bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of the ADP/ATP carrier in permeabilization processes of the inner mitochondrial membrane
Authors: Vaz de MACEDO Denise , NEPOMUCENO Maria de Fátima , PEREIRA‐DA‐SILVA Lucia
Published: March 2005

The effect of different agents on inner‐mitochondrial‐membrane permeabilization and lipoperoxidation induced by Ca2+ and the pyridine‐nucleotide oxidant t‐butylhydroperoxide or inorganic phosphate was investigated. Comparing the protection conferred by ADP, a substrate of the ADP/ATP carrier, dithiothreitol, a disulfide reductant and butylhydroxytoluene, a radical scavenger, it was found that ADP was always the most effective against mitochondrial damage, when present in the incubation medium from the beginning. Moreover, carboxyatractyloside, a specific inhibitor of the ADP/ATP carrier, abolished completely the protective effect of ADP on both the lipoperoxidation and mitochondrial swelling processes. Experiments where deenergized mitochondria were previously incubated with Ca2+ showed a decrease in the content of active ADP/ATP carrier, indicating a direct involvement of this protein in the formation of a non‐specific Ca2+‐dependent pore. Our results also eliminate the possibility of an attack of oxygen radicals on lipids or proteins of the mitochondrial membrane as the primary event triggering the permeability transition of the inner mitochondrial membrane.
Abundance and subcellular distribution of GTP‐binding proteins in 3T3‐L1 cells before and after differentiation to the insulin‐sensitive phenotype
Authors: HUPPERTZ Christine , SCHÜRMANN Annette , JOOST Hans‐Georg
Published: March 2005

The abundance and the subcellular distribution of GTP‐binding proteins was studied in membrane fractions (plasma membranes and low‐density microsomes) from 3T3‐L1 cells before and after differentiation to the insulin‐sensitive phenotype. After differentiation, the abundance of αi (α subunit of GTP‐binding protein Gi), αi (α subunit of GTP‐binding protein Gi), and of a 47‐kDa αs, (α subunit of GTP‐binding protein Gs) as detected by immunoblotting with specific antisera was reduced by 10–50% when normalized per membrane protein. In contrast, a 43‐kDa αs was increased about threefold after differentiation. Furthermore, cholera‐toxin‐catalyzed ADP‐ribosylation of both 43‐kDa and 47‐kDa αs was disproportionately increased ninefold and threefold, respectively, possibly reflecting the increased production of an ADP‐ribosylation factor in the differentiated cells. The small GTP‐binding protein Ha‐ras was reduced by 50%, whereas rab1 and other small GTP‐binding proteins tentatively identified as rab‐isoforms (ras‐homologous gene products from brain) were increased by 100% and 70%, respectively. Since the total protein content of 3T3‐L1 cells was increased threefold after differentiation, the observed increase of the 43‐kDa αs, rab1 and of the other rab isoforms was eightfold, sixfold and fivefold, respectively, when normalized/cell count. With the exception of the rab isoforms, all GTP‐binding proteins were predominantly, if not exclusively, located in the plasma membrane; comparable amounts of the rab isoforms were found in plasma membranes and low‐density microsomes. Insulin induced the characteristic redistribution of glucose transporters GLUT4 from low‐density microsomes to the plasma membranes, but failed to alter the subcellular distribution of any of the GTP‐binding proteins investigated. These data suggest that the increase in the abundance of the 43‐kDa αs subunit and of several rab isoforms might be related to specific functions of the adipocyte‐like phenotype, but that none of the investigated guanine‐nucleotide‐binding regulatory (G)‐proteins appears to be tightly associated with the GLUT4.
Retrobiosynthetic analysis of carbon fixation in the phototrophic eubacterium Chloroflexus aurantiacus
Authors: EISENREICH Wolfgang , STRAUSS Gerhard , WERZ Udo , FUCHS Georg , BACHER Adelbert
Published: March 2005

The phototrophic bacterium Chloroflexus aurantiacus does not use any of the known autotrophic CO2 fixation pathways. There is evidence for a new cyclic autotrophic pathway in which acetyl‐CoA is converted to 3‐hydroxypropionate and further to succinate and malate. This hypothesis was tested by feeding growing cultures during several generations with 3‐hydroxy[1‐13C]propionate, [1‐13C]acetate, or [2‐13C]acetate, in addition to unlabeled CO2. The relative 13C content of individual carbon atoms in biosynthetic amino acids and nucleosides was determined by 1H‐ and 13C‐NMR spectroscopy. 13C coupling patterns were analyzed by two‐dimensional 13C‐TOCSY experiments which were optimized for the analysis of multiply 13C‐labeled biosynthetic samples. From the 13C enrichments of amino acids and nucleosides, the labeling patterns of central metabolic intermediates were evaluated by a retrobiosynthetic approach. Both 3‐hydroxypropionate and acetate were incorporated into all central metabolic pools. The 13C labeling and coupling patterns suggest a novel carbon fixation pathway via 3‐hydroxypropionate. Specifically, we propose that acetyl‐CoA is carboxylated to malonyl‐CoA which is reduced under formation of 3‐hydroxypropionyl‐CoA. Dehydration and reduction yield propionyl‐CoA which is converted to succinate by a second carboxylation reaction. The net product of autotrophic carbon fixation appears to be glyoxylate. However, it is not yet known how glyoxylate is channeled into anabolic metabolism. Assimilation of acetate can proceed via the CO2 fixation pathway, but also via the glyoxylate pathway.
Molecular characterization of HES‐2, a mammalian helix‐loop‐helix factor structurally related to Drosophila hairy and Enhancer of split
Authors: ISHIBASHI Makoto , SASAI Yoshiki , NAKANISHI Shigetada , KAGEYAMA Ryoichiro
Published: March 2005

