bluuebunny/crossref_metadata_2025 huggingface dataset:
Neurotensin and neuromedin N undergo distinct catabolic processes in murine astrocytes and primary cultured neurons
Authors: VINCENT Bruno , VINCENT Jean‐Pierre , CHECLER Frédéric
Published: March 2005

We examined the occurence of various endopeptidases and exopeptidases and their subcellular partition within soluble and membrane‐associated compartments of 15‐day‐old astrocytes and 4‐day‐old primary cultured neurons. Peptidases were monitored with chromogenic of fluorimetric substrates and identified by means of specific inhibitors. We assessed the contribution of these peptidases in the catabolism of two related neuropeptides, neurotensin and neuromedin N. Metabolites were separated by HPLC and the identity of the proteolytic activities involved in their formation was established using specific inhibitors. Neuromedin N and neurotensin undergo both quantitative and qualitative differential proteolysis. Initial maximal rates of neuromedin N degradation were higher than those of neurotensin in both cell types. Furthermore, the two peptides were inactivated much more rapidly by the soluble than by the membrane‐associated fractions prepared from both cell cultures. Neuromedin N was rapidly broken down by an aminopeptidase M/leucine aminopeptidase attack, leading to the functionally silent Des‐Lys1‐neuromedin N metabolite. In the astrocytic membrane‐associated fraction, neuromedin N underwent an additional minor endoproteolytic cleavage at the Pro3‐Tyr4 bond elicited by endopeptidase 24.11, as suggested by the protective effect of its blocking agent phosphoramidon. Unlike neuromedin N, neurotensin totally resisted hydrolysis by aminopeptidases. Primary inactivating cleavages detected in both cell types appeared mainly located at the Arg8‐Arg9 and Pro10‐Tyr11 bonds, leading to the formations of neurotensin‐(1–8) and neurotensin‐(1–10) as the major biologically inactive neurotensin catabolites. Endopeptidase 24.15 appeared mainly responsible for neurotensin‐(1–8) formation by the soluble fraction of neurons and astrocytes. In contrast, endopeptidase 24.16 was involved in neurotensin‐(1–10) formation by both soluble and membrane‐associated fractions of the two cell types. An additional cleavage leading to neurotensin‐(1–11) formation and ascribed to endopeptidase 24.11 was detected mainly in the membrane‐associated fraction from astrocytes. Finally, the secondary processing of neurotensin degradation products indicated that: (a) neurotensin‐(1–11) was converted into neurotensin‐(1–8) in the membrane fraction prepared from astrocytes; (b) neurotensin‐(1–10) was transformed into neurotensin‐(1–8) by an unidentified peptidase belonging to the class of metalloenzymes. The significance of distinct quantitative and qualitative catabolic fates of neuromedin N and neurotensin in cultured astrocytes and neurons is discussed.
Multistep regulation mechanisms for tolerance induction to lipopolysaccharide lethality in the tumor‐necrosis‐factor‐α‐mediated pathway
Authors: MATSUURA Motohiro , KISO Makoto , HASEGAWA Akira , NAKANO Masayasu
Published: March 2005

Lipid A is the active principle of lipopolysaccharide (LPS). Synthetic lipid A analogues with monosaccharide backbones, GLA‐60, GLA‐69 and GLA‐58, which exhibit potent, weak and scarce agonistic activities of LPS, respectively, induced tolerance against LPS lethality in galactosamine‐(GalN)‐sensitized mice while none of them were pyrogenic in rabbits. The tolerance‐inducing mechanisms were investigated focusing on the regulation of tumor‐necrosis‐factor‐α(TNF‐α)‐mediated lethal pathway of LPS. Induction of serum TNF‐α in LPS‐challenged mice was suppressed by prior administration of these analogues as well as LPS. Prior treatment of murine macrophages with the substances suppressed LPS‐stimulated TNF‐α production in the culture supernatant and TNF‐α mRNA expression in the cells as well. Lethal toxicity to TNF‐α in GalN‐sensitized mice was effectively suppressed by prior treatment with LPS, GLA‐60 and GLA‐69 but not by GLA‐58. This protective effect was suggested to be mediated by endogenous TNF‐α, which was induced by prior treatment with the effective substances, because either neutralization of endogenously induced TNF‐α activity with an antibody or deletion of its induction by using LPS‐resistant C3H/HeJ mice reduced the protective effect, and a detectable amount of TNF‐α was produced by stimulating macrophages with the effective substances but not with GLA‐58. These results indicated that multiple regulation steps (one is prior to and the others are following TNF‐α production) are participating in the tolerance induction by LPS and some lipid A analogues and that GLA‐58 is a characteristic compound which induces the tolerance by only blocking the step prior to TNF‐α production.
Catabolism of aromatics in Pseudomonas putida U
Authors: OLIVERA Elias R. , REGLERO Angel , MARTÍNEZ‐BLANCO Honorina , FERNÁNDEZ‐MEDARDE Alberto , MORENO Miguel A. , LUENGO José M.
Published: March 2005

Phenylacetic acid (PhAcOH) and 4‐hydroxyphenylacetic acid (4HOPhAcOH) are catabolized in Pseudomonas putida U through two different pathways. Mutation carried out with the transposon Tn5 has allowed the isolation of several mutants which, unlike the parental strain, are unable to grow in chemically defined medium containing either PhAcOH or 4HOPhAcOH as the sole carbon source. Analysis of these strains showed that the ten mutants unable to grow in PhAcOH medium grew well in the one containing 4HOPhAcOH, whereas four mutants handicapped in the degradation of 4HOPhAcOH were all able to utilize PhAcOH. These results show that the degradation of these two aromatic compounds in P. putida U is not carried out as formerly believed through a single linear and common pathway, but by two unrelated routes. Identification of the blocked point in the catabolic pathway and analysis of the intermediate accumulated, showed that the mutants unable to utilize 4HOPhAcOH corresponded to two different groups: those blocked in the gene encoding 4‐hydroxyphenylacetic acid‐3‐hydroxylase; and those blocked in the gene encoding homoprotocatechuate‐2,3‐dioxygenase. Mutants unable to use PhAcOH as the sole carbon source have been also classified into two different groups: those which contain a functional PhAc‐CoA ligase protein; and those lacking this enzyme activity.
Use of monoclonal antibodies to study the structure and function of eukaryotic protein synthesis initiation factor eIF‐2B
Authors: OLDFIELD Susan , JONES Bridget L. , TANTON Deborah , PROUD Christopher G.
Published: March 2005

