bluuebunny/crossref_metadata_2025_split huggingface dataset:
The membrane‐bound hydrogenase from Paracoccus denitrificans
Authors: KNÜTTEL Karlheinz , SCHNEIDER Klaus , SCHLEGEL Hans Günter , MÜLLER Achim 
Published: March 2005

The membrane‐bound hydrogenase from Paracoccus dentitrificans was purified 68‐fold with a yield of 14.6%. The final preparation had a specific activity of 161.9 μmol H2 min−1 (mg protein)−1 (methylene blue reduction). Purification involved solubilzation by Triton X‐114, phase separation, chromatography on DEAE‐Sephacel, ammonium‐sulfate precipitation and chromatography on Procion‐red HE‐3B‐Sepharose. Gel electrophoresis under denatruing conditions revealed two non‐identical subunits with molecular masses of 64 kDa and 34 kDa. The molecular mass of the native enzyme was 100kDa, as estimated by FPLC gel filtration in the presence of Chaps, a zwitterionic detergent. The isoelectric point of the Paracoccus hydrogenase was 4.3. Metal analysis of the purified enzyme indicated a content of 0.6 nickel and 7.3 iron atoms/molecule. ESR spectra of the reduced enzyme exhibited a close similarity to the membrane‐bound hydrogenase from Alcaligenes eutrophus H16 with g values of 1.86, 1.92 and 1.98. The half‐life for inactivation under air at 200C was 8 h.The Praracoccus hydrogenase reduced several electron acceptors, namely methylene blue, benzyl viologen, methyl viologen, menadione, cytochrome c, FMN, 2,6‐dichloriondophenol, ferricyanide and phenazine methosulfate. The highest activity was measured with methylene blue (V= 161.9 U/mg; Km= 0.04 mM), whereas benzyl and methyl viologen were reduced at distinctly lower rates (16.5 U/mg and 12.1 U/mg, respectively).The native hydrogenase from P. denitrificans cross‐reacted with purified antibodies raised aginst the membrane‐bound hydrogense from A. eutrophus H16. The corresponding subunits from both enzymes also showed immunological relationship. All reactions were of partial identity.
Human plasma inter‐α‐trypsin inhibitor is encoded by four genes on three chromosomes
Authors: DIARRA‐MEHRPOUR Maryam , BOURGUIGNON Jeannette , SESBOÜE Richard , MATTEÏ Marie‐Geneviève , PASSAGE Edith , SALIER Jean‐Philippe , MARTIN Jean‐Pierre 
Published: March 2005

Human inter‐α‐trypsin inhibitor is a plasma protein of Mr 180000 which has long been described as a single polypeptide chain. However, we have previously demonstrated that it is synthesized in liver by two different mRNA populations coding for heavy or light polypeptide chains [Bourguignon, J. et al. (1983) FEBS Lett. 162, 379–383] and cDNA clones for the heavy or light chains have recently been isolated and characterized [Bourguignon, J. et al. (1985) Biochem. Biophys. Res. Commun. 131, 1146–1153; Salier, J. P. et al. (1987) Proc. Natl Acad. Sci. USA 84, 8272–8276].In the present study, we show that human poly(A)‐rich RNAs hybrid‐selected with various heavy‐chain‐encoding cDNA clones translate three different heavy chains, designated H1 (Mr 98000) and H3 (Mr 107000). We previously characterized two heavy‐chain cDNA clones. We now report that they correspond to H1 and H2 chains. We have also determined the sequence of an additional cDNA clone which codes for H3 chain. Its insert size is 1.79 kb with a single open reading frame and a poly(A) tail. The deduced amino acid sequence of the H3 chain is highly similar to those of the H1 (54%) and H2 (44%) chains. Northern analysis of human liver poly(A)‐rich RNAs with the three heavy‐chain cDNAs as probes clearly identified a single major mRNA population of 3.3 ± 0.1 kb.Chromosomal localization by in situ hybridization shows that inter‐α‐trypsin inhibitor genes are located on three different human chromosomes. The H1 and H3 genes are located in the p211–p212 region of chromosome 3, whereas the H2 gene resides in the p15 band of chromosome 10. The light‐chain gene is located in the q32–q33 region of chromosome 9.These results indicate that heavy and light chains of inter‐α‐trypsin inhibitor are encoded by at least four functional genes.
Carbohydrate structure of glycoprotein 65 encoded by the polycythemia‐inducing strain of Friend spleen focus‐forming virus
Authors: STRUBE Karl‐Hermann , GEYER Rudolf 
Published: March 2005

