study is presented on the role of F0 and F1 subunits in oligomycin‐sensitive H+ conduction and energy transfer reactions of bovine heart mitochondrial F0F1 H+‐ATP synthase.

Mild treatment with azodicarboxylic acid bis(dimethylamide) (diamide) enhanced oligomycin‐sensitive H+ conduction in submitochondrial particles containing F1 attached to F0. This effect was associated with stimulation of the ATPase activity, with no effect on its inhibition by oligomycin, and depression of the 32Pi—ATP exchange. The stimulatory effect of diamide on H+ conduction decreased in particles from which F1 subunits were partially removed by urea. The stimulatory effect exerted by diamide in the submitochondrial particles with F1 attached to F0 was directly correlated with a decrease of the original electrophoretic bands of a subunit of F0 (F0I‐PVP protein) and the γ subunit of F1, with corresponding formation of their cross‐linking product. In F0 liposomes, devoid of γ subunit, diamide failed to stimulate H+ conduction and to cause disappearance of F0I‐PVP protein, unless purified γ subunit was added back. The addition to F0 liposomes of γ subunit, but not that of α and β subunits, caused per se inhibition of H+ conduction.

It is concluded that F0I‐PVP and γ subunits are directly involved in the gate of the F0F1 H+‐ATP synthase. Data are also presented indicating contribution to the gate of oligomycin‐sensitivity conferral protein and of another protein subunit of F0, F6.
Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper‐binding residues
Authors: ZUMFT Walter G. , DREUSCH Andreas , LÖCHELT Sabine , CUYPERS Heinrich , FRIEDRICH Bärbel , SCHNEIDER Beate
Published: March 2005

The nosZ genes encoding the multicopper enzyme nitrous oxide reductase of Alcaligenes eutrophus H16 and the type strain of Pseudomonas aeruginosa were cloned and sequenced for structural comparison of their gene products with the homologous product of the nosZ gene from Pseudomonas stutzeri [Viebrock, A. & Zumft, W. G. (1988) J. Bacteriol. 170, 4658–4668] and the subunit II of cytochrome‐c oxidase (COII). Both types of enzymes possess the CuA binding site. The nosZ genes were identified in cosmid libraries by hybridization with an internal 1.22‐kb PstI fragment (NS220) of nosZ from P. stutzeri. The derived amino acid sequences indicate unprocessed gene products of 70084 Da (A. eutrophus) and 70695 Da (P. aeruginosa). The N‐terminal sequences of the NosZ proteins have the characteristics of signal peptides for transport.

A homologous domain, extending over at least 50 residues, is shared among the three derived NosZ sequences and the CuA binding region of 32 COII sequences. Only three out of nine cysteine residues of the NosZ protein (P. stutzeri) are invariant. Cys618 and Cys622 are assigned to a binuclear center, A, which is thought to represent the CuA site of NosZ and is located close to the C terminus. Two conserved histidines, one methionine, one aspartate, one valine and two aromatic residues are also part of the CuA consensus sequence, which is the domain homologous between the two enzymes. The CuA consensus sequence, however, lacks four strictly conserved residues present in all COII sequences. Cys165 is likely to be a ligand of a second binuclear center, Z, for which we assume mainly histidine coordination. Of 23 histidine residues in NosZ (P. stutzeri), 14 are invariant, 7 of which are in regions with a degree of conservation well above the 50% positional identity between the Alcaligenes and Pseudomonas sequences. Conserved tryptophan residues are located close to several potential copper ligands. Trp615 may contribute to the observed quenching of fluorescence when the CuA site is occupied.
The three‐dimensional structure of guanine‐specific ribonuclease F1 in solution determined by NMR spectroscopy and distance geometry
Authors: NAKAI Takahisa , YOSHIKAWA Wataru , NAKAMURA Haruki , YOSHIDA Hiroshi
Published: March 2005