The eukaryotic protein synthesis initiation factor, eIF‐2B, is a multimeric protein of five different subunits termed α, β, γ, δ and ɛ, which facilitates recycling of a further factor, eIF‐2, and is an important control point in the initiation process. In order to investigate the structure and function of eIF‐2B, monoclonal antibodies have been prepared to the β, δ and ɛ subunits of the factor from rabbit reticulocytes. All three antibodies are active in Western blotting, ELISA and immunoprecipitation. The anti‐ɛ antibody inhibits both the guanine nucleotide exchange activity of eIF‐2B and protein synthesis in the rabbit reticulocyte lysate at the level of initiation. The other two antibodies do not inhibit either guanine nucleotide exchange or protein synthesis. The monoclonal antibodies and a polyclonal anti‐(rabbit reticulocyte eIF‐2B) serum were used to investigate the subunit size and the antigenic structure of eIF‐2B from a variety of rabbit tissues and from a variety of mammalian species. eIF‐2B from all rabbit tissues tested was indistinguishable from that prepared from rabbit reticulocytes. Quantitative studies showed substantial variation in the relative concentrations of eIF‐2 and eIF‐2B between different rabbit tissues. Marked variation in both the sizes of the subunits and their reaction with the antibodies was observed between eIF‐2B from rabbit, rat, guinea pig and man.
Characterisation of two intronic nuclear‐matrix‐attachment regions in the human DNA topoisomerase I gene
Authors: ROMIG Helmut , RUFF Jürgen , FACKELMAYER Frank O. , PATIL Manohar S. , RICHTER Arndt
Published: March 2005

We identify two high‐affinity matrix‐attachment regions (MAR elements) located in two introns of the human DNA topoisomerase I gene (TOP1). These intronic MAR elements, designated MI and MII, are specifically bound by the nuclear matrix and partition with scaffolds in vitro. One of these MAR elements, MII, is part of a genomic region which is hypersensitive for endogeneous nucleases. We have sequenced both DNA elements and have characterized their mode of binding to the nuclear matrix. Experiments with the minor‐groove‐binding ligands distamycin and chromomycin indicate that the A+T‐rich regions, most likely homopolymeric A tracts, are responsible for binding of these DNA elements to the nuclear matrix. MII contains an alu‐like element and a segment of curved DNA. Analysis of subfragments of MII show that the curved DNA region itself shows only weak nuclear‐matrix binding, and that the high‐affinity binding sites are located on subfragments on the 5′ side of the curved DNA. In addition, we found that the alu‐like sequence does not contribute significantly to the binding of MII and of subfragments of MII to nuclear‐matrix proteins. Comparing the distribution of repetitive sequences in the cloned parts of human DNA topoisomerase I gene with the location of high‐affinity matrix‐binding sites we find no evidence that repetitive DNA may be located close to MAR elements as has been previously suggested.
Substrate and inhibitor specificity of mevalonate kinase determined with substrate analogues
Authors: WILDE Jürgen , EGGERER Hermann
Published: March 2005

Substrate analogues of mevalonate kinase were prepared and their kinetic properties determined with the enzyme from pig liver. The analogues are derived from mevalonate by substitutions of hydroxyl and/or methyl at C3(group 1), by systematic prolongation of C6 to yield 3‐alkyl‐3,5‐dihydroxyvalerates (group 2) and by methylation at C2, C6 and C4 (group 3).

Compounds of group 1 were inhibitors of mevalonate kinase, their Ki values ranging over 140 mM to 2.5 mM and decreasing in the series CH2 > C(CH3)2 > CH(OH) > CH(CH3) > C(OH)‐CO2H > C(OH)CH2OH. Three of these analogues, the competitive inhibitors 3,5‐dihydroxyvalerate, 3‐methyl‐5‐hydroxy‐valerate and 3,3‐dimethyl‐5‐hydroxyvalerate are also (poor) substrates of the kinase.

Substances of group 2 contain an additional hydrophobic group and are noncompetitive inhibitors with Ki values decreasing systematically from 7.7 mM to 0.7 mM in the sequence 3‐n‐propyl, 3‐n‐butyl, 3‐n‐pentyl and 3‐n‐hexyl derivative of 3,5‐dihydroxyvalerate.

The methylated mevalonates of group 3 are competitive inhibitors as well as good substrates of the kinase, their Ki=Km values ranging over 4.3–0.2 mM and their maximal velocities related to mevalonate varying between 7–65%.