The secondary envelope‐gene product, glycoprotein 65 (gp65), of the polycythemia‐inducing variant of Friend spleen focus‐forming virus (F‐SFFVp) was isolated from F‐SFFVp‐infected normal rat kidney cells cultivated in the presence or absence (–Glc) of glucose. Oligosaccharide side chains present were sequentially liberated by treatment of tryptic glycopeptides with endo‐β‐N‐acetylglucosaminidase H and peptide N‐glycosidase F and fractionated by high‐performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, by chromatographic comparison with oligosaccharide standards and by methylation analysis.The results demonstrate that gp65 contains oligomannosidic, hybrid and N‐acetyllactosaminic glycans. The oligomannosidic glycans represent the same partially glucosylated species with six to nine mannose residues present in F‐SFFVp gp52, the biosynthetic precursor of gp65 [Strube, K.‐H. Schott, H.‐H. and Geyer, R. (1988) J. Biol. Chem. 263, 3762–3771]. Oligosaccharides of the hybrid type were found to comprise one sialylated lactosamine unit and three or four α‐linked mannose residues. Analysis of the N‐acetyllactosaminic glycans revealed that gp65 carries fucosylated, partially sialylated bi‐antennary, tri‐antennary and tetra‐antennary oligosaccharides, in addition to incomplete species. The glycosylation of gp65(–Glc) is characterized by the presence of oligomannosidic glycans with five to nine mannose residues, similar hybrid‐type species and by increased amounts of incomplete N‐acetyllactosaminic oligosaccharides, a decrease in sialylation and the lack of tetra‐antennary species.
Effect of papain digestion on polypeptide subunits and electron‐transfer pathways in mitochondrial b‐c1 complex
Authors: LORUSSO Michele , COCCO Tiziana , BOFFOLI Domenico , GATTI Domenico , MEINHARDT Steven , OHNISHI Tomoko , PAPA Sergio 
Published: March 2005

Papain digestion of subunits of mitochondrial b‐c1 complex (ubiquinol–cytochrome‐c reductase) isolated from bovine heart and its impact on redox and proton‐motive activity of the whole complex were investigated. A 5‐min incubation of the oxidized enzyme with papain resulted in digestion of core protein II and the 14‐kDa subunit, and limited digestion of the iron‐sulfur protein. This was accompanied by a small inhibition of the rate of electron flow and a marked inhibition of proton translocation with decrease of the H+/e− ratio for proton pumping. When papain treatment was performed on the b‐c1 complex pre‐reduced with ascorbate, partial proteolysis of the iron‐sulfur protein and the 14‐kDa subunit was greatly accelerated and the electron transfer activity was more markedly inhibited. In all the conditions tested, digestion of the Rieske iron‐sulfur protein paralleled the inhibition of reductase activity. Under ascorbate‐reduced conditions, papain digestion of the complex gave rise to an alteration of the EPR line shape of the iron‐sulfur cluster, namely a broadening and shift of the gx negative peak and destabilization of the protein‐bound antimycin‐sensitive semiquinone. The latter paralleled the decrease in electron transfer activity and inhibition of antimycin‐sensitive cytochrome‐b reduction. The results obtained indicate the following.1. Core protein II and the 14‐kDa protein may contribute to the proton‐conducting pathway(s) from the matrix aqueous phase to the primary protolytic redox center (protein‐bound semiquinone/quinone couple).2. The iron‐sulfur protein contributes, together with other protein(s) (the 14‐kDa subunit), to the stabilization of the protein bound antimyein‐sensitive semiquinone species in a protein pocket in the complex.3. Reduction of the high‐potential redox centers induces a change in the quaternary structure of the complex which results in an enhanced surface exposure of segments of the 14‐kDa protein and the iron‐sulfur protein.
Purification and characterization of a methionine‐specific aminopeptidase from Salmonella typhimurium
Authors: WINGFIELD Paul , GRABER Pierre , TURCATTI Gerardo , MOVVA N. Rao , PELLETIER Michel , CRAIG Stewart , ROSE Keith , MILLER Charles G. 
Published: March 2005