Drosophila hairy (h) plays a crucial role in early development as a pair‐rule segmentation gene. h and its structurally related gene Enhancer of split [E(spl)] are also required for normal sensory neurogenesis in late development. To analyze the molecular mechanisms of mammalian development, we recently characterized three rat helix‐loop‐helix (HLH) factors that show structural homology to the Drosophila h and E(spl) gene products, and found that rat factors exhibit distinct spatiotemporal expression patterns and act as a negative regulator. Here, we report the molecular characterization of another member of this family, designated HES‐2. Rat HES‐2 protein has a basic HLH domain homologous to h and E(spl) as well as the carboxy‐terminal Trp‐Arg‐Pro‐Trp sequence conserved among this family. The HES‐2 mRNA is present as early as embryonic day 9.5 and is detected in a variety of tissues of both embryos and adults. DNase‐I‐footprinting analyses indicate that HES‐2 binds to all E box sequences (CANNTG) we tested as well as to the N‐box sequences (CACNAG). Further studies of gel‐mobility‐shift assays show that HES‐2 has a higher affinity for the E box than for the N box. Transient transfection analyses suggest that HES‐2 decreases the transcription originating from the promoters containing either the E box or the N box. These results indicate that HES‐2 acts as a negative regulator through interaction with both E‐box and N‐box sequences.
Sequence of extracellular mouse protein BM‐90/fibulin and its calcium‐dependent binding to other basement‐membrane ligands
Authors: PAN Te‐Cheng , KLUGE Matthias , ZHANG Rui‐Zhu , MAYER Ulrike , TIMPL Rupert , CHU Mon‐Li
Published: March 2005

Partial sequence comparisons have recently indicated that two extracellular components, fibulin from human placenta and BM‐90 from a basement‐membrane‐producing mouse tumor, are either identical or closely related proteins. In this study, a complete sequence analysis of mouse BM‐90 cDNA showed a 539‐residue N‐terminal core structure (domains I and II), which was 85% identical with the same core structure of human fibulin. A 137‐residue C‐terminal sequence (domain III) was unique for BM‐90 and could also be identified by Edman degradation. This suggested a novel splice product, variant D, which is characteristic for the mouse tumor. A second 117‐residue C‐terminal sequence (domain III) was identified in additional mouse cDNA clones and showed 91% identity with the region specific for variant C of fibulin. Northern blots using mouse cells demonstrated two mRNA species, 2.7 kb and 2.3 kb, which encoded the variants D and C, respectively. The sequence of BM‐90/fibulin indicates the presence of nine epidermal‐growth‐factor‐like repeats in the core domain‐II structure, eight of which contain consensus motifs for calcium binding. This binding is apparently important for the interaction of BM‐90 with laminin and nidogen and for some weaker interactions with collagen IV. Further binding of BM‐90 was demonstrated to fibronectin and BM‐90 itself, but did not depend on calcium. Major binding sites for BM‐90 were identified at a C‐terminal segment of laminin A chain and at the N‐terminus of nidogen. The broad interaction repertoire of BM‐90 is comparable to that of nidogen and both proteins may have similar roles as connecting elements in the extracellular matrix.
Reaction of imidazole‐citrate‐deformed glycogen phosphorylase with amino acids
Authors: PAN Peng , SCHINZEL Reinhard , PALM Dieter , CHRISTEN Philipp
Published: March 2005

Incubation of phosphorylase with l‐valine in the presence of 0.4 M imidazole citrate results in a time‐dependent decrease in the absorption of the enzyme‐bound cofactor pyridoxal 5′‐phosphate at 333 nm and the generation of a new absorption maximum at 415 nm which appears to be due to an enzyme‐bound coenzyme–amino‐acid aldimine adduct. Consequently, the enzyme is inactivated to less than 10% of its initial activity. The formation of the adduct of phosphorylase b with l‐valine (0.1 M) proceeds with t1/2∼8 min at pH 6.8 and 25°C and is slightly inhibited by AMP. Phosphorylase a reacts five times more slowly than phosphorylase b. The decrease in enzymic activity is linked to the formation of the coenzyme–amino‐acid adduct and is not due to resolution of the enzyme. Both the original absorption spectrum and phosphorylase activity are restored by gel filtration in the absence of l‐valine and imidazole citrate. Similar reactions occur with other l‐amino acids, an exception being l‐cysteine which leads to resolution of the enzyme [Shaltiel, S., Hedrick, J. L. & Fischer, E. H. (1966) Biochemistry 5, 2108–2116]. No reaction is observed with d‐amino acids or in the absence of imidazole citrate. Pyridoxal‐reconstituted phosphorylase rapidly produces with amino acids not only the aldimine adduct but also a species absorbing at 318 nm. Enzyme‐bound pyridoxal 5′‐phosphate and pyridoxal exhibit a positive CD signal in the region of 333 nm; in contrast, the absorption bands of the coenzyme–amino‐acid adducts at 415 nm and 318 nm are optically inactive. Neither pyridoxal‐5′‐phosphate‐reconstituted nor pyridoxal‐reconstituted phosphorylase in imidazole citrate catalyses any of the common pyridoxal‐5′‐phosphate‐mediated reactions of amino acids, e.g. transamination, decarboxylation or racemization, thus testifying to the high degree of reaction specificity of phosphorylase.
Structural similarity between lung surfactant protein D and conglutinin
Authors: LU Jinhua , WIEDEMANN Hanna , HOLMSKOV Uffe , THIEL Steffen , TIMPL Rupert , REID Kenneth B. M.
Published: March 2005