All of these analogues have in common the carbon chain and 5‐hydroxy group of the substrate and could, therefore, fit to the active site of mevalonate kinase and be phosphorylated by ATP. The results, however, indicate a strong dependence of such interaction on the ligands at C3 of the substrate. Enzymic activity as well as affinity are lost if both of these ligands are replaced by hydrogen; partial restoration occurs at a low level if only one of the ligands is concerned. In the presence of the ligands even monomethylation at C2 and C4 yields enzymically active products. Oxidation of C6 of the substrate yields inhibitors but methylation yields a reasonably good substrate. Thus 3,5‐dihydroxy‐3‐ethylvalerate fits into the active site whereas higher C6 homologues are only inhibitory and probably bind to a hydrophobic region of the enzyme. Some of these results are shown to have parallels in the enzymic behaviour of fluorinated mevalonates.
The kinetics and thermodynamics of the binding of cytochalasin B to sugar transporters
Authors: WALMSLEY Adrian R. , LOWE Allan G. , HENDERSON Peter J. F.
Published: March 2005

The kinetics of the binding of cytochalasin B to the proton‐linked l‐arabinose (AraE) and d‐galactose (GalP) symporters from Escherichia coli and to the human erythrocyte glucose transporter (GLUT1) have been investigated by exploiting the changes in protein fluoresence that occur upon binding the ligand. Steady‐state mesurements yielded Kd values of 1.1, 1.9 and 0.14 μM for the AraE, GalP and GLUT1 proteins, respectively. The association and dissociation rate constants for the binding of cytochalasin B have been determined by stopped‐flow spectroscopy. In each case, the apparent Kd was calculated from the corresponding rate constants, yielding values of 1.5, 0.4 and 1.6 μM for AraE, GalP and GLUT1, respectively. The differences between these apparent Kd values and those measured by fluorescence titration is interpreted in terms of the following three step mechanism where CB represents cytochalasin B:

The transporter is proposed to alternate between two different conformational forms (T1 and T2), with cytochalasin B binding only to the T2 conformation, to induce a further conformational transition of the transporter to the T3 form. The values for the overall dissociation constants show that the T1 conformation is favoured by AraE and GalP in the absence of ligands, but the T2 conformation is favoured by GLUT1. Thus, the binding of cytochalasin B to GLUT1 alters the equilibrium towards the T3(CB) conformational state, producing the observed tight binding, in contrast to the changes in the equilibrium observed with the binding of cytochalasin B to AraE and GalP. A thermodynamic analysis of these conformational transitions has been performed. The T1 and T2 conformations may represent transporter states in which the binding site is facing outwards and inwards, respectively.
Purification and characterization of three isolectins of soybean agglutinin
Authors: MANDAL Dipak K. , NIEVES Edward , BHATTACHARYYA Lokesh , ORR George A. , ROBOZ John , YU Qui‐tao , BREWER C. Fred
Published: March 2005

Soybean agglutinin (SBA) is a tetrameric dD‐Gal/d‐GalNAc‐specific lectin possessing one Man9 oligomannose‐type chain/monomer. SBA exists as multiple isolectins having similar binding and immunochemical properties. The present study shows that native SBA consists of at least five isolectins. Three of these isoforms have been purified by chromatofocusing and designated as SBA‐I, SBA‐II and SBA‐III in order of their elution from a chromatofocusing column. The pI of the isolectins are 7.0, 6.85 and 6.7, respectively, as determined by isoelectric focusing. Each isolectin was denatured in 6 M guanidine hydrochloride into their individual subunits which were separated by reverse‐phase high performance liquid chromatography (RP‐HPLC). The HPLC profiles were similar for all three isoforms which showed two major peaks (peak 1 and peak 3) along with a minor peak (peak 2). The first peak of SBA‐II existed as a doublet labeled as 1 a and 1 b. Each peak was analyzed by electrospray ionization mass spectrometry to characterize each isoform and determine their structural differences. The calculated mass of an intact lectin monomer from the amino acid sequence (253 residues) derived from cDNA of the lectin including a Man9 oligomannose chain is 29438 Da. The present results show that peak 3 of each isoform corresponds to an intact subunit (α) while peak 1 of each isoform shows lower masses which are assigned to C‐terminal fragmentation of the protein. Peak 1 of SBA‐I has a molecular mass of 28000Da corresponding to a fragmented subunit (β) consisting of 240 residues (calculated molecular mass 28001Da). Peak 1 a of SBA‐II shows a molecular mass of 28000Da corresponding to a fragmented β subunit, while peak 1 b showed two major species: a 28000‐Da (β subunit) and a 28327‐Da subunit which corresponds to 243 residues (calculated mass 28326Da) designated as a γ subunit. In addition, peak 1 b showed the presence of a molecular species of 28627Da corresponding to a 246‐residue subunit (γ′). Peak 1 of SBA‐III showed a major molecular species corresponding to a fragmented γ subunit. The minor peak in the HPLC profile (peak 2) represented a subunit of 252 residues for all three isoforms. The results suggest that the subunit compositions of SBA‐I, SBA‐II and SBA‐III are approximately α2β2, α2βγ and α2γ2, respectively. The results account for the decreasing pI of SBA‐I to SBA‐III since the 240‐residue β subunit involves loss of two acidic amino acids (Asp243 and Glu251) while the 243‐residue γ subunit involves loss of one acidic amino acid (Glu251) from the intact subunit. The present findings thus demonstrate that three isolectins of SBA arise from different combinations of intact and C‐terminal fragmented subunits.
Stimulation of cGMP‐dependent protein kinase Iα by a peptide from its own sequence
Authors: HUGGINS John P. , GANZHORN Axel J. , SAUDEK Vladimir , PELTON John T. , ATKINSON R. Andrew
Published: March 2005

The structure of cGMP‐dependent protein kinase Iα‐(546–576)‐peptide amide (peptide‐546) and its effects on cGMP‐dependent protein kinase Iα (G‐kinase) have been studied. By primary sequence analysis and analogy to a peptide that stimulates protein kinase C, peptide‐546 was predicted to form part of the protein/peptide binding site of G‐kinase, and it was proposed that it would stimulate the enzyme by interaction with an autoinhibitory site. The portion of cAMP‐dependent protein kinase analogous to peptide‐546 forms part of the peptide substrate binding site, interacting with the peptide inhibitor residues Argp‐2 and Phep‐11 (where p is the pseudophosphorylation site), through residues at positions corresponding to Glu4, Pro10 and Ser13 in peptide‐546. Peptide‐546 is a reasonably potent G‐kinase activator, increasing the turnover number with the peptide substrate Arg‐Lys‐Arg‐Ser‐Arg‐Lys‐Glu by about threefold with an activation constant that is about fivefold lower than the Km value of this peptide substrate.

