bluuebunny/crossref_metadata_2025 huggingface dataset:
Characteristics of an exochitinase from Streptomyces olivaceoviridis, its corresponding gene, putative protein domains and relationship to other chitinases
Authors: BLAAK Harald , SCHNELLMANN Jürgen , WALTER Stefan , HENRISSAT Bernard , SCHREMPF Hildgund
Published: March 2005

Streptomyces olivaceoviridis efficiently degrades chitin. Shotgun cloning of partially Sau3A‐cleaved DNA using the multicopy vector pIJ702 and Streptomyces lividans 66 as host resulted in the identification of the plasmid pCHI O1 which harbours an insert of 4.6 kb. In the presence of chitin as sole carbon source, transformants of S. lividans 66 carrying pCHI O1 or its derivatives with smaller inserts overproduced an exochitinase which was purified to homogeneity. The chitininducible enzyme with an isoelectric point of 4.0 shows optimal activity at pH 7.3 and 55°C, has an apparent molecular mass of 47 kDa and is competitively inhibited by the pseudosugar allosamidin. The enzyme was identified as an exochitinase since it generates exclusively chitobiose from chitotetraose, chitohexaose, and colloidal high‐molecular mass chitin. Sequence analysis of a reading frame of 1794 base pairs and comparison of the deduced amino‐acid sequence allowed the identification of the putative catalytic domain, one region with significant similarity to the type‐III module of fibronectin and one domain of unknown function. Multiple sequence alignment and hydrophobiccluster analysis of 25 chitinolytic enzymes from bacteria, fungi and plants allowed the identification of their characteristic domains. The exochitinase from S. olivaceoviridis shares highest similarity with the chitinase D from Bacillus circulans.
Hydrogen‐bonding pattern of methyl β‐lactoside binding to the Ricinus communis lectins
Authors: SOLÍS Dolores , FERNÁNDEZ Paloma , DÍAZ‐MAURIÑO Teresa , JIMÉNEZ‐BARBERO Jesús , MARTÍN‐LOMAS Manuel
Published: March 2005

The binding of O‐methyl and fluorodeoxy derivatives of methyl β‐lactoside to the Ricinus communis toxin (RCA60) and agglutinin (RCA120) was studied in order to determine the donor/acceptor relationships of the hydrogen bonds between the hydroxyl groups of methyl β‐lactoside and the binding sites of the lectins. Free energy contributions of the hydrogen bonds at each position have been estimated from these data and from those previously reported for the monodeoxy derivatives [Rivera‐Sagredo, A., Solís, D., Díaz‐Mauriño, T., Jiménez‐Barbero, J. & Martín‐Lomas, M. (1991) Eur. J. Biochem. 197, 217–228; Rivera‐Sagredo, A., Jiménez‐Barbero, J., Martín‐Lomas, M., Solís, D. & Díaz‐Mauriño, T. (1992) Carbohydr. Res. 232, 207–226]. The nature of the groups of the lectins involved in hydrogen bonding has been predicted on the basis of the free energy data. Analysis of the results indicates that both the C‐3′ and C‐4′ hydroxyl groups act as hydrogen‐bond donors to charged groups of both RCA60 and RCA120. The C‐6′ and probably also the C‐2′ hydroxyl groups participate both as donors and as acceptors of two hydrogen bonds with neutral groups of the lectins. And finally, the C‐6 hydroxyl group possibly acts as a donor of a weak hydrogen bond to a neutral group in RCA60, but not in RCA120. The results provide a molecular basis to explain some features of the binding specificity of the lectins. Comparison of RCA60 binding data with the recently refined X‐ray crystal structure of the RCA60‐lactose complex shows similarities but also some discrepancies that can be attributed to the marked influence of the pH on the carbohydrate—lectin interaction.
Structural analysis of two oligosaccharide bisphosphates isolated from the lipopolysaccharide of a recombinant strain of Escherichia coli F515 (Re chemotype) expressing the genus‐specific epitope of Chlamydia lipopolysaccharide
Authors: HOLST Otto , BROER Wim , THOMAS‐OATES Jane E. , MAMAT Uwe , BRADE Helmut
Published: March 2005

The lipopolysaccharide of the recombinant strain Escherichia coli F515–207, expressing the genus‐specific epitope of Chlamydia lipopolysaccharide, was sequentially de‐O‐ and de‐N‐acylated by mild hydrazinolysis and treatment with 4 M KOH, respectively, yielding two oligosaccharide bisphosphates which were isolated by high‐performance anion‐exchange chromatography and gelpermeation chromatography. Their structures were determined by chemical analysis, NMR spectroscopy, and mass spectrometry as α‐Kdo‐(2‐4)‐α‐Kdo‐(2‐6)‐β‐d‐GlcN‐(1‐6)‐α‐d‐GlcN 1,4′‐P2 (tetrasaccharide bisphosphate) and α‐Kdo‐(2‐8)‐α‐Kdo‐(2‐4)‐α‐Kdo‐(2‐6)‐β‐d‐GlcN‐(1–6)‐α‐d‐GlcN 1,4′‐P2 (pentasaccharide bisphosphate).
31P‐NMR analysis of Entamoeba histolytica
Authors: MARTIN Jean‐Baptiste , BAKKER‐GRUNWALD Tilly , KLEIN Gérard
Published: March 2005

Perchloric‐acid extracts of axenic Entamoeba histolytica were investigated by 31P‐NMR spectroscopy. All major 31P resonances observed were assigned to specific compounds. The cells contained inorganic phosphate (1039 nmol/g wet cells), pyrophosphate (16 nmol/g wet cells), nucleoside diphosphates (91 nmol/g wet cells), nucleoside triphosphates (275 nmol/g wet cells), NAD(P) (60 nmol/g wet cells), phosphocholine (184 nmol/g wet cells), phosphoethanolamine (214 nmol/g wet cells), cytidine 5′‐diphosphocholine (41 nmol/g wet cells) and cytidine 5′‐diphosphoethanol‐amine (55 nmol/g wet cells). The latter four compounds may act as intermediates in the salvage pathway for the synthesis of phosphatidylethanolamine and phosphatidylcholine. E. histolytica trophozoites also contained two inositol phosphates in large quantities, InsP3 (0.26 μmol/g wet cells) and InsP7 (0.11 μmol/g wet cells). These components were identified by 31P‐NMR, using homonuclear J‐resolved and two‐dimensional 1H‐31P correlative, analyses as myo‐inositol trisphosphate, Ins(2,4,6)P3, and pentakisphospho‐myo‐inositol diphosphate, Ins(1,2,3,4,6)P5(5)P2.
Isolation and characterization of microsomal acyl‐CoA thioesterase
Authors: ALEXSON Stefan E. H. , MENTLEIN Rolf , WERNSTEDT Christer , HELLMAN Ulf
Published: March 2005