Preparations of bovine lung surfactant D (SP‐D) and conglutinin were examined by electron microscopy, gel‐filtration and SDS/PAGE. SP‐D is composed of non‐covalently linked subunits, of 160 kDa, which each contain three, disulphide‐linked, 44‐kDa polypeptide chains. In the electron microscope a single 160‐kDa subunit of SP‐D appears as a 45.8 ± 3‐nm‐long rod connected to a small globular ‘head’. Particles were also scen which correspond to non‐covalently linked dimers, trimers and tetramers of the 160‐kDa monomer subunit of SP‐D. The tetramer structure contains 12 polypeptide chains and is very similar to the electron microscopy images and model reported by Strang et al. [Strang, C. J., Slayter, US., Lachmann, P. J. and Davis, A. E. (1986) Biochem. J. 236, 3811–389] for bovine conglutinin in which four 160‐kDa subunits are disulphide‐linked to give a molecule of expected molecular mass of 528 kDa. This study confirmed the findings by Strang et al. in the above paper for intact conglutinin and also emphasised that the rod‐like structures, of length 37.6 ± 3.7 nm, seen in the conglutinin subunits were significantly shorter than those in SP‐D despite the close similarity in amino acid sequence (79% identity) and chain length between the two proteins. In addition, a truncated form of conglutinin was found in the conglutinin preparations, due to limited proteolysis of the Arg‐Ala bond at position 54 in the 44‐kDa chains. These truncated conglutinin chains yield a subunit composed of three shortened, non‐disulphide‐linked, chains and this subunit appears as a monomer with a rod length of 34.2 ± 2.8 nm in the electron microscope. On gel‐filtration, a proportion of the SP‐D preparation behaved, as expected, as a molecule with an apparent molecular mass of 600 kDa. The remainder of the SP‐D preparation behaved as aggregated material with a molecular mass greater than 900 kDa which yielded no distinct structures in the electron microscope. Intact conglutinin was eluted at a position greater than 900 kDa but yet provided clear electron microscopy images of the tetramer structure described above. Although it is difficult to explain fully the anomolous behaviour of SP‐D and conglutinin on gel‐filtration, it is proposed that the natural form of both SP‐D and conglutinin is the highest distinct oligomer form seen in the electron microscope, i.e. a tetramer of the 160‐kDa subunits in which the four rod‐like elements are linked in a tail‐to‐tail fashion to a central core.
Nitric oxide synthase
Authors: RIVEROS‐MORENO Valentina , BEDDELL Christopher , MONCADA Salvador
Published: March 2005

The amino acid sequence for the constitutive rat brain nitric oxide (NO) synthase was analysed by a set of computer programs that estimate and display physicochemical properties such as hydrophilicity, flexibility, accessibility, hydrophilic periodicity and conformation [Comerford, S. A., McCance, D. J., Dougan, G. & Tite, J. P. (1991) J. Virol. 65, 4681–4690]. Overall, they allow prediction of whether each peptide region will be an α‐helix, a β‐strand or a less regular coil and also whether the region will be buried in the protein core or exposed to water at the surface of the protein molecule. Ten peptide regions were chosen; the majority were predicted to be exposed areas of the molecule and therefore likely to be immunogenic. The peptides were chemically synthesised, coupled to keyhole limpet haemocyanin carrier protein and injected into rabbits to raise antibodies.

These antibodies have been used by us and others to locate the NO synthase in different tissues and species. Here we present the characterisation of the antibodies in relation to the possible conformation of the enzyme and an immunological comparison between two isoforms of NO synthase: constitutive (rat brain) and inducible (macrophage). Peptide regions predicted to be exposed, flexible or substantially in core, have produced antibodies that were able to recognise the native protein. Peptides of mixed characteristics possibly involved in the binding site tended to produce antibodies with low recognition for the tertiary structure of the native, purified NO synthase, although these peptides were all highly immunogenic. We postulate that either the peptides when conjugated to the carrier protein attain a different conformation to that in the native NO synthase, or alternatively the accessibility of the antibodies to substrate binding sites is highly restricted by steric hindrance. This latter seems to be more likely since a mixture of antibodies against this area of the protein molecule was able to achieve a similar neutralisation of the enzyme activity as the antibodies against the whole enzyme (∼ 50%).

Most of the selected anti‐peptide antibodies were not able to cross‐react with the inducible macrophage enzyme; only two that have 60% sequence identity showed a weak reaction in Western blot. The polyclonal antibody against the complete brain enzyme showed cross‐reaction in a Western blot with inducible enzyme. The macrophage enzyme was able to compete weakly with the binding of the brain enzyme to its own antibody, but 10 times more inducible protein was required. We propose that conformational epitopes are responsible for the cross‐reaction, suggesting that the overall tertiary structures of the two enzymes share a degree of similarity.
Pigments induce folding of light‐harvesting chlorophyll a/b‐binding protein
Authors: PAULSEN Harald , FINKENZELLER Bärbel , KÜHLEIN Nicola
Published: March 2005

