bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quinoproteins: enzymes containing the quinonoid cofactor pyrroloquinoline quinone, topaquinone or tryptophan‐tryptophan quinone
Authors: DUINE Johannis A. 
Published: March 2005

The presently best known and largest group of quinoproteins consists of enzymes using the cofactor 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐f]quinoline‐4,5‐dione (PQQ), a compound having a pyrrole ring fused to a quinoline ring with an o‐quinone group in it. Representatives of this group are found among the bacterial, NAD(P)‐independent, periplasmic dehydrogenases. Despite their high midpoint redox potential, the overall behaviour of quinoprotein dehydrogenases is similar to that of their counterparts, those using a flavin cofactor or a nicotinamide coenzyme. Apart from an exceptional Gram‐positive one, the sole organisms where the presence of PQQ has really been established are Gram‐negative bacteria. Evidence for the occurrence of covalently bound PQQ is lacking since it has now been shown that several enzymes previously considered to contain this prosthetic group do not in fact do so.Another group of quinoproteins, consisting of amine oxidoreductases, has a protein chain containing one of the following quinonoid aromatic amino acids: 6‐hydroxy‐phenylalanine‐3,4‐dione (TPQ) or 4‐(2′‐tryptophyl)‐tryptophan‐6,7‐dione (TTQ). There is no doubt that these o‐quinones play a role as cofactor, in the case of TPQ in prokaryotic as well as eukaryotic amine oxidases. It appears, therefore, that a novel calass of amino‐acid‐derived cofactors is emerging, ranging from the free radical form of tyrosine and tryptophan to those containing a dicarbonyl group (like the already known pyryvoyl group and the o‐quinones here described).
Glycosphingolipid specifity of the human sulfatide activator protein
Authors: VOGEL A. , SCHWARZMANN G. , SANDHOFF K. 
Published: March 2005

The interaction of the sulfatide activator protein with different glycosphingolipids has been studied in detail. The following findings were made.1. The sulfatide activator protein forms water‐soluble complexes with sulfatides [Fischer, G. and Jatzkewitz, H. (1977) Hoppe‐Seyler's Z. Physiol. Chem. 356, 6588–6591] and various other glycosphingolipids.2. In the absence of degrading enzymes the activator protein acts in vitro as a glycosphingolipid transfer protein, transporting glycosphingolipids from donor to acceptor liposomes. Lipids having less than three hexoses, e.g. galactosylceramide, sulfatide and ganglioside GM3 were transferred at very slow rates, whereas complex lipids such as gangliosides GM2, GM1 and GD1a were transferred much faster than the former. The transfer rate increased with increasing length of the carbohydrate chain of the lipid molecules.3. Both the acyl residue in the ceramide moiety and the nature of the carbohydrate chain are significant for recognition of the glycosphingolipids by the sulfatide activator protein. Apparently, both residues serve as an anchor and the longer they are the better they are recognized by the protein.4. In the absence of activator protein, degradation rates of sulfatide derivatives by arylsulfatase A, and of ganglioside GM1 derivatives by β‐galactosidase, increase with decreasing length of acyl residues in their hydrophobic ceramide moiety. Addition of activator protein stimulates the degradation of only those GM1 and sulfatide derivatives that have long‐chain fatty acids in their hydrophobic ceramide anchor.
Protein‐kinase‐C‐independent activation of arachidonate release and prostaglandin E2 formation in macrophages interacting with certain bacteria
Authors: SVENSSON Ulf , HOLST Elisabet , SUNDLER Roger 
Published: March 2005

Certain bacterial species, of which we selected Fusobacterium nucleatum, Gardnerella vaginalis, Peptostreptococcus anaerobius and Propionibacterium acnes, were found to induce release of arachidonic acid in a dose‐ and time‐dependent manner in mouse macrophages. The release of arachidonic acid showed a characteristic lag period of approximately 10 min and was accompanied by selective transformation into prostaglandin E2. Bacteria killed by various methods caused a similar response, indicating that bacteiral surface structures rather than secreted products were involved. Down‐regulation of protein kinase C by treatment of macrophages with 4β‐phorbol 12‐myristate 13‐acetate hardly affected the response to bacteria at all, except for a partial inhibition in the case of P. acnes. Furthermore, the generation of prostaglandin E2 was synergistically enhanced when macrophages were exposed to both bacteria and phorbol ester. It is also unlikely that bacterial activation was mediated exclusively via a rise in cytosolic [Ca2+], since bacteria stimulated the release of arachidonic acid also when [Ca2+] was clamped at various levels and since the response to bacteria was enhanced in an additive to synergistic manner when combined with calcium ionophore.Changes in protein phosphorylation in macrophages exposed to F. nucleatum (Gram‐negative) were virtually identical to those seen with bacterial lipopolysaccharide, while P. anaerobius (Gram‐positive) induced enhanced labeling of a single detectable phosphoprotein. In both cases, the changes in protein phosphorylation showed a time lag of 4–8 min and occurred independently of protein kinase C, consistent with a possible role in signal transduction. These results demonstrate that certain bacteria cause activation of arachidonic acid release and prostaglandin E2 formation in mouse macrophages; that the response is independent of protein kinase C and that it is not wholly mediated via a rise in cytosolic [Ca2+].
NMR studies on p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida
Authors: VERVOORT Jacques , VAN BERKEL Willem J. H. , MÜLLER Franz , MOONEN Chrit T. W. 
Published: March 2005