We have isolated and characterized an acyl‐CoA thioesterase from rat liver microsomes. The enzyme consists mainly of a monomer of 59 kDa. However, the final preparation was found to contain minor amounts of a trimeric form of the protein. The enzyme was purified more than 85‐fold from isolated microsomes and used for NH2‐terminal sequence analysis and for analysis of peptides isolated after proteolytic digestion. The NH2‐terminal sequence was unique but highly conserved compared to those of other carboxylesterases. Internal sequence data, covering almost 20% of the protein, showed high similarity to the deduced amino acid sequences from a cDNA encoding a carboxylesterase synthesized in the liver and subsequently secreted to the blood [Alexson, S. E. H., Finlay, T. H., Hellman, U., Diczfalusy, U. & Eggertsen, G., unpublished results] and nonspecific rat liver microsomal carboxylesterase with isoelectric point of 6.1 [Robbi, M., Beaufay, H. & Octave, J.‐N. (1990) Biochem. J. 269, 451–458], thus confirming earlier suggestions that this enzyme is a member of the microsomal carboxylesterase multigene family. The peptide sequences contained two of the four conserved cysteic acid residues found in other carboxylesterases. Amino acid analysis indicated that the protein contains five cysteine residues in contrast to most other described carboxylesterases which contain four highly conserved cysteins.

The purified protein was used for immunization and the antiserum was used to detect the protein as well as its trimeric form, which is a minor component, in isolated rat liver microsomes. The antiserum recognized proteins of similar sizes in microsomes and 100000Xg supernatant prepared from hamster brown adipose tissue, a tissue known to contain very high activity of carboxylesterase, and to recognize carboxylesterases isolated from porcine and rabbit liver.
Isomalto‐oligosaccharide‐containing lipoteichoic acid of Streptococcus sanguis
Authors: KOCHANOWSKI Bernd , LEOPOLD Klaus , FISCHER Werner
Published: March 2005

The lipoteichoic acid of Streptococcus sanguis DSM 20567 contains a poly(glycerophosphate) chain, with 49% of the glycerophosphate residues being substituted with D‐alanine ester, 35% with α‐d‐glucopyranosyl and α‐isomalto‐oligosaccharide residues. Analysis of molecular species by affinity chromatography on concanavalin A showed all chains to be substituted and alanine ester and glycosyl residues to be present on the same rather than on separate chains. Molecular species varied in the length of the poly(glycerophosphate) chain, the extent of glycosylation, and had a constant alanine‐ester content. An alkali‐hydrolysis procedure revealed a distribution pattern between random and regular for the glycosyl substituents and suggested a similar distribution for the alanyl residues which occupy the free positions between the glycosyl substituents.
Two different genes coding for fibronectin‐binding proteins from Streptococcus dysgalactiae
Authors: LINDGREN Per‐Eric , McGAVIN Martin J. , SIGNÄS Christer , GUSS Bengt , GURUSIDDAPPA Sivashankarappa , HÖÖK Magnus , LINDBERG Martin
Published: March 2005

The binding of Streptococcus dysgalactiae to fibronectin involves fibronetcin‐binding protein(s) present on the bacterial surface. Previously, we reported the cloning of two different genes coding for cell‐wall‐associated fibronectin‐binding proteins from S. dysgalactiae strain S2 [Lindgren, P.‐E., Speziale, P., McGavin, M. J., Monstein, H.‐J., Höök, M., Visai, L., Kostiainen, T., Bozzini, S. & Lindberg, M. (1992) J. Biol. Chem. 267, 1924–1931]. The two genes, fnbA and fnbB, have now been sequenced and the primary amino acid sequences of the two fibronectin‐binding proteins, FnBA and FnBB, have been deduced. The two proteins have predicted molecular masses of 117 kDa and 122 kDa, respectively, and are organized in a similar way. The fibronectin‐binding activities are localized in repeated motifs, 32–37 amino acids long, in the COOH‐terminal regions of the proteins. The two fibronectin‐binding proteins have heterologous amino acid sequences, except for the COOH‐terminal ends which include the fibronectin‐binding repeats. The fibronectin‐binding regions of the genes have been fused to IgG‐binding domains of protein A, utilizing the IgG‐binding capacity of the resulting fusion proteins, to facilitate isolation of the fibronectin‐binding domains.
Dipeptidyl‐peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon‐like peptide‐1(7–36)amide, peptide histidine methionine and is responsible for their degradation in human serum
Authors: MENTLEIN Rolf , GALLWITZ Baptist , SCHMIDT Wolfgang E.
Published: March 2005