The conformational behaviour of the light‐harvesting chlorophyll a/b‐binding protein (LHCP), the apoprotein of the major light‐harvesting complex (LHCII) of photosystem II in plants, has been studied. According to the circular dichroism in the ultraviolet range measured with isolated LHCII, the protein in the complex adopts a folded structure with a high content of α helix (about 60%), whereas the non‐pigmented, solubilized protein has a less ordered structure (about 20%α helix). LHCP‐pigment complexes that have been reconstituted from the overexpressed protein and isolated pigments in the presence of detergents display a protein CD signal similar to that of authentic LHCII, indicating that LHCP folds into the native structure during the reconstitution procedure. Renaturation of LHCP in these experiments is dependent on the presence of pigments and the formation of stable LHCP‐pigment complexes. Pigment‐induced engagement of LHCP in a compact structure has also been shown by two additional experimental approaches. (a) Upon complex formation, LHCP or its precursor (pLHCP) becomes resistant to trypsin digestion with the exception of an N‐terminal segment of the protein; the same protection of LHCP is known to occur in intact thylakoids. (b) Pigment binding renders a cysteine residue within the N‐proximal hydrophobic domain of the protein as well as a newly introduced cysteine four amino acid positions from the C terminus inaccessible to modification with a sulfhydryl‐specific label whereas the N terminus stays susceptible to specific labelling. These observations support the notion that only the N terminus protrudes from a compact protein‐pigment structure in LHCII. The fact that the major part of LHCP is trypsin‐resistant in pigmented complexes reconstituted in the absence of a membrane or even lipids justifies caution in using protection against trypsin as a criterion for the integration of LHCP into the thylakoid membrane.
Steady‐state kinetics and analysis of pH dependence on wild‐type and a modified allosteric Pseudomonas aeruginosa ornithine carbamoyltransferase containing the replacement of glutamate 105 by alanine
Authors: TRICOT Catherine , NGUYEN Van Thanh , STALON Victor
Published: March 2005

The substitution of alanine for glutamate at position 105 (E105 A) of the allosteric ornithine carbamoyltransferase (OTCase) of Pseudomonas aeruginosa abolishes the carbamoylphosphate (CP) cooperativity observed in the wild‐type enzyme. A kinetic analysis of [E105 A]OTCase was performed in order to determine the mechanism of the reaction. The results of initial velocity and inhibition studies are consistent with an ordered mechanism with CP as the first substrate to add to the enzyme. In addition, similar studies have been made using the wild‐type enzyme in the presence of the activator, phosphate. The results are similar to those obtained with [E105 A]OTCase indicating that the residue E105 is critical for the allosteric transition of the wild‐type enzyme.

The activities of the wild‐type allosteric OTCase and of [E105 A]OTCase have been studied in the pH range 5.8–8.2 in the absence and in the presence of positive and negative effectors. The sigmoid saturation of OTCases by CP has been analyzed according to the Hill equation. At low pH values, CP cooperativity is low in the wild‐type enzyme but cooperativity and [S]CP0.5 values increase markedly with pH. For [E105 A]OTCase, the saturation by CP is hyperbolic at all pH values; in this modified enzyme, the presence of spermidine, an allosteric inhibitor of the wild‐type enzyme, results in an inhibition which induces CP cooperativity. Thus, the ionization of the residue E105 apparently results in a conformational change in the wild‐type enzyme which modifies the catalytic site. Since the [E105 A]enzyme retains the heterotropic effects of the wild‐type enzyme, other structural features are required for the allosteric transition in the wild‐type catabolic OTCase.
Teichoic acid and lipoteichoic acid of Streptococcus pneumoniae possess identical chain structures
Authors: FISCHER Werner , BEHR Thomas , HARTMANN Rudolf , PETER‐KATALINIĆ Jasna , EGGE Heinz
Published: March 2005

Teichoic acid (C polysaccharide) was extracted and purified from Streptococcus pneumoniae R6 with standard procedures except that lipoteichoic acid was extracted first. The dephosphorylated repeating unit was isolated after hydrolysis with 48% (by mass) HF, the bis(phosphocholine)‐containing repeating unit was isolated by alkali hydrolysis, anion‐exchange chromatography and phosphomonoester cleavage. On the basis of compositional analysis, fast‐atom‐bombardment mass spectrometry and NMR spectroscopy the following structure is proposed:

image

where AATGal is 2‐acetamido‐4‐amino‐2,4,6‐trideoxy‐d‐galactose. The repeating units are linked to each other by phosphodiester bonds between O5 of the ribitol and O6 of the glucopyranosyl residue of adjacent units. This chain structure is identical with that previously established for pneumococcal lipoteichoic acid [Behr, T., Fischer, W., Peter‐Katalinić, J. & Egge, H. (1992) Eur. J. Biochem. 207, 1063–1075]. This represents a unique situation because in other Gram‐positive bacteria teichoic and lipoteichoic acids are structurally unrelated.
Microenvironment changes in human blood platelet membranes associated with binding of tissue‐type plasminogen activator
Authors: WATALA Cezary , PIETRUCHA Tadeusz , GWOZDZINSKI Krzysztof , KRALISZ Urszula , CIERNIEWSKI Czeslaw S.
Published: March 2005

Whole washed platelets were labelled with the free radicals [2‐(14‐carboxytetradecyl)‐2‐ethyl‐4,4‐dimethyl‐3‐oxazolidinyloxy] (16‐DOXYL‐Ste) or [2‐(3‐carboxypropyl)‐4,4‐dimethyl‐2‐tridecyl‐3‐oxazolidinyloxy] (5‐DOXYL‐Ste) and incubated with recombinant tissue‐type plasminogen activator (rt‐PA). Changes in the membrane fluidity caused by rt‐PA were detected by alterations in h+1/ho calculated from the ESR spectra for 16‐DOXYL‐Ste and 5‐DOXYL‐Ste incorporated into the lipid bilayer (h+1 and ho are the heights of the low‐field and middle‐field lines of the spectra, respectively). Interaction of rt‐PA with both resting and stimulated platelets resulted in increased rigidity of the membrane lipid bilayer as indicated by the reduced value of h+1/ho. This phenomenon can be explained either by conformational changes of membrane receptors caused by the attachment of rt‐PA and the subsequent rearrangement of the lipid matrix of platelet membranes, or by the direct association of rt‐PA with membrane phospholipids and thus partial embedding of protein molecules into the lipid bilayer restraining lipid mobility.
Glucose administration induces the premature expression of liver glucokinase gene in newborn rats
Authors: BOSSARD Pascale , PARSA Ramine , DECAUX Jean‐François , IYNEDJIAN Patrick , GIRARD Jean
Published: March 2005