Peptide‐546 does not appear to change the affinity of the enzyme for the above substrate, ATP or cGMP and does not affect the binding of [3H]cGMP to G‐kinase. The activation does not seem to result from an interaction between peptide‐546 and peptide substrates, and a kinetic scheme is proposed which is compatible with an action of peptide‐546 on G‐kinase independent of substrates. The activation is additive with that given by cGMP and causes the enzyme to enter a hitherto unrecognised superactive state. Peptide conformation has been monitored in mixed 2,2,2‐trifluoroethanol/H2O solvents by circular dichroism: helical structure is observed in these mixtures when the 2,2,2‐trifluoroethanol content is above 25%. The structure is lost only gradually on raising the temperature to 80°C with no clear melting transition.

Assignment of the resonances in the 1H‐NMR spectrum has allowed the identification of elements of secondary structure from detected nuclear Overhauser effects. In particular, a helical segment from Met18 to Arg26 is observed. The four proline residues (Pro10, Pro11, Pro15 and Pro17) are all seen to be in the trans conformation, although additional, weaker peaks in the spectra may correspond to a minor conformer in which one or more of the prolines is in a cis conformation. The N‐terminal residues are less structured but show some helical character.

Calculation of the structure of the peptide using nuclear Overhauser effects (NOEs) as distance constraints in distance geometry and molecular dynamics simulations revealed a well defined helical structure in the C‐terminal region preceded by a less well defined proline‐rich segment and N‐terminal region. On the basis of these results, peptide‐546 may adopt a conformation similar to that expected by analogy with cAMP‐dependent protein kinase, and we propose that residues 546–576 of G‐kinase are involved in enzyme autoinhibition.
Differential signal transduction of epidermal‐growth‐factor receptors in hormone‐dependent and hormone‐independent human breast cancer cells
Authors: MUELLER Heinz , LOOP Petra , LIU Rong , WOSIKOWSKI Katja , KUENG Willy , EPPENBERGER Urs
Published: March 2005

In breast cancer, hormone dependency is inversely correlated with the number of surface epidermal‐growth‐factor (EGF) receptors on the tumor cells. In vitro, EGF stimulated only hormone‐dependent immortalized human breast cancer cells to grow with an increased rate whereas hormone‐independent cells were not affected by EGF. The number of EGF surface receptors is about 5–10‐times smaller on hormone‐dependent cells than on hormone‐independent cells. Two cell lines representing the two cell types were used to demonstrate the signal‐transduction capabilities of the EGF receptors. The two cell lines were the hormone‐dependent MCF‐7 cells and the hormone‐independent MDA‐MB‐231 cells. Incubation at 37°C for 15 min with 10−8 M EGF increased the surface EGF‐receptor density substantially on MCF‐7 cells (50%) and reduced the number of these receptors on MDA‐MB‐231 cells to about 65% of the control. Both cell lines internalized a fluoresceinisothiocyanate‐labeled EGF with similar kinetics. EGF triggered tyrosine phosphorylation of several targets in isolated MCF‐7 cell membranes. One of these targets was shown by immuno‐precipitation to be the EGF receptor. In MDA‐MB‐231 cell membranes, the EGF receptor was demonstrated to be the main target for tyrosine phosphorylation. The mRNA expression of the immediate early proto‐oncogene c‐fos was stimulated by EGF only in MCF‐7 cells. In contrast, the mRNA of the EGF receptors was stimulated by EGF in both cell lines. These results demonstrate that, although EGF‐binding sites are present on both cell lines, their signal‐transduction capacity and activities are substantially different and resulted in a divergent response of the two cell types to EGF.
Distinct effects of glutathione disulphide on the nuclear transcription factors κB and the activator protein‐1
Authors: GALTER Dagmar , MIHM Sabine , DRÖGE Wulf
Published: March 2005

Oxidative conditions potentiate the activation of the nuclear transcription factor κB (NFκB) and the activator protein‐1 (AP‐1) in intact cells, but inhibit their DNA binding activity in vitro. We now show that both the activation of NFκB and the inhibition of its DNA binding activity is modulated in intact cells by the physiological oxidant glutathione disulphide (GSSG). NFκB activation in human T lineage cells (Molt‐4) by 12‐O‐tetradecanoyl‐phorbol 13‐acetate was inhibited by dithiothreitol, and this was partly reversed by the glutathione reductase inhibitor 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea (BCNU) or by hydrogen peroxide, indicating that GSSG may be required for NFκB activation. These effects of BCNU and hydrogen peroxide were not seen in glutathione‐depleted cells. However, NFκB and AP‐1 activation were potentiated by dithiothreitol if added to cell cultures 1 h after the phorbol ester, indicating that a shift of redox conditions may support optimal oxidative activation with minimal inhibition of DNA binding. The elevation of intracellular GSSG levels by BCNU before stimulation suppressed the chloramphenicol acetyltransferase expression dependent on NFκB but increased that dependent on AP‐1. This selective suppression of NFκB was also demonstrable by electrophoretic mobility shift assays. In vitro, GSSG inhibited the DNA binding activity of NFκB more effectively than that of AP‐1, while AP‐1 was inhibited more effectively by oxidized thioredoxin.
Sialidase in rabbit blood
Authors: CHEN Xiao‐Guang , NAGAI Takayuki , YAMADA Haruki
Published: March 2005