An aminopeptidase specific for methionine (peptidase M) has been purified from wild‐type and mutant Salmonella typhimurium strains. Recombinant peptidase M was also purified from Escherichia coli. These preparations were characterized with respect to their physicochemical properties using analytical ultracentrifugation. SDS/PAGE, isoelectric focusing, titration curve analysis, amino acid analysis, N‐ and C‐terminal sequencing and various spectroscopic methods.Peptidase M activity is stimulated by Co2+, in agreement with previous studies using crude extracts of Salmonella. The purified preparations did not contain significant amounts of any metal. Enzymically important metal is loosely associated and lost during enzyme purification.Peptidase M was shown to contain seven free sulphydryl residues none of which are involved in either intraor inter‐molecular disulphide bonds. Most appear solvent‐accessible as evidenced by their reactivity under native conditions. Limited modification of the sulphydryl residues with either iodoacetamide or 5.5′‐dithiobis(2‐nitrobenzoic acid) led to inactivation. Several cysteines were shown to be labelled to various degrees by peptide mapping of inactivated S‐[14C]carboxymethylated protein. Whether cysteine modification affects enzymic activity directly (blocking an active site) or indirectly (by causing conformational change) remains to be established.
Primary structures of rat and bovine liver mitochondrial aldehyde dehydrogenases deduced from cDNA sequences
Authors: FARRÉS Jaume , GUAN Kun‐Liang , WEINER Henry 
Published: March 2005

The cDNA coding for rat liver mitochondrial aldehyde dehydrogenase was cloned and sequenced. It contained an open reading frame of 1557 bp. Of the deduced 519 amino acid residues, 19 were proposed to correspond to the signal peptide necessary to allow the protein to enter the mitochondria [Farrés, J., Guan, K.‐L. and Weiner, H. (1987) Biochem. Biophys. Res. Commun. 150, 1083–1087]. The sequence of the 500 amino acid residues comprising the mature subunit was 96% identical to that of the corresponding human liver mitochondrial enzyme.The longest cDNA isolated coding for the bovine liver enzyme contained sequence information corresponding to residues 72‐500 of the rat or human enzyme. The deduced protein sequence of these residues was 94% identical to those of the human enzyme.Over 60% of the bases in the coding region of the rat cDNA were G and C. These residues were clustered non‐randomly and two potentially stable stem‐loop structures could be calculated as existing in the mRNA. One would be found in the region coding for amino acid residues 64‐79; a similar secondary structure could be responsible for the existing truncated cDNAs from the bovine‐cDNA libraries. Comparisons with the amino acid sequences of other aldehyde dehydrogenases support the suggestion that the NAD‐binding domain may be located in the middle portion of the mature enzyme.
Neutral glycolipids of atherosclerotic plaques and unaffected human aorta tissue
Authors: PROKAZOVA Nina V. , MUKHIN Dmitry N. , OREKHOV Alexander N. , GLADKAYA Elena M. , VASILEVSKAYA Valentina V. , MIKHAILENKO Irina A. , SADOVSKAYA Valentina L. , BUSHUEV Valery N. , BERGELSON Lev D. 
Published: March 2005

The composition, structure and localization of neutral glycosphingolipids of human aorta taken from subjects who had died after myocardial infarction were studied. Individual glycosphingolipids were purified by high‐performance liquid chromatography and were characterized on the basis of their chromatographic mobility, carbohydrate composition, methylation analysis and by 1H‐NMR spectroscopy. The main aortic glycosphingolipids were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. Significant differences in the neutral glycosphingolipid composition of intima and media were detected.The neutral glycosphingolipid profile of medial plaques resembled that of unaffected media; however, significant differences were detected between intimal plaques and unaffected intima. Whereas the latter contained trihexosylceramide and globoside as the only neutral glycolipids, the intimal plaque glycolipids consisted mainly of glucosylceramide and also contained appreciable amounts of lactosylceramide which were completely absent in the unaffected intima. In comparison to intimal plaques, unaffected intima is characterized by a much higher content of cerebrosides terminating by β‐galactosyl residues which are known to interact with growth factors and other external stimuli.It thus seems possible that the proliferative activity of smooth muscle cells in atherosclerotic diseases is to some extent associated with their neutral glycolipid profile.
Transfection of hepatic genes into adult rat hepoatocytes in primary culture and their tissue‐specific expression
Authors: GINOT Frédéric , DECAUX Jean‐François , COGNET Mireille , BERBAR Tsouria , LEVRAT François , KAHN Axel , WEBER Anne 
Published: March 2005

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested: an optimal level of CAT activity can be obtained using a serum‐free, hormonally defined medium. Using these techniques, we have investigated the expression of liver‐specific genes transferred into hepatocytes. We show that the lv‐pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts.Moreover, the use of serum‐free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.
Study of the arrangement of the functional domains along the yeast cytoplasmic aspartyl‐tRNA synthetase
Authors: PREVOST Gilles , ERIANI Gilbert , KERN Daniel , DIRHEIMER Guy , GANGLOFF Jean 
Published: March 2005