Glucokinase first appears in the liver of the rat 2 weeks after birth and its activity rapidly increases after weaning on to a high‐carbohydrate diet. The appearance of glucokinase is principally due to the increase of plasma insulin and to the decrease of plasma glucagon concentrations. Oral glucose administration to 1‐ or 10‐day‐old suckling rats induced an increase in plasma insulin and a fall in plasma glucagon and allowed a rapid accumulation of liver glucokinase mRNA, secondarily to a stimulation of gene transcription. When unrestrained late pregnant rats were infused with glucose during 36 h to induce an increase in fetal plasma insulin and a decrease in fetal plasma glucagon concentrations, glucokinase mRNA was detectable in fetal liver but the level was 100‐fold lower than that observed in 1‐ or 10‐day‐old suckling rats. It is suggested that the hormonal environment did not allow glucokinase gene expression to be induced in fetal liver and that the absence of expression of glucokinase in suckling rat liver is due to the presence of low plasma insulin and high plasma glucagon levels.

The chromatin structure of the glucokinase gene was examined during development by identification of DNase‐I‐hypersensitive sites from the region comprised between −8 kb upstream and +4 kb downstream of the cap site. Five hypersensitive sites were found: four liver‐specific sites upstream of the cap site and one non‐specific site in the first intron. These sites are already present in term fetus but the intensity of the two proximal sites located upstream of the cap site increase markedly after birth. This suggests that these sites could be implicated in the regulation of glucokinase gene expression by insulin and glucagon. Full DNase‐I‐hypersensitivity of these two proximal sites seems necessary for the mature response of glucokinase gene in response to changes in pancreatic hormones concentrations.
Ferredoxin binding site on ferredoxin: NADP+ reductase
Authors: JELESAROV Ilian , De PASCALIS Antonio R. , KOPPENOL Willem H. , HIRASAWA Masakazu , KNAFF David B. , BOSSHARD Hans Rudolf
Published: March 2005

The chloroplast enzyme ferredoxin:NADP+ reductase (FNR) catalyzes the reduction of NADP+ by ferredoxin (Fd). FNR and Fd form a 1:1 complex that is stabilized by electrostatic interactions between acidic residues of Fd and basic residues of FNR. To localize lysine residues at the Fd binding site of FNR, the FNR:Fd complex (both proteins from spinach) was studied by differential chemical modification. In a first set of experiments, free FNR and the FNR:Fd complex were reacted with the N‐hydroxysuccinimidyl ester of biotin. Biotinylated peptides and non‐biotinylated peptides were separated on monovalent avidin‐Sepharose and purified by high‐performance liquid chromatography. Two peptides containing Lys18 and Lys153, respectively, were less biotinylated in complexed FNR than in free FNR. In a second set of experiments, free and complexed FNR were treated with 4‐N,N‐dimethylaminoazobenzene‐4′‐isothiocyano‐2′‐sulfonic acid (S‐DABITC) to obtain coloured lysine‐modified FNR. Protection of Lys153 was again found by modification with S‐DABITC. In addition, Lys33 and Lys35 were less labelled in the S‐DABITC‐modified, Fd‐bound enzyme. FNR modified in the presence, but not in the absence, of Fd was still able to bind Fd, indicating that the Fd‐protected residues are involved in the formation of the Fd:FNR complex.

The lysine residues disclosed by differential modification surround the positive end of the molecular dipole moment (558 Debye ∼ 1.85 × 10−27 Cm) and are located in a domain of strong positive potential on the surface of the FNR molecule. This domain we had proposed to belong to the binding site of FNR for Fd [De Pascalis, A. R., Jelesarov, I., Ackermann, F., Koppenol, W. H., Hirasawa, M., Knaff, D. B. & Bosshard, H. R. (1993) Protein Science 2, 1126–1135]. The prediction was based on the complementarity of shape between positive and negative potential domains of FNR and Fd, respectively.
Non‐cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei
Authors: BIENEN E. Jay , MATURI Raj. K. , POLLAKIS Georgios , CLARKSON Allen B.
Published: March 2005

The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has been suggested that glycolysis is the sole source of ATP in all bloodstream forms. However, earlier results indicated that in the mitochondria of the transitional intermediate/short stumpy bloodstream forms, the biochemical pathways are present that could allow intra‐mitochondrial production of ATP. Using a high mannitol buffer to enhance permeability, we confirm previous observations showing that transitional forms maintain motility and respiratory activity with 2‐oxoglutarate as the sole substrate. Using a luminometer to measure intracellular ATP levels via the luciferin/luciferase chemiluminescence assay, we show that these same transitional forms, but not long slender forms, maintain high levels of intracellular ATP in the presence of 2‐oxoglutarate. Further, in the presence of bongkrekic acid, an inhibitor of the mitochondrial adenine nucleotide translocase, ATP levels are reduced with subsequent death and lysis of the cells when 2‐oxoglutarate, but not glucose, is used as sole substrate. These data are direct evidence of ATP production by transitional bloodstream form mitochondria.
Evidence for transformation‐related increase in CTP synthetase activity in situ in human lymphoblastic leukemia
Authors: van den BERG A. André , van LENTHE Henk , BUSCH Sandra , de KORTE Dirk , ROOS Dirk , van KUILENBURG André B. P. , van GENNIP Albert H.
Published: March 2005