Sialidase activities of rabbit blood cells and serum were measured. The leucocyte particulate fraction showed the highest specific activity of sialidase towards mixed gangliosides and sialyllactose, and the cytosolic fraction showed for fetuin. Predominant sialidase activity in the blood was detected in erythrocyte particulate fraction when mixed gangliosides were used as substrate. The sialidase for ganglioside was solubilized from the erythrocyte ghosts by using Triton X‐100. The solubilized sialidase was purified 1886‐fold by sequential chromatographies on DEAE‐cellulose, EAH‐Sepharose 4B, Octyl‐Sepharose CL‐4B, Sephadex G‐100, concanavalin‐A‐Sepharose, N‐(p‐aminophenyl)oxamic acid‐agarose and Heparin‐Sepharose CL‐6B. The optimum pH of purified sialidase was 4.5 for ganglioside mixture, and this enzyme exhibited Mr= 48000 by gel filtration. When the purified sialidase was subjected to SDS/PAGE, a major sialidase‐active protein band at Mr= 54000 and another fainter inactive protein band with Mr= 115000 were observed. The purified enzyme was active towards oligosaccharides, gangliosides, fetuin glycopeptide and 4‐methylumbelliferyl α‐d‐N‐acetylneuraminic acid except for glycoproteins tested. Fe2+, Fe3+ and dithiothreitol significantly inhibited the enzyme activity, while Triton X‐100 activated the enzyme. Inside‐out vesicles and unsealed ghosts of rabbit erythrocyte showed the sialidase activity for mixed gangliosides but not for resealed ghosts or intact erythrocytes. These results indicate that the active site of this sialidase is oriented mainly on the inside of the erythrocyte membrane and not on the outside. Treatment of rabbit erythrocyte unsealed ghosts with phosphatidylinositol‐specific phospholipase C liberated no sialidase activity toward mixed gangliosides from the ghosts.
Characterization of nuclear factors involved in 202 gene induction by interferon‐α in murine leukemia cells
Authors: GARIGLIO Marisa , GABOLI Mirella , MANA Cristina , YING Guo‐Guang , GRIBAUDO Giorgio , CAVALLO Rossana , LANDOLFO Santo
Published: March 2005

The 5′ terminal flanking region of the interferon‐inducible gene, 202, contains an interferon‐stimulable response element (ISRE), called a GA box, that confers inducibility by interferon(IFN)‐α, but not by IFN‐γ, on a reporter gene, such as the chloramphenicol acetyltransferase (CAT). Nuclear extracts from L1210 murine leukemia cells, stimulated for various periods of time with IFN‐α, were mixed with 32P‐labeled GA box and analyzed for the presence of retarded complexes in electrophoretic‐mobility‐shift assays. In addition to a few constitutive retarded complexes, an inducible GA box‐binding activity (GAbf‐1) appeared after 5 min, peaked at about 2 h, and was still abundant 12 h after IFN‐α treatment. In the cytoplasmic fraction GAbf‐1 was not detectable before 30 min, continued to increase up to 2 h, but had disappeared whithin 12 h. GAbf‐1 activity was not observed in nuclear extracts treated with IFN‐γ, and was not inhibited by prior treatment with the protein‐synthesis inhibitor cycloheximide. When the binding properties of GAbf‐1 were compared with those of ISGF‐3, the primary transcriptional activator for IFN‐α‐induced genes, a different pattern of retarded complexes was observed. Moreover, as observed by immunoblotting analysis, nuclear extracts from IFN‐α‐treated L1210 cells did not contain the p91/84 subunit of the ISGF3, the best characterized nuclear complex activated by IFN‐α. Altogether these results indicate that GAbf‐1 may be a novel transcription factor exploited by IFN‐α to activate the 202 inducible gene in murine pre‐B leukemia cells.
Nucleic‐acid‐binding properties of hnRNP‐U/SAF‐A, a nuclear‐matrix protein which binds DNA and RNAin vivoandin vitro
Authors: FACKELMAYER Frank O. , DAHM Kirsten , RENZ Andrea , RAMSPERGER Uwe , RICHTER Arndt
Published: March 2005

We show that SAF‐A, a nuclear protein which specifically binds vertebrate scaffold‐attachmentregion (SAR) elements with high affinity is identical with hnRNP‐U, assumed to be involved in packaging of hnRNA in ribonucleoprotein particles. Ultraviolet cross‐linking experiments show that the protein, referred to as hnRNP‐U/SAF‐A, is bound to chromosomal DNAin vivo. In vitro, the isolated protein binds to double‐stranded and single‐stranded DNA and forms higher ordered nucleic‐acid–protein complexes. Filter‐binding experiments performed with different types of natural and synthetic nucleic acids as substrates show that the protein binds DNA and RNA with different affinities and most likely at different binding sites. We conclude that hnRNP‐U/SAF‐A thus may have functions in the organisation of chromosomal DNA in addition to its suggested role in hnRNA metabolism.
Use of transferred nuclear‐Overhauser‐effect spectroscopy to measure the bound conformation of a disulphide‐replaced analogue of glutathione disulphide as an inhibitor of yeast glutathione reductase
Authors: EMBREY Kevin J. , MEHTA Anita , CARRINGTON Simon J. , JAOUHARI Rabih , McKIE James H. , DOUGLAS Kenneth T.
Published: March 2005