Aspartyl‐tRNA synthetase from yeast (AspRS) was screened for functional domains by measuring the effect of two types of amino acid mutations on its catalytic properties: (a) insertion of a dipeptide or a tetrapeptide along the polypeptide chain, (b) deletion of various lenghts from the enzyme C‐terminal. It was shown that insertion mutations significantly affect the kinetic properties of AspRS only when occurring in the second quarter of the molecule and the two centrally located mutations even inactivate the enzyme completely. Analysis of kinetic data stronly suggests that, in fact, all the observed activity modifications result from alteration of the activation reaction rate constant, Kcat only. This led to the conclusion that the domain involved in aspartic acid activation should be located in the second quarter of the molecule.Furthermore, a deletion mutant with a modification of the last five amino acid residues was isolated. This mutant is fully active in the activation step, but has lost 80% of the wild‐type aminoacylation activity. This involvement of the C‐terminus in acylation implies that it has to be folded towards strategic regions of the enzyme, thus favouring conformations required for catalysis or maintaining the tRNA in a functional position.
Acridine dimers: influence of the intercalating ring and of the linking‐chain nature on the equilibrium and kinetic DNA‐binding parameters
Authors: MARKOVITS Judith , GARBAY‐JAUREGUIBERRY Christiane , ROQUES Bernard P. , Le PECQ Jean‐Bernard 
Published: March 2005

The rigidity of the linking chain of bifunctional intercalators in the ditercalinium series was shown to be critical for antitumor activity. In order to study the influence of the rigidity of the linking chain on the DNA‐binding properties of DNA bifunctional intercalators, fluorescent 9‐aminoacridine and 2‐methoxy‐6‐chloro‐9‐aminoacridine analogues with chains of variable rigidity were synthesized. 1H‐NMR studies show that the conformation of 9‐aminoacridine dimers is almost independent of the nature of the linking chain. A strong self‐stacking of the aromatic rings of the 2‐methoxy‐6‐chloro‐9‐aminoacridine is observed for dimers with flexible chains but not for those with rigid chains.All the dimers having a linking chain long enough to bisintercalate in DNA according to the excluded site model are indeed bisintercalators. The kinetic association constant of all monomers and dimers for poly‐[d(A‐T)] poly[d(A‐T)] are in the same range (2–4 × 107 M−1 s−1). The large increase of DNA binding affinity observed for the dimers is always associated with the expected decrease of the dissociation rate constant.The effect of chain rigidity and pH on the calf thymus DNA binding of 9‐aminoacridine and 2‐methoxy‐6‐chloro‐9‐aminoacridine dimers is quite different. In the series of 9‐aminoacridine the pKa of the dimers remains high and therefore no difference of DNA‐binding affinity is observed between pH 5 and 7.4. The rigidity of the linking chain does not significantly alter the DNA‐binding affinity.In the 2‐methoxy‐6‐chloro‐9‐aminoacridine series. the pKa of all dimers became smaller than the physiological pH and a dramatic decrease of DNA‐binding affinity is observed when the pH is increased from pH 5 to 7.4. This decrease appears significantly smaller for dimers with rigid chains. A similar dramatic decrease of binding affinity at pH 7.4 is not observed for poly[d(A‐T)] · poly[d(A‐T)]. This factor makes these dimers strongly specific for the alternating polymer at pH 7.4.
A human melanoma‐derived cell line (IGR39) with a very high number of vasoactive‐intestinal‐peptide (VIP) receptors
Authors: LUIS José , MARTIN Jean‐Michel , EL BATTARI Assou , REYNIER Max , MARVALDI Jacques , PICHON Jacques 
Published: March 2005