Two‐dimensional 1H‐NMR studies have been performed on ribonuclease F1 (RNase F1), which contains 106 amino acid residues. Sequence‐specific resonance assignments were accomplished for the backbone protons of 99 amino acid residues and for most of their side‐chain protons. The three‐dimensional structures were constructed on the basis of 820 interproton‐distance restraints derived from NOE, 64 distance restraints for 32 hydrogen bonds and 33 φ torsion‐angle restraints. A total of 40 structures were obtained by distance geometry and simulated‐annealing calculations. The average root‐mean‐square deviation (residues 1–106) between the 40 converged structures and the mean structure obtained by averaging their coordinates was 0.116±0.018 nm for the backbone atoms and 0.182±0.015 nm for all atoms including the hydrogen atoms. RNase F1 was determined to be an α/β‐type protein. A well‐defined structure constitutes the core region, which consists of a small N‐terminal β‐sheet (β1, β2) and a central five‐stranded β‐sheet (β3‐β7) packed on a long helix. The structure of RNase F1 has been compared with that of RNase T1, which was determined by X‐ray crystallography. Both belong to the same family of microbial ribonucleases. The polypeptide backbone fold of RNase F1 is basically identical to that of RNase T1. The conformation‐dependent chemical shifts of the Cα protons are well conserved between RNase F1 and RNase T1. The residues implicated in catalysis are all located on the central β‐sheet in a geometry similar to that of RNase T1.
Molecular dynamics study of a complex between the human histocompatibility antigen HLA‐A2 and the IMP58‐66 nonapeptide from influenza virus matrix protein
Authors: ROGNAN Didier , ZIMMERMANN Norbert , JUNG Günther , FOLKERS Gerd
Published: March 2005

The structure of the influenza‐virus‐matrix‐protein (IMP) 58‐66 nonapeptide, bound to the major‐histocompatibility‐complex‐encoded human leukocyte antigen (HLA) A2 protein was studied by molecular dynamics simulation. Starting from the extra electron density map of peptides co‐crystallized with HLA‐A2, the nonapeptide IMP58‐66 was docked residue by residue in the protein binding cleft. The complex was simulated for 100 ps in a shell of 1372 water molecules. The averaged simulated HLA‐A2 conformation was found to be similar to the crystal structure (0.182 nm RMS deviation, for the backbone atoms of the α1‐α2 domain). Nine out of the 14 hydrogen bonds observed in the antigen‐binding site were reproduced in the simulation.

The IMP58‐66 peptide exhibits an extended conformation with kinks at positions 3 and 5. The side chains of residues 2, 3 and 9 develop van der Waals' interactions with hydrophobic pockets of HLA‐A2, corresponding to polymorphic residues of the major‐histocompatibility‐complex‐encoded proteins. Both the N‐terminus and C‐terminus of the nonapeptide were anchored in the antigen‐binding groove by hydrogen bonds with conserved amino acids. The N‐terminus was more flexible and contacts four HLA‐A2 conserved tyrosines (Tyr7, Tyr59, Tyr159 and Tyr171) and Glu63 by direct or water‐relayed hydrogen bonds. Water intercalation occurred only around the N‐terminus of the peptide, the C‐terminal carboxylate forming strong hydrogen bonds with polar residues (Tyr84 and Thr143) and a salt bridge with Lys146 all over the molecular dynamics simulation.

This model is fully compatible with the recently published crystal structure of the HLA‐B27 protein, complexed by a mixture of self nonapeptides [15]
7‐Substituted pterins in humans with suspected pterin‐4a‐carbinolamine dehydratase deficiency
Authors: ADLER Carmen , GHISLA Sandro , REBRIN Igor , HAAVIK Jan , HEIZMANN Claus W. , BLAU Nenad , KUSTER Thomas , CURTIUS Hans‐Christoph
Published: March 2005

A recently described new form of hyperphenylalaninemia is characterized by the excretion of 7‐substituted isomers of biopterin and neopterin and 7‐oxo‐biopterin in the urine of patients. It has been shown that the 7‐substituted isomers of biopterin and neopterin derive from L‐tetrahydrobiopterin and D‐tetrahydroneopterin and are formed during hydroxylation of phenylalanine to tyrosine with rat liver dehydratase‐free phenylalanine hydroxylase.