Peptides of the glucagon/vasoactive‐intestinal‐peptide (VIP) peptide family share a considerable sequence similarity at their N‐terminus. They either start with Tyr‐Ala, His‐Ala or His‐Ser which might be in part potential targets for dipeptidyl‐peptidase IV, a highly specialized aminopeptidase removing dipeptides only from peptides with N‐terminal penultimate proline or alanine. Growthhormone‐releasing factor(1–29)amide and gastric inhibitory peptide/glucose‐dependent insulinotropic peptide (GIP) with terminal Tyr‐Ala as well as glucagon‐like peptide‐1(7–36)amide/insulinotropin [GLP‐1(7–36)amide] and peptide histidine methionine (PHM) with terminal His‐Ala were hydrolysed to their des‐Xaa‐Ala derivatives by dipeptidyl‐peptidase IV purified from human placenta. VIP with terminal His‐Ser was not significantly degraded by the peptidase. The kinetics of the hydrolysis of GIP, GLP‐1(7–36)amide and PHM were analyzed in detail. For these peptides Km values of 4–34 μM and Vmax values of 0.6–3.8 μmol · min−1· mg protein−1 were determined for the purified peptidase which should allow their enzymic degradation also at physiological, nanomolar concentrations. When human serum was incubated with GIP or GLP‐1(7–36)amide the same fragments as with the purified dipeptidyl‐peptidase IV, namely the des‐Xaa‐Ala peptides and Tyr‐Ala in the case of GIP or His‐Ala in the case of GLP‐1(7–36)amide, were identified as the main degradation products of these peptide hormones. Incorporation of inhibitors specific for dipeptidylpeptidase IV, 1 mM Lys‐pyrrolidide or 0.1 mM diprotin A (Ile‐Pro‐Ilc), completely abolished the production of these fragments by serum. It is concluded that dipeptidyl‐peptidase IV initiates the metabolism of GIP and GLP‐1(7–36)amide in human serum. Since an intact N‐terminus is obligate for the biological activity of the members of the glucagon/VIP peptide family [e. g. GIP(3–42) is known to be inactive to release insulin in the presence of glucose as does intact GIP], dipeptidyl‐peptidase‐IV action inactivates these peptide hormones. The relevance of this finding for their inactivation and their determination by immunoassays is discussed.
Myristoyl‐CoA: protein N‐myristoyltransferase activity in cancer cells
Authors: BOUTIN Jean A. , FERRY Gilles , ERNOULD Anne‐Pascale , MAES Pierrette , REMOND Georges , VINCENT Michel
Published: March 2005

Myristoylation is a co‐translational maturation process of proteins. It is extremely specific for the cosubstrate (myristoyl‐CoA) and for the substrate protein that should bear a glycine at the N‐terminus of the protein to be myristoylated. This acylation is catalyzed by the myristoyl‐CoA:protein N‐myristoyltransferase. Most of the molecular biochemistry and biology concerning this enzyme has been done on Saccharomyces cerevisiae. Because of the major importance of this pathway in several types of pathology, it is essential to study intensively the enzyme(s) isolated from mammalian tissue(s) to confirm that the enormous amount of work done on the yeast enzyme can be transposed to mammalian tissues. In earlier studies, we demonstrated the existence of a microsomal N‐myristoyltransferase from the murine leukemia cell line L1210 [Boutin, J. A., Clarenc, J.‐P., Ferry, G., Ernould, A. P., Remond, G., Vincent, M. & Atassi, G. (1991) Eur. J. Biochem. 201, 257–263], a feature which is not shared by yeast, and examined the N‐myristoyltransferase activities associated with L1210 cytosol. In the present work, we purified to homogeneity one of the isoforms (A) of the transferase from L1210 cytosol. The purified enzyme showed on SDS/PAGE an apparent molecular mass of 67.5 kDa, distinct from the 53‐kDa yeast cytosolic enzyme. The purified enzyme from L1210 cytosol could be labeled with [14C]myristoyl‐CoA. Rabbit antibodies were raised against the A isoform and used to immunoprecipitate the enzyme and immunoinhibit the activity from the same source. A survey of the specificity of the partially and completely purified isoforms was performed using peptides derived from the NH2‐terminus of 42 proteins which are potential substrates for myristoylation, including oncogene products and virus structural proteins. We synthesized a series of compounds capable of inhibiting the cytosol activities of the enzyme. For example, a myristoyltetrahydroquinolein derivative showed an IC50 of about 0.1 μM. Based on both biophysical and biochemical evidence, the N‐myristoyltransferases extracted from mammalian cell cytosols seem to be different from the extensively studied yeast enzyme.
Yeast seryl‐tRNA synthetase expressed in Escherichia coli recognizes bacterial serine‐specific tRNAs in vivo
Authors: WEYGAND‐DURAŠEVIĆ Ivana , BAN Nenad , JAHN Dieter , SÖLL Dieter
Published: March 2005

The Saccharomyces cerevisiae serS gene which encodes seryl‐tRNA synthetase (SerRS) was expressed in Escherichia coli from the promoter and the ribosome binding sequences contained in its own 5′‐flanking region. The low level of yeast SerRS in the prokaryotic host was sufficient to permit in vivo complementation of two temperature‐sensitive E. coli serS mutants at the nonpermissive temperature. Thus, yeast SerRS can aminoacylate E. coli tRNASer species in vivo.

Yeast SerRS, isolated from an overexpressing E. coli strain by a rapid two‐step purification on FPLC, aminoacylated E. coli tRNA with serine much more poorly (relative kcat/Km= 2 × 10−4) than its homologous tRNAs. DL‐Serine hydroxamate, an inhibitor of E. coli SerRS, inhibits yeast SerRS in vivo and in vitro with an inhibition constant (K1) of 2.7 mM, a value 90‐fold higher than that for E. coli SerRS.
Topological study of PSI‐A and PSI‐B, the large subunits of the photosystem‐I reaction center
Authors: VALLON Olivier , BOGORAD Lawrence
Published: March 2005

The core of the photosystem‐I reaction center is formed by polypeptides PSI‐A and PSI‐B, the products of the homologouspsaAandpsaBgenes. Based on hydropathy analyses, models have been proposed for the folding of these polypeptide chains in the membrane [Fish, L. E., Kück, U. & Bogorad, L. (1985), inMolecular biology of the photosynthetic apparatus, pp. 111–120, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY]. To test these models, we have tried to identify regions of PSI‐A that are exposed to the surrounding medium, on the stromal or lumenal surface of the membrane. Immunogold labeling of thylakoid vesicles, with antibodies to synthetic peptides, shows that residues 413–421 of PSI‐A are exposed on the stromal surface of the membrane, and that the accessibility of this region is enhanced by NaSCN treatment, which removes extrinsic polypeptides. This treatment also enhances a trypsin‐cleavage site which may lie just after residues 413–421. Immunogold labeling also indicates that residues 371–379 and 497–505 are exposed on the lumenal surface. These results establish the conformation of the central portion of the polypeptide. Assuming that the transmembrane regions are correctly predicted by the 11‐helix model, the N‐terminal domain, as well as the conserved region proposed to bind the iron‐sulfur center Fx, would be expected to be on the stromal surface.
Reversal of the double‐stranded‐RNA‐induced inhibition of protein synthesis by a catalytically inactive mutant of the protein kinase PKR
Authors: SHARP Tyson V. , XIAO Qiurong , JEFFREY Ian , GEWERT Dirk R. , CLEMENS Michael J.
Published: March 2005

The interferon‐inducible double‐stranded‐RNA(dsRNA)‐dependent protein kinase PKR has been implicated in both the antiviral and cell growth‐regulatory effects of the interferons. Over‐expression of the wild‐type form of this protein inhibits cell proliferation, whereas over‐expression of inactive mutant forms transforms cells to a tumourigenic phenotype. It has been suggested that mutant PKR exerts a dominant negative effect on the activity of the wild‐type protein kinase. We have investigated this possibility using the rabbit reticulocyte cell‐free translation system in which protein synthesis is inhibited by dsRNA due to activation of PKR and phosphorylation of initiation factor eIF‐2. Addition of a highly purified inactive PKR mutant, synthesised in a baculovirus‐infected insect cell system, rescues protein synthesis from inhibition by low concentrations of dsRNA in a dose‐dependent manner. The PKR mutant has no effect on protein synthesis in the absence of dsRNA or in the presence of another inhibitory protein kinase, the haem‐controlled repressor. Inhibition of translation can be re‐established in the presence of the mutant PKR by adding a higher concentration of dsRNA. These results suggest that inactive mutant PKR does exert a dominant negative effect on wild‐type PKR and that this may be due to competition for dsRNA binding.
Localization of two myosin‐subfragment‐1 binding contacts in the 96–132 region of actin subdomain‐1
Authors: LABBÉ Jean‐Pierre , BOYER Mireille , MÉJEA Catherine , ROUSTAN Claude , BENYAMIN Yves
Published: March 2005

Many direct observations and indirect experimental approaches have pin‐pointed two segments (sequences 1–28 and 360–372) in actin subdomain‐1 which bind to myosin subfragment‐1. In a previous investigation [Labbé, J. P., Méjean, C., Benyamin, Y. & Roustan, C. (1990) Biochem. J. 271, 407–413], we have observed competition between myosin subfragment‐1 and anti‐actin anti‐bodies specific to epitopes including Thr103. A multisite interface model has also been proposed to take into account myosin‐head binding to the N‐terminal and C‐terminal regions and to more central 40–113 sequence of actin.

In the present study, two limited actin segments encompassing residues 96–103 and 112–125 were identified as myosin‐head‐binding sites. Myosin subfragment‐1 competed for monomeric actin with the antibodies directed against sequences 96–105 and 114–120 and its binding to the tryptic 96–113 and synthetic 112–125 actin peptides was prevented by magnesium pyrophosphate but not by calcium pyrophosphate. In the presence of ATP‐Mg2+, myosin subfragment‐1 was dissociated by filamin from its complex with monomeric actin or with peptide 105–120. Contact points of filamin on actin were previously located in the 105–120 and 360–372 actin sequence [Méjean, C., Lebart, M. C., Boyer, M., Roustan, C. & Benyamin, Y. (1992) Eur. J. Biochem. 209, 555–562].

The in vitro inhibitory effect of filamin on actin‐activated Mg2+‐ATPase would thus be explained by this competition. Furthermore, the (27‐kDa–50‐kDa–20‐kDa) trypsin‐split myosin subfragment‐1 which could no longer be activated by actin, did not bind at all to the two sites located in the 96–125 region, but it still interacted with the 360–372 segment.

Our results regarding the position of the myosin head on actin monomers in rigor conditions provide evidence on the presence of two topologically independant contact points in the myosin‐head/actin interface. One group exposed residues in the 1–7, 21–29, 77–95 and 96–103 actin segment, another, on the opposite side of subdomain‐1, included residues from 112–125 and 360–372 sequences.
Equilibrium and transient kinetic studies of the binding of cytochalasin B to the l‐arabinose–H+ symport protein of Escherichia coli
Authors: WALMSLEY Adrian R. , PETRO Kathleen R. , HENDERSON Peter J. F.
Published: March 2005

The kinetics of the binding of cytochalasin B to the l‐arabinose–H+ symport protein of Escherichia coli have been investigated, using a strain that over‐produces the symport protein in the cytoplasmic membrane. Equilibrium binding studies revealed a single set of binding sites (2.9–8.9 nmol/mg protein) with a Kd of 0.7–1.0 μM at 22°C. It proved possible to follow the transient kinetics of cytochalasin B binding by measuring the changes in the fluorescence of the l‐arabinose–H+ symporter upon binding the ligand, by stopped‐flow fluorescence spectroscopy. The association and dissociation rate constants thus determined were confirmed by rapid filtration measurements, using [3H]cytochalasin B, yielding values of 4.5–6.5 μM−1·s−1, and 4–5 s−1, respectively, consistent with Kd values obtained by measuring equilibrium binding of [3H]cytochalasin B by dialysis at 22°C. Titration of the protein fluorescence with cytochalasin B yielded a similar binding site concentration and Kd value to those obtained in equilibrium binding studies. All the measurements of binding site concentration are consistent with a stoichiometry of 1 mol cytochalasin B binding sites/mol l‐arabinose–H+ symport protein. Inhibition of both the rate and equilibrium binding of cytochalasin B by sugars indicated the following order of substrate binding 5‐thio‐d‐d‐glucose > d‐fucose > l‐arabinose > 6‐deoxy‐6‐fluoro‐d‐galactose > d‐xylose ≫ 6‐deoxy‐d‐glucose > d‐galactose > d‐glucose > d‐ribose. Neither d‐arabinose nor l‐fucose had any significant inhibitory effect upon cytochalasin B binding.
The existence of two mitochondrial isoforms of 2,4‐dienoyl‐CoA reductase in the rat
Authors: HAKKOLA Elina H. , HILTUNEN J. Kalervo
Published: March 2005

Isoforms of 2,4‐dienoyl‐CoA reductase (EC 1.3.1.34), which is the key enzyme in the β‐oxidation of fatty acids with double bonds, have been studied in rat heart and liver. Electrofocusing and adsorption chromatography on hydroxyapatite were used to separate the reductase activity in tissue homogenates into two peaks, one peak in each experiment being identified as the previously purified mitochondrial reductase.

The novel activity was partially purified from rat liver by means of ammonium sulphate precipitation, anion‐exchange chromatography on DEAE‐cellulose (DE‐52), hydrophobic chromatography on Phenyl‐Sepharose and dye‐ligand binding chromatography (Blue Sepharose). Taking into account the contribution of the different reductases to the total activity in rat liver, the overall purification for the novel isoform was 1900‐fold. Ultracentrifugation on a sucrose gradient gave an Mr of 50000 and size‐exclusion chromatography on Superdex 200 an Mr of 60000.

The antibody against the previously characterised reductase did not cross‐react with this novel isoform, but the distribution of the activity peaks in heart and liver tissue, and an electrofocusing experiment with isoled mitochondria, both pointed to a mitochondrial origin. The novel reductase was estimated to account for 80% (50%) of the total reductase activity in rat heart (liver) homogenate measured with 2,4‐hexadienoyl‐CoA. The present results, together with those previously published, suggest that mammals have at least three reductase isoforms: two in mitochondria and a third one in peroxisomes, but the peroxisomal activity has not been characterised so far.
X‐ray and primary structure of horse serum albumin (Equus caballus) at 0.27‐nm resolution
Authors: HO Joseph X. , HOLOWACHUK Eugene W. , NORTON Elizabeth J. , TWIGG Pamela D. , CARTER Daniel C.
Published: March 2005

The amino‐acid sequence and three‐dimensional structure of equine serum albumin have been determined. The amino‐acid sequence was deduced from cDNA isolated from equine liver. Comparisons of the primary structure of equine serum albumin with human serum albumin and bovine serum albumin reveal 76.1% and 73.9% sequence identity, respectively. The three‐dimensional structure was determined crystallographically by the molecular‐replacement method using molecular coordinates from the previously determined structure of human serum albumin, to a resolution of 0.27 nm. In accordance with the primary structure, the three‐dimensional structures are highly conserved. There is a root‐mean‐square difference between α‐carbons of the two structures of 0.201 nm. The association constants (Ka) for the binding of 2,3,5‐triiodobenzoic acid were determined by ultrafiltration methods for equine and human serum albumins to be approximately 104M−1 and 105 M−1, respectively. Crystallographic studies of equine serum albumin reveal two binding sites for 2,3,5‐triiodobenzoic acid identical with those previously reported for human serum albumin which are located within subdomains IIA and IIIA. Details and comparisons of the binding chemistry are discussed.
Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster
Authors: HORODYSKI Frank M. , EWER John , RIDDIFORD Lynn M. , TRUMAN James W.
Published: March 2005

Eclosion hormone (EH) is a neuropeptide that triggers the performance of ecdysis behaviors at the end of a molt. We have isolated the EH gene from Drosophila melanogaster, and localized the gene to the right arm of chromosome 3 at band position 90B1‐2. The 97‐amino‐acid translation product contains a signal peptide followed by a 73‐amino‐acid prohormone. The N‐terminus of the prohormone has diverged from lepidopteran EH both in its length and amino acid composition, and contains a potential endoproteolytic cleavage site. The deduced sequence of Drosophila EH is 58% identical (36 of 62 amino acid) to that of Manduca EH. The EH gene is expressed as a 0.8‐kb transcript in a single pair of brain neurons which extend their processes the entire length of the central nervous system and also to the corpora cardiaca portion of the ring gland. These cells show massive depletion of immunoreactive EH at ecdysis.
NMR assignments and solution conformation of the DNA · RNA hybrid duplex d(GTGAACTT) · r(AAGUUCAC)
Authors: LANE Andrew N. , EBEL Susanne , BROWN Tom
Published: March 2005

Nearly complete assignments of the 1H‐NMR spectrum of the DNA · RNA hybrid d(GTGAACTT) · r(AAGUUCAC) have been obtained by two‐dimensional methods. Three‐bond coupling constants measured from cross‐sections of two‐dimensional NOE spectra and double quantum‐filtered correlation spectra showed that the sugars in the DNA strand are predominantly in the S domain of puckers, whereas the ribofuranoses are mainly C3′ ‐endo. Analysis of time‐dependent NOE intensities from one‐and two‐dimensional experiments showed that the glycosidic torsion angles in the DNA strand are near −120°, whereas those in the RNA strand are in the range –140° to –160°. These nucleotide conformations correspond to those typically found in B‐DNA and A‐RNA, respectively.

The circular dichroism of the duplex is similar to that of A‐form RNA, consistent with a global A‐like conformation. A large number of duplex structures was generated in which the nucleotides were fixed in the experimentally determined conformations. A subset of these structures was found that satisfied the internucleotide NOE intensities, backbone constraints and had acceptable Lennard‐Jones energies. The base pairs in the duplex were found to have positive inclinations, a translation (Dx) of about 0.4 nm from the helix axis, and more than 10bp/turn on average. This implies a helical structure in the A family of conformations.
The isopenicillin‐N acyltransferase of Penicillium chrysogenum has isopenicillin‐N amidohydrolase, 6‐aminopenicillanic acid acyltransferase and penicillin amidase activities, all of which are encoded by the single penDE gene
Authors: ALVAREZ Emilio , MEESSCHAERT Boudewijn , MONTENEGRO Eduardo , GUTIÉRREZ Santiago , DÍEZ Bruno , BARREDO Jose L. , MARTÍN Juan F.
Published: March 2005