To determine the role of the enzyme CTP synthetase (EC 6.3.4.2) in the synthesis in situ of CTP in normal and in malignant lymphoblastic cells, the metabolism of radiolabeled pyrimidine ribonucleosides was studied in proliferating normal T lymphocytes and was compared with that of proliferating MOLT‐3 cell‐line cells and differentiated (non‐proliferating) MOLT‐3 cells. Both the incorporation of [14C]uridine into UTP and CTP and the incorporation of [14C]cytidine in CTP, as well as the fluxes of these labeled nucleosides through the nucleotide pools into nucleic acids, were elevated in proliferating MOLT‐3 cells compared to proliferating T lymphocytes. Furthermore, the conversion of UTP into CTP was enhanced in proliferating MOLT‐3 cells compared to proliferating T lymphocytes, indicating a higher activity of CTP synthetase in the leukemic cells. In non‐proliferating MOLT‐3 cells, the pyrimidine ribonucleotide fluxes were decreased compared to proliferating MOLT‐3 cells and proliferating T lymphocytes. However, the decreased ratio of uracil/cytosine ribonucleotides that was found in proliferating T lymphocytes and proliferating MOLT‐3 cells compared to non‐proliferating blood lymphocytes, was preserved in the differentiated MOLT‐3 cells. Moreover, although the fluxes had decreased, most CTP was still synthesized by CTP synthetase in the differentiated MOLT‐3 cells. Thus, the elevated activity of CTP synthetase in MOLT‐3 cells was independent of the cell growth and maturation stage. We conclude that the increased activity of CTP synthetase is associated with the process of malignant transformation in MOLT‐3 cells. Therefore, CTP synthetase offers an attractive target for selective therapy in human acute T‐lymphoid leukemia.
Recombinant soluble human interleukin‐6 receptor
Authors: STOYAN Tanja , MICHAELIS Uwe , SCHOOLTINK Heidi , VAN DAM Marc , RUDOLPH Rainer , HEINRICH Peter C. , ROSE‐JOHN Stefan
Published: March 2005

The recombinant soluble human interleukin‐6 receptor (srhIL‐6R) was expressed in Escherichia coli as a non‐glycosylated protein comprising the first 339 amino acids after the signal peptide. The protein accumulated within the cells as insoluble protein aggregates (inclusion bodies). After solubilization, 10% of the denatured srhIL‐6R could be renaturated by an in vitro folding procedure using l‐arginine and the glutathione‐redox system. The native receptors were purified to near homogeneity by affinity chromatography on an IL‐6–Sepharose column.

The functional features of the recombinant soluble receptor were further analysed. A part of the extracellular domain (amino acids 145–345) of the human interleukin‐6 receptor (IL‐6R) was expressed in E. coli and the purified protein was used to raise antibodies in rabbits. Characterization of the antiserum obtained indicated that an epitope of 13 amino acids close to the transmembrane region is needed for recognition by the antibodies. Since the antiserum obtained did not interfere with IL‐6 binding, it could be used to establish a cell‐free IL‐6‐binding assay, In this assay, the srhIL‐6R bound IL‐6 with an affinity of Kd= 1.5 nM as measured by Scatchard‐plot analysis. When 125I‐IL‐6 was chemically cross‐linked to the purified srhIL‐6R and analyzed by SDS/PAGE, several 125I‐IL‐6‐containing bands were detected, indicating the possible existence of a multimeric structure of the natural IL‐6/IL‐6R complex. The srhIL‐6R was shown to exhibit biological acitivity, i.e. it stimulated acute‐phase protein synthesis in the recently established human hepatoma cell line HepG2‐IL‐6 which does not express the IL‐6‐binding subunit of the IL‐6R complex on the cell surface.
Identification of digitalis‐like compounds in human cataractous lenses
Authors: LICHTSTEIN David , GATI Irith , SAMUELOV Sarah , BERSON David , ROZENMAN Yaacov , LANDAU Louis , DEUTSCH Joseph
Published: March 2005

Human cataractous lens nuclei extract inhibited, in a dose‐dependent fashion, [3H]ouabain binding to rat brain synaptosomes and microsomal Na+‐ and K+‐dependent adenosine triphosphate (Na+, K+‐ATPase) activity and interacted with anti‐digoxin antibodies. The compounds responsible for these activities, termed digitalis‐like compounds (DLC), were also detected in bovine, rat, cat and rabbit, normal, transparent lenses, but the levels were only 0.7–5.4% of the average levels in the cataractous human lenses. DLC from the human cataractous lenses were purified by a procedure consisting of organic extractions and batch chromatography followed by filtration through a 3000 Da cut‐off filter and subsequent separations using reverse‐phase high‐performance liquid chromatography. The presence of DLC in the different fractions obtained in the chromatograms was monitored by their ability to inhibit [3H]ouabain binding and Na+, K+‐ATPase activity. Based on chemical ionization mass spectrometry together with ultraviolet spectrometry and biological characterization, it is suggested that new bufodienolides, 19‐norbufalin and 19‐norbufalin peptide derivatives are responsible for the endogenous DLC activity. It is proposed that these compounds may regulate Na+, K+‐ATPase activity in the lens under some physiological and pathological conditions.
Isolation and identification of two proto‐oncogene products related to c‐fgr and fyn in a tyrosine‐protein‐kinase fraction of rat spleen
Authors: BRUNATI Anna Maria , JAMES Peter , DONELLA‐DEANA Arianna , MATOSKOVA Brona , ROBBINS Keith C. , PINNA Lorenzo A.
Published: March 2005

TPK‐III, a tyrosine‐protein‐kinase fraction previously isolated from rat spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451–457] has been further purified and resolved by Mono‐Q FPLC into two homogeneous compounds, Q1 and Q2, with molecular mass approximately 52 kDa, exhibiting high specific activities for phosphate incorporation into the phosphoacceptor substrate poly(Glu80Tyr20) (1194 nmol · min−1· mg−1 and 579 nmol · min−1· mg−1, respectively). Both Q1 and Q2 appear to be scr‐related enzymes since they are readily recognised by anti‐SEEP serum raised against the highly conserved segment at positions 330–345 of p60c‐scr. Q1, but not Q2, interacts with a specific antibody raised against the N‐terminal segment of p55c‐fgr. Microse‐quence analysis of tryptic fragments generated from Q1 revealed five peptides which exactly overlap the expected segments of p55c‐fgr. Two of these peptides were not entirely conserved in any of the other src‐related tyrosine protein kinases. A sixth fragment is very similar, albeit not identical, to the C‐terminus of p55c‐fgr.