p‐Hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida have been reconstituted with 13C‐ and 15N‐enriched FAD. The protein preparations were studied by 13C‐NMR, 15N‐NMR and 31P‐NMR techniques in the oxidized and in the two‐electron‐reduced states. The chemical shift values are compared with those of free flavin in water or chloroform. It is shown that the π electron distribution in oxidized free p‐hydroxybenzoate hydroxylase is comparable to free flavin in water, and it is therefore suggested that the flavin ring is solvent accessible. Addition of substrate has a strong effect on several resonances, e. g. C2 and N5, which indicates that the flavin ring becomes shielded from solvent and also that a conformational change occurs involving the positive pole of an α‐helix microdipole.In the reduced state, the flavin in p‐hydroxybenzoate hydroxylase is bound in the anionic form, i. e. carrying a negative charge at N1. The flavin is bound in a more planar configuration than when free in solution. Upon binding of substrate the resonances of N1, C10a and N10 shift upfield. It is suggested that these upfield shifts are the result of a conformational change similar, but not identical, to the one observed in the oxidized state.The 13C chemical shifts of FAD bound to apo(salicylate hydroxylase) indicate that in the oxidized state the flavin ring is also fairly solvent accessible in the free enzyme. Addition of substrate has a strong effect on the hydrogen bond formed with O4α. It is suggested that this is due to the exclusion of water from the active site by the binding of substrate. In the reduced state, the flavin is anionic. Addition of substrate forces the flavin ring to adopt a more planar configuration, i.e. a sp2‐hybridized N5 atom and a slightly sp3‐hybridized N10 atom. The NMR results are discussed in relation to the reaction catalyzed by the enzymes.
Pterocarpan phytoalexin biosynthesis in elicitor‐challenged chickpea (Cicer arietinum L.) cell cultures
Authors: TIEMANN Karin , INZÉ Dirk , VAN MONTAGU Marc , BARZ Wolfgang 
Published: March 2005

NADPH:isoflavone oxidoreductase (IFR) is the first soluble enzyme of the pterocarpan‐specific part of phytoalexin biosynthesis in chickpea (Cicer arietinum L.). The enzyme was purified to apparent homogeneity by a five‐step procedure from chickpea cell cultures treated with yeast extract as elicitor. Analysis by gel filtration and SDS/PAGE showed that the enzyme consists of a single polypeptide with a molecular mass of 36 kDa. Km values for the substrates 2′‐hydroxyformononetin, 2′‐hydroxypseudobaptigenin and NADPH were 6, 6 and 20 μM, respectively. The IFR showed pronounced specificity for the substitution pattern of isoflavones. We found a 2′‐hydroxy group and a 4′,5′‐methylenedioxy or 4′‐methoxy function to be essential for acceptance as substrate. The isoelectric point of the protein was determined as 6.3 by IEF and there is no evidence for the existence of isoenzymes.Partial amino acid sequences of IFR were determined from internal peptides obtained by tryptic digestion of the protein and corresponding oligonucleotides were synthesized. A λgt10 cDNA library was constructed using poly(A)‐rich RNA isolated from chickpea cell cultures treated with Ascochyta rabiei elicitor. 150 positive clones were obtained by screening 2 × 105 clones with an IFR‐specific oligonucleotide. The identity of sequenced clones was confirmed by comparison of the deduced amino acid sequence with the internal peptide sequences of purified IFR. The sequence of a 1183‐bp clone contained a continuous open reading frame of 954 bases encoding a polypeptide of 318 amino acids with a calculated molecular mass of 35.4 kDa, indicating that a full‐length cDNA coding for IFR was isolated.
Mathematical analysis of enzymic reaction systems using optimization principles
Authors: HEINRICH Reinhart , SCHUSTER Stefan , HOLZHÜTTER Hermann‐Georg 
Published: March 2005