Using mono[125I]iodinated vasoactive intestinal peptide (125I‐VIP), a very high number of specific binding sites for VIP were identified at the surface of the human melanoma cell line IGR39. The Scatchard analysis of competitive displacement experiments between native VIP and 125I‐VIP was consistent with the existence of two classes of VIP‐binding sites. IGR39 cells possess 0.54 × 106 high‐affinity sites with a dissociation constant (Kd) of 0.66 nM and 1.3 × 106 sites of moderate affinity with a Kd of 4.7 nM. Pharmacological studies indicated that the order of potency in inhibiting 125I‐VIP binding of the VIP secretin family peptides was VIP Gt; peptide histidine methioninamide > human growth‐hormone‐releasing factor(1–44) > secretin. Glucagon has no effect on the binding of the labelled peptide.By means of photoaffinity labelling a polypeptide of Mr 63000 was characterized. The labelling of this species was completely abolished by native VIP. The order of potency of VIP‐related peptides in inhibiting 125I‐VIP cross‐linking to its receptor was the same as in the competition experiments. The glycoprotein nature of the VIP‐binding sites of IGR39 cells has been investigated by affinity chromatography on wheat‐germ‐agglutinin–Sepharose.
The G · C base‐pair preference of 2‐N‐methyl 9‐hydroxyellipticinium
Authors: SCHWALLER Marc‐Antoine , AUBARD Jean , AUCLAIR Christian , PAOLETTI Claude , DODIN Guy 
Published: March 2005

Among the DNA‐intercalating drugs in the ellipticinium series, 9‐hydroxy derivatives elicit the highest antitumoral properties. In water these drugs display a very low fluorescence quantum yield. Replacement of H2O by D2O partially restores the fluorescence of the ellipticinium chromophore. The possibility that such a proton‐exchange mechanism could be involved in a base‐recognizing process at the DNA level (and therefore be responsible for some base preference) was examined by direct fluorescence titration in deuterated buffer and DNA/drug fluorescence energy transfer. These experimental approaches provide mutually consistent results showing that the 9‐hydroxylated drug recognizes specific DNA sites that are not recognized by the non‐hydroxylated drug.When compared to 2‐N‐methyl ellipticinium, the 2‐N‐methyl 9‐hydroxyellipticinium presents: (1) higher binding constants for each DNA studied; (2) a base dependence of the fluorescence properties of the bound form (fluorescence increment upon DNA binding varying over 5–11); (3) a base dependence of its DNA affinity constants (1.1–3.3 × 106 M−1) and of its site size (exclusion parameters varying over 3.0‐4.4); (4) a base dependence of its energy transfer from DNA bases.Analysis of the binding data suggests that the 9‐hydroxyl group of 2‐N‐methyl ellipticinium is responsible for a G · C base‐pair preference, the preferred binding site being a doublet sequence of two adjacent G · C which could be flanked either by a additional G · C base pair or by an A · T base pair.
Substrate‐modulated reactions of putidamonooxin
Authors: WENDE Peter , BERNHARDT Frithjof‐Hans , PFLEGER Karl 
Published: March 2005

4‐Methoxybenzoate monooxygenase is fairly nonspecific. The enzyme system with putidamonooxin as its oxygen‐activating component catalyses:(a) O‐, S‐ and N‐demethylation; (b) the oxygenation of 4‐methylbenzoate and 4‐methylmercaptobenzoate, with formation of 4‐carboxybenzyl alcohol and 4‐carboxyphenylmethyl sulfoxide, respectively, and (c) attack of the aromatic ring of 4‐and 3‐hydroxybenzoate and 4‐aminobenzoate, yielding 3,4‐dihydroxybenzoate and 4‐amino‐3‐hydroxybenzoate, respectively.
Compounds which are bound by the active sites of putidamonooxin have two essential features in common: a planar aromatic ring system, and a free carboxyl group attached to it.
By a substrate‐modulated reaction putidamonooxin can be induced to function not only as a monoxygenase but also as a peroxotransferase, i.e. it incorporates both atoms of the activated oxygen molecule into a substrate molecule. This finding supports the hypothesis that a mesomeric state of the iron · peroxo complex, [FeO2]+, is indeed the active oxygenating species of putidamonooxin.
The lifetime of the ternary complex consisting of enzyme · iron‐peroxo‐complex · substrate is significantly prolonged by uncoupling and partially uncoupling substrates, except when it is inactivated by protonation of the iron · peroxo complex by a proton transported into the active sites by a special kind of substrate (i.e. isomers of monoaminobenzoate), with the direct formation of H2O2.
The lifetime of the active oxygen species is determined by (a) the rate of the oxygenation reaction in the presence of tight‐coupling substrates and (b) the rate of the oxygenation reaction as well as detoxification by the availability of a dissociable proton in the presence of partial uncoupling (and uncoupling) substrate analogues.
The rate of the oxygenation reaction depends on the lifetime of the active oxygen species, [FeO2]+, in the presence of partial uncoupling substrates.
The iron · peroxo complex attacks an aromatic ring system according to the empiric rules of electrophilic substitution, whereas the attack of aliphatic substituents at the aromatic ring is controlled by steric criteria.