Microsequence analysis of two tryptic fragments from the other TPK‐III fraction, Q2, provided the sequences FQILNSSE and LTTQETGYIPSNY, which are identical to two segments of fyn not entirely conserved in any of the other src‐related tyrosine protein kinases.

These results provide evidence that the spleen tyrosine‐protein‐kinase fraction, conventionally designated TPK‐III, is composed of products from two proto‐oncogenes, one of which corresponds to fyn, while the other is either identical or very closely related to c‐fgr.
Molecular cloning and expression of mouse and human cDNA encoding AES and ESG proteins with strong similarity to Drosophila enhancer of split groucho protein
Authors: MIYASAKA Hitoshi , CHOUDHURY Barun K. , HOU Esther W. , LI Steven S.‐L.
Published: March 2005

Mouse and human cDNA encoding AES (amino‐terminal enhancer of split) and ESG (enhancer of split groucho) proteins with strong similarity to Drosophila enhancer of split groucho protein were isolated and sequenced. Mouse AES‐1 and AES‐2 proteins, probably resulting from alternative splicing, contain 202 and 196 amino acids, respectively, while mouse ESG protein consists of 771 amino acids. The amino acid sequences of mouse and human AES proteins were found to exhibit approximately 50% identity to the amino‐terminal region of Drosophila groucho, mouse ESG and human transducin‐like enhancer of split (TLE) proteins. Mouse AES transcripts of 1.5 kb and 1.2 kb were abundantly expressed in muscle, heart and brain. Human AES transcripts of 1.6 kb and 1.4 kb were predominantly present in muscle, heart and placenta. Mouse ESG (homolog of human TLE 3) transcripts of 3.3 kb and 4.0 kb were found only in testis, while human TLE 1 transcripts of 4.5 kb was more abundant in muscle and placenta compared to heart, brain, lung, liver, kidney and pancreas. Human AES, TLE 1 and TLE 3 genes were mapped to chromosomes 19, 9 and 15, respectively, using human and Chinese hamster hybrid cell lines.
A universal method for achieving increases in metabolite production
Authors: KACSER Henrik , ACERENZA Luis
Published: March 2005

To increase the in vivo output of a chosen metabolite requires the increase of a number of enzyme concentrations. The enzymes are identified as those leading directly from output back to the input(s). This identification will reveal a number of branch points leading to other parts of metabolism which should not be perturbed. A simple calculation gives the enzyme multipliers, factors by which the identified enzyme concentrations should be increased to generate a given increase in the output flux. The net result of this procedure will be to extract any desired increase in flux while leaving the rest of metabolism to growth and other important functions of the cell unchanged. The method is entirely general.
A modified uridine in the first position of the anticodon of a minor species of arginine tRNA, the argU gene product, from Escherichia coli
Authors: SAKAMOTO Kensaku , KAWAI Gota , NIIMI Tatsuya , SATOH Takahiko , SEKINE Mitsuo , YAMAIZUMI Ziro , NISHIMURA Susumu , MIYAZAWA Tatsuo , YOKOYAMA Shigeyuki
Published: March 2005

The argU (dnaY) gene product, a minor tRNAArg, from Escherichia coii has the anticodon N*CU with an unidentified modified nucleoside N* in position 34 [Kiesewetter, S., Fisher, W. & Sprinzl, M. (1987) Nucleic Acids Res. 15, 3184]. In the present study, argU tRNA was purified from E. coli A 19 strain and nucleoside N* was characterized by the TLC and HPLC analyses. Nucleoside N* was found to be different from any naturally occurring modified nucleosides. From unfractionated E. coli tRNA species, nucleoside N* was prepared in an amount sufficient for 1H‐NMR experiments. By the analyses of one‐dimensional and two‐dimensional NMR spectra, nucleoside N* was suggested to be 5‐methylaminomethyluridine (mnm5U), which was confirmed by comparison with a chemically synthesized preparation of mnm5U. Thus, the occurrence of mnm5U in mature tRNA was found for the first time. Further, the modification of U(34) to mnm5U in this tRNA was found to contribute to the strict recognition of two degenerate codons terminating in A and G.
Solution structures of the lantibiotics duramycin B and C
Authors: ZIMMERMANN Norbert , FREUND Stefan , FREDENHAGEN Andreas , JUNG Günther
Published: March 2005