The isopenicillin‐N acyltransferase of Penicillium chrysogenum catalyzes the conversion of the biosynthetic intermediate isopenicillin N to the hydrophobic penicillins. The isopenicillin‐N acyltransferase copurified with the acyl‐CoA:6‐aminopenicillanic acid (6‐APA) acyltransferase activity which transfers an acyl residue from acyl‐CoA derivatives (e.g. phenylacetyl‐CoA, phenoxyacetyl‐CoA) to 6‐APA. Other thioesters of phenylacetic acid were also used as substrates. An amino acid sequence similar to that of the active site of thioesterases was found in the isopenicillin‐N acyltransferase, suggesting that this site is involved in the transfer of phenylacetyl residues from phenylacetyl thioesters. Purified isopenicillin‐N acyltransferase also showed isopenicillin‐N amido‐hydrolase, penicillin transacylase and penicillin amidase activities. The isopenillin‐N amidohydrolase (releasing 6‐APA) showed a much lower specific activity than the isopenicillin‐N acyltransferase of the same enzyme preparation, suggesting that in the isopenicillin‐N acyltransferase reaction the 6‐APA is not released and is directly converted into benzylpenicillin. Penicillin transacylase exchanged side chain between two hydrophobic penicillin molecules; or between one penicillin molecule and 6‐APA. The penicillin amidase activity is probably the reverse of the biosynthetic acyl‐CoA:6‐APA acyltransferase. Four P. chrysogenum mutants deficient in acyl‐CoA:6‐APA acyltransferase lacked the other four related activities. Transformation of these mutants with the penDE gene restored all five enzyme activities.
L‐Serine and L‐threonine dehydratase from Clostridium propionicum Two enzymes with different prosthetic groups
Authors: HOFMEISTER Antje E. M. , GRABOWSKI Reiner , LINDER Dietmar , BUCKLER Wolfgang
Published: March 2005

l‐Serine dehydratase from the Gram‐positive bacterium Peptostreptococcus asaccharolyticus is novel in the group of enzymes deaminating 2‐hydroxyamino acids in that it is an iron‐sulfur protein and lacks pyridoxal phosphate [Grabowski, R. and Buckel, W. (1991) Eur. J. Biochem. 199, 89–94]. It was proposed that this type of l‐serine dehydratase is widespread among bacteria but has escaped intensive characterization due to its oxygen lability. Here, we present evidence that another Gram‐positive bacterium, Clostridium propionicum, contains both an iron‐sulfur‐dependent l‐serine dehydratase and a pyridoxal‐phosphate‐dependent l‐threonine dehydratase. These findings support the notion that two independent mechanisms exist for the deamination of 2‐hydroxyamino acids.

l‐Threonine dehydratase was purified 400‐fold to apparent homogeneity and revealed as being a tetramer of identical subunits (m= 39kDa). The purified enzyme exhibited a specific activity of 5 μkat/mg protein and a Km for l‐threonine of 7.7 mM. l‐Serine (Km= 380 mM) was also deaminated, the V/Km ratio, however, being 118‐fold lower than the one for l‐threonine. l‐Threonine dehydratase was inactivated by borohydride, hydroxylamine and phenylhydrazine, all known inactivators of pyridoxal‐phosphate‐containing enzymes. Incubation with NaB3H4 specifically labelled the enzyme. Activity of the phenylhydrazine‐inactivated enzyme could be restored by pyridoxal phosphate.

l‐Serine dehydratase was also purified 400‐fold, but its extreme instability did not permit purification to homogeneity. The enzyme was specific for l‐serine (Km= 5mM) and was inhibited by l‐cysteine (Ki= 0.5 mM) and d‐serine (Ki= 8 mM). Activity was insensitive towards borohydride, hydroxylamine and phenylhydrazine but was rapidly lost upon exposure to air. Fe2+ specifically reactivated the enzyme. l‐Serine dehydratase was composed of two different subunits (α, m= 30kDa; βm= 26 kDa), their apparent molecular masses being similar to the ones of the two subunits of the iron‐sulfur‐dependent enzyme from P. asaccharolyticus. Moreover, the N‐terminal sequences of the small subunits from these two organisms were found to be 47% identical. In addition, 38% identity with the N‐terminus of one of the two l‐serine dehydratase of Escherichia coli was detected.
The variants of the protein toxins abrin and ricin
Authors: HEGDE Ramesh , KARANDE Anjali A. , PODDER Sunil K.
Published: March 2005

Kinetic data on inhibition of protein synthesis in thymocyte by three abrins and ricin have been obtained. The intrinsic efficiencies of A chains of four toxins to inactivate ribosomes, as analyzed by k1‐versus‐concentration plots were abrin II, III > ricin > abrin I. The lag times were 90, 66, 75 and 105 min at a 0.0744 nM concentration of each of abrin I, II, III and ricin, respectively. To account for the observed differences in the dose‐dependent lag time, functional and structural variables of toxins such as binding efficiency of B chains to receptors and low‐pH‐induced structural alterations have been analyzed. The association constants obtained by stopped flow studies showed that abrin‐I (4.13 × 105 M−1 s−1) association with putative receptor (4‐methylumbelliferyl‐α‐D‐galactoside) is nearly two times more often than abrin III (2.6 × 105 M−1 s−1) at 20°C. Equillibrium binding constants of abrin I and II to thymocyte at 37°C were 2.26 × 107 M−1 and 2.8 × 107 M−1 respectively. pH‐induced structural alterations as studied by a parallel enhancement in 8‐anilino‐L‐naphthalene sulfonate fluorescence revealed a high degree of qualitative similarity. These results taken with a nearly identical concentration‐independent lag time (minimum lag of 41–42 min) indicated that the binding efficiencies and internalization efficiencies of these toxins are the same and that the observed difference in the dose‐dependent lag time is causally related to the proposed processing event. The rates of reduction of inter‐subunit disulfide bond, an obligatory step in the intoxication process, have been measured and compared under a variety of conditions. Intersubunit disulfide reduction of abrin I is fourfold faster than that of abrin II at pH 7.2. The rate of disulfide reduction in abrin I could be decreased 1 I‐fold by adding lactose, compared to that without lactose. The observed differences in the efficiencies of A chains, the dose‐dependent lag period, the modulating effect of lactose on the rates of disulfide reduction and similarity in binding properties make the variants a valuable tool to probe the processing events in toxin transport in detail.
Purification and properties of an iron‐sulfur and FAD‐containing 4‐hydroxybutyryl‐CoA dehydratase/vinylacetyl‐CoA3‐2‐isomerase from Clostridium aminobutyricum
Authors: SCHERF Uwe , BUCKEL Wolfgang
Published: March 2005