The analogue of glutathione disulphide (GSSG) in which the disulphide bridge of GSSG is replaced by ‐CH2‐S‐ was synthesised from L‐cystathionine usingt‐butoxycarbonyl andt‐butyl ester protection with triethylsilane‐promoted deprotection. This analogue (GCSG) was found to be a linear, competitive inhibitor of yeast glutathione reductase (Kivalue 981 μM at pH 7.0), a very poor substrate and not to act as an irreversible inhibitor of glutathione reductase. The weak binding of GCSG to glutathione reductase permitted the use of transferred nuclear Overhauser effect spectroscopy (TRNOESY) to investigate the bound conformation of GCSG in its complex with glutathione reductase. The solution structure of free GCSG was investigated by NMR spectroscopy using a range of NMR techniques. The TRNOESY experiment allowed a range of conformations to be determined for the central bridge region (containing the ‐CH2‐S‐ replacement) of GCSG bound to yeast glutathione reductase. Using the nuclear Overhauser effect constraints thus derived, in combination with molecular graphics and energy minimisation based on the known crystal coordinates of glutathione disulphide (GSSG) bound to human erythrocyte glutathione reductase, allowed an explanation of the lack of substrate activity of GCSG, its inactivity as a suicide inactivator and its relatively weak binding in terms of the enforced mislocation of the ‐CH2‐S‐ bridge with respect to the catalytic residues (relative to GSSG). Thus, the simple replacement of ‐S‐ by ‐CH2‐, common in medicinal chemistry, can lead to poor receptor binding if the replacement occurs in a central, rather than peripheral, part of the ligand under modification.
Stability and proteolytic domains of Nef protein from human immunodeficiency virus (HIV) type 1
Authors: FREUND Jens , KELLNER Roland , HOUTHAEVE Tony , KALBITZER Hans Robert
Published: March 2005

Proteolytic experiments in conjunction with 1H‐NMR spectroscopy show that the Nef (negative factor) protein from human immunodeficiency virus type 1 probably consists of two main domains, the N‐terminal anchor domain at amino acid positions 2–65 and the C‐terminal core domain at positions 66—206. The N‐terminal domain is likely to be located at the surface of the protein, while the C‐terminal domain has a compactly folded core and is stable in the absence of the anchor domain. It is conceivable that the core domain represents a functional domain of the Nef protein, activated after the removal of the membrane anchor by the human‐immunodeficiency‐virus protease or cellular proteases.

Nef is stable at pH 5–12 and denatures at 317–322 K. The Nef protein remains in its native conformation in dimethyl‐sulfoxide/water mixtures up to 35% (by vol.), and in acetonitrile/water up to 14% (by vol.). Nef refolds spontaneously after denaturation with urea or guanidinium hydrochloride. The 1H‐NMR parameters and pKa values of five of the nine histidine residues and one of the seven tyrosine residues were determined and were found in four cases to be typical for residues which are not located in the interior of the protein.
On the iron‐sulfur cluster of adenosine phosphosulfate reductase from Desulfovibrio vulgaris (Hildenborough)
Authors: VERHAGEN Marc F. J. M. , KOOTER Ingeborg M. , WOLBERT Ronnie B. G. , HAGEN Wilfred R.
Published: March 2005

Adenosine phosphosulfate reductase from Desulfovibrio vulgaris Hildenborough has been purified to homogeneity and was found to consist of two subunits. The α and β subunits have molecular masses of 67.8 kDa and 25.6 kDa, respectively. The apparent molecular mass of the protein is dependent on the ionic strength of the buffer. At low ionic strength, a high molecular‐mass multimer is formed, which reversibly changes into smaller units upon addition of salt. The smallest catalytically active unit of the enzyme has a molecular‐mass of 186 kDa, as determined by gel‐filtration chromatography and, therefore, an α2β2 stoichiometry is proposed. The protein was found to contain 5.6±1.1 iron and 4.4±0.6 acid‐labile sulfur atoms/αβ heterodimer. The reduced protein exhibits a single, rhombic S= 1/2 signal with g values 2.070, 1.932 and 1.891. Lowering the ionic strength of the buffer reversibly changes this spectrum into a complex EPR spectrum, indicating intermolecular, dipolar magnetic coupling. Spin quantification of the reduced protein either at low or at high ionic strength never resulted in more than 1 spin/αβ heterodimer. Hence, it follows that the iron and sulfur atoms are arranged in one single cluster. The reduction potential of the iron sulfur cluster, measured in an EPR‐monitored redox titration, was found to be −19 mV versus the normal hydrogen electrode (NHE) at pH 7.5. The reduction potential of the flavin measured in an optical titration was found to be −59 mV against NHE at pH 7.5. The flavin behaves as a two‐electrontransferring group; no evidence was obtained for a stabilization of the intermediate semiquinone state in the enzyme. Determination of the kinetic parameters of adenosine 5′‐phosphosulfate (Ado‐PSO4) reductase for its substrates resulted in Km values for sulfite and AMP of 130 μM and 50 μM, respectively. It is proposed that AdoPSO4 reductase contains a single novel Fe/S structure, possibly with an iron‐nuclearity greater than four.
Characterisation and purification of ribulose‐bisphosphate carboxylase from heterotrophically grown halophilic archaebacterium, Haloferax mediterranei
Authors: RAJAGOPALAN Rema , ALTEKAR Wijaya
Published: March 2005

The CO2‐fixing enzyme of Calvin cycle ribulose‐1,5‐bisphosphate‐carboxylase/oxygenase has been isolated from a halophilic bacterium, Haloferax mediterranei grown heterotrophically. A homogeneous preparation was obtained from sonicated extract of the cells by three steps, resulting in a specific activity of 52 nmol · min−1· mg protein−1. The physicochemical and catalytic properties of the enzyme were studied. The halobacterial ribulose‐bisphosphate carboxylase is an oligomer of 54‐kDa and 14‐kDa subunits as detected by SDS/PAGE. By sucrose‐density‐gradient centrifugation, the molecular mass of the enzyme was estimated as approximately 500 kDa indicating a hexadecameric nature. No evidence for an additional form of the enzyme devoid of small subunits was obtained. The enzyme required Mg2+ for activity, KCl for activity and stability, and an optimal pH of 7.8. In contrast to many halophilic proteins, ribulose‐bisphosphate carboxylase from H. mediterranei is not an acidic protein. From the comparison of amino acid composition of halobacterial enzyme with its counterparts from a few eukaryotic and eubacterial sources, the SΔQ values showed that these proteins share some compositional similarities.
Polymorphonuclear neutrophils release 35S‐labelled proteoglycans into cartilage during frustrated phagocytosis
Authors: GARDINER Elizabeth E. , MOK Su San , SRIRATANA Absorn , ROBINSON H. Clem , VEITCH Barry J. A. , LOWTHER Dennis A. , HANDLEY Christopher J.
Published: March 2005