We have now obtained identical results using human phenylalanine hydroxylase. The identity of the pterin formed in vitro and derived from L‐tetrahydrobiopterin as 7‐(1′,2′‐dihydroxypropyl)pterin was proven by gas‐chromatography mass spectrometry. Tetrahydroneopterin and 6‐hydroxymethyltetrahydropterin also are converted to their corresponding 7‐substituted isomers and serve as cofactors in the phenylalanine hydroxylase reaction. Dihydroneopterin is converted by dihydrofolate reductase to the tetrahydro form which is biologically active as a cofactor for the aromatic amino acid monooxygenases. The 6‐substituted pterin to 7‐substituted pterin conversion occurs in the absence of pterin‐4a‐carbinolamine dehydratase and is shown to be a nonenzymatic process. 7‐Tetrahydrobiopterin is both a substrate (cofactor) and a competitive inhibitor with 6‐tetrahydrobiopterin (Ki∼ 8 μM) in the phenylalanine hydroxylase reaction. For the first time, the formation of 7‐substituted pterins from their 6‐substituted isomers has been demonstrated with tyrosine hydroxylase, another important mammalian enzyme which functions in the hydroxylation of phenylalanine and tyrosine.
Integration of early light‐inducible proteins into isolated thylakoid membranes
Authors: KRUSE Elisabeth , KLOPPSTECH Klaus
Published: March 2005

An in‐vitro system has been established to study the integration of early light‐inducible proteins (ELIP) into isolated thylakoid membranes. The in‐vitro‐expressed ELIP precursor proteins exist in two forms, a high‐molecular‐mass aggregate which is accessible to trypsin but no longer to the stromal processing protease and a soluble form which is readily cleaved to the mature form by the stromal protease. The mature form of ELIP is integrated into thylakoid membranes; its correct integration can be deduced from the observation that the posttranslationally transported products and the in‐vitro integrated ELIP species are cleaved by trypsin to products of the same apparent molecular mass. Trypsin‐resistant fragments of high‐molecular‐mass and low‐molecular‐mass ELIP appear to have the same size. The processed ELIP species, as well as an engineered mature form of ELIP, are integrated into isolated thylakoid membranes. Integration of the mature protein occurs in the absence of stroma, into sodium‐chloride‐washed, and trypsin‐treated thylakoid membranes. The process of integration is almost temperature independent over 0–30°C. Analysis of the time course of integration leads to the conclusion that, under in‐vitro conditions, processing but not integration into membranes is the rate‐limiting step. In the absence of stroma, the ELIP precursor is bound to the thylakoid membranes, however, it is no longer accessible to the stromal maturating protease when added after binding has occurred. In conclusion, integration of ELIP differs in many essential details from that of its relatives, the light‐harvesting chlorophyll a/b protein family.
Ligand‐independent reduction of cAMP receptors in Dictyostelium discoideum cells over‐expressing a mutated ras gene
Authors: LUDÉRUS M. E. Eva , KESBEKE Fanja , KNETSCH Menno L. W. , VAN DRIEL Roel , REYMOND Christophe D. , SNAAR‐JAGALSKA B. Ewa
Published: March 2005

Drug‐resistance selection in Dictyostelium discoideum transformants resulted in up to eight‐times‐higher ras protein levels. Over‐production of the wild‐type ras protein did not lead to an aberrant phenotype. Increased levels of the mutated [G12T]ras protein, however, were correlated with severe deficiencies in aggregation and development. This aberrant phenotype is associated with reduced cAMP binding, due to a lower number of cell‐surface receptors. We show that both RNA and cAMP‐receptor‐protein levels are reduced. These results indicate that ras in Dictyostelium discoideum seems to be involved in regulating cAMP‐receptor‐gene expression.