4‐Hydroxybutyryl‐CoA dehydratase, the key enzyme in the metabolism of γ‐aminobutyrate in Clostridium aminobutyricum, represents approximately 15–25% of the soluble protein. The enzyme was purified to homogeneity under anaerobic conditions to a specific activity of 209 nkat mg−1. The dehydratase catalyses the reversible conversion of 4‐hydroxybutyryl‐CoA (Km= 50 μM) to crotonyl‐CoA and possesses a probably intrinsic vinylacetyl‐CoA 3‐2‐isomerase with a specific activity of 223 nkat mg−1. The equilibrium of the reversible dehydration was determined from both sides as K= [crotonyl‐CoA]/[4‐hydroxybutyryl‐CoA] = 4.2±0.3. Cyclopropylcarboxyl‐CoA was not converted to crotonyl‐CoA. The native enzyme has an apparent molecular mass of 232 kDa and is composed of four apparently identical subunits (molecular mass = 56 kDa), indicating a homotetrameric structure. Under anaerobic conditions the active enzyme revealed a brown colour and contained 2±0.2 mol FAD (64±5% oxidized). 16±0.8 mol Fe and 14.4±1.2 mol inorganic sulfur, which probably form iron‐sulfur clusters. Exposure to air resulted initially in a slight activation followed by irrevesible inactivation. Concomitantly the vinylacetyl‐CoA ‐isomerase activity was lost and the colour of the enzyme changed to yellow. Reduction by sodium dithionite yielded inactive enzyme which could be completely reactivated by oxidation with potassium hexacyanofer‐rate(III). The data indicate that the active enzyme contains oxidized FAD despite its sensitivity towards oxygen. During the dehydration the dehydration a non activated C‐H bond at C‐3 of 4‐hydroxybutyryl‐CoA has to be cleaved. A putative mechanism for 4‐hydroxybutyryl‐CoA dehydratase is proposed in which this cleavage is achieved by a FAD‐dependent oxidation of 4‐hydroxybutyryl‐CoA to 4‐hydroxycrotonyl‐CoA. In a second step the hydroxyl group is substituted by a hydride derived from the now reduced FAD in an SN2′ reaction leading to vinylacetyl‐CoA. Finally isomerisation yields crotonyl‐CoA.

4‐Hydroxybutyryl‐CoA dehydratase is quite distinct from 3‐hydroxyacyl‐CoA dehydratase (crotonase) and 2‐hydroxyacyl‐CoA dehydratases. Contrary to the latter enzymes [e.g. (R)‐lactyl‐CoA dehydratase and (R)‐2‐hydroxyglutaryl‐CoA dehydratase] which are composed of three different subunits and similarly catalyse the cleavage of a non activated C‐H bond at C‐3, 4‐hydroxybutyryl‐CoA dehydratase does not require ATP, MgCl2 and Ti(III)citrate for activity. Furthermore 4‐hydroxybutyryl‐xybutyryl‐CoA dehydratase is not inactivated by oxidants such as 5 mM 4‐nitrophenol, 5 mM chloramphenicol and 5 mM hydroxylamine.
Cell Swelling and the sensitivity of autophagic proteolysis to inhibition by amino acids in isolated rat hepatocytes
Authors: MEIJER Alfred J. , GUSTAFSON Lori A. , LUIKEN Joost J. F. P. , BLOMMAART Pietjan J. E. , CARO L. Heleen P. , VAN WOERKOM George M. , SPRONK Claire , BOON Louis
Published: March 2005

In the isolated perfused rat liver, autophagic proteolysis is inhibited by hypo‐osmotic perfusion media [Häussinger, D., Hallbrucker, C., vom Dahl, S., Lang, F. & Gerok, W. (1990) Biochem. J. 272, 239–242]. Here we report that in isolated hepatocytes, incubated in the absence of amino acids to ensure maximal proteolytic flux, proteolysis was not inhibited by hypo‐osmolarity while the synthesis of glycogen from glucose, a process known to be very sensitive to changes in cell volume [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955–959], was stimulated under identical conditions. However, in isolated hepatocytes, hypo‐osmolarity increased the sensitivity of autophagic proteolysis to inhibition by low concentrations of amino acids. The anti‐proteolytic effect of hypo‐osmolarity in our experiments was not due to stimulation of amino‐acid transport into the hepatocytes: neither the consumption of most amino acids, nor the rate of urea synthesis was appreciably affected by hypo‐osmotic incubation conditions. In the course of these studies we also found that hypo‐osmolarity increased the affinity of protein synthesis for amino acids.

In the presence of amino acids the intracellular level of ATP was not much affected. However, because of cell swelling under these conditions the intracelluar concentration of ATP decreased. It is proposed that a small part of the inhibition of proteolysis by amino acids may be due to this fall in ATP concentration.
Distinct mechanisms of phospholipase D activation and attenuation utilized by different mitogens in NIH‐3T3 fibroblasts
Authors: BEN‐AV Pazit , ELI Yona , SCHMIDT Uta‐Susanne , TOBIAS Karin E. , LISCOVITCH Mordechai
Published: March 2005