The solution structures of the lantibiotics duramycin B in H2O/2H2O (9:1) and duramycin C in (2H3)acetonitrile/H2O (1:1) have been determined by NMR followed by distance‐geometry and restrained‐molecular‐mechanics calculations. The constitution and location of three thioether bridges and a lysinoalanine ring system could be established by unambiguously assigned NOE contacts between the bridging side chains. Model building based on NMR constraints resulted in a U‐shaped topology of the tetracyclic 19‐peptides with a turn around Pro9 and a kink along a virtual line from residues 5 to 13. This clamp‐like conformation is stabilized by the thioether bridges and is additionally supported by an antiparallel ß‐strand‐like structure of the N‐termini and C‐termini and the inherent amphiphilicity of duramycin‐type lantibiotics. The duramycins B and C differ mainly in the relative mobilities of their rings A, C and D. Duramycin B is closely related to cinnamycin with an exchange of Phe 10 to leucine, whereas duramycin C differs from duramycin B by three conserved and two non‐conserved amino‐acid exchanges.
Sequences in the rev‐responsive element responible for premature translational arrest in the human‐immunodeficiency‐virus‐type‐1 envelope
Authors: ELLERBROK Heinz , SERPENTE Norberto , PANCINO Gianfranco , VANHÉE Christine , D'AURIOL Luc , SITBON Marc , VAQUERO Catherine
Published: March 2005

Cell‐free translation in the presence of pancreatic microsomal membranes of the full‐lenght envelope transcript of the human immunodeficiency virus type 1 (HIV‐1) yielded the expected extensively glycosylated and immunologically reactive gp 160 envelope‐protein precursor. In addition to this gp160, a shorter glycoprotein, which we designated gp120*, was produced due to a premature translation arrest. Utilizing kinetic experiments, pulse‐chase and analyses and various gp160 envelope RNA mutants, we demonstrated that the in‐vitro‐produced gp120* was not formed by cleavage of the gp160 precursor or by internal initiation of translation. A gp120 produced before gp160 synthesis was completed, and, independent of the gp160 proteolytic processing, has been shown to be produced and sequestered in the endoplasmic reticulum of HIV‐1‐infected cells [Willey, R. L., Klimkait, T., Frucht, D. M., Bonifacino, J. S. & Martin, M. A. (1991) Virology 184, 319–329]. The specific translational arrest shown to occur in vitro was found to be dependent on the Rev‐responsive element, since deletion of this highly structured sequence abolished the production of gp120*. We found that the combination of two contiguous putative stem loops of the Rev‐responsive element, located at nucleotides 7494–7522 and 7525–7550 of the HIV‐1 Rev‐responsive‐element sequence, was responsible for the production of this truncated protein. To our knowledge, these stem‐loop structures, distinct from that known to bind the Rev protein, represent the first example responsible for the production of alternative products by premature translational arrest in higher eukarytes.
Cloning and nucleotide sequence of the gcv operon encoding the Escherichia coli glycine‐cleavage system
Authors: OKAMURA‐IKEDA Kazuko , OHMURA Yosuke , FUJIWARA Kazuko , MOTOKAWA Yutaro
Published: March 2005

P‐protein, H‐protein and T‐protein of the glycine cleavage system have been purified from Escherichia coli. Their N‐terminal amino acid sequences were determined, and a set of oligonucleotide probes was designed for gene cloning. The nucleotide sequence of a fragment of DNA around the 62‐min region of the E. coli chromosome, containing genes for the components of the glycinecleavage system has been determined. The sequence includes three structural genes encoding T‐protein (363 amino acid, 40013 Da), H‐protein (128 amino acids, 13679 Da) and P‐protein (956 amino acids, 104240 Da). These genes are named gcvT, gcvH and gcvP, respectively. They are organized in the above‐mentioned order on the same strand of DNA with short intercistronic sequences. The presence of a potential promoter preceding gcvT and a typical rho‐independent terminator sequence following gcvP indicated that the three genes constitute a single operon. Each component of the E. coli glycine‐cleavage system exhibits considerable amino acid sequence similarity with the animal and plant counterparts. When the plasmid containing the gcv operon was transfected in E. coli cells, the gene products of gcvT, gcvH and gcvP were overexpressed under the direction of the promoter of the gcv operon. However, bacteria harboring the plasmid that contained the gcv operon without the promoter region and the 5′ terminal portion of gcvT failed to overexpress any of the three components.
The electrochemical‐proton‐gradient‐activated states of F0F1 ATPase in plant mitochondria as revealed by detergents
Authors: VALERIO Marie , DIOLEZ Philippe , HARAUX Francis
Published: March 2005

ATP hydrolysis, triggered by the addition of polyoxyethylene‐9‐lauryl ether (Lubrol) or lauryldimethylamine oxide (LDAO) to energized plant mitochondria was studied in some details. The membrane disruption was quasi‐instantaneous (2–3 s) with both detergents, as shown by the decrease of turbidity and the stopping of respiration. In pea leaf mitochondria, Lubrol triggered ATP hydrolysis in almost the same way as valinomycin plus nigericin, except that the activity was slightly stimulated and became insensitive to carboxyatractyloside. This allowed investigations of ATP hydrolysis without any interference of the ATP/ADP antiporter or the phosphate carrier. Lubrol did not prevent the ATPase from deactivating in pea leaf mitochondria, and did not trigger any ATP hydrolysis in potato tuber mitochondria. At variance with Lubrol, LDAO changed the properties of the F0F1 ATPase. It made the enzyme oligomycin insensitive and froze it in an activated state. The activity was also 5–8‐times stimulated in pea leaf mitochondria. Moreover, LDAO revealed an important ATP hydrolase activity when added to energized potato tuber mitochondria. Despite the specific effect of LDAO, the activity triggered by this detergent strongly depended on the energized state of the organelles before detergent addition.

From this study, it is concluded that the electrochemical proton gradient is completely necessary to activate the F0F1‐ATPase in intact plant mitochondria, as known in chloroplasts and suggested by some reports in animal mitochondria. Moreover, it is suggested that the main difference between the enzymes of pea leaf and potato tuber mitochondria is their rate of deactivation after the collapse of the transmembrane electrochemical potential difference. Finally, when properly used, detergents appear to be a powerful tool to probe the state of the ATPase in intact mitochondria, and maybe in more integrated systems.