bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of a highly phosphorylated troponin from bovine heart
Authors: BEIER Norbert , JAQUET Kornelia , SCHNACKERZ Klaus , HEILMEYER Ludwig M. G.
Published: March 2005

A modified procedure for isolation of troponin from bovine heart is described, which results in a stable and highly phosphorylated protein. 31P‐NMR spectra show up to four phosphoserine signals indicating that at least four serine residues of cardiac troponin are phosphorylated in the intact organ. The hydrodynamic parameters of phosphotroponin are almost identical to those previously published. Characteristically cardiac troponin shows a strong tendency to associate that is dependent on protein concentration. Mg2+ may specifically induce an aggregation, which can be observed during sedimentation. This phenomenon seems to be analogous to the Mg2+ ‐ induced dimerization of cardiac troponin C [Jaquet, K. and Heilmeyer, L. M. G., Jr (1987) Biochem. Biophys. Res. Commun. 145, 1390 – 13961]. Upon Mg2+ saturation a shift of one of the four 31P‐NMR signals is observed. The affinity of troponin to Ca2+ is reduced when the protein concentration is enhanced only in the presence of Mg2+. This effect of Mg2+ suggests a model for the regulation of the Ca2+ ‐binding affinity of cardiac troponin.
Syntheses of the sulfoconjugated isomers of norepinephrine and dopamine, controlled by HPLC with ultraviolet detection
Authors: STROBEL Günther , WERLE Egon , HELFINGER Heike , GRIEBEL Dieter , WEICKER Helmut
Published: March 2005

The physiological significance of sulfoconjugated catecholamines and their involvement in clinical disorders, e.g. hypertension and Parkinsonism, is poorly investigated. For this reason, the sulfoconjugated isomers of dopamine as well as of norepinephrine were synthesized by modified methods. All isomers and their intermediates could be detected by a reversed‐phase high‐performance liquid chromatography with ultraviolet detection (HPLC‐UV) with short retention times and a good reproducibility. Ion‐exchange chromatography with an extended column length improved the separation of the reaction products, and the immediate control by HPLC‐UV enabled precise cutting of the fractions. The selection of the fractions with the optimum ratios of product/by‐product resulted in improved yields and highest purity. All by‐products, e.g. dopamine sulfonic acids, were less than 0.04%, as detected by HPLC‐U V and, in addition, the contamination by free catecholamines was only 41×10−4 ‐87×104%, as measured by HPLC with electrochemical detection (HPLC‐ED). The purity was further demonstrated in two highly sensitive biological assays: cAMP production in human mononuclear leukocytes and aggregation of human platelets. The sulfoconjugated catecholamines were characterized by melting point, thin‐layer chromatography, infrared spectrum, HPLC‐UV, elemental analysis, and unequivocally identified by 1H‐NMR.
Nucleotide sequence of the carA gene and regulation of the carAB operon in Salmonella typhimurium
Authors: KILSTRUP Mogens , LU Chung‐Dar , ABDELAL Ahmed , NEUHARD Jan
Published: March 2005

The carAB operon of Salmonella typhimurium encoding carbamoyl‐phosphate synthetase (CPSase) has been cloned, and the nucleotide sequence of the first gene of the operon, carA, together with 760 base pairs of the 5′‐flanking region was determined. The product of the carA gene is the small subunit of CPSase. It catalyzes the transfer of the amide group from glutamine to an NH3‐site on the heavy subunit. Primer extension and S1 niiciease mapping of in vivo carAB transcripts revealed that transcription is similar to that of Escherichia coli [Piette, J. et al. (1984) Proc. Natl Acad. Sci. USA 81, 4134‐4138] in its initiation at two promoters, P1 and P2, controlled by pyrimidines and arginine, respectively. The arginine control is mediated through binding to the arginine repressor (argR). The involvement of titratable regulatory elements is indicated by the escape from both arginine and pyrimidine control, when the operon is present in multicopies on a plasmid. Measurements of CPSase levels in mutants which allows independent manipulation of the intracellular uraoy and cytosine nucleotide pools show, that both uracil and cytosine nucleotides are required for full repression and that limitation of either nucleotide results in derepression of CPSase synthesis. Deletion analyses indicate that regions upstream of the PI promoter are required for normal expression from this promoter but not from P2.
Structural and immunochemical studies of the lipopolysaccharide from a new provisional serotype of Shigella flexneri
Authors: WEHLER Thomas , CARLIN Nils I. A.
Published: March 2005

The chemical structure of the O‐antigen of a proposed new provisional serotype of Shigella flexneri has been determined. Methylation analysis, GLC‐MS, 1H‐NMR and 13C‐NMR showed that the linear O‐antigenic polysaccharide is the same as for all S. flexneri [Kenne, L., Lindberg, B., Petersson, K. & Romanowska, E. (1977) Carbohydr. Res. 56, 363‐370]. A novel structural feature is that the disaccharide α‐D‐Glcp‐(1→2)‐α‐D‐Glcp is linked to O4 of the N‐acetyl‐glucosamine residue.

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Western blotting of the lipopolysaccharide with an E. coli R3 core‐specific monoclonal antibody, suggested the presence of anE. coli R3 core.
The poly(A)‐binding protein facilitates in vitro translation of poly(A)‐rich mRNA
Authors: GROSSI de SA Maria‐Fatima , STANDART Nancy , MARTINS de SA Cezar , AKHAYAT Omar , HUESCA Michèle , SCHERRER Klaus
Published: March 2005

To investigate the role of the 73‐kDa poly(A)‐binding protein in protein synthesis, the effect of the addition of homo‐polyribonucleotides on the translation of polyadenylated and non‐adenylated mRNA was studied in the rabbit reticulocyte lysate. Poly(A) was found to be the most effective polynucleotide in inhibiting duck‐globin mRNA translation, whereas it had no effect on the translation of polyribosomal duck‐globin mRNP, or on the endogenous synthesis of the rabbit reticulocyte lysate. The translation of poly(A)‐free mRNA was not affected by the addition of poly(A).

Furthermore, we found that the inhibiting effect of poly(A) can be reversed by addition of purified poly(A)‐binding protein. It is thus likely that the 73‐kDa poly(A)‐binding protein is an essential factor necessary for poly(A)‐rich mRNA translation.
The function of β‐N‐acetyl‐D‐glucosaminyl monophosphorylundecaprenol in biosynthesis of lipoteichoic acids in a group of Bacillus strains
Authors: SHIMADA Akira , OHTA Midori , IWASAKI Hiroyoshi , ITO Eiji
Published: March 2005

Membrane preparations, obtained from Bacillus strains which have N‐acetylglucosamine‐linked lipoteichoic acids in their membranes, were shown to catalyze the transfer of N‐[14C]acetylglucosamine (GlcNAc) from β‐[14C]GlcNAc‐P‐undecaprenol to endogenous polymer. In this reaction, α‐GlcNAc‐P‐undecaprenol or α‐GlcNAc‐PP‐undecaprenol could not substitute for β‐GlcNAc‐P‐undecaprenol as the N‐acetylglucosamine donor. This enzyme was most active at pH 6.0 and in the presence of 40 mM MgCl2. The apparent Km for β‐GlcNAc‐P‐undecaprenol was 2 μM. The radioactive polymer products, solubilized by hot phenol treatment, coincided with lipoteichoic acids in chromatographic behavior. Hydrogen fluoride treatment of the polymer products gave a major fragment identical with GlcNAc(α→2)glycerol, which corresponded to the dephosphorylated repeating units of the lipoteichoic acids in the examined strains. Thus it is concluded that β‐GlcNAc‐P‐undecaprenol serves as the donor of N‐acetylglucosamine in the biosynthesis of lipoteichoic acids in a group of Bacillus strains.
Assembly of the OmpF porin of Escherichia coli B
Authors: PAGES Jean‐Marie , BOLLA Jean‐Michel
Published: March 2005

The different conformations of the outer membrane protein OmpF of Escherichia coli B were studied with immunological probes. The antigenic determinants recognized by one monoclonal (MoF3) and two polyclonal antibodies were investigated under various conditions of solubilization which modify the association of OmpF with other membrane components, such as lipopolysaccharide. Several polymeric forms of the protein could be detected after extraction at 37°C or 56°C. The monoclonal antibody, which is specific to an exposed region of native OmpF, recognized various trimeric forms in an immunoprecipitation assay. Under the same conditions, the binding of polyclonal antibodies apparently induced strong conformational rearrangements, since the pattern of trimeric forms detected was greatly modified. The conversion of newly synthesized monomers of OmpF to the various trimer forms was investigated using these antibodies. The trimerization occurred rapidly but the appearance of the native conformation of OmpF was delayed. Some additional step was required to expose the MoF3‐specific antigenic site at the surface of the trimeric form. These results are discussed in relation to the structure of OmpF and its association with lipopolysaccharide in the outer membrane.
Multiplicity of interactions between dromedary hemoglobin and solvent components
Authors: BERTOLLINI Alberto , SANTUCCI Roberto , AMICONI Gino , ASCOLI Franca , CURRELL Douglas L. , MURSAL Omar , BRUNORI Maurizio
Published: March 2005

The interaction of dromedary hemoglobin with various solvent components [2‐(p‐chlorophenoxy)‐2‐methylpropionic acid (CFA), 2,3‐bisphospho‐D‐glycerate (glycerate‐2,3‐P2) and chloride] has been studied.

CFA greatly lowers the oxygen affinity of dromedary hemoglobin.

The oxygen‐linked CFA binding sites are probably located in the deoxy derivative at the α cleft, while in the oxy form and in the presence of two other effectors (glycerate‐2,3‐P2 and chloride) additional, structurally and possibly functionally relevant binding site(s) should be considered.

Both CFA and glycerate‐2,3‐P2 stabilize the deoxy‐like tertiary structure in the oxy derivative.

Chloride appears to be fundamental to obtain quaternary structural changes.

Interaction energy, retained in the protein when the three ligands (CFA, glycerate‐2,3‐P2 and chloride) are bound to the oxy form, favours intermediates not stable if only one or two allosteric effector(s) is (are) present on the protein.

The oxygen affinity appears to be related to both tertiary and quaternary structural changes, while cooperativity is largely invariant with solvent conditions.

In conclusion, the functional properties of dromedary hemoglobin do not depend in any simple way on the variety of stabilized conformations.
Amino acid sequences of two nonspecific lipid‐transfer proteins from germinated castor bean
Authors: TAKISHIMA Kunio , WATANABE Shinichiro , YAMADA Mitsuhiro , SUGA Tatsuko , MAMIYA Gunji
Published: March 2005

The amino acid sequence of two nonspecific lipid‐transfer proteins (nsLTP) B and C from germinated castor bean seeds have been determined. Both the proteins consist of 92 residues, as for nsLTP previously reported, and their calculated Mr values are 9847 and 9593 for nsLTP‐B and nsLTP‐C, respectively. The sequences of nsLTP‐B and nsLTP‐C, compared to the known sequence of nsLTP‐A from the same source, are 68% and 35% similar, respectively. No variation was found at the positions of the cysteine residues, indicating that they might be involved in disulfide bridges.
S‐Adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus.
Authors: PORCELLI Marina , CACCIAPUOTI Giovanna , CARTENÌ‐FARINA Maria , GAMBACORTA Agata
Published: March 2005

Two isoforms of methionine adenosyltransferase (S‐adenosylmethionine synthetase), A and B, have been partially purified from Sulfolobus solfataricus, a thermophilic archaebacterium optimally growing at 87°C. The chromatographic procedure, involving hydrophobic chromatography on a phenyl‐Sepharose column as a major step. results in 330‐fold and 150‐fold purification of adenosylmethionine synthetase A and B respectively. The apparent molecular masses, estimated by gel filtration, are 180 kDa for A and 75 kDa for B.

The A and B isoforms follow Michaelis‐Menten kinetics with apparent Km values of 10 μM nd 20 μM for L‐methionine and of 50 μM and 150 μM for ATP respectively. Adenosylmethionine, a product of the reaction, acts as a powerful non‐competitive inhibitor (Ki= 50 μM) of the A isoform while it inhibits only slightly the B isoform. Both isozymes exhibit tripolyphosphatase activity but only that associated with the form A is stimulated by 5 μM adenosylmethionine concentration. The two enzymes absolutely require a divalent cation for the activity, but are not affected by monovalent ions and reducing agents.

The optimum temperature is 90°C and no significant loss of activity is observable after incubation of the two isoforms at 100°C in the presence of ATP. The Arrhenius plots observed for both isozymes are biphasic, indicating different activation energies below and above 75°C.

The cytoplasmic levels of ATP, methionine and adenosylmethionine are evaluated.
cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of β‐naphthoflavone‐induced rabbit liver P‐450 LM4 and LM6
Authors: POMPON Denis
Published: March 2005

A cDNA library was constructed from liver mRNA of a β‐naphthoflavone‐induced rabbit. Two clones pLM4‐1 and pLM6‐1 containaing 2.2‐kbp inserts that hybridized at low stringincy with a mouse P1 P‐450 probe were selected. The clone pLM4‐1 was fully sequenced and found to contain a full‐length cDNA coding for cytochrome P‐450 LM4. Partial sequence and restriction mapping made it possible to identify pLM6‐1 as coding for the major part of cytochrome P‐450 LM6. Cloned LM4‐1 cDNA was reformed by deletion of the 5′ and 3′ non‐coding regions before insertion into yeast expression vectors PYe DP1/10. A similar operation was performed on pLM6–1 cDNA after replacement of the missing N‐terminus‐coding sequences by homologous sequences form the pLM4–1 clone resulting in a chimeric cytochrome P‐450 coding sequence.

Expression of cloned rabbit cytochrome P‐450 into transformed yeast was optimized by studying the effect of the nature of the DNA sequence just preceding the initiation codon on the level of cytochrome P‐450 production. Yeast synthesized cytochromes P‐450 were characterized by immunoblotting, spectra and catalytic activity determinations. Cloned cytochrome P‐450 LM4 was found by all criteria to be identical to the authentic rabbit one. The chimeric cytochrome P‐450 that contains the 143 N‐terminal amino acids of cytochrome P‐450 LM4 and the remaining 375 amino acids of cytochrome P‐450 LM6 was found to exhibit most of the authentic cytochrome P‐450 LM6 catalytic properties. Enzymatic and evolutionary implications of these results are discussed.
Plasma clearance, organ distribution and target cells of interleukin‐6/hepatocyte‐stimulating factor in the rat
Authors: CASTELL José V. , GEIGER Thomas , GROSS Volker , ANDUS Tilo , WALTER Eicke , HIRANO Toshio , KISHIMOTO Tadamitsu , HEINRICH Peter C.
Published: March 2005

The plasma half‐life of recombinant human interleukin‐6 (rhIL‐6) was determined in rats by measuring the disappearance of the biological activity as well as of the radioactivity of 125I‐rhIL‐6 from the circulation. The kinetics of clearance were biphasic. It consisted of a rapid initial disappearance corresponding to a half‐life of 3 min, and of a second slow one corresponding to a half‐life of about 55 min. By cellulose‐acetate electrophoresis it was shown that rhIL‐6 binds to a plasma protein resulting in a complex migrating in the β–γ region; 20 min after intravenous injection, about 80% of the 125I‐rhIL‐6 that had disappeared from the circulation was found in the liver. 125I‐rhIL‐6 was exclusively‐localized on the surface of parenchymal cells suggesting the existence of an interleukin‐6 receptor on the hepatocytes.
Tyrosine phenol‐lyase from Citrobacter intermedius
Authors: FALEEV Nikolai G. , RUVINOV Sergei B. , DEMIDKINA Tatyana V. , MYAGKIKH Igor V. , GOLOLOBOV Mikhail Yu. , BAKHMUTOV Vladimir I. , BELIKOV Vassili M.
Published: March 2005

L‐Amino acids are competitive inhibitors of tyrosine phenol‐lyase from Citrobacter intermedius. For non‐branched amino acids the correlation exists between –RTInKi and side‐chain hydrophobicity. Aspartic and glutamic acids are anomalously potent inhibitors taking into account low hydrophobicity of their side chains. This suggests the presence of an electrophilic group in the active site which interacts with the terminal carboxylic group of aspartic or glutamic acids. Tyramine, β‐phenylethylamine and tryptamine do not display detectable inhibition. The esters and amides of aromatic L‐amino acids, D‐phenylalanine and D‐tryptophan are competitive inhibitors. The enzymatic isotope exchange of the α‐proton in 2H2O was observed only in the case of L‐amino acids. For L‐phenylalanine and L‐tryptophan it was shown to proceed with complete retention of configuration. The substrate specificity of tyrosine phenol‐lyase is controlled during the stage of phenol elimination. The OH group in the para position of the ring is necessary for this stage to proceed. The same stage is also sensitive to the steric parameters of the substituent in the ring which ensures the second factor of control. When all the requirements of substrate specificity are fulfilled (L‐tyrosine, 3‐fluoro‐L‐tyrosine) the ‘key’ phenol‐elimination step is not the rate‐limiting one, the reaction velocity being determined by the preceding α‐proton abstraction.
Structure and biogenesis ofChlamydomonas reinhardtiiphotosystem I
Authors: SCHUSTER Gadi , NECHUSHTAI Rachel , FERREIRA Paulo. C. G. , THORNBER J. Philip , OHAD Itzhak
Published: March 2005

The photosystem I complex of the green algaChlamydomonas reinhardtiiwas isolated and fractionated into its two subcomplex components: the core complex (CC I), which contained the reaction center (P‐700) and had four polypeptide subunits, and the light‐harvesting complex (LHC I) which contained four polypeptides of about 22, 25, 26 and 27 kDa. The 22‐kDa apoprotein was isolated as a chlorophyllaandbbinding protein. In the isolated photosystem I holocomplex, about ten copies of the 22‐kDa LHC I apoprotein are present for each CC I unit. The 22‐kDa polypeptide as well as the other three polypeptides of this complex and the subunit II of CC I are translated on 80S cytoplasmic ribosomes, and therefore are coded in the nucleus. During the greening process of theChlamydomonas reinhardtiiy‐1 mutant the 22‐kDa LHC I polypeptide, which cross‐reacts with polyclonal antibodies raised against theLemna gibba20‐kDa LHC I apoprotein, accumulates in thylakoids at a late stage of their development, and about 2–3 h after the LHC II and CC I subunit II polypeptides have accumulated. Accumulation of the 22‐kDa protein during greening is inhibited by cycloheximide but not by chloramphenicol.
Possible control of transcript levels by chlorophyll precursors in Chlamydomonas
Authors: JOHANNINGMEIER Udo
Published: March 2005

Steady‐state mRNA levels of the three nuclear genes cab1, rbcS1 and rbcS2 (coding for the light‐harvesting chlorophyll‐binding protein (LHCP) and the small subunit of ribulose 1,5‐bisphosphate carboxylase, respectively) and of the two plastid‐encoded genes rbcL and psaA2 (coding for the large subunit of the carboxylase and a member of the P700 chlorophyll a protein, respectively) have been investigated in synchronized Chlamydomonas cells in response to light and inhibitors interfering with chlorophyll synthesis. The accumulation of cab1, rbcS1 and psaA2 transcripts is light‐dependent, whereas transcripts from rbcS2 and rbcL genes are present in high amounts in the light and in the dark. Dioxoheptanoic acid, an inhibitor blocking chlorophyll synthesis prior to porphyrin formation, does not affect the accumulation of all five mRNAs. However, inhibition of chlorophyll synthesis by incubating cells with dipyridyl, cycloheximide or nitrogen promotes the accumulation of porphyrin compounds, but specifically prevents the accumulation of light‐dependent transcripts. Although functionally unrelated, these inhibitors are known to block an Fe‐dependent oxygenase, which is involved in the formation of the isocyclic ring in the chlorophyll molecule. The data are explained as a control by chlorophyll precursors over the accumulation of light‐dependent transcripts.
Binding of Ca2+ influences susceptibility of laminin to proteolytic digestion and interactions between domain‐specific laminin fragments
Authors: PAULSSON Mats , SALADIN Katrina , LANDWEHR Ruth
Published: March 2005

Ca2+ was found to influence the patterns of limit digests of laminin obtained with various neutral proteases. In the presence of Ca2+, larger fragments were obtained from the central part of laminin than in its absence. This was interpreted as being due to a stabilization of the central short‐arm domains of laminin by bound Ca2+. When proteolytic fragments were tested for their ability to aggregate, only large fragments containing intact short arms were active, indicating an important role for these domains in laminin self‐aggregation.
TGGCA protein is present in erythroid nuclei and binds within the nuclease‐hypersensitive sites 5′ of the chicken βH‐ and βA‐globin genes
Authors: RUPP Ralph A. W. , NICOLAS Robert H. , BORGMEYER Uwe , LOBANENKOV Victor V. , PLUMB Marc A. , SIPPEL Albrecht E. , GOODWIN Graham H.
Published: March 2005

The developmentally regulated 5′‐flanking DNase‐I‐hypersensitive site of the chicken βH‐globin gene in nuclei contains a subregion which is resistant to DNase I and which disappears when nuclei are extracted with 0.3 M NaCl, suggesting that there are salt‐extractable proteins bound to sequences within this region. The 0.3 M NaCl extract contains two proteins which bind in vitro to these sequences. One of the binding sequences has an inverted repeat very similar to that bound by TGGCA protein. Partially purified TGGCA protein from chicken liver binds to this sequence in vitro giving exactly the same footprint as that obtained with erythroid nuclear proteins. Similarly TGGCA protein binds to an inverted repeat with the βA‐globin 5′‐hypersensitive site giving a footprint identical to that obtained with erythroid nuclear protein extracts. From competition footprinting experiments and the electrophoretic mobility of the protein‐DNA complex, it is concluded that the erythroid proteins previously described as binding to the βH‐ and βA‐globin inverted repeats within the 5′‐flanking hypersensitive sites both belong to the TGGCA protein family.
The complete sequence of a chicken‐muscle cDNA encoding the acidic ribosomal protein P1
Authors: FERRO Jesus A. , REINACH Fernando C.
Published: March 2005

We have isolated and sequenced a complete cDNA encoding the acidic phosphoprotein P1 from chicken. The analysis of the deduced protein sequence and its comparison with the known sequence of P proteins from human, rat and Artemia salina indicates that the central, alanine‐rich region of these proteins was probably generated by internal duplications of the gene followed by modifications within each repeat. This observation explains the length heterogeneity and sequence divergence of this particular region when compared with the highly conserved structure of the remaining segments of the protein.
Properties of a 19‐kDa Zn2+‐binding protein and sequence of the Zn2+‐binding domains
Authors: BRAND Ingeborg Anke , HEINICKEL Antje , KRATZIN Hartmut , SÖLING Hans‐Dieter
Published: March 2005

A novel 19‐kDa protein has been described recently [Brand, I. A. and Söling, H.‐D. (1986) J. Biol. Chem. 261, 5895 – 5900] which is able to inactivate 6‐phosphofructo‐1‐kinase reversibly in a Zn2+‐dependent manner. We present now additional biochemical and physicochemical data concerning this protein. It is extremely acidic with 40% glutamic and 15% aspartic acid residues. It contains no sulfur, aromatic amino acids, histidine or isoleucine. The protein has four binding sites for Zn2+ with an apparent Kd of about 6 μM. Two of these binding sites are called unspecific as Zn2+ is displaced from these binding sites at physiological concentrations of free Mg2+ (0.75 mM) and at high salt concentrations (100 mM NaCl). Whereas Mg2+‐binding to the two other so‐called specific Zn2+‐binding sites occurs only at Mg2+ concentrations at about 5 mM. The four Zn2+‐binding sites were detected on a tryptic peptide (T8) of 43 amino acid residues, which still possessed biological activity. This peptide has been sequenced and is characterized by four clusters of acidic amino acids separated by only a few neutral amino acids. The two specific Zn2+‐binding sites could be detected in the C‐terminal portion of T8, the two unspecific Zn2+‐binding sites must therefore be located at the N‐terminal portion.

The Zn2+‐binding domains of the 19‐kDa Zn2+‐binding protein described here are completely different from those of the ‘zinc finger’ discovered in several DNA‐binding proteins.
Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8‐azido‐AMP
Authors: PATIL Rajkumar V. , DATTA Prasanta
Published: March 2005

The photoreactive AMP analog, 8‐azido‐AMP, stimulated the activity of biodegradative threonine dehydratase of Escherichia coli in a reversible manner and, like AMP, decreased the Km for threonine. The concentrations required for half‐maximal stimulation by AMP and 8‐azido‐AMP were 40 μM and 1.5 μM, respectively, and the maximum stimulation by 8‐azido‐AMP was 25% of that seen with AMP. Gel‐filtration experiments revealed that 8‐azido‐AMP stabilized a dimeric form of the enzyme, whereas AMP promoted a tetrameric species. When present together, AMP and 8‐azido‐AMP showed mutual competition in influencing catalytic activity as well as the conformational state of the protein. Photolabeling of AMP‐free dehydratase with 8‐azido‐[2‐3H]AMP resulted in a time and concentration‐dependent enzyme inactivation and concomitant incorporation of 8‐azido‐AMP into protein. At low 8‐azido‐AMP concentrations, incorporation of about 1 mol 8‐azido‐AMP/mol dehydratase tetramer was correlated with almost complete inactivation of the enzyme. The presence of AMP in the photolabeling reaction greatly reduced the extent of enzyme inactivation and 8‐azido‐AMP binding. Ultraviolet irradiation with 20 μM 3H‐labeled 8‐azido‐AMP revealed one tryptic peptide, Thr230‐Thr‐Gly‐Thr‐Leu‐Ala‐Asp‐Gly‐Cys‐Asp‐Val‐Ser‐Arg242, with bound radioactivity. This peptide, labeled at low concentration of 8‐azido‐AMP, most likely represents the AMP‐binding region on the dehydratase molecule.
Novel transcellular interaction: conversion of granulocyte‐derived leukotriene A4 to cysteinyl‐containing leukotrienes by human platelets
Authors: EDENIUS Charlotte , HEIDVALL Katarina , LINDGREN Jan Åke
Published: March 2005

Human platelets dose‐dependently converted exogenous leukotriene A4 to leukotriene C4 and efficiently metabolized this compound to leukotrienes D4 and E4. Neither of these compounds were produced after stimulation of human platelet suspensions with ionophore A23187. After LTA4 incubation of subcellular fractions, formation of leukotriene C4 was exclusively observed in the particulate fraction and was separable from the classical glutathione S‐transferase activity. This suggested the presence of a specific leukotriene C4 synthase in human platelets. Addition of physiological amounts of autologous platelets to human granulocyte suspensions significantly increased ionophore A23187‐induced formation of leukotriene C4. In contrast, the production of leukotriene B4 was decreased. After preincubation of platelets with [35S]cysteine, 35S‐labeled leukotriene C4 was produced by A23187‐stimulated platelet‐granulocyte suspensions, strongly indicating a transcellular biosynthesis of this compound.
Oxidation of glycolipids in liposomes by galactose oxidase
Authors: LAMPIO Anja , SIISSALO Ilkka , GAHMBERG Carl G.
Published: March 2005

Small unilamellar phosphatidylcholine vesicles containing globo‐series glycolipids were labeled by the galactose oxidase/NaB[3H]4 procedure. The major glycolipid of human red cells, globoside, was the best substrate for galactose oxidase both in vesicles and in tetrahydrofuran‐containing buffer. The oxidation rates of membrane‐bound ceramide trihexoside and Forssman glycolipid were one‐fourth and one‐tenth, respectively, of the oxidation rate of globoside. Membrane‐bound ceramide dihexoside was not a substrate for galactose oxidase, although it was readily oxidized in tetrahydrofuran‐containing buffer. Soluble sialoglycoproteins and membrane‐incorporated glycophorin A stimulated the oxidation of globoside‐containing vesicles, whereas membrane‐bound GD1a ganglioside had no effect on globoside oxidation.
Comparison of the metabolism of dodecanedioic acid in vivo in control, riboflavin‐deficient and clofibrate‐treated rats
Authors: DRAYE Jean‐Pierre , VEITCH Keith , VAMECQ Joseph , VAN HOOF François
Published: March 2005

Intravenous administration of dodecanedioate (or hexadecanedioate) to anaesthetized rats resulted in the urinary excretion of medium‐chain dicarboxylic acids (adipic, suberic and sebacic acids). In control animals, the recovery of infused dodecanedioate in the form of urinary medium‐chain dicarboxylic acids corresponded to 30% of the infused dose (22 μmol/100 g body mass). This excretion was markedly increased in riboflavin‐deficient rats (75% of the infused dose) while it was severely decreased in clofibrate‐treated animals (< 5%). The initial velocity of this process was similar in both control and riboflavin‐deficient rats. In control animals, halving the infused dose of dodecanedioate to 11 μmol/100 g body mass resulted in a halving of the initial rate of the urinary appearance of medium‐chain dicarboxylates, while doubling the amount of dicarboxylate administered to 44 μmol/100 g body mass did not further modify this velocity, but rather prolonged the duration of the excretion of the resulting products. In riboflavin‐deficient and clofibrate‐treated rats, the hepatic peroxisomal dicarboxylyl‐CoA β‐oxidation activity measured as dicarboxylyl‐CoA H2O2‐generating oxidase and cyanide‐insensitive dicarboxylyl‐CoA‐dependent NAD+ reduction was increased about threefold and tenfold, respectively. Dicarboxylyl‐CoA synthetase activity was normal in the clofibrate‐treated rat livers but was increased more than tenfold in the livers from the riboflavin‐deficient animals. This work provides evidence that in the rat both mitochondria and peroxisomes are involved in the catabolism of dicarboxylates.
Purification, characterization and induction of L‐phenylalanine ammonia‐lyase in Phaseolus vulgaris
Authors: da CUNHA Antoinette
Published: March 2005

The enzyme L‐phenylalanine ammonia‐lyase was purified from leaves of Phaseolus vulgaris by Sephacryl S‐200 gel filtration and Sepharose‐4‐B–succinyl‐aminoethyl‐L‐phenylalanine affinity chromatography. L‐Phenylalanine ammonia‐lyase was specifically eluted from the affinity matrix with its substrate L‐phenylalanine at 20–25°C. The purified enzyme was shown to be homogeneous by gel electrophoresis both in presence and absence of SDS. Its Mr, determined by gel filtration and non‐denaturing gel electrophoresis, was 320000 ± 9000 and 330000 ± 4000 respectively. After SDS electrophoresis only one band of Mr 83000 ± 4000 was detected, indicating that the enzyme is an oligomer containing four subunits. The pH optimum of enzyme activity was 8.8–9.2. Ampholyte isoelectrofocusing in polyacrylamide demonstrated the presence of a single charged species at pH 4.2. The homogeneous enzyme catalyzed the deamination of L‐phenylalanine to trans‐cinnamate but did not catalyze the transamination of L‐phenylalanine to L‐phenylpyruvate. The enzyme showed Km 1.25 mM for L‐phenylalanine. Antibodies to homogeneous L‐phenylalanine ammonia‐lyase recognised specific epitopes on L‐phenylalanine aminotransferase as demonstrated by immunoaffinity purification and immunoblotting. The induction of L‐phenylalanine ammonia‐lyase activity during phaseollin biosynthesis in the Phaseolus vulgaris – Colletotrichum lindemuthianum interaction was regulated by an increase in enzyme concentration resulting from an increase in de novo synthesis of L‐phenylalanine ammonia‐lyase protein.
Separation and characterization of four enzyme forms of β‐galactosidase from Saccharomyces lactis
Authors: MBUYI‐KALALA Alafuele , SCHNEK Arthur G. , LÉONIS José
Published: March 2005

β‐Galactosidase from Saccharomyces lactis has been purified to serve as a model for the kinetic behavior of human lactase in adult lactase deficiency. Enzymes from both species are neutral and follow Michaelis‐Menten kinetics. β‐Galactosidase of S. lactis is more readily available than the human lactase. An enzyme preparation from S. lactis (Maxilact 40000), which is used commercially to hydrolyze lactose in milk, has been found to contain four isozymes of β‐galactosidase. Methods have been developed for the separation and purification of each of the four enzymes. The enzymes were found to differ in molecular mass, kinetic behavior, isoelectric point, response to pH, specific volume and sensitivity to metal ions. The four enzymes had apparent molecular masses of 630 kDa, 550 kDa, 41 kDa and 19 kDa. Their specificity constants (kcat/Km) were found to be 42.0, 355.2, 0.38 and 0.48 mM−1 s−1, respectively. The techniques of reiterated ultrafiltration used for the isolation of these isozymes may be applicable to other purification processes.
Altered relationship between protonmotive force and respiration rate in non‐phosphorylating liver mitochondria isolated from rats of different thyroid hormone status
Authors: HAFNER Roderick P. , NOBES Catherine D. , McGOWN Anne D. , BRAND Martin D.
Published: March 2005

We have determined the relationship between rate of respiration and protonmotive force in oligomycininhibited liver mitochondria isolated from euthyroid, hypothyroid and hyperthyroid rats. Respiration rate was titrated with the respiratory‐chain inhibitor malonate. At any given respiration rate mitochondria isolated from hypothyroid rats had a protonmotive force greater than mitochondria isolated from euthyroid controls, and mitochondria isolated from hyperthyroid rats had a protonmotive force less than mitochondria isolated from euthyroid controls. In the absence of malonate mitochondrial respiration rate increased in the order hypothyroid < euthyroid < hyperthyroid, while protonmotive force increased in the order hyperthyroid < euthyroid < hypothyroid. These findings are consistent with a thyroid‐hormone‐induced increase in the proton conductance of the inner mitochondrial membrane or a decrease in the H+/O ratio of the respiratory chain at any given protonmotive force. Thus the altered proton conductance or H+/O ratio of mitochondria isolated from rats of different thyroid hormone status controls the respiration rate required to balance the backflow of protons across the inner mitochondrial membrane. We discuss the possible relevance of these findings to the control of state 3 and state 4 respiration by thyroid hormone.
Characterization of an inhibitor of actin polymerization in vinculin‐rich fraction of turkey gizzard smooth muscle
Authors: MIRON Talia , WILCHEK Meir , GEIGER Benjamin
Published: March 2005

We report here on the purification and characterization of a new 25‐kDa inhibitor of actin polymerization from turkey gizzard smooth muscle. The protein was purified by chromatography on DEAE‐cellulose and hydroxyapatite, as well as by affinity chromatography on an immobilized‐antibody column. The purified polypeptide reduced the low‐shear viscosity of actin, apparently due to its inhibitory effect on actin polymerization. We demonstrate that this protein is largely responsible for the apparent inhibitory activity previously reported to be associated with smooth muscle vinculin preparations. Three independent monoclonal antibodies prepared against the 25‐kDa inhibitor of actin polymerization can effectively adsorb the inhibiting activity of actin polymerization from the crude vinculin preparation or inhibit it. We also show here that the 25‐kDa inhibitor of actin polymerization tends to undergo dimerization when maintained in non‐reducing buffers, concomitant with the loss of its inhibitory activity. Immunohistochemical labeling of frozen sections, as well as immunoblotting analyzes, indicated that the 25‐kDa inhibitor of actin polymerization is particularly enriched in smooth muscle cells and that its distribution is apparently homogenous throughout the cytoplasm showing no apparent enrichment in the vinculin‐rich dense plaques located along the endofacial surface of the plasma membrane.
Low‐density‐lipoprotein‐receptor mRNA content of fibroblasts from normal and familial hypercholesterolaemic subjects
Authors: KNIGHT Brian L. , PATEL Dilip D. , GAVIGAN Sean J. P. , SOUTAR Anne K.
Published: March 2005

The amount of mRNA for the low‐density‐lipoprotein (LDL) receptor in cultured human fibroblasts was estimated by hybridization of the poly(A)‐rich RNA fraction with a DNA probe, using the recovery of β‐actin mRNA to correct for losses. During incubation of the cells with lipoprotein‐deficient serum (LPDS) both the LDL‐receptor mRNA content and the rate of receptor protein synthesis increased fourfold during the first 16 h and then fell by approximately 50% during the next 24 h. The content of β‐actin mRNA fell by a similar amount, so that the ratio of receptor/β‐actin mRNAs rose and then remained constant. The fall in β‐actin mRNA content during incubation with LPDS was not prevented by the addition of cholesterol to the medium.

In cells from a homozygous familial hypercholesterolaemic (FH) subject that bound 20% of the normal amount of LDL, the content of LDL‐receptor mRNA and the changes during incubation with LPDS or free sterols were similar to normal. Cells from a familial hypercholesterolaemic subject that produced no immunodetectable receptor protein produced a small amount of receptor mRNA of apparently normal size which responded in the same way as in normal cells to LPDS and free sterols.
The binding of double‐stranded RNA and adenovirus VAI RNA to the interferon‐induced protein kinase
Authors: GALABRU Julien , KATZE Michael G. , ROBERT Nadine , HOVANESSIAN Ara G.
Published: March 2005

The protein kinase from human cells dependent on double‐stranded (ds) RNA is a 68‐kDa protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by low concentrations of dsRNA, the p68 kinase becomes phosphorylated and thereby catalyzes the phosphorylation of the protein‐synthesis initiation factor, eIF2. Here, we have purified the p68 kinase to homogeneity using a specific monoclonal antibody to investigate its capacity to bind dsRNA, poly(I). poly(C). Our study suggest that p68 kinase has high‐ and low‐affinity binding sites: the high‐affinity binding site is responsible for the activation and the low‐affinity binding site for the inhibition of kinase activity. This is in accord with the fact that autophosphorylation of p68 kinase occurs at low concentrations of dsRNA whereas high concentrations of dsRNA inhibit its autophosphorylation.

We have also investigated the binding of adenoviral VAI RNA to the purified p68 kinase and have found that the affinity of this binding is lower than that of poly(I). poly(C). We show that VAI RNA can activate or inhibit autophosphorylation of p68 kinase in a dose‐dependent manner, i.e. activation at ≤1 μg/ml or inhibition at >1 μg/ml of VAI RNA. In spite of its lower affinity of binding, VAI RNA cannot be displaced by poly(I). poly(C) or reovirus dsRNA. These data confirm our previous results to illustrate that VAI RNA can bind p68 kinase and cause its inactivation irreversably.
Thermostable DNA polymerase from the archaebacterium Sulfolobus acidocaldarius
Authors: ELIE Christiane , DE RECONDO Anne Marie , FORTRRE Patrick
Published: March 2005

We have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a polypeptide of 100 kDa. On the basis of a Stokes radius of 4.2 nm and a sedimentation coefficient of 6 S, the purified enzyme has an estimated molecular mass of 109 kDa. These results are consistent with the enzyme being a monomer of 100 kDa. In addition a polyclonal antiserum, obtained by injection of the electroeluted 100‐kDa polypeptide into a rabbit, specifically neutralized the DNA‐polymerase activity. The enzyme is sensitive to both N‐ethylmaleimide and 2′,3′‐dideoxyribosylthymine triphosphate and resistant to aphidicolin. The purified DNA polymerase has neither exonuclease nor primase activities. In our in vitro conditions, the enzyme is thermostable up to 80°C and is active between 55°C and 85°C in the presence of activated calf‐thymus DNA.
A crystallographic study of the glutathione binding site of glutathione reductase at 0.3‐nm resolution
Authors: KARPLUS P. Andrew , PAI Emil F. , SCHULZ Georg E.
Published: March 2005

The binding of glutathione, some related molecules and two redox compounds to crystals of glutathione reductase has been investigated by X‐ray crystallography at 0.3‐nm resolution. Models for several bound ligands have been built and subjected to crystallographic refinement. The results clearly show the residues involved in glutathione binding as well as the geometry of the disulfide exchange. Glutathione‐I is bound in a V‐shaped conformation, while glutathione‐II is extended. The zwitterionic glutamyl end of glutathione‐II appears to be the most tightly bound part of the substrate. All glutathione conjugates and derivatives studied show binding dominated by the interactions at this site. In the reduced enzyme, glutathione‐I forms a mixed disulfide intermediate with Cys58. Other structural changes are observed on reduction of the enzyme, and it is demonstrated that the carboxamidomethylated enzyme is a good model for the reduced species. Lipoate, a weak substrate of the enzyme, assumes a defined binding site where its disulfide is available for being attacked by Cys58‐Sγ. A second region with affinity for a number of compounds has been found in a large cavity at the dimer interface of the enzyme. No functional role of this site is known.
Structural and physicochemical requirements of endotoxins for the activation of arachidonic acid metabolism in mouse peritoneal macrophages in vitro
Authors: LÜDERITZ Thomas , BRANDENBURG Klaus , SEYDEL Ulrich , ROTH Achim , GALANOS Chris , RIETSCHEL Ernst Th.
Published: March 2005

Lipopolysaccharides of different wild‐type and mutant gram‐negative bacteria, as well as synthetic and bacterial free lipid A, were studied for their ability to activate arachidonic acid metabolism in mouse peritoneal macrophages in vitro. It was found that lipopolysaccharides of deep‐rough mutants of Salmonella minnesota and Escherichia coli (Re to Rc chemotypes) stimulated macrophages to release significant amounts of leukotriene C4 (LTC4) and prostaglandin E2 (PGE2). Lipopolysaccharides of wild‐type strains (S. abortus equi, S. friedenau) only induced PGE2 and not LTC4 formation. Unexpectedly, free bacterial and synthetic E. coli lipid A were only weak inducers of LTC4 and PGE2 production. Deacylated Re‐mutant lipopolysaccharide preparations were inactive. However, co‐incubation of macrophages with both deacylated lipopolysaccharide and lipid A lead to the release of significant amounts of LTC4 and PGE2, similar to those obtained with Re‐mutant lipopolysaccharide. The significance of the lipid A portion of lipopolysaccharide for the induction of LTC4 was indicated by demonstrating that peritoneal macrophages of endotoxin‐low‐responder mice or of mice rendered tolerant to endotoxin did not respond with the release of arachidonic acid metabolites on stimulation with Re‐mutant lipopolysaccharide and that polymyxin B prevented the Re‐lipopolysaccharide‐induced LTC4 and PGE2 release. Physical measurements showed that the phase‐transition temperatures of both free lipid A and S‐form lipopolysaccharide were above 37°C while those of R‐mutant lipopolysaccharides were significantly lower (30–35°C). Thus, with the materials investigated, an inverse relationship between the phase‐transition temperature and the capacity to elicit LTC4 production was revealed.
Inhibition of mammalian spermine synthase by N‐alkylated‐1,3‐diaminopropane derivatives in vitro and in cultured rat hepatoma cells
Authors: BAILLON Jean G. , KOLB Michael , MAMONT Pierre S.
Published: March 2005

A number of N‐alkylated‐1,3‐diaminopropane derivatives[H2N‐(CH2)3‐NH‐(CH2)nH, where n= 1–9] have been tested as potential inhibitors of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase.

Among the compounds described in this paper, the most potent competitive inhibitor of spermine synthase with respect to spermidine, is N‐butyl‐1,3‐diaminopropane with Ki values of 11.9 nM and 10.4 nM for the HTC cell and bovine spleen enzymes respectively. Inhibition of spermine synthase by this alkylated amine is selective since spermidine synthase activity is not affected up to 100 μM N‐butyl‐1,3‐diaminopropane at a range of 5–200 μM putrescine.

Added to the culture medium of growing HTC cells, N‐butyl‐1,3‐diaminopropane causes the expected changes in the polyamine levels with a marked decrease of spermine and an increase of spermidine. Under these conditions cell growth continues unabated.

Such N‐alkylated‐1,3‐diaminopropane derivatives may have considerable potential as tools for studying the role of polyamines and in particular the functions of spermine in cell multiplication and differentiation.
The multiplicity of troponin T isoforms
Authors: HÄRTNER Karl‐Thomas , KIRSCHBAUM Bernhard J. , PETTE Dirk
Published: March 2005

Polyclonal antibodies were raised in guinea pigs against troponin‐T (TnT) isoforms purified from fast‐ and slow‐twitch rabbit muscles. With the use of these antibodies and immunoblots of one‐ and two‐dimensional electrophoreses, the distribution of fast and slow TnT isoforms was investigated in normal and chronically stimulated hindlimb muscles of the rabbit. According to differences in their apparent molecular masses, six fast TnT isoforms (TnTcf, TnT1f, TnT2f, TnT3f, TnT4f, TnT5f) were distinguished in normal tibialis anterior and extensor digitorum longus muscles. These muscles also contained low amounts of TnT1s and TnT2s which were the predominant TnT isoforms in slow‐twitch soleus muscle. Fast and slow TnT isoforms were found to exist in several charge variants, i.e. one for TnTcf, three different charge forms for TnT1f, seven for TnT2f, four for TnT3f, three for TnT4f, one for TnT5f, four for TnT1s, and three for TnT2s. Some charge variants were phosphorylated isoforms because treatment with alkaline phosphatase reduced the number of the 19 fast and 7 slow variants to 12 and 3, respectively. The stimulation‐induced fast‐to‐slow transition caused progressive decreases in fast and increases in slow isoforms. The decrease and the disappearance of the major fast isoforms followed a sequence of TnT2f, TnTcf, TnT4f, TnT1f, and TnT3f. This decrease in fast isoforms fits well with the reduction of fast TnT mRNAs assessed by Northern blot analysis. Prolonged stimulation ultimately created a TnT isoform pattern similar to that found in normal slow‐twitch muscle. Stimulation also induced changes in the tropomyosin subunit pattern with a decrease in the fast and an increase in the slow α‐tropomyosin subunit without altering the α/β‐tropomyosin subunit ratio. Similar to slow‐twitch soleus muscle, long‐term stimulated muscles contained appreciable amounts of the fast α‐tropomyosin subunit, but only traces of fast TnT isoforms. This combination indicated that the predominant slow TnT isoforms may be capable of interacting with fast tropomyosin in these muscles.