ContentsIntroductionOptimization of the catalytic properties of single enzymesIrreversible two‐step kinetic mechanismReversible three‐step kinetic mechanismCooperative enzymesOptimization of multienzyme systemsBasic equations for unbranched reaction chainsMaximization ofsteady‐state fluxesTransient timesSensitivity to external signalsImplications of the solvent capacity and osmotic balanceMetabolic channellingThermodynamic principlesOptimal stoichiometriesTime hierarchy in biochemical networksFinal remarks
The lux gene of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish
Authors: LEE Chan Yong , SZITTNER Rose B. , MEIGHEN Edward A. 
Published: March 2005

The lux genes required for light expression in the luminescent bacterium Photobacterium leiognathi (ATCC 25521) have been cloned and expressed in Escherichia coli and their organization and nucleotide sequence determined. Transformation of a recombinant 9.5‐kbp chromosomal DNA fragment of P. leiognathi into an E. coli mutant (43R) gave luminescent colonies that were as bright as those of the parental strain. Moreover, expression of the lux genes in the mutant E. coli was strong enough so that not only were high levels of luciferase detected in crude extracts, but the fatty‐acid reductase activity responsible for synthesis of the aldehyde substrate for the luminescent reaction could readily be measured.Determination of the 7.3‐kbp nucleotide sequence of P. leiognathi DNA, including the genes for luciferase (luxAB) and fatty‐acid reductase (luxCDE) as well as a new lux gene (luxG) found recently in luminescent Vibrio species, showed that the order of the lux genes was luxCDABEG. Moreover, luxF, a gene homologous to luxB and located between luxB and luxE in Photobacterium but not Vibrio strains, was absent. In spite of this different lux gene organization, an intergenic stem‐loop structure between luxB and luxE was discovered to be highly conserved in other Photobacterium species after luxF.
Microcalorimetric investigation of the interactions between the subunits of the bovine pancreatic procarboxypeptidase A‐S6 complex
Authors: MICHON Thierry , SARI Jean‐Claude , GRANON Simone , KERFELEC Brigitte , CHAPUS Catherine 
Published: March 2005

A complete microcalorimetric investigation of the interactions between the native subunits of the bovine pancreatic procarboxypeptidase A‐S6 ternary complex has been performed. All the association constants and thermodynamic parameters associated with the reactions forming the various complexes have been determined.The influence of pH and ionic strength on the binding reactions has been investigated. Interestingly, the affinity between the subunits is not significantly modified by varying the ionic strength. In this respect, an enthalpy/ entropy compensatory effect is observed for the binding of subunit III to subunit I when the ionic strength is increased, suggesting a physiological function for the association.The various pathways for formation of the ternary complex have been studied. Binding of subunit II (or III) to subunit I, the central element of the ternary complex, does not significantly modify the affinity of the other subunit for subunit I. From a thermodynamic point of view, the same final state is obtained whatever the pathway of ternary complex formation.This study is the first step of a kinetic investigation of the associated subunits.
Cloning and expression of human ciliary neurotrophic factor
Authors: NEGRO Alessandro , TOLOSANO Emanuela , SKAPER Stephen D. , MARTINI Irene , CALLEGARO Lanfranco , SILENGO Lorenzo , FIORINI Ferdinando , ALTRUDA Fiorella 
Published: March 2005

Ciliary neurotrophic factor (CNTF) is a survival factor for avian ciliary ganglion neurons and a variety of other neuronal cell types in vitro. We report here the cloning of the entire genomic sequence encoding human CNTF and its primary structure.Biologically active CNTF has been expressed in Chinese hamster ovary cells from a human genomic DNA clone. Human CNTF has no significant sequence similarity to any previously reported protein, although approximately 84% similarity exists compared with rat and rabbit CNTF. The lack of both an N‐terminal signal sequence and consensus sequences for glycosylation or hydrophobic regions, and the fact that active CNTF is expressed but not released into the culture medium of transfected cells, argue in favour of human CNTF as a cytosolic protein. These data provide a basis for understanding the role of CNTF in nervous system physiology and pathology.
Malic enzyme in human liver
Authors: ŻELEWSKI Mateusz , ŚWIERCZYŃSKI Julian 
Published: March 2005