Rabbit peritoneal polymorphonuclear neutrophils (PMN), incubated in medium containing [35S]sulphate, incorporated 35S into proteoglycan and protein fractions. Approximately 46% of the 35S‐labelled macromolecules associated with the PMN cells after 1 h of incubation were recovered in a cytoplasmic granule extract, the majority being present in azurophil granules. Analysis of the azurophil granule fraction showed that approximately 90% of the 35S‐labelled macromolecules were proteoglycans. When challenged with heat‐aggregated rabbit γ‐globulin in the presence of cytochalasin B and cGMP, PMN were induced to release granular enzymes but did not release 35S‐labelled proteoglycans into the incubation medium. When incubated with articular cartilage slices, PMN released their granule 35S‐labelled proteoglycan into the medium and into the cartilage matrix. Granule enzymes and 35S‐labelled granule proteoglycan were extracted from the cartilage tissue after incubation and 35S‐labelled macromolecules were detected in the cartilage tissue by autoradiography.
Characterization of the human gene (PTGS2) encoding prostaglandin‐endoperoxide synthase 2
Authors: KOSAKA Tetsuya , MIYATA Atsuro , IHARA Hayato , HARA Shuntaro , SUGIMOTO Tamiko , TAKEDA Osamu , TAKAHASHI Ei‐ichi , TANABE Tadashi
Published: March 2005

The human gene (PTGS2) encoding an inducible isozyme of prostaglandin‐endoperoxide synthase (prostaglandin‐endoperoxide synthase 2) that is distinct from the well‐characterized and constitutive isozyme (prostaglandin‐endoperoxide synthase 1), was isolated using a polymerase‐chain reaction‐generated cDNA fragment probe for human prostaglandin‐endoperoxide synthase 2. Nucleotide sequence analysis of the entire human prostaglandin‐endoperoxide‐synthase‐2 gene demonstrated that it is more than 8.3 kb in size and consists of ten exons; this gene is very similar to the murine and chicken prostaglandin‐endoperoxide‐synthase‐2 genes. The structures of exons in the human prostaglandin‐endoperoxide‐synthase‐2 gene were also similar to those of the human prostaglandin‐endoperoxide‐synthase‐1 gene (PTGS1). However, the sizes of introns in the human prostaglandin‐endoperoxide‐synthase‐2 gene were generally smaller than those of the human prostaglandin‐endoperoxide‐synthase‐1 gene. Primer‐extension analysis indicated that the transcriptional‐start site is 134 bases upstream of the translational‐initiation site. The sequence of the 1.69‐kb region of nucleotides preceding the transcriptional‐start site and the first 0.8‐kb intron contained a canonical TATA box and various transcriptional‐regulatory elements (CArG box, NF‐IL6, PEA‐1, myb, GATA‐1, xenobiotic‐response element, cAMP‐response element, NF‐κB, PEA‐3, Sp‐1 and 12‐O‐tetradecanoyl‐phorbol‐13‐acetate‐response element). The nucleotide sequence of the 5′‐flanking region (275 bp) of the human prostaglandin‐endoperoxide‐synthase‐2 gene showed 63% similarity to the sequence of murine prostaglandin‐endoperoxide‐synthase‐2/TIS10 gene, but essentially no homology to the chicken prostaglandin‐endoperoxide‐synthase‐2 gene, and human and murine prostaglandin‐endoperoxide‐synthase‐1 genes. A fluorescence in situ hybridization study showed that the human genes coding for prostaglandin‐endoperoxide synthase 1 (PTGS1) and prostaglandin‐endoperoxidase synthase 2 (PTGS2) were mapped to distinct chromosomes 9q32‐q33.3 and 1q25.2‐q25.3, respectively, indicating that these genes are not genetically linked.
Expression, purification, and substrate specificity of isocitrate dehydrogenase from Thermus thermophilus HB8
Authors: MIYAZAKI Kentaro , YAOI Takuro , OSHIMA Tairo
Published: March 2005

Isocitrate dehydrogenase (ICDH) from an extreme thermophile, Thermus thermophilus HB8, was overexpressed in Escherichia coli. The enzyme was easily purified to homogeneity by a combination of heat treatment (70°C, 20 min) and column chromatography. The N‐terminal sequence of the protein thus purified coincided with that of the protein extracted from the thermophile. The substrate specificity of the enzyme was mutationally analyzed and engineered to recognize 3‐alkylmalate as a substrate. Based on the three‐dimensional structure of E. coli isocitrate dehydrogenase, Ser97 qnd Asn99 of the thermophile enzyme were speculated to participate in the substrate recognition, and these residues were replaced with threonine and leucine, respectively. Molecular recognition of the mutant enzymes, [S97T]ICDH, [N99L]ICDH, and [S97T, N99L]ICDH, were studied using isocitrate, 3‐isopropylmalate, and 3‐ethylmalate. The affinity toward isocitrate was reduced in the cases of [S97T]ICDH and [N99L]ICDH, confirming the importance of the residues for the reaction. Though none of the mutants acted on 3‐isopropylmalate, [N99L]ICDH was competitively inhibited by 3‐isopropylmalate with a higher affinity than that of the wild‐type enzyme. [N99L]ICDH showed an approximately 103‐fold higher value of (kcat/Km)3‐ethylmalate/(kcat/Km)isocitrate than the wild‐type enzyme, indicating that the single mutation of Asn99 to leucine switched the substrate specificity of the enzyme away from isocitrate and toward 3‐ethylmalate.
Molecular characterization of human TRPM‐2/clusterin, a gene associated with sperm maturation, apoptosis and neurodegeneration
Authors: WONG Paul , TAILLEFER Daniel , LAKINS Jonathon , PINEAULT Jean , CHADER Gerald , TENNISWOOD Martin
Published: March 2005