The activation of phospholipase D (PLD) by platelet‐derived growth factor (PDGF), prostaglandin F2α and 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) was studied in NIH‐3T3 fibroblasts. PLD activation was determined by measuring the production of both [3H]phosphatidic acid and [3H]phosphatidylpropanol (products of the PLD‐catalyzed hydrolysis and transphosphatidylation reactions, respectively), in cells that were metabolically pre‐labeled with [3H]oleic acid. All mitogens caused a rapid (within 2 min) activation of PLD. Activation of PLD by prostaglandin F2α and PDGF was transient and declined to near basal levels by 15 min and 55 min, respectively. In contrast, TPA‐induced activation of PLD was sustained for at least 60 min of incubation. A combination of maximally effective concentrations of PDGF and TPA stimulated PLD activity in a non‐additive manner, while the effect of prostaglandin F2α was additional to that of either PDGF or TPA. The protein kinase inhibitor staurosporine inhibited PLD activation by PDGF or TPA with almost identical dose/response curves. In contrast, staurosporine potentiated prostaglandin‐F2α‐induced PLD activation. The specific protein kinase C inhibitor GF109203X (a bisindolylmaleimide) inhibited PLD activation by prostaglandin F2α and PDGF at concentrations higher than those required for inhibition of PLD activation induced by TPA. Depletion of cellular protein kinase C abolished PLD activation by all three mitogens without affecting in vitro activity of membrane‐bound PLD. The distinct kinetics of PLD activation and its differential susceptibility to protein kinase inhibitors suggest the existence of agonist‐specific activation and/or inactivation mechanisms. The results indicate also that protein kinase C participates in the mechanism of PLD activation via PDGF, while the effect of prostaglandin F2α involves a pathway independent of protein kinase C.
Refolding and single‐step purification of porcine interferon‐γ from Escherichia coli inclusion bodies
Authors: VANDENBROECK Koen , MARTENS Erik , D'ANDREA Sabine , BILLIAU Alfons
Published: March 2005

Recombinant porcine interferon‐γ, overexpressed in Escherichia coli, was found to accumulate in cytoplasmic inclusion bodies. The influence of various physicochemical parameters on refolding was investigated using 6 M guanidine/HCI‐solubilised inclusion bodies which had been purified by ultracentrifugation on a sucrose step gradient. It appeared that the yield of reconstitution of denatured protein reached 60–70% under optimum conditions. i.e. at an intermediary guanidine/HCI concentration of 0.5M and at a protein concentration of 10–20μ (0°C). Since intermediary guanidine/HCI concentration at 0.5–1.65 M increasingly promoted off‐pathway formation of soluble aggregates and at 0.5–0.2 M progressively promoted precipitation, maximal recovery of biologically active protein required a twofold transition in the surrounding guanidine/HCI concentration (6 M → 0.5 M → OM). A single additional size‐exclusion chromatographic step yielded a final product that was >99.5% pure, had specific antiviral activity >107 U/mg protein and contained ≤25 pg/ml endotoxin. Cross‐linking by means of disulfosuccinimidyl tartarate revealed that the refolded protein possessed a dimeric structure. Furthermore, we have characterized three different molecular species of recombinant porcine interferon‐γ that are formed under non‐optimal refolding conditions (1 M guanidine/HCI) and that differ from each other in specific activity, size and stability. One of these converts irreversibly into dimeric interferon‐γ in a temperature‐dependent manner and is therefore considered as a productive folding intermediate.
Distribution of alcohol and sorbitol dehydrogenases
Authors: ESTONIUS Mats , DANIELSSON Olle , KARLSSON Christina , PERSSON Håkan , JÖRNVALL Hans , HÖÖG Jan‐Olov
Published: March 2005

The tissue distribution of mRNA of alcohol dehydrogenases of classes I, II and III, and sorbitol dehydrogenase, was studied. mRNA from 19 different rat tissues was purified and analyzed by Northern blots, utilizing cDNA probes specific for the four dehydrogenases. Class‐I alcohol‐dehydrogenase mRNA was shown to be of widespread occurrence, detectable in all tissues including brain, but with pronounced differences in amounts. Hybridization revealed the pattern of occurrence of class‐II alcohol‐dehydrogenase mRNA to be unique, with transcripts only in the liver, duodenum, kidney, stomach, spleen and testis. Abundant levels of class‐III alcohol‐dehydrogenase (glutathione‐dependent formaldehyde dehydrogenase) mRNA were present in all tissues analyzed, reflecting the general need for scavenging of formaldehyde in physiological cytoprotection. Sorbitol dehydrogenase mRNA was detected in all tissues except small intestine, in agreement with sorbitol resorbtion by passive diffusion in this tissue. In addition, evidence for a sex‐specific expression, in the liver, of class‐II alcohol dehydrogenase was obtained.
DNA‐sequence binding preference of the GC‐selective ligand mithramycin
Authors: CARPENTER Mark L. , MARKS Jonathan N. , FOX Keith R.
Published: March 2005

We have used hydroxy‐radical and deoxyribonuclease‐I footprinting to probe the interaction of mithramycin with DNA fragments containing the sequences (AT)10X(AT)10 (X = CCCG, CCGC or CGGC) and A14GCCCT15. As expected the drug produces clear footprints located around the central four GC base pairs. The exact position of the footprint is different for the four sequences; the footprint with CCCG is displaced by two base pairs in the 5′ direction relative to GCCC. These variations are explained by suggesting that mithramycin avoids the dinucleotide CG and binds better to GG/CC than GC. Although there is little change in deoxyribonuclease‐I cleavage of the surrounding blocks of (AT)n. cleavage by deoxyribonuclease II is markedly enhanced and certain thymines on the 5′ side of the ligand‐binding site become hyperreactive to hydroxy‐radical attack. Adjacent regions of An· Tn show enhanced rates of deoxyribonuclease‐I cleavage in the presence of the antibiotic.
1H‐ and15N‐NMR assignment and solution structure of the chemotacticEscherichia coliChe Y protein
Authors: BRUIX Marta , PASCUAL Jaime , SANTORO Jorge , PRIETO Jesús , SERRANO Luis , RICO Manuel
Published: March 2005

Che Y is a 129‐residue parallel α/β protein involved in bacterial chemotaxis. We have used this protein as a model to study the folding reaction of parallel α/β proteins. As a first step we carried out the complete assignment of the1H and15N spectra fromEscherichia coliChe Y protein on the basis of two‐dimensional1H homonuclear and1H‐15N heteronuclear experiments by using sequence‐specific methods. Our assignments differ from the preliminary assignments made by Kar et al. [Kar, L., Matsumura, P. & Johnson, M. E. (1992)Biochem. J. 287, 521–531] of aromatic residues obtained by comparison of NOEs with short proton–proton distances in the crystal structure of Che Y. The analysis of the extension of the secondary elements, as well as a preliminary calculation of the three‐dimensional structure, indicate that the solution structure is closely coincident with the single crystal structure determined by X‐ray diffraction.