In human liver, almost 90% of malic enzyme activity is located within the extramitochondrial compartment, and only approximately 10% in the mitochondrial fraction.Extramitochondrial malic enzyme has been isolated from the post‐mitochondrial supernatant of human liver by (NH4)2SO4 fractionation, chromatography of DEAE‐cellulose, ADP–Sepharose‐4B and Sephacryl S‐300 to apparent homogenity, as judged from polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was 56 μmol · min−1· mg protein−1, which corresponds to about 10000‐fold purification.The molecular mass of the native enzyme determined by gel filtration is 251 kDa. SDS/polyacrylamide gel electrophoresis showed one polypeptide band of molecular mass 63 kDa. Thus, it appears that the native protein is tetramer composed of identical‐molecular‐mass subunits.The isoelectric point of the isolated enzyme was 5.65.The enzyme was shown to carboxylate pyruvate with at least the same rate as the forward reaction.The optimum pH for the carboxylation reaction was at pH 7.25 and that for the NADP‐linked decarboxylation reaction varied with malate concentration.TheKm values determined at pH 7.2 for malate and NADP were 120 μM and 9.2 μM, respectively. TheKm values for pyruvate, NADPH and bicarbonate were 5.9 mM, 5.3 μM and 27.9 mM, respectively.The enzyme converted malate to pyruvate (at optimum pH 6.4) in the presence of 10 mM NAD at approximately 40% of the maximum rate with NADP. TheKm values for malate and NAD were 0.96 mM and 4.6 mM, respectively. NAD‐dependent decarboxylation reaction was not reversible.The purified human liver malic enzyme catalyzed decarboxylation of oxaloacetete and NADPH‐linked reduction of pyruvate at about 1.3% and 5.4% of the maximum rate of NADP‐linked oxidative decarboxylation of malate, respectively.The results indicate that malic enzyme from human liver exhibits similar properties to the enzyme from animal liver.
The catalytic domain of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex From Azotobacter vinelandii and Escherichia coli
Authors: SCHULZE Egbert , WESTPHAL Adrie H. , OBMOLOVA Galya , MATTEVI Andrea , HOL Wim G. J. , de KOK Arie 
Published: March 2005

Partial sequences of the dihydrolipoyl transacetylase componenent (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli, containing the catalytic domain, were cloned in pUC plasmids and over‐expressed in E. coli TG2. A high expression of a homogeneous protein was only detectable for E2p mutants consisting of the catalytic domain and the alanine‐proline‐rich sequence between a putative binding region for the peripheral components and the catalytic domain (apa‐4). Most of the catalytic domain from A. vinelandii without the apa‐4 sequence was degraded intracellularly, probably due to incorrect folding.Fusion proteins of six amino acids from β‐galactosidase, the apa‐4 region and the catalytic domains of A. vinelandii or E. coli E2p could be highly purified. Both catalytic domains were assembled in 24‐subunit structures with a molecular mass of approximately 670 kDa. The expression of catalytic domain from A. vinelandii E2p is more than twice as high as found for wild‐type E2p. This can be explained by intracellular degradation of overexpressed wild‐type E2p, whereas the catalytic domains are stable against proteolysis in vivo and in vitro.The interaction of the peripheral components pyruvate dehydrogenase (E1p) and dihydrolipoamide dehydrogenase (E3) with the catalytic domains was studied, using gel filtration on Superose‐6 and sedimentation velocity experiments. No binding of either E1p or E3 to the catalytic domain of either organism was detectable.Crystals of the catalytic domain of A. vinelandii E2p could be grown to a maximum size of 0.6 × 0.6 × 0.4 mm. They diffract up to a resolution of 0.28 nm.
Purification and characteriation of 3‐hydroxyphenylacetate 6‐hydroxylase: a novel FAD‐dependent monooxygenase from a Flavobacterium species
Authors: VAN BERKEL Willem J. H. , VAN DEN TWEEL Will J. J. 
Published: March 2005

3‐Hydroxyphenylacetate 6‐hyroxylase was purified 70‐fold from a Flavobacterium sp. grown upon phenylactic acid as its sole carbon and energy source. The presence of FAD and dithiothreitol during purification is essential for high recovery of active enzyme.SDS/PAGE of purified enzyme reveals a single band with a minimum molecuarl mass of 63 KDa. Analytical gel‐filtration, sedimentation‐equilibrium and sedimentation‐velocity experiments indicate that the purified enzyme exists in solultion manily as a dimer, containing 1 molecule non‐covalently bound FAD/sununit.3‐Hydroxyphenylacetate 6‐hydroxylase utilizes NADH and NADPH as external electron donors with similar efficiency. The enzyme shows a narrow substrate specificity. Only the primary substrate 3‐hydroxyphenylacetate is hydroxylated efficiently, yielding 2.5‐dihydroxyphenylacetate as a product. During turnover, the substrate analogues 3,4‐dihydroxyphenylacetate and 4‐hydroxyphenylacetate are partially hydroxylated, exculsively at the 6′ (2′) positition. The physiological product 2.5‐dihydroxyphenylacetate acts as an effector, strongly stimulating NAD(P)H oxidation. The activity of 3‐hydroxyphenylacetate 6‐hydroxylase is severely inhibited by chloride ions, competitive to the aromatic substrate.In the native state of enzyme, two sulfhydryl groups are accessible to 5.5′‐dithiobis(2‐nitrobenzoate). Titration with Stoichiometric amounts of either 5.5′‐dithiobis(2‐nitrobenzoate) or mercurial reagents completely blocks enzyme activity. Inactivation by cysteine reagents is inhibited by the substrate 3‐hydroxyphenylacetate. The original activity is fully restored by treatment of the modified enzyme with dithiothreitol.The N‐terminal amino acid sequence of the enzyme lacks the consenusus sequence GXGXXG, found at the termini of all faavin‐dependent external mnooxygenses sequenced so far. The amino acid composition of 3‐hydroxyphenylacetate 6‐hydroxylase is also presented.
Effect of retinoic acid on the synthesis of tissue‐type plasminogen activator and plasminogen activator inhibitor‐1 in human endothelial cells
Authors: THOMPSON E. Anne , NELLES Luc , COLLEN Désiré 
Published: March 2005

The synthesis of plasminogen activators and inhibitors in endothelial cells is highly regulated by hormones, drugs and growth factors. The present study evaluates the effect of retinoic acid on the synthesis of tissue‐type plasminogen activator (t‐PA) and of plasminogen activator inhibitor‐1 (PAI‐1) by cultured human umbilical vein endothelial cells (HUVEC).Retinoic acid produced a time‐ and concentration‐dependent increase in the secretion of t‐PA‐related antigen but not of PAI‐1 related antigen into the culture medium. A maximal sevenfold increase of t‐PA antigen after 24 h was observed with 10 μM and a half‐maximal increase with 0.1 μM retinoic acid. Retinoic acid induced a time‐dependent increase of the t‐PA mRNA, with a maximum at 8 h and returning to normal at 24 h.The protein kinase inhibitor H7 decreased the t‐PA antigen induced by both retinoic acid and phorbol 12‐myristate 13‐acetate. These results suggest that treatment of HUVEC with retinoic acid increases t‐PA production by a pathway which, at some level, involves protein kinases. Thus, retinoic acid induces t‐PA synthesis in the absence of altered PAI‐1 synthesis, which may enhance the fibrinolytic potential of the endothelium.
1,25‐Dihydroxycholecalciferol‐related Na+/D‐glucose transport in brush‐border membrane vesicles from embryonic chick jejunum
Authors: DEBIEC Hanna , CROSS Heide S. , PETERLIK Meinrad 
Published: March 2005

1,25‐Dihydroxycholecalciferol, when present at and above 10nM in an organ‐culture system of embryonic chick jejunum, approximately doubled the rate of Na+ ‐gradient‐driven D‐glucose uptake by brush‐border membrane vesicles, but had no effect on Na+ ‐independent D‐glucose transfer. The sterol also had no effet on Na+ influx along an outside/inside Na+ gradient ([Na+]o= 100mM; [Na+]i= 0 mM). This renders it unlikely that in embryonic intestine, calcitriol raises Na+ ‐dependent D‐glucose transport through changes in the electrochemical Na+ gradient. D‐[U‐14C] Glucose tracer exchange, measured under voltage‐clamp condition at Na+/D‐glucose equilibrium, revealed that addition of calcitriol to the culture medium approximately doubled the activity of the Na+/D‐glucose transporter in the brush‐border membrane. This was also reflected by an corresponding increase in the maximal velocity of the transfer process. Increased [3H]phlorizin binding after calcitriol treatment suggests that the steroid hormone activates Na+/D‐glucose transport through increasing the number of carrier molecules in the brush‐border membrane.10 nM triiodothyronine, which by itself has no effect on Na+ ‐dependent D‐glucose transport, potentiated the effect of 1,25‐dihydroxycholecalciferol such that in the presence of both hormones, Na+/D‐glucose‐carrier activity was increased fourfold above control levels.