The TRPM‐2/clusterin gene and its cognate protein has been characterized in a number of species. Although the functional role, or roles, of the TRPM‐2/clusterin protein remains to be firmly established, the gene has been implicated in a variety of physiological processes, including sperm maturation, lipid transport, membrane remodelling and inhibition of the complement cascade. TRPM‐2/clusterin is induced de novo during the regression of the prostate and other hormone‐dependent tissues after hormone ablation, and is over‐expressed in several human neurodegenerative diseases including Alzheimer's disease, epilepsy and retinitis pigmentosa. We describe the genomic structure of the human TRPM‐2/clusterin gene which is organized into nine exons, ranging in size from 47 bp (exon I) to 412 bp (exon V), spanning a region of 16580 bp. Comparison with sequences registered in the databases shows that it has extensive similarity to the human protein designated as SP‐40,40 or complement‐lysis inhibitor (CLI), a protein that appears to block the membrane‐attack complex of complement. However, the cDNA sequences reported for SP‐40,40 and CLI diverge significantly in the 5′ untranslated region of the mRNA (coded for by exon I), raising the possibility that the TRPM‐2/clusterin gene is present in the human genome as a small multi‐gene family or that there are several alternate exon I sequences in the TRPM‐2 gene. Southern analysis and fluorescent in situ hybridization suggest that the clusterin gene is a single‐copy gene, and that, if alternative exon I sequences are present in the genome, they lie outside of the λ clones that have been characterized. Analysis of the promoter region of the human TRPM‐2/clusterin gene shows many similarities to the rat TRPM‐2/clusterin promoter including a putative control region containing several potential regulatory elements that may regulate the complex tissue‐specific control of the gene which must be constitutively expressed in some tissues but is inducible in others.
Solution structure of the ɛ‐aminohexanoic acid complex of human plasminogen kringle 1
Authors: REJANTE Marita R. , LLINÁS Miguel
Published: March 2005

The solution structure of the human plasminogen kringle 1 domain complexed to the antifibrinolytic drug 6‐aminohexanoic acid (ɛAhx) was obtained on the basis of 1H‐NMR spectroscopic data and dynamical simulated annealing calculations. Two sets of structures were derived starting from (a) random coil conformations and (b) the (mutated) crystallographic structure of the homologous prothrombin kringle 1. The two sets display essentially the same backbone folding (pairwise root‐mean‐square deviation, 0.15 nm) indicating that, regardless of the initial structure, the data is sufficient to locate a conformation corresponding to an essentially unique energy minimum. The conformations of residues connected to prolines were localized to energetically preferred regions of the Ramachandran map. The Pro30 peptide bond is proposed to be cis. The ligand‐binding site of the kringle 1 is a shallow cavity composed of Pro33, Phe36, Trp62, Tyr72 and Tyr74. Doubly charged anionic and cationic centers configured by the side chains of Asp55 and Asp57, and Arg34 and Arg71, respectively, contribute to anchoring the zwitterionic ɛAhx molecule at the binding site. The ligand exhibits closer contacts with the kringle anionic centers (∼0.35 nm average O…H distance between the Asp55/Asp57 carboxylate and ligand amino groups) than with the cationic ones (∼0.52 nm closest O…H distances between the ligand carboxylate and the Arg34/Arg71 guanidino groups). The ɛAhx hydrocarbon chain rests flanked by Pro33, Tyr64, Tyr72 and Tyr74 on one side and Phe36 on the other. Dipolar (Overhauser) connectivities indicate that the ligand aliphatic moiety establishes close contacts with the Phe36 and Trp62 aromatic rings. The computed structure suggests that the ɛAhx molecule adopts a kinked conformation when complexed to kringle 1, effectively shortening its dipole length to ɛ0.65 nm.
Purification and characterization of a thermostable carboxylesterase from the thermoacidophilic eubacterium Bacillus acidocaldarius
Authors: MANCO Giuseppe , DI GENNARO Spartaco , DE ROSA Mario , ROSSI Mosè
Published: March 2005

A thermostable carboxylesterase from the thermoacidophilic eubacterium Bacillus acidocaldarius was isolated, purified 1800‐fold to homogeneity, and characterised. The apparent molecular mass was 36.5 ± 2.5 kDa when determined by SDS/PAGE and 37.5 kDa when determined by analytical gel filtration, suggesting a monomeric structure. The pure enzyme regained activity on removal of SDS after SDS/PAGE. Several esterase activities were revealed in crude extracts by PAGE and activity staining, although only one was detected after SDS/PAGE and detergent removal. The esterase showed optimal activity at around 70°C and pH 8, and was thermostable. p‐Nitrophenyl esters of fatty acids from C2 to C12 were used as substrates; Vmax and Km values were determined at three different temperatures. The enzyme was able to hydrolyse tributyrylglycerol and trihexanoyl‐glycerol dissolved in 0.8% acetonitrile, but neither lipase activity toward [14C]trioleoylglycerol nor proteolytic activity could be detected. Inactivation by diethyl p‐nitrophenyl phosphate, by phenyl‐methansulfonyl fluoride and physostigmine, and by diethylpyrocarbonate suggested that the enzyme contained a catalytic triad Ser‐His‐Asp/Glu in the active site, similar to that demonstrated for other serine‐type enzymes. The amino acid composition and the sequence of 19 amino acid residues at the N‐teminus were determined. These data, together with substrate preference and inhibition pattern, allowed us to classify this enzme as a B‐type carboxylesterase (EC 3.1.1.1).