bluuebunny/crossref_metadata_2025 huggingface dataset:
Myticin, a novel cysteine‐rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis
Authors: Mitta Guillaume , Hubert Florence , Noël Thierry , Roch Philippe
Published: March 2003

We report here the isolation of two isoforms of a novel cysteine‐rich peptide from haemocytes (isoform A of 4.438 Da and B of 4.562 Da) and plasma (isoform A) of the mussel, Mytilus galloprovincialis. The two molecules display antibacterial activity against gram‐positive bacteria, whereas only isoform B is active against the fungus Fusarium oxysporum and a gram‐negative bacteria Escherichia coli D31. Complete peptide sequences were determined by a combination of Edman degradation, mass spectrometry and cDNA cloning using a haemocyte cDNA library. The mature molecules, named myticins, comprise 40 residues with four intramolecular disulfide bridges and a cysteine array in the primary structure different to that of the previously characterized cysteine‐rich antimicrobial peptides. Sequence analysis of the cloned cDNAs revealed that myticin precursors consist of 96 amino acids with a putative signal peptide of 20 amino acids, the antimicrobial peptide sequence and a 36‐residue C‐terminal extension. This structure suggests that myticins are synthesized as preproproteins and then processed by various proteolytic events before storage of the active peptide in the haemocytes. Myticin precursors are expressed mainly in the haemocytes as revealed by Northern blot analysis.
Aspartyl proteases in Caenorhabditis elegans
Authors: Geier Gebhard , Banaj Hans‐Jörg , Heid Hans , Bini Luca , Pallini Vitaliano , Zwilling Robert
Published: March 2003

Crude homogenates of the nematode Caenorhabditis elegans exhibit maximal proteolytic activity under acidic pH conditions. About 90% of this activity is inhibited by the oligopeptide pepstatin, which specifically inhibits the activity of aspartyl proteases such as pepsin, cathepsins D and E or renin.

 We have purified enzymes responsible for this proteolytic activity by a single‐step affinity chromatography on pepstatin–agarose. Analysis of the purified fraction by 1D SDS gel electrophoresis revealed six bands ranging from 35 to 52 kDa. After electrotransfer to poly(vinylidene difluoride) membranes, all bands were successfully subjected to N‐terminal microsequencing.

 On 2D gels, the purified protein bands split into 19 spots which, after renewed microsequencing, were identified as isoelectric variants of the six proteins already described. The N‐termini obtained for these proteins could be correlated to genomic DNA sequences determined in the course of the C. elegans genome sequencing project. All these sequences were predicted to code for expressed proteins as collected in the WORMPEP database. Five of the six coding sequences identified in this study were found to contain the typical active‐site consensus sequence of aspartyl proteases and displayed an overall amino acid identity between 25 and 66% as compared to aspartyl proteases from other organisms. In addition to the five aspartyl proteases detected at the protein level, we have identified the coding sequences for seven other enzymes of this protease family by a similarity search in the genomic DNA of C. elegans which has recently been completely sequenced.
Formation of complexes involving RasGAP and p190 RhoGAP during morphogenetic events of the gastrulation in Xenopus
Authors: Dupont Henri , Blancq Marie
Published: March 2003

In relation to the activation of the Src‐family of tyrosine kinases during early morphogenetic events of gastrulation in Xenopus, we have identified two multiprotein complexes. The first complex, including RasGAP, p190 RhoGAP and p62, was previously characterized in murine fibroblasts overexpressing c‐Src or transformed by v‐Src and has been correlated with cytoskeleton remodelling. A second complex, not identified in other models includes tyrosine‐phosphorylated p66SHC, Grb2, RasGAP and p190 RhoGAP. The association with p66SHC, considered as a negative regulator of ERK (extracellular signal‐regulated kinase), p120RasGAP and p190RhoGAP, suggests a possible mechanism for coupling Ras and Rho signalling pathways. The interaction of RasGAP and p190 RhoGAP in two multiprotein complexes could constitute an additional level of Rho regulation during morphogenetic events of gastrulation.
Binding of ATP to human DNA topoisomerase I resulting in an alteration of the conformation of the enzyme
Authors: Chen Hui‐Jye , Hwang Jaulang
Published: March 2003

It has been known for some time that ATP inhibits the DNA relaxation activity of human DNA topoisomerase I. However, the underlying mechanism of this inhibitory effect remains largely unknown. Using filter binding assays, the binding of human DNA topoisomerase I to DNA was decreased in the presence of ATP. This result suggests that the inhibition of DNA relaxation activity of human DNA topoisomerase I by ATP is at the binding step rather than at the nicking or resealing step. DNA topoisomerase I cleavage assay further supports this notion. ATP‐agarose binding and UV cross‐linking assays also demonstrate that ATP directly and specifically binds human DNA topoisomerase I. To address whether the ATP binding results in conformational changes in human DNA topoisomerase I, various proteases were employed for detecting potential protein conformational changes. Our results indicated that the proteolytic susceptibilities of trypsin and chymotrypsin were altered in the presence of ATP. The result suggests that the conformation of human DNA topoisomerase I was altered upon ATP binding. In addition, the binding between ATP and human DNA topoisomerase I was also reduced by increasing concentrations of DNA. Our data suggests that human DNA topoisomerase I exhibits at least two incompatible conformations. One conformation is in the form of a topoisomerase I–ATP complex, which inhibits DNA relaxation activity of human DNA topoisomerase I, and the other, a topoisomerase I–DNA complex, which exerts DNA relaxation activity. Our studies identify the role of ATP in the regulation of human DNA topoisomerase I and provide a substantial implication of how human DNA topoisomerase I compromises its versatile functions.
Cloning and characterization of new orphan nuclear receptors and their developmental profiles during Tenebrio metamorphosis
Authors: Mouillet Jean‐François , Bousquet François , Sedano Nathalie , Alabouvette Josiane , Nicolaï Maryse , Zelus Dominique , Laudet Vincent , Delachambre Jean
Published: March 2003

Five PCR fragments corresponding to a part of the DNA‐binding domain of different hormone nuclear receptors were isolated from Tenebrio molitor mRNAs. The sequence identity of three of them with known Drosophila nuclear receptors strongly suggests that they are the Tenebrio orthologs of seven‐up, DHR3 and β‐FTZ‐F1, and thus named Tmsvp, TmHR3 and TmFTZ‐F1. The full‐length sequences of the other two were established. TmHR78 is either a new receptor of the DHR78 family or the same gene which has evolved rapidly, particularly in the E domain. TmGRF belongs to the GCNF1 family and its in vitro translated product binds to the extended half site TCAAGGTCA with high affinity. The periods of expression of the corresponding transcripts in epidermal cells during Tenebrio metamorphosis were analyzed as a function of 20‐hydroxyecdysone titers measured in the hemolymph of the animals taken for RNA extraction. Comparison of the expression profiles of these nuclear receptors with those observed during Drosophila metamorphosis revealed similar temporal correlations as a function of ecdysteroid variations, which further supported the sequence identity data for TmSVP, TmHR3, TmFTZ‐F1 and TmHR78.
Pyruvate dehydrogenase from Azotobacter vinelandii
Authors: Hengeveld Annechien F. , Schoustra Siemen E. , Westphal Adrie H. , de Kok Aart
Published: March 2003

The pyruvate dehydrogenase multienzyme complex (PDHC) catalyses the oxidative decarboxylation of pyruvate and the subsequent acetylation of coenzyme A to acetyl‐CoA. Previously, limited proteolysis experiments indicated that the N‐terminal region of the homodimeric pyruvate dehydrogenase (E1p) from Azotobacter vinelandii could be involved in the binding of E1p to the core protein (E2p) [Hengeveld, A. F., Westphal, A. H. & de Kok, A. (1997) Eur J. Biochem.250, 260–268]. To further investigate this hypothesis N‐terminal deletion mutants of the E1p component of Azotobacter vinelandii pyruvate dehydrogenase complex were constructed and characterized. Up to nine N‐terminal amino acids could be removed from E1p without effecting the properties of the enzyme. Truncation of up to 48 amino acids did not effect the expression or folding abilities of the enzyme, but the truncated enzymes could no longer interact with E2p. The 48 amino acid deletion mutant (E1pδ48) is catalytically fully functional: it has a Vmax value identical to that of wild‐type E1p, it can reductively acetylate the lipoamide group attached to the lipoyl domain of the core enzyme (E2p) and it forms a dimeric molecule. In contrast, the S0.5 for pyruvate is decreased. A heterodimer was constructed containing one subunit of wild‐type E1p and one subunit of E1pδ48. From the observation that the heterodimer was not able to bind to E2p, it is concluded that both N‐terminal domains are needed for the binding of E1p to E2p. The interactions are thought to be mainly of an electrostatic nature involving negatively charged residues on the N‐terminal domains of E1p and previously identified positively charged residues on the binding and catalytic domain of E2p.
Deficient coacervation of two forms of human tropoelastin associated with supravalvular aortic stenosis
Authors: Wu Wendy J. , Weiss Anthony S.
Published: March 2003

Human tropoelastin associates by coacervation and is subsequently cross‐linked to make elastin. In Williams syndrome, defective elastin deposition is associated with hemizygous deletion of the tropoelastin gene in supravalvular aortic stenosis (SVAS). Remarkably, point‐mutation forms of SVAS correspond to incomplete forms of tropoelastin which include in‐frame termination by nonsense mutations, yet the resulting phenotype of these disorders is not explained because expression variably occurs from both normal and mutant alleles. Proteins corresponding to two truncated tropoelastin mutants were expressed and purified to homogeneity. Coacervation of these proteins occurred as expected with increasing temperature, but substantially contrasted with that of the performance of a normal tropoelastin. Significantly, association by coacervation of the truncated SVAS tropoelastin molecules was negligible at 37 °C, which contrasted with the substantial coacervation seen for normal tropoelastin. Furthermore their midpoints of coacervation increased and correlated with the extent of deletion, in accord with the loss of hydrophobic regions required for tropoelastin association. Their secondary structures are similar, as evidenced by CD studies. We propose a model for point‐mutation SVAS in which aberrant tropoelastin molecules are incompetent and are mainly excluded from participation in coacervation and consequently in elastogenesis. These forms of SVAS may consequently be considered functionally similar to a hemizygous deletion, and mark point‐mutation SVAS as a disorder of defective coacervation.
A cinnamoyl esterase from Aspergillus niger can break plant cell wall cross‐links without release of free diferulic acids
Authors: Garcia‐Conesa Maria‐Teresa , Kroon Paul A. , Ralph John , Mellon Fred A. , Colquhoun Ian J. , Saulnier Luc , Thibault Jean‐François , Williamson Gary
Published: March 2003

A cinnamoyl esterase, ferulic acid esterase A, from Aspergillus niger releases ferulic acid and 5‐5‐ and 8‐O‐4‐dehydrodiferulic acids from plant cell walls. The breakage of one or both ester bonds from dehydrodimer cross‐links between plant cell wall polymers is essential for optimal action of carbohydrases on these substrates, but it is not known if cinnamoyl esterases can break these cross‐links by cleaving one of the ester linkages which would not release the free dimer. It is difficult to determine the mechanism of the reaction on complex substrates, and so we have examined the catalytic properties of ferulic acid esterase A from Aspergillus niger using a range of synthetic ethyl esterified dehydrodimers (5‐5‐, 8‐5‐benzofuran and 8‐O‐4‐) and two 5‐5‐diferulate oligosaccharides. Our results show that the esterase is able to cleave the three major dehydrodiferulate cross‐links present in plant cell walls. The enzyme is highly specific at hydrolysing the 5‐5‐ and the 8‐5‐benzofuran diferulates but the 8‐O‐4‐is a poorer substrate. The hydrolysis of dehydrodiferulates to free acids occurs in two discrete steps, one involving dissociation of a monoesterified intermediate which is negatively charged at the pH of the reaction. Although ferulic acid esterase A was able to release monoesters as products of reactions with all three forms of diesters, only the 5‐5‐ and the 8‐O‐4‐monoesters were substrates for the enzyme, forming the corresponding free diferulic acids. The esterase cannot hydrolyse the second ester bond from the 8‐5‐benzofuran monoester and therefore, ferulic acid esterase A does not form 8‐5‐benzofuran diferulic acid. Therefore, ferulic acid esterase A from Aspergillus niger contributes to total plant cell wall degradation by cleaving at least one ester bond from the diferulate cross‐links that exist between wall polymers but does not always release the free acid product.
Striatum‐ and testis‐specific phosphodiesterase PDE10A
Authors: Fujishige Kotomi , Kotera Jun , Omori Kenji
Published: March 2003

PDE10A, a phosphodiesterase (PDE) exhibiting properties of a cAMP PDE and a cAMP‐inhibited cGMP PDE, was cloned and investigated in detail in rats. PDE10A transcripts were abundant in the brain and testis. In situ hybridization analysis using a PDE10A riboprobe demonstrated the presence of PDE10A transcripts in the neurons of the striatum and the olfactory tubercle regions of the brain. Rat PDE10A cDNAs were isolated from a brain cDNA library and nucleotide sequence analysis revealed several N‐terminal variants. The deduced amino‐acid sequence of one of the major variant forms contained 794 amino acids, and it was 96% identical to that of the human PDE10A2. The other major form has a distinct N‐terminal sequence that is not found in humans. PDE10A was partially purified from rat striatum and testis, and characterized with respect to Km, inhibitor sensitivity and immunoreactivity to an anti‐PDE10A serum. These findings indicate that PDE10A functions in these tissues.
Structural analysis of photosystem II in far‐red‐light‐adapted thylakoid membranes
Authors: Stoylova Svetla , Flint Toby D. , Ford Robert C. , Holzenburg Andreas
Published: March 2003

We studied two‐dimensional crystals of the major pigment–protein complex, photosystem II, in far‐red‐light‐adapted thylakoid membranes of the viridis‐zb63 mutant of barley. Significantly larger grana membranes were produced with an increased synthesis of the entire photosystem II complex. These red‐light‐adapted membranes also contained two‐dimensional crystals with a high frequency. Three different crystal forms of photosystem II were observed, providing the following data which further our understanding of the architecture of the native complex. (a) The oligomeric form of photosystem II in the membrane was monomeric in all crystal forms, but with a clear non‐crystallographic pseudo‐twofold symmetry. This was more apparent on the lumenal face of the complex. (b) The variability of unit cell contacts in different crystal forms implied that the peripheral light‐harvesting antenna complex and the core of the complex were loosely connected. These peripheral subunits were predicted to rearrange so that they can either encircle the core complex or associate in parallel channels separated by lines of core complexes. (c) Grana membranes were found to retain a double‐layered inside‐out character, with a stromal face‐to‐stromal face packing. However, the presence of a crystal in one membrane did not necessarily impose crystallinity on its pair.
The relative importance of passive and P‐glycoprotein mediated anthracycline efflux from multidrug‐resistant cells
Authors: Wielinga Peter R. , Westerhoff Hans V. , Lankelma Jan
Published: March 2003

For the four anthracyclines idarubicin, daunorubicin, epirubicin and doxorubicin the passive and active efflux rates in intact multidrug resistant cells were compared. Although highly similar structurally, these anti‐tumor agents differ in lipophilicity and membrane permeability (k). The method we used was based on the continuous measurement of the cellular efflux and determination of the ratio (RVp) of transport rates just before and just after inhibition of the active transport with verapamil (Vp). Hence, RVp − 1 should reflect the active transport rate relative to the passive transport rate. If cells were single, well‐stirred compartments, RVp − 1 should equal Vmax/(k·Km), where Vmax is the maximal pumping rate and Km is the Michaelis constant. However, using the plasma membrane permeabilizing agent digitonin, we found an effective intracellular anthracycline store. Particularly, when the efflux was fast, e.g. with idarubicin or in intensively pumping cells, the intracellular transport began to control the cellular efflux. Under these conditions, k underestimated the true plasma membrane permeability (k0) and RVp − 1 underestimated Vmax/(k·Km). Based on the effects of digitonin on the efflux rates in pumping and nonpumping cells, we developed an index (RVp,corrected − 1) which should equal Vmax/(k0·Km). The term Vmax/(k0·Km) varied substantially between the drugs. It appears that differences in lipophilicity between the drugs do not affect passive efflux and pumping equally. This demonstrates that passive permeation plays a substantial and independent role in determining the drug resistance for these anthracyclines. The methods developed here enable dissection of this role from that of drug pumping and intracellular subcompartmentation.
Characterization of a nif‐regulated flavoprotein (FprA) from Rhodobacter capsulatus
Authors: Jouanneau Yves , Meyer Christine , Asso Marcel , Guigliarelli Bruno , Willison John C.
Published: March 2003

A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6‐tag fusion from an Escherichia coli clone over‐expressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48‐kDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 – Em1 of > 80 mV. The reduction of SQ by dithionite was significantly accelerated when the [2Fe–2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be −275 ± 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to cross‐react when incubated together with the 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of ≈ 60 000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Fe–2S] ferredoxins, including FdV and FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross‐reacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprA‐deleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wild‐type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions.
Interpretation of biological activity data of bacterial endotoxins by simple molecular models of mechanism of action
Authors: Frecer Vladimir , Ho Bow , Ding Jeak L.
Published: March 2003

Lipid A moiety has been identified as the bioactive component of bacterial endotoxins (lipopolysaccharides). However, the molecular mechanism of biological activity of lipid A is still not fully understood. This paper contributes to understanding of the molecular mechanism of action of bacterial endotoxins by comparing molecular modelling results for two possible mechanisms with the underlying experimental data. Mechanisms of action involving specific binding of lipid A to a protein receptor as well as nonspecific intercalation into phospholipid membrane of a host cell were modelled and analysed. As the cellular receptor for endotoxin has not been identified, a model of a peptidic pseudoreceptor was proposed, based on molecular structure, symmetry of the lipid A moiety and the observed character of endotoxin‐binding sites in proteins. We have studied the monomeric form of lipid A from Escherichia coli and its seven synthetic analogues with varying numbers of phosphate groups and correlated them with known biological activities determined by the Limulus assay. Gibbs free energies associated with the interaction of lipid A with the pseudoreceptor model and intercalation into phospholipid membrane calculated by molecular mechanics and molecular dynamics methods were used to compare the two possible mechanisms of action. The results suggest that specific binding of lipid A analogues to the peptidic pseudoreceptor carrying an amphipathic cationic binding pattern BHPHB (B, basic; H, hydrophobic; P, polar residue, respectively) is energetically more favourable than intercalation into the phospholipid membrane. In addition, binding affinities of lipid A analogues to the best minimum binding sequence KFSFK of the pseudoreceptor correlated with the experimental Limulus activity parameter. This correlation enabled us to rationalize the observed relationship between the number and position of the phosphate groups in the lipid A moiety and its biological activity in terms of specific ligand–receptor interactions. If lipid A–receptor interaction involves formation of phosphate‐ammonium ion‐pair(s) with cationic amino‐acid residues, the specific mechanism of action was fully consistent with the underlying experimental data. As a consequence, recognition of lipid A variants by an amphipathic binding sequence BHPHB of a host‐cell protein receptor might represent the initial and/or rate‐determining molecular event of the mechanism of action of lipid A (or endotoxin). The insight into the molecular mechanism of action and the structure of the lipid A‐binding pattern have potential implications for rational drug design strategies of endotoxin‐neutralizing agents or binding factors.
Structural comparison of psychrophilic and mesophilic trypsins
Authors: Schrøder Leiros Hanna‐Kirsti , Willassen Nils P. , Smalås Arne O.
Published: March 2003

Structural rationalizations for differences in catalytic efficiency and stability between mesophilic and cold‐adapted trypsins have been suggested from a detailed comparison of eight trypsin structures. Two trypsins, from Antarctic fish and Atlantic cod, have been constructed by homology modeling techniques and compared with six existing X‐ray structures of both cold‐adapted and mesophilic trypsins. The structural analysis focuses on the cold trypsin residue determinants found in a more extensive comparison of 27 trypsin sequences, and reveals a number of structural features unique to the cold‐adapted trypsins. The increased substrate affinity of the psychrophilic trypsins is probably achieved by a lower electrostatic potential of the S1 binding pocket particularly arising from Glu221B, and from the lack of five hydrogen bonds adjacent to the catalytic triad. The reduced stability of the cold trypsins is expected to arise from reduced packing in two distinct core regions, fewer interdomain hydrogen bonds and from a destabilized C‐terminal α‐helix. The helices of the cold trypsins lack four hydrogen bonds and two salt‐bridges, and they have poorer van der Waals packing interactions to the body of the molecule, compared to the mesophilic counterparts.
Effects of cadmium on manganese peroxidase
Authors: Youngs Heather L. , Sundaramoorthy Munirathinam , Gold Michael H.
Published: March 2003

Inhibition of manganese peroxidase by cadmium was studied under steady‐state and transient‐state kinetic conditions. CdII is a reversible competitive inhibitor of MnII in the steady state with Ki ≈ 10 µm. CdII also inhibits enzyme‐generated MnIII–chelate‐mediated oxidation of 2,6‐dimethoxyphenol with Ki ≈ 4 µm. CdII does not inhibit direct oxidation of phenols such as 2,6‐dimethoxyphenol or guaiacol (2‐methoxyphenol) in the absence of MnII. CdII alters the heme Soret on binding manganese peroxidase and exhibits a Kd ≈ 8 µm, similar to Mn (Kd ≈ 10 µm). Under transient‐state conditions, CdII inhibits reduction of compound I and compound II by MnII at pH 4.5. However, CdII does not inhibit formation of compound I nor does it inhibit reduction of the enzyme intermediates by phenols in the absence of MnII. Kinetic analysis suggests that CdII binds at the MnII‐binding site, preventing oxidation of MnII, but does not impair oxidation of substrates, such as phenols, which do not bind at the MnII‐binding site. Finally, at pH 4.5 and 55 °C, MnII and CdII both protect manganese peroxidase from thermal denaturation more efficiently than CaII, extending the half‐life of the enzyme by more than twofold. Furthermore, the combination of half MnII and half CdII nearly quadruples the enzyme half‐life over either metal alone or either metal in combination with CaII.
Affinity for the cognate monoclonal antibody of synthetic peptides derived from selection by phage display
Authors: Ferrières Gaëlle , Villard Sylvie , Pugnière Martine , Mani Jean‐Claude , Navarro‐Teulon Isabelle , Rharbaoui Faïza , Laune Daniel , Loret Erwann , Pau Bernard , Granier Claude
Published: March 2003

Randomized peptide sequences displayed at the surface of filamentous phages are often used to select antibody ligands. The selected sequences are generally further used in the form of synthetic peptides; however, as such, their affinity for the selecting antibody is extremely variable and factors influencing this affinity have not been fully deciphered. We have used an f88.4 phage‐displayed peptide library to identify ligands of mAb 11E12, an antibody reactive to human cardiac troponin I. A majority of the sequences thus selected showed a (T/A/I/L) EP(K/R/H) motif, homologous to the Y‐TEPH motif identified by multiple peptide synthesis as the critical motif recognized by mAb 11E12 in the peptide epitope. A set of 15‐mer synthetic peptides derived from the phage‐selected sequences was used in BIACORE to characterize their interaction with mAb 11E12. Most peptides exhibited affinities in the 7–26 nm range. These affinities represented, however, only 1.9–7.5% of the affinity of the 15‐mer peptide epitope. In circular dichroism experiments, the peptide epitope showed a propensity to have some stabilized conformation, whereas a low‐affinity peptide selected by phage‐display did not. To try to decipher the molecular basis of this difference in affinity, new peptides were prepared by grafting the N‐ or the C‐terminal sequence of the peptide epitope to the Y‐TEPK motif of a low‐affinity peptide selected by phage‐display. These hybrid peptides showed marked increases both in affinity (as assessed using BIACORE) and in inhibitory potency (as assessed in competition ELISA), compared with the parent sequence. Thus, the sequences flanking the motif, although not containing critical residues, convey some determinants necessary for high affinity. The affinity of a given peptide strongly depends on its capacity to maintain the antigenically reactive structure it has on the phage, implying that it is impossible to predict whether high‐ or low‐affinity peptides will be obtained from phage display.
Cloning and over‐expression of thermostable Bacillus sp. YM55‐1 aspartase and site‐directed mutagenesis for probing a catalytic residue
Authors: Kawata Yasushi , Tamura Koichi , Kawamura Michiyo , Ikei Kazuyo , Mizobata Tomohiro , Nagai Jun , Fujita Miyuki , Yano Shigeru , Tokushige Masanobu , Yumoto Noboru
Published: March 2003

A thermostable aspartase gene (aspB) from Bacillus sp. YM55‐1 was cloned and the gene sequenced. The aspB gene (1407 bp ORF) encodes a protein with a molecular mass of 51 627 Da, consisting of 468 amino‐acid residues. An amino‐acid sequence comparison revealed that Bacillus YM55‐1 aspartase shared 71% homology with Bacillus subtilis aspartase and 49% with Escherichia coli and Pseudomonas fluorescens aspartases. The E. coli TK237/pUCASPB strain, which was obtained by transforming E. coli TK237 (aspartase‐null strain) with a vector plasmid (pUCASPB) containing the cloned aspB gene, produced a large amount of the enzyme corresponding to > 10% of the total soluble protein. The over‐expressed recombinant enzyme (native molecular mass: 200 kDa) was purified effectively and rapidly using heat treatment and affinity chromatography. In order to probe the catalytic residues of this enzyme, two conserved amino‐acid residues, Lys183 and His134, were individually mutated to alanine. Although the tertiary structure of each mutant was estimated to be the same as that of wild‐type aspartase in CD and fluorescence measurements, the Lys183Ala mutant lost its activity completely, whereas His134Ala retained full activity. This finding suggests that Lys183 may be involved in the catalytic activity of this thermostable Bacillus YM55‐1 aspartase.
Substrate specificity of a maize ribosome‐inactivating protein differs across diverse taxa
Authors: Krawetz Julie E. , Boston Rebecca S
Published: March 2003

The superfamily of ribosome‐inactivating proteins (RIPs) consists of toxins that catalytically inactivate ribosomes at a universally conserved region of the large ribosomal RNA. RIPs carry out a single N‐glycosidation event that alters the binding site of the translational elongational factor eEF1A and causes a cessation of protein synthesis that leads to subsequent cell death. Maize RIP1 is a kernel‐specific RIP with the unusual property of being produced as a zymogen, proRIP1. ProRIP1 accumulates during seed development and becomes active during germination when cellular proteases remove acidic residues from a central domain and both termini. These deletions also result in RIP activation in vitro. However, the effectiveness of RIP1 activity against target ribosomes remains species‐dependent. To determine the potential efficiency of maize RIP1 as a plant defense protein, we used quantitative RNA gel blots to detect products of RIP activity against intact ribosomal substrates from various species. We determined the enzyme specificity of recombinant maize proRIP1 (rproRIP1), papain‐activated rproRIP1 and MOD1 (an active deletion mutant of rproRIP1) against ribosomal substrates with differing levels of RIP sensitivity. The rproRIP1 had no detectable enzymatic activity against ribosomes from any of the species assayed. The papain‐activated rproRIP1 was more active than MOD1 against ribosomes from either rabbit or the corn pathogen, Aspergillus flavus, but the difference was much more marked when rabbit ribosomes were used as a substrate. The papain‐activated rproRIP1 was much more active against rabbit ribosomes than homologous Zea mays ribosomes and had no detectable effect on Escherichia coli ribosomes.
An anti‐idiotypic antibody with an internal image of human interferon‐γ and human interferon‐γ‐like antiviral activity
Authors: Depraetere Hilde , Depla Erik , Haelewyn Joost , De Ley Marc
Published: March 2003

D9D10, a monoclonal antibody that inhibits the biological activity of human interferon‐γ (IFN‐γ), was used to generate monoclonal anti‐idiotypic antibodies. After a first selection, the monoclonal anti‐idiotypic antibody AA1E5 was chosen to be fully characterized. To the best of our knowledge this is the first description of a monoclonal antibody with an IFN‐γ‐like antiviral activity; AA1E5 competed with IFN‐γ for binding to D9D10 indicating its anti‐idiotypic character. However, AA1E5 also fully mimics HuIFN‐γ as it not only binds to the HuIFN‐γ‐receptor, where it competes with HuIFN‐γ, but more importantly AA1E5 and its Fv fragment, cloned and expressed in Escherichia coli, mimic the antiviral activity of HuIFN‐γ. Indeed, 15 µg of AA1E5 and 2.5 µg of its Fv fragment had an effect comparable to that of 10 IU of HuIFN‐γ in an antiviral assay on A549 cells. Sequence comparison between the complementarity determination regions of the antibody and the sequence of HuIFN‐γ revealed that both the heavy chain variable domain, VH , and the kappa light chain variable domain, Vκ , have epitopes of 3–4 amino acids that are present in the HuIFN‐γ sequence, some of which contribute to receptor binding, as identified by Walter et al. [M. R. Walter, W. T. Windsor, T. L. Nagabhushan, D. J. Lundell, C. A. Lunn, P. J. Zauodny & S. K. Narula (1995) Nature376, 230–235].
The N‐terminal portion of the main cytosolic loop mediates K+sensitivity in the retinal rod Na+/Ca2+‐K+‐exchanger
Authors: Seiler Elena P. , Guerini Danilo , Guidi Fabrizio , Carafoli Ernesto
Published: March 2003

Two types of Na+/Ca2+‐exchangers have been characterized in the literature: The first is the cardiac, skeletal muscle and brain type, which exchanges 1 Ca2+for 3 Na+, the second, found in retinal photosensor cells, transports 1 Ca2+and 1 K+in exchange for 4 Na+. The present work describes the properties of chimeric constructs of the two exchanger types. Ca2+gel overlay experiments have identified a high affinity (Kdin the 1 µmrange) Ca2+‐binding domain between Glu601 and Asp733 in the main cytosolic loop of the retinal protein, just after transmembrane domain 5. Insertion of the retinal Ca2+‐binding domain in the cytosolic loop of the cardiac exchanger conferred K+‐dependence to the Ca2+uptake activity of the chimeric constructs expressed in HeLa cells. The apparentKmof the K+effect was about 1 mm. Experiments with C‐terminally truncated versions of the retinal insert indicated that the sequence between Leu643 and Asp733 was critical in mediating K+sensitivity of the recombinant chimeras. Thus, the high affinity Ca2+‐binding domain in the main cytosolic loop of the retinal exchanger may regulate the activity of the retinal protein by binding Ca2+, and by conferring to it K+sensitivity.
Interaction between the lipoamide‐containing H‐protein and the lipoamide dehydrogenase (L‐protein) of the glycine decarboxylase multienzyme system
Authors: Neuburger Michel , Polidori Ange M. , Piètre Emmanuel , Faure Magali , Jourdain Agnés , Bourguignon Jacques , Pucci Bernard , Douce Roland
Published: March 2003

Lipoamide dehydrogenase or dihydrolipoamide dehydrogenase (EC 1.8.1.4) is the E3‐protein component of the mitochondrial 2‐oxoacid dehydrogenase multienzyme complexes. It is also the L‐protein component of the glycine decarboxylase system. Although the enzymology of this enzyme has been studied exhaustively using free lipoamide as substrate, no data are available concerning the kinetic parameters of this enzyme with its physiological substrates, the dihydrolipoyl domain of the E2 component (dihydrolipoyl acyltransferase) of the 2‐oxoacid dehydrogenase multienzyme complexes or the dihydrolipoyl H‐protein of the mitochondrial glycine decarboxylase. In this paper, we demonstrate that Tris(2‐carboxyethyl)phosphine, a specific disulfide reducing agent, allows a continuous reduction of the lipoyl group associated with the H‐protein during the course of the reaction catalysed by the L‐protein. This provided a valuable new tool with which to study the catalytic properties of the lipoamide dehydrogenase. The L‐protein displayed a much higher affinity for the dihydrolipoyl H‐protein than for free dihydrolipoamide. The oxidation of the dihydrolipoyl H‐protein was not affected by the presence of structurally related analogues (apoH‐protein or octanoylated H‐protein). In marked contrast, these analogues strongly and competitively inhibited the decarboxylation of the glycine molecule catalysed by the P‐protein component of the glycine decarboxylase system. Small unfolded proteolytic fragments of the H‐protein, containing the lipoamide moiety, displayed Km values for the L‐protein close to that found for the H‐protein. On the other hand, these fragments were not able to promote the decarboxylation of the glycine in the presence of the P‐protein. New highly hydrophilic lipoate analogues were synthesized. All of them showed Km and kcat/Km values very close to that found for the H‐protein. From our results we concluded that no structural interaction is required for the L‐protein to catalyse the oxidation of the dihydrolipoyl H‐protein. We discuss the possibility that one function of the H‐protein is to maintain a high concentration of the hydrophobic lipoate molecules in a nonmicellar state which would be accessible to the catalytic site of the lipoamide dehydrogenase.
Membranolytic selectivity of cystine‐stabilized cyclic protegrins
Authors: Tam James P. , Wu Chengwei , Yang Jin‐Long
Published: March 2003

To correlate conformational rigidity with membranolytic selectivity of antimicrobial activity and cytotoxicity, we prepared six cyclic analogs of protegrin‐1 (PG‐1), an 18‐residue cationic peptide with a broad‐spectrum antimicrobial activity. These cyclic protegrins bear end‐to‐end peptide bonds together with varying numbers (zero to three) of cross‐strand disulfide constraints. The most constrained analog is a cyclic tricystine protegrin (ccPG 3) containing three evenly spaced, parallel disulfide bonds. Antimicrobial assays against 10 organisms in low‐ and high‐salt conditions showed that these cyclic protegrins were broadly active with different antimicrobial profiles against Gram‐positive and Gram‐negative bacteria, fungi and one tested virus, HIV‐1. Compared to PG‐1, the cyclic tricystine ccPG 3 displayed approximately a 10‐fold decrease in hemolytic activity against human cells and 6‐ to 30‐fold improvement of membranolytic selectivity against six of the 10 tested organisms. In contrast, [ΔSS]cPG 8, a cyclic protegrin with no disulfide bond, and [ΔCys6,15]cPG 5, a cyclic mimic of PG‐1 with one disulfide bond, exhibited activity spectra, potency, and cytotoxicity similar to PG‐1. Circular dichroism showed that cyclic protegrins containing with one to three cystine bonds displayed some degree of β‐strand structures in water/trifluoroethanol or phosphate‐buffered solutions. Collectively, our results indicate that cyclic structures are useful in the design of antimicrobial peptides and that an increase in the conformational rigidity of protegrins may confer membranolytic selectivity that dissociates antimicrobial activity from hemolytic activity.
The role of individual lysine residues in the basic patch on turnip cytochrome f for electrostatic interactions with plastocyanin in vitro
Authors: Gong Xiao‐Song , Wen Jiang Qi , Fisher Nicholas E. , Young Simon , Howe Christopher J. , Bendall Derek S. , Gray John C.
Published: March 2003

The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild‐type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long‐range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f–plastocyanin complex determined by NMR.
Crystal structure of cambialistic superoxide dismutase from Porphyromonas gingivalis
Authors: Sugio Shigetoshi , Hiraoka B. Yukihiro , Yamakura Fumiyuki
Published: March 2003

The crystal structure of cambialistic superoxide dismutase (SOD) from Porphyromonas gingivalis, which exhibits full activity with either Fe or Mn at the active site, has been determined at 1.8‐Å resolution by molecular replacement and refined to a crystallographic R factor of 17.9% (Rfree 22.3%). The crystals belong to the space group P212121 (a = 75.5 Å, b = 102.7 Å, c = 99.6 Å) with four identical subunits in the asymmetric unit. Each pair of subunits forms a compact dimer, but not a tetramer, with 222 point symmetry. Each subunit has 191 amino‐acid residues most of which are visible in electron density maps, and consists of seven α helices and one three‐stranded antiparallel β sheet. The metal ion, a 3 : 1 mixture of Fe and Mn, is coordinated with five ligands (His27, His74, His161, Asp157, and water) arranged at the vertices of a trigonal bipyramid. Although the overall structural features, including the metal coordination geometry, are similar to those found in other single‐metal containing SODs, P. gingivalis SOD more closely resembles the dimeric Fe‐SODs from Escherichia coli rather than another cambialistic SOD from Propionibacterium shermanii, which itself is rather similar to other tetrameric SODs.
Differential modulation of α, β and γ enolase isoforms in regenerating mouse skeletal muscle
Authors: Merkulova Tatyana , Dehaupas Michèle , Nevers Marie‐Claire , Créminon Christophe , Alameddine Hala , Keller Angélica
Published: March 2003

Nothing is known about the expression of the glycolytic enzyme enolase in skeletal muscle alterations such as myofiber degeneration and regeneration. Enolase is a dimeric enzyme which exhibits cell type specific isoforms. The embryonic form, αα, remains expressed in most adult tissues, whereas a transition towards specific isoforms occurs during ontogenesis in two cell types with high energy requirements: αγ and γγ in neurons, αβ and ββ in striated muscle cells. During murine myogenesis, β enolase transcripts are detected early in the forming muscles, and the β gene is further upregulated at specific stages of muscle development. The α and β subunits exhibit characteristic developmental microheterogeneity patterns. High levels of β enolase subunits characterize the glycolytic fast‐twitch fibers of adult muscles. We have investigated the expression of enolase subunits in a mouse experimental model of muscle regeneration. Following a single intramuscular injection of the necrotic agent cardiotoxin, we observed a rapid decrease in the level of the major muscle enolase subunit β, accounting for the drop in total enolase activity that correlated with the degeneration of myofibers. Concomitant with the regeneration of new fibers, β subunit levels began to increase, reaching normal values by 30 days after injury. Changes in the embryonic and ubiquitous subunit, α, mimicked those occurring during development by two aspects: modifications in electrophoretic variants and redistribution between soluble and insoluble compartments of muscle extracts. Imunocytochemical analyses of α and β enolase subunits first revealed a homogeneous labeling within myofibers. Striations characteristic of normal adult muscle tissue were visible again by day 14 after injury. A perinuclear α and β immunoreactivity was often observed in regenerating myofibers but its functional significance remains to be elucidated. Double labeling experiments with anti‐γ enolase and FITC‐α bungarotoxin allowed us to follow the neuromuscular junction remodeling that occurs during muscle regeneration despite the absence of nerve injury.
F1 and F0 connections in the bovine mitochondrial ATP synthase
Authors: Xu Ting , Zanotti Franco , Gaballo Antonio , Raho Gabriella , Papa Sergio
Published: March 2003

We have studied the functional effect of limited proteolysis by trypsin of the constituent subunits in the native and reconstituted F1F0 complex and isolated F1 of the bovine heart mitochondrial ATP synthase (EC 3.6.1.34). Chemical cross‐linking of oligomycin‐sensitivity conferring protein (OSCP) with other subunits of the ATP synthase and the consequent functional effects were also investigated.

The results obtained show that the α subunit N‐terminus is essential for the correct, functional connection of F1 to F0. The α‐subunit N‐terminus contacts OSCP which, in turn, contacts the F0I‐PVP(b) and the F0‐d subunits. The N‐terminus of subunit α, OSCP, a segment of subunit d and the C‐terminal and central region of F0I‐PVP(b) subunits are peripherally located with respect to subunits γ and δ which are completely shielded in the F1F0 complex against trypsin digestion. This qualifies the N‐terminus of subunit α, OSCP, subunit d and F0I‐PVP(b) as components of the lateral element of the stalk. These subunits, rather than being confined at one side of the complex which would leave most of the central part of the γ subunit uncovered, surround the γ and the δ subunits located in the central stalk.
Processing of synthetic pro‐islet amyloid polypeptide (proIAPP) ‘amylin’ by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro
Authors: Higham Claire E. , Hull Rebecca L. , Lawrie Laura , Shennan Kathleen I. J. , Morris John F. , Birch Nigel P. , Docherty Kevin , Clark Anne
Published: March 2003

Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67‐residue propeptide in islet β‐cells and colocalized with insulin in β‐cell granules. The mature 37‐amino acid peptide is produced by proteolysis at pairs of basic residues at the C‐ and N‐termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5–16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N‐terminal–IAPP junction and later at the C‐terminus, whereas initial PC2 activity was at the IAPP–C‐terminal junction. Processing at the basic residues within the C‐terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B‐chain–C‐peptide junction, followed by PC2 at A chain–C‐peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.