bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium‐binding properties and molecular organization of bradykinin
Authors: Gaggelli Elena , D’Amelio Nicola , Maccotta Antonella , Valensin Gianni
Published: March 2003

The NMR features of bradykinin were investigated in dimethylsulfoxide containing 1% water. The temperature dependence of chemical shifts and ROESY maps were monitored for the major species where all X–Pro bonds are trans. The occurrence of a head‐to‐tail ionic interaction and intramolecular hydrogen bonds stabilizing a pseudo cyclic arrangement was inferred, a β turn at the C‐terminus being the main feature of the secondary structure. Calcium was shown to bind to the peptide with a dissociation constant Kd = 2.8 + 0.2 mm. 2Pro and 3Pro carbonyls, as well as the 9Arg carboxyl, were assigned as the metal‐binding sites. A molecular model of the 1 : 1 metal–complex was obtained. In light of conformational changes experienced by the peptide upon interaction with calcium, a role for the metal was hypothesized in the process of conformational selection from the free to the receptor‐bound state of bradykinin.
The A26G replacement in the consensus sequence A‐X‐X‐X‐X‐G‐K‐[T,S] of the guanine nucleotide binding site activates the intrinsic GTPase of the elongation factor 2 from the archaeon Sulfolobus solfataricus
Authors: De Vendittis Emmanuele , Adinolfi Bianca Stella , Amatruda Maria Rosa , Raimo Gennaro , Masullo Mariorosario , Bocchini Vincenzo
Published: March 2003

A recombinant form of the elongation factor 2 from the archaeon Sulfolobus solfataricus (SsEF‐2), carrying the A26G substitution, has been produced and characterized. The amino acid replacement converted the guanine nucleotide binding consensus sequences A‐X‐X‐X‐X‐G‐K‐[T,S] of the elongation factors EF‐G or EF‐2 into the corresponding G‐X‐X‐X‐X‐G‐K‐[T,S] motif which is present in all the other GTP‐binding proteins. The rate of poly(U)‐directed poly(Phe) synthesis and the ribosome‐dependent GTPase activity of A26GSsEF‐2 were decreased compared to SsEF‐2, thus indicating that the A26G replacement partially affected the function of SsEF‐2 during translocation. In contrast, the A26G substitution enhanced the catalytic efficiency of the intrinsic SsEF‐2 GTPase triggered by ethylene glycol [Raimo, G., Masullo, M., Scarano, G., & Bocchini, V. (1997) Biochimie78, 832–837]. Surprisingly, A26GSsEF‐2 was able to hydrolyse GTP even in the absence of ethylene glycol; furthermore, the alcohol increased the affinity for GTP without modifying the catalytic constant of A26GSsEF‐2 GTPase. Compared to SsEF‐2, the affinity of A26GSsEF‐2 for [3H]GDP was significantly reduced. These findings suggest that A26 is a regulator of the biochemical functions of SsEF‐2. The involvement of this alanine residue in the guanine nucleotide‐binding pocket of EF‐2 or EF‐G is discussed.
Structural studies of the BstVI restriction–modification proteins by fluorescence spectroscopy
Authors: Saavedra Claudia , Vásquez Claudio , Encinas María Victoria
Published: March 2003

Structural studies of the proteins of the BstVI restriction–modification system of Bacillus stearothermophilus V were carried out using intrinsic fluorescence techniques. The exposure and environments of their tryptophanyl residues were determined using collisional quenchers. Quenching of BstVI endonuclease by iodide suggested a heterogeneous class of tryptophan residues, while the results obtained with M.BstVI methylase were consistent with a rather exposed tryptophan population. A comparison of the quenching efficiencies at 20 °C and 55 or 60 °C showed that their structures are more flexible and open at the temperature at which they exhibit maximal activity. The endonuclease reached its active conformation only after 1 h of incubation at 60 °C. Fluorescence changes were observed upon Mn2+ and Mg2+ binding, with Kd values in the range 3–5 μm. The binding of S‐adenosyl‐l‐methionine to the methylase produced conformational changes, which were consistent with binding to a single site of Kd 550 and 680 μm at 20 °C and 55 °C, respectively. Quenching experiments with iodide showed that the presence of S‐adenosyl‐l‐methionine leads to different conformational states at 20 °C and 55 °C. These results were interpreted in terms of differences in the structural characteristics of these restriction–modification proteins as well as in terms of differences in the conformational states that these enzymes exhibit at 20 °C and at the temperature at which they are most active.
Autocatalytic processing of the streptococcal cysteine protease zymogen
Authors: Doran John D. , Nomizu Motoyoshi , Takebe Sachiko , Ménard Robert , Griffith Douglas , Ziomek Edmund
Published: March 2003

The autocatalytic processing of the streptococcal cysteine protease zymogen (proSCP) to active streptococcal cysteine protease (SCP) was investigated in vitro using purified protein from Streptococcus pyogenes strain B220. It was found that the autocatalytic maturation of the zymogen proceeds through the sequential appearance of at least six intermediates, five of which were characterized through a combination of N‐terminal sequencing and MS. Intermediates were identified as resulting from cleavages after Lys26, Asn41, Lys101, Ala112, and Lys118. Time‐course studies of the proSCP processing gave a sigmoidal activity profile and indicated that proSCP catalyses its own transformation, mainly via an intermolecular processing mechanism. A similar sequential appearance of intermediates was observed when inactive Cys192Ser proSCP was treated with native, enzymatically active SCP, thus demonstrating that the maturation can exclusively proceed by a bimolecular mechanism. It was shown that proSCP, but not mature SCP, immobilized on a Sepharose resin is capable of liberating itself from the column, indicating that the zymogen is also capable of intramolecular processing. In order to test whether the amino acid sequences at the processing sites could be used for developing new, specific substrates, 3‐amino benzoic acid octapeptide derivatives based on all five characterized amino acid sequences from the autoprocessing cleavage sites were synthesized and tested for activity. The 3‐amino benzoic acid derivatives have kcat/KM values ranging from 1200 to 7700·m−1·s−1, making them very good endopeptidase substrates for SCP.
The nuclear localization signal (NLS) of PDX‐1 is part of the homeodomain and represents a novel type of NLS
Authors: Hessabi Behnam , Ziegler Patrick , Schmidt Ines , Hessabi Claudia , Walther Reinhard
Published: March 2003

The β‐cell homeodomain transcription factor PDX‐1 has vital functions both in controlling the expression of pancreatic polypeptide hormones and in the development of the pancreas. The transactivating and DNA‐binding properties of PDX‐1 have been well characterized, but nuclear transport is still undefined. Here we show that PDX‐1 bears a nuclear localization signal (NLS) that is part of helix 3 of the homeodomain. PDX‐1 deletion mutants were tagged with enhanced green fluorescent protein (EGFP) and expressed in COS‐7 cells. Subcellular localization of the respective PDX‐1–EGFP fusion proteins was analyzed by direct fluorescence microscopy and Western immunoblotting using an anti‐(GFP). As a result we were able to demonstrate that the homeodomain or helix 3 alone was sufficient and necessary for transport into the nucleus. Point mutations of basic amino acid residues within helix 3 led to identification of an NLS with six amino acids being crucial for nuclear transport of PDX‐1. Because this NLS does not match known examples of NLSs, the PDX‐1 NLS may represent a novel class of NLS.
Structure and dynamics of the B12‐binding subunit of glutamate mutase from Clostridium cochlearium
Authors: Hoffmann Bernd , Konrat Robert , Bothe Harald , Buckel Wolfgang , Kräutler Bernhard
Published: March 2003

Glutamate mutase (Glm) is an adenosylcobamide‐dependent enzyme that catalyzes the reversible rearrangement of (2S)‐glutamate to (2S,3S)‐3‐methylaspartate. The active enzyme from Clostridium cochlearium consists of two subunits (of 53.6 and 14.8 kDa) as an α2β2 tetramer, whose assembly is mediated by coenzyme B12. The smaller of the protein components, GlmS, has been suggested to be the B12‐binding subunit. Here we report the solution structure of GlmS, determined from a heteronuclear NMR‐study, and the analysis of important dynamical aspects of this apoenzyme subunit. The global fold and dynamic behavior of GlmS in solution are similar to those of the corresponding subunit MutS from C. tetanomorphum, which has previously been investigated using NMR‐spectroscopy. Both solution structures of the two Glm B12‐binding subunits share striking similarities with that determined by crystallography for the B12‐binding domain of methylmalonyl CoA mutase (Mcm) from Propionibacterium shermanii, which is B12 bound. In the crystal structure a conserved histidine residue was found to be coordinated to cobalt, displacing the endogenous axial ligand of the cobamide. However, in GlmS and MutS the sequence motif, Asp‐x‐His‐x‐x‐Gly, which includes the cobalt‐coordinating histidine residue, and a predicted α‐helical region following the motif, are present as an unstructured and highly mobile loop. In the absence of coenzyme, the B12‐binding site apparently is only partially formed. By comparing the crystal structure of Mcm with the solution structures of B12‐free GlmS and MutS, a consistent picture on the mechanism of B12 binding has been obtained. Important elements of the binding site only become structured upon binding B12; these include the cobalt‐coordinating histidine residue, and an α helix that forms one side of the cleft accommodating the nucleotide ‘tail’ of the coenzyme.
6‐Hydroxycyclohex‐1‐ene‐1‐carbonyl‐CoA dehydrogenase and 6‐oxocyclohex‐1‐ene‐1‐carbonyl‐CoA hydrolase, enzymes of the benzoyl‐CoA pathway of anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica
Authors: Laempe Diana , Jahn Martina , Fuchs Georg
Published: March 2003

Benzoyl‐CoA is a common intermediate in the anaerobic bacterial metabolism of many aromatic substrates. Two enzymes and ferredoxin of the central benzoyl‐CoA pathway in Thauera aromatica have been purified so far. Benzoyl‐CoA reductase reduces the aromatic ring with reduced ferredoxin yielding cyclohexa‐1,5‐diene‐1‐carbonyl‐CoA [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem.234, 921–933]. Dienoyl‐CoA hydratase subsequently adds one molecule of water and thereby produces 6‐hydroxycyclohex‐1‐ene‐1‐carbonyl‐CoA [Laempe, D., Eisenreich, W., Bacher, A., & Fuchs, G. (1998) Eur. J. Biochem.255, 618–627]. Here two new enzymes, which convert this intermediate to the noncyclic product 3‐hydroxypimelyl‐CoA, were purified from T. aromatica and studied. 6‐Hydroxycyclohex‐1‐ene‐1‐carbonyl‐CoA dehydrogenase is an NAD+‐specific β‐hydroxyacyl‐CoA dehydrogenase that catalyzes 6‐hydroxycyclohex‐1‐ene‐1‐carbonyl‐CoA + NAD+ → 6‐oxocyclohex‐1‐ene‐1‐carbonyl‐CoA + NADH + H+. 6‐Oxocyclohex‐1‐ene‐1‐carbonyl‐CoA hydrolase acts on the β‐oxoacyl‐CoA compound and catalyzes the addition of one molecule of water to the double bound and the hydrolytic C–C cleavage of the alicyclic ring, 6‐oxocyclohex‐1‐ene‐1‐carbonyl‐CoA + 2 H2O → 3‐hydroxypimelyl‐CoA. The genes for both enzymes, had and oah, were cloned, had was overexpressed in Escherichia coli and the recombinant protein was purified. Hence, presumably all enzymes of the central benzoyl‐CoA pathway of anaerobic aromatic metabolism from this organism have now been purified and studied and the corresponding genes have been cloned and sequenced.
Actin and nucleotide induced conformational changes in the vicinity of Lys553 in myosin subfragment 1
Authors: Peyser Y. Michael , Muhlrad Andras
Published: March 2003

Bertrand et al. [Bertrand, R., Derancourt, J. & Kassab, R. (1995) Biochemistry34, 9500–9507] reported that 6‐[fluoresceine‐5(and 6)‐carboxamido] hexanoic acid succinimidyl ester (FHS) selectively modifies Lys553, which is part of the strong actin‐binding site of myosin subfragment 1 (S1). We found that the reaction of FHS with Lys533 is accompanied by a decrease in the fluorescence intensity of the reagent. The rate of the FHS reaction increased with increasing pH implying that the unprotonated form of the ε‐amino group of Lys553 reacts with FHS. Addition of 0.4 m KCl reduced the rate of reaction significantly, which indicates ionic strength‐dependent changes in the structure of S1. Limited trypsinolysis of S1 before the FHS reaction also decreased the rate of the reaction showing that the structural integrity of S1 is needed for the reactivity of Lys553. ATP, ADP, ADP·BeFx, ADP·AlF4, ADP·Vi  and pyrophosphate significantly decreased the rate of Lys553 labelling, suggesting nucleotide‐induced conformational changes in the environment of Lys553. The fluorescence emission spectrum of the Lys553‐bound FH moiety and the quenching of its fluorescence by nitromethane was not influenced by nucleotides, implying that the chemical reactivity but not the accessibility of Lys553 was decreased by the nucleotide‐induced conformational change. In the presence of ATP when the M**ADP·Pi state of the ATPase cycle is predominantly populated, the reaction rate decreased more than in the case of the S1·ADP·AlF4− and S1·ADP·Vi  complexes, which are believed to mimic the M**ADP.Pi state. This indicates that the conformation of the S1–ADP·AlF4− and S1·ADP·Vi  complexes in the vicinity of Lys553 does not resemble the structure of the M**ADP·Pi state. The rate of Lys553 labelling decreased strongly in the presence of actin. The nitromethane quenching of the Lys553‐bound FHS was not influenced by actin, which indicates that the reduced reaction rate is not due to steric hindrance caused by the bulky protein but by actin induced conformational changes in the vicinity of Lys553.
Secretory expression and site‐directed mutagenesis studies of the winter flounder skin‐type antifreeze polypeptides
Authors: Lin Qingsong , Ewart K. Vanya , Yan Qing , Wong W. K. Raymond , Yang Daniel S. C. , Hew Choy L.
Published: March 2003

Winter flounder contains both liver‐type, extracellular antifreeze polypeptides (wflAFPs) and less active skin‐type, intracellular antifreeze polypeptides (wfsAFPs). The lower activity of wfsAFPs might be due to their lack of complete ice‐binding motifs ‘‐K–DT‐’. In order to test the functional role of this putative ice‐binding motif, mutations were introduced into the N‐terminal or C‐terminal regions of wfsAFP‐2, which lack any presumptive ice‐binding motifs. The wild‐type and mutant wfsAFP‐2 were secreted in Escherichia coli culture media as mature antifreeze proteins and purified to homogeneity. Surprisingly, the antifreeze activity decreased with the introduction of ice‐binding motifs. However, there was a corresponding decrease in α‐helical content as well as thermal stability and this would suggest a compromise in retaining helical structure with the presence of ice‐binding motifs. These studies have brought new definitions of the roles of ice‐binding motif residues in type I antifreeze proteins.
Structural and functional implications of the amino acid sequences of dimeric, cytoplasmic and octameric mitochondrial creatine kinases from a protostome invertebrate
Authors: Pineda Agustin O. , Ellington W. Ross
Published: March 2003

The cDNA and deduced amino‐acid sequences for dimeric and octameric isoforms of creatine kinase (CK) from a protostome, the polychaete Chaetopterus variopedatus, were elucidated and then analysed in the context of available vertebrate CK sequences and the recently determined crystal structure of chicken sarcomeric mitochondrial CK (MiCK). As protostomes last shared a common ancestor with vertebrates roughly 700 million years ago, observed conserved residues may serve to confirm or reject contemporary hypotheses about the roles of particular amino acids in functional/structural processes such as dimer/octamer formation and membrane binding. The isolated cDNA from the dimeric CK consisted of 1463 nucleotides with an open reading frame of 1116 nucleotides encoding a 372‐amino‐acid protein having a calculated molecular mass of 41.85 kDa. The percentage identity of C. variopedatus dimeric CK to vertebrate CK is as high as 69%. The octameric MiCK cDNA is composed of 1703 nucleotides with an open reading frame of 1227 nucleotides. The first 102 nucleotides of the open reading frame encode a 34‐amino‐acid leader peptide whereas the mature protein is composed of 375 amino acids with a calculated molecular mass of 42.17 kDa. The percentage identity of C. variopedatus MiCK to vertebrate CK is as high as 71%. This similarity is also evident in residues purported to be important in the structure and function of dimeric and octameric CK: (a) presence of seven basic amino acids in the C‐terminal end thought to be important in binding of MiCK to membranes; (b) presence of a lysine residue (Lys110 in chicken MiCK) also thought to be involved in membrane binding; and (c) presence of a conserved tryptophan thought to be important in dimer stabilization which is present in all dimeric and octameric guanidino kinases. However, C. variopedatus MiCK lacks the N‐terminal heptapeptide present in chicken MiCK, which is thought to mediate octamer stabilization. In contrast with vertebrate MiCK, polychaete octamers are very stable indicating that dimer binding into octamers may be mediated by additional and/or other residues. Phylogenetic analyses showed that both octamer and dimer evolved very early in the CK lineage, well before the divergence of deuterostomes and protostomes. These results indicate that the octamer is a primitive feature of CK rather than being a derived and advanced character.
Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat‐2 fibroblasts by cell‐permeable ceramides
Authors: Bladergroen Bellinda A. , Bussière Miguel , Klein Wil , Geelen Math J H. , Van Golde Lambert M. G. , Houweling Martin
Published: March 2003

Phospholipids and sphingolipids are important precursors of lipid‐derived second messengers such as diacylglycerol and ceramide, which participate in several signal transduction pathways and in that way mediate the effects of various agonists. The cross‐talk between glycerophospholipid and sphingolipid metabolism was investigated by examining the effects of cell‐permeable ceramides on phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) synthesis in Rat‐2 fibroblasts. Addition of short‐chain C6‐ceramide to the cells resulted in a dose‐ and time‐dependent inhibition of the CDP‐pathways for PtdCho and PtdEtn synthesis. Treatment of cells for 4 h with 50 µM C6‐ceramide caused an 83% and a 56% decrease in incorporation of radiolabelled choline and ethanolamine into PtdCho and PtdEtn, respectively. Exposure of the cells for longer time‐periods (≥ 16 h) to 50 µM C6‐ceramide resulted in apoptosis. The structural analogue dihydro‐C6‐ceramide did not affect PtdCho and PtdEtn synthesis. In pulse–chase experiments, radioactive choline and ethanolamine accumulated in CDP‐choline and CDP‐ethanolamine under the influence of C6‐ceramide, suggesting that synthesis of both PtdCho and PtdEtn were inhibited at the final step in the CDP‐pathways. Indeed, cholinephosphotransferase and ethanolaminephosphotransferase activities in membrane fractions from C6‐ceramide‐treated cells were reduced by 64% and 43%, respectively, when compared with control cells. No changes in diacylglycerol mass levels or synthesis of diacylglycerol from radiolabelled palmitate were observed. It was concluded that C6‐ceramide affected glycerophospholipid synthesis predominantly by inhibition of the step in the CDP‐pathways catalysed by cholinephosphotransferase and ethanolaminephosphotransferase.
A prokaryotic‐type cytidine deaminase from Arabidopsis thaliana
Authors: Faivre‐Nitschke S. Emmanuelle , Grienenberger Jean M. , Gualberto José M.
Published: March 2003

The gene and cDNA of an Arabidopsis thaliana cytidine deaminase (CDA) were cloned and sequenced. The gene, At‐cda1, is located on chromosome 2 and is expressed in all plant tissues tested, although with quantitative differences. Expression analysis suggest that At‐cda1 probably codes for the housekeeping cytidine deaminase of Arabidopsis. The gene was functionally expressed in Escherichia coli and the protein, At‐CDA1, shows similar enzymatic and substrate specificities as conventional cytidine deaminases: it deaminates cytidine and deoxycytidine and is competitively inhibited by cytosine‐containing compounds. Because the protein shows no affinity to RNA, it is not likely to be involved in RNA‐editing by C‐to‐U deamination.

 When compared to cytidine deaminases from other organisms, it becomes clear that At‐CDA1 is related, both in sequence and structure, to the CDA of E. coli and other gram‐negative bacteria. The eubacterial nature of the Arabidopsis CDA suggests that it is an additional example of a plant gene of endosymbiotic origin.
Regulated transcription of the histone H2B genes of Trypanosoma brucei
Authors: García‐Salcedo José A. , Gijón Purificación , Pays Etienne
Published: March 2003

In Trypanosoma brucei, the genes encoding histone H2B are organized in a cluster of about 10–15 tandemly linked copies per haploid genome. The H2B transcripts are processed by trans‐splicing and polyadenylation, and encode a polypeptide of 111 residues with a molecular mass of 12.5 kDa. H2B mRNAs are differentially expressed during the parasite life‐cycle and are present at higher levels in dividing procyclic and bloodstream slender forms than in the nondividing bloodstream stumpy forms. Analysis of H2B mRNA levels during the synchronous differentiation from stumpy to procyclics forms revealed that the abundance of these transcripts is regulated through the cell‐cycle, reaching maximum levels during S‐phase. Addition of hydroxyurea to procyclic forms in culture specifically decreased H2B mRNA levels by about twofold, an effect not linked to its 3′ untranslated region. Inhibition of protein synthesis prevented this decrease.
Expression and activity of cyclic and linear analogues of esculentin‐1, an anti‐microbial peptide from amphibian skin
Authors: Ponti Donatella , Mignogna Giuseppina , Mangoni Maria L. , De Biase Daniela , Simmaco Maurizio , Barra Donatella
Published: March 2003

Esculentin‐1 is a potent anti‐microbial peptide present in minute amounts in skin secretions of Rana esculenta. It contains 46 amino‐acid residues and a C‐terminal disulfide bridge. We have explored the possibility of producing analogues of this peptide by recombinant expression in Escherichia coli of a fusion protein which is sequestered in inclusion bodies. The peptide of interest has been inserted at the N‐terminus of the protein, from which it can be released by cyanogen bromide cleavage. The anti‐microbial activities of the recombinant peptide as well as that of a mutant linear form devoid of the disulfide bridge are presented. The recombinant analogues retain the biological activity of the natural peptide, as tested with an inhibition zone assay against a variety of microorganisms. However, experiments on the rate of bacterial killing show that gram‐negative bacteria are more sensitive to the peptides than the gram‐positive bacterium, the effect of the cyclic peptide being in all cases faster than that of the linear molecule. Moreover, the activity against gram‐negative bacteria for both peptides is not affected by salts, whereas the activity against Staphylococcus aureus is lost at high salt concentration.
Asparaginyl endopeptidase (VmPE‐1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH‐EP)
Authors: Okamoto Takashi , Yuki Atsushi , Mitsuhashi Naoto , Mimamikawa Takao
Published: March 2003

A vacuolar cysteine proteinase, designated SH‐EP, is synthesized in cotyledons of germinated Vigna mungo seeds and is responsible for degradation of the seed proteins accumulated in protein bodies (protein storage vacuoles). SH‐EP belongs to the papain proteinase family and has a large N‐terminal prosegment consisting of 104 amino acid residues and a C‐terminal prosegment of 10 amino acid residues. It has been suggested that an asparaginyl endopeptidase, V. mungo processing enzyme 1 (VmPE‐1), is involved in the N‐terminal post‐translational processing of SH‐EP. The recombinant proform of SH‐EP (rSH‐EP) was produced in Escherichia coli cells, purified to homogeneity and refolded by stepwise dialysis. 31P‐NMR analysis of intact germinated cotyledons revealed that the vacuolar pH of cotyledonary cells changes from 6.04 to 5.47 during seed germination and early seedling growth. rSH‐EP was converted in vitro to the mature form through autocatalytic processing at a pH mimicking the vacuolar pH at the mid and late stages of seed germination, but not at the pH of the early stage. VmPE‐1 accelerated the rate of processing of rSH‐EP in vitro at the pH equivalent to the vacuolar pH at the early and mid stages of germination. In addition, the cleavage sites of the in vitro processed intermediates and the mature form of SH‐EP were identical to those of SH‐EP purified from germinated cotyledons of V. mungo. We propose that the asparaginyl endopeptidase (VmPE‐1)‐mediated processing mainly functions in the activation of proSH‐EP at the early stage of seed germination, and both VmPE‐1‐mediated and autocatalytic processings function synergistically in the activation of proSH‐EP in cotyledons at the mid and late stages.
Mapping of an epitope recognized by a neutralizing monoclonal antibody specific to toxin Cn2 from the scorpion Centruroides noxius, using discontinuous synthetic peptides
Authors: Calderon‐Aranda Emma S. , Selisko Barbara , York Eunice J. , Gurrola Georgina B. , Stewart John M. , Possani Lourival D.
Published: March 2003

The Na+‐channel‐affecting toxin Cn2 represents the major and one of the most toxic components of the venom of the Mexican scorpion Centruroides noxius Hoffmann. A monoclonal antibody BCF2 raised against Cn2 has been shown previously to be able to neutralize the toxic effect of Cn2 and of the whole venom of C. noxius. In the present study the epitope was mapped to a surface region comprising the N‐ and C‐terminal segments of Cn2, using continuous and discontinuous synthetic peptides, designed on the basis of the sequence and a three‐dimensional model of Cn2. The study of peptides of varying length resulted in the identification of segments 5–14 and 56–65 containing residues essential for recognition by BCF2. The peptide (abbreviated SP7) with the highest affinity to BCF2 (IC50 = 5.1 µm) was a synthetic heterodimer comprising the amino acid sequence from position 3–15 (amidated) of Cn2, bridged by disulfide to peptide from position 54–66, acetylated and amidated. Similar affinity was found with peptide SP1 [heterodimer comprising residues 1–14 (amidated) of Cn2, bridged with synthetic peptide 52–66 (acetylated)]. SP1 and SP7 were used to induce anti‐peptide antibodies in mouse and rabbit. Both peptides were highly immunogenic. The sera obtained were able to recognize Cn2 and to neutralize Cn2in vitro. The most efficient protection (8.3 µg Cn2 neutralized per mL of serum) was induced by rabbit anti‐SP1 serum.
Alteration of the lateral organization of the plasma membrane of Chinese hamster ovary cells by synthetic lipopeptide, Pam3Cys‐Ser‐Lys4
Authors: Vergne Isabelle , Cézanne Laurence
Published: March 2003

The cationic lipohexapeptide (S)‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐N‐palmitoyl‐(R)‐Cys‐(S)‐Ser‐(S)‐Lys4‐OH, trihydrochloride (Pam3Cys‐Ser‐Lys4) is a synthetic analog of the triacylated N‐terminal part of bacterial lipoproteins. In this study we addressed the question of whether Pam3Cys‐Ser‐Lys4 could modify the organization of the plasma membrane of Chinese hamster ovary cells. 1‐Acyl‐2‐[6‐(7‐nitro‐2–1,3‐benzoxadiazol‐4‐yl)amino]caproyl]‐sn‐glycero‐3‐phosphocholine (C6‐NBD‐PC) diffusion was followed by fluorescence recovery after photobleaching experiments carried out on the plasma membrane of Chinese hamster ovary cells. Incubation of cells in the presence of Pam3Cys‐Ser‐Lys4 induced an increase in the lateral diffusion coefficient and in the immobile fraction of C6‐NBD‐PC probes. Various control experiments have shown that the increase in the immobile fraction was not due to probe internalization induced by Pam3Cys‐Ser‐Lys4. Back‐exchange experiments showed that a good correlation exists between the fractions of immobilized probes and nonextractable probes in the plasma membrane of Chinese hamster ovary cells. A useful way to analyze the origin of probe immobilization (micrometer‐sized domains or aggregated patches of proteins) is to carry out fluorescence recovery after photobleaching experiments at variable observation radii. This type of experiment, carried out on the plasma membrane of Chinese hamster ovary cells incubated with Pam3Cys‐Ser‐Lys4, confirmed that the lipopeptide induced the aggregation of proteins of Chinese hamster ovary plasma membrane. Lipids which were trapped inside these aggregates were thus prevented from diffusing at long range in the plasma membrane plane and behave as an immobile fraction.
Activation of active‐site cysteine residues in the peroxiredoxin‐type tryparedoxin peroxidase of Crithidia fasciculata
Authors: Montemartini Marisa , Kalisz Henryk M. , Hecht Hans‐Jürgen , Steinert Peter , Flohé Leopold
Published: March 2003

Tryparedoxin peroxidase (TXNPx), recently identified as the hydroperoxide‐detoxifying enzyme of trypanosomatidae [Nogoceke, E., Gommel, D. U., Kiess, M., Kalisz, H. M. & Flohé, L. (1997) Biol. Chem.378, 827–836], is a member of the peroxiredoxin family and is characterized by two VCP motifs.

 Based on a consensus sequence of TXNPx and peroxiredoxin‐type peroxidases, eight TXNPx variants were designed, heterologously expressed in Escherichia coli, checked for α‐helix content by CD and kinetically analysed. The variant Q164E was fully active, C52S, W87D and R128E were inactive and C173S, W87H, W177E and W177H showed reduced activity. Wild‐type TXNPx and Q164E exhibit ping‐pong kinetics with infinite maximum velocities, whereas saturation kinetics were observed with C173S and W177E. The data comply with a mechanism in which C52, primarily activated by R128 and possibly by W87, is first oxidized by hydroperoxide to a sulfenic acid derivative. C173, supported by W177, then forms an intersubunit disulfide bridge with C52. If C173 is exchanged with a redox‐inactive residue (Ser) or is insufficiently activated, the redox shuttle remains restricted to C52. The shift in the kinetic pattern and decrease in specific activity of C173S and W177E may result from a limited accessibility of the oxidized C52 to tryparedoxin, which in the oxidized wild‐type TXNPx presumably attacks the C173 sulfur of the disulfide bridge. The proposed mechanism of action of TXNPx is consistent with that deduced for the homologous thioredoxin peroxidase of yeast [Chae, H. Z., Uhm, T. B. & Rhee, S. G. (1994) Proc. Natl Acad. Sci. USA91, 7022–7026] and is supported by molecular modelling based on the structure of the human peroxiredoxin ‘hORF6’[Choi, H.‐J., Kang, S. W. Yang, C.‐H., Rhee, S. G. & Ryu, S.‐E. (1998) Nat. Struct. Biol.5, 400–406].
The effects of hydrostatic pressure on the conformation of plasminogen
Authors: Kornblatt Jack A. , Kornblatt M. Judith , Clery Cecile , Balny Claude
Published: March 2003

Plasminogen undergoes a large conformational change when it binds 6‐aminohexanoate. Using ultraviolet absorption spectroscopy and native PAGE, we show that hydrostatic pressure brings about the same conformational change. The volume change for this conformational change is −33 mL·mol−1. Binding of ligand and hydrostatic pressure both cause the protein to open up to expose surfaces that had previously been buried in the interior.
Characterization of the dihydrolipoamide acetyltransferase of the mitochondrial pyruvate dehydrogenase complex from potato and comparisons with similar enzymes in diverse plant species
Authors: Millar A. Harvey , Leaver Christopher J. , Hill Steven A.
Published: March 2003

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) can be disassociated in 1 m NaCl and 0.1 m glycine into a large dihydrolipoamide acetyltransferase (E2) complex and smaller pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3) complexes. The E2 complex consists of 55 and 78‐kDa polypeptides which are reversibly radiolabelled to a similar degree in the intact mPDC by [2‐14C]pyruvate. Affinity‐purified antibodies against the 55‐kDa protein do not cross‐react with the 78‐kDa protein and the two proteins show different peptide patterns following partial proteolysis. The 78 and 55‐kDa proteins are present in approximately equal abundance in the E2 complex and incorporate a similar amount of [14C] on incubation with [2‐14C]pyruvate. Native mPDC and the E2 complex have sedimentation coefficients of 50S and 30S, respectively. Titration of electro‐eluted polypeptides against the intact mPDC and E2 complex revealed that each mg of mPDC contains 0.4 mg of E1, 0.4 mg of E2 and 0.2 mg of E3. Labelling of partially purified mPDC from potato, pea, cauliflower, maize and barley, with [2‐14C]pyruvate, suggest that a 78‐kDa acetylatable protein is only found in the dicotyledonous species, while all plant species tested contained a smaller 52–60 kDa acetylatable protein.
Mutational analysis of Ser14 and Asp157 in the nucleotide‐binding site of β‐actin
Authors: Schüler Herwig , Korenbaum Elena , Schutt Clarence E. , Lindberg Uno , Karlsson Roger
Published: March 2003

This paper compares wild‐type and two mutant β‐actins, one in which Ser14 was replaced by a cysteine, and a second in which both Ser14 and Asp157 were exchanged (Ser14→Cys and Ser14→Cys,Asp157→Ala, respectively). Both of these residues are part of invariant sequences in the loops, which bind the ATP phosphates, in the interdomain cleft of actin. The increased nucleotide exchange rate, and the decreased thermal stability and affinity for DNase I seen with the mutant actins indicated that the mutations disturbed the interdomain coupling. Despite this, the two mutant actins retained their ATPase activity. In fact, the mutated actins expressed a significant ATPase activity even in the presence of Ca2+ ions, conditions under which actin normally has a very low ATPase activity. In the presence of Mg2+ ions, the ATPase activity of actin was decreased slightly by the mutations. The mutant actins polymerized as the wild‐type protein in the presence of Mg2+ ions, but slower than the wild‐type in a K+/Ca2+ milieu. Profilin affected the lag phases and elongation rates during polymerization of the mutant and wild‐type actins to the same extent, whereas at steady‐state, the concentration of unpolymerized mutant actin appeared to be elevated. Decoration of mutant actin filaments with myosin subfragment 1 appeared to be normal, as did their movement in the low‐load motility assay system. Our results show that Ser14 and Asp157 are key residues for interdomain communication, and that hydroxyl and carboxyl groups in positions 14 and 157, respectively, are not necessary for ATP hydrolysis in actin.
Oligomerization and structural changes of the pore‐forming Pseudomonas aeruginosa cytotoxin
Authors: Sliwinski‐Korell Annette , Engelhardt Harald , Kampka Marita , Lutz Frieder
Published: March 2003

Pseudomonas aeruginosa produces a pathogenic factor, the 29‐kDa pore‐forming protein cytotoxin. Nonspecific oligomers of cytotoxin up to the hexamer, induced by oxidative crosslinking or detergent micellae, were based on intermolecular disulfide bridges. SDS induced tetramer, hexamer and mainly pentamers that were resistant to reducing conditions, indicating an additional oligomerization mechanism. Functional oligomerization after incubation with different membranes resulted in an oligomer of approximately 145 kDa that was identified as the pentamer by comparison with the SDS‐induced oligomers. Covalent modification with diethylpyrocarbonate showed that histidine residues are indispensable for functional pentamerization. Pentamer formation was not influenced by the lipid composition of the liposomes tested, indicating that rising membrane fluidity did not increase oligomerization. The secondary structure of cytotoxin determined by spectroscopy is characterized by approximately 50% β‐sheet, 20% β‐turn, 10% α‐helix and 20% remaining structure. Contact with detergent micellae or liposomes induced a reorganization of β‐structure associations, as observed by attenuated total reflection–Fourier transform infrared spectroscopy. Electron microscopy and principle component analysis of the cytotoxin monomer demonstrated a tapered molecule of 11 nm in length and a maximum width of 3.5 nm. These results classify the cytotoxin as a pore‐forming toxin, rich in antiparallel β‐structure, that needs to oligomerize and inserts into membranes; it is very similar to the Staphylococcus aureusα‐toxin.
Physarum amoebae express a distinct fragmin‐like actin‐binding protein that controls in vitro phosphorylation of actin by the actin–fragmin kinase
Authors: T’Jampens Davy , Bailey Juliet , Cook Lynnette J. , Constantin Bruno , Vandekerckhove Joël , Gettemans Jan
Published: March 2003

Amoebae and plasmodia constitute the two vegetative growth phases of the Myxomycete Physarum. In vitro and in vivo phosphorylation of actin in plasmodia is tightly controlled by fragmin P, a plasmodium‐specific actin‐binding protein that enables actin phosphorylation by the actin–fragmin kinase. We investigated whether amoebal actin is phosphorylated by this kinase, in spite of the lack of fragmin P. Strong actin phosphorylation was detected only following addition of recombinant actin–fragmin kinase to cell‐free extracts of amoebae, suggesting that amoebae contain a protein with properties similar to plasmodial fragmin. We purified the complex between actin and this protein to homogeneity. Using an antibody that specifically recognizes phosphorylated actin, we demonstrate that Thr203 in actin can be phosphorylated in this complex. A full‐length amoebal fragmin cDNA was cloned and the deduced amino acid sequence shows 65% identity with plasmodial fragmin. However, the fragmins are encoded by different genes. Northern blots using RNA from a developing Physarum strain demonstrate that this fragmin isoform (fragmin A) is not expressed in plasmodia. In situ localization showed that fragmin A is present mainly underneath the plasma membrane. Our results indicate that Physarum amoebae express a fragmin P‐like isoform which shares the property of binding actin and converting the latter into a substrate for the actin–fragmin kinase.
Phosphorylation of cytosolic phospholipase A2 in platelets is mediated by multiple stress‐activated protein kinase pathways
Authors: Börsch‐Haubold Angelika G. , Ghomashchi Farideh , Pasquet Sophie , Goedert Michel , Cohen Philip , Gelb Michael H. , Watson Steve P.
Published: March 2003

Stress‐activated protein kinases (SAPKs) are stimulated by cell damaging agents as well as by physiological receptor agonists. In this study we show that human platelets contain the isoforms SAPK2a, SAPK2b, SAPK3 and SAPK4 as determined by immunoblotting with specific antibodies. All four kinases were activated in thrombin‐stimulated platelets whereas only SAPK2a and SAPK2b were significantly stimulated by collagen. All four isoforms were able to phosphorylate wild‐type human cPLA2in vitro, although to different extents, but not cPLA2 mutants that had Ser505 replaced by alanine. Phosphorylation at Ser505 was confirmed by phosphopeptide mapping using microbore HPLC. SAPK2a and 42‐kDa mitogen‐activated protein kinase incorporated similar levels of phosphate into cPLA2 relative to the ability of each kinase to stimulate phosphorylation of myelin basic protein. SAPK2b and SAPK4 incorporated less phosphate, and cPLA2 was a poor substrate for SAPK3. The inhibitor of SAPK2a and SAPK2b, SB 202190, completely blocked collagen‐induced phosphorylation of cPLA2 at its two phosphorylation sites in vivo, Ser505 and Ser727. We have also reported previously that SB 202190 partially (≈ 50%) blocks phosphorylation at both sites and to a similar extent in thrombin‐stimulated platelets. Inhibition of phosphorylation resulted in a two‐ to threefold shift to the right in the concentration response curves for arachidonic acid release from thrombin‐ and collagen‐stimulated platelets. Our data suggest that cPLA2 is a substrate for several SAPK cascades and that phosphorylation of cPLA2 augments arachidonic acid release.
Characterization of theRhodobacter sphaeroides5‐aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinantEscherichia coli
Authors: Bolt Edward L. , Kryszak Luiza , Zeilstra‐Ryalls Jill , Shoolingin‐Jordan Peter M. , Warren Martin J.
Published: March 2003

ThehemAandhemTgenes encoding 5‐aminolaevulinic acid synthase (ALAS) from the photosynthetic bacteriumRhodobacter sphaeroides, were cloned to allow high expression inEscherichia coli. Both HemA and HemT appeared to be activein vivoas plasmids carrying the respective genes complemented anE. coli hemAstrain (glutamyl‐tRNA reductase deficient). The over‐expressed isoenzymes were isolated and purified to homogeneity. Isolated HemA was soluble and catalytically active whereas HemT was largely insoluble and failed to show any activityex vivo. Pure HemA was recovered in yields of 5–7 mg·L−1of starting bacterial culture and pure HemT at 10 mg·L−1. HemA has a final specific activity of 13 U·mg−1with 1 unit defined as 1 µmol of 5‐aminolaevulinic acid formed per hour at 37 °C. TheKmvalues for HemA are 1.9 mmfor glycine and 17 µmfor succinyl‐CoA, with the enzyme showing a turnover number of 430 h−1.

 In common with other ALASs the recombinantR. sphaeroidesHemA requires pyridoxal 5′‐phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor resulted in inactive apo‐ALAS. Similarly, reduction of the HemA–PLP complex using sodium borohydride led to > 90% inactivation of the enzyme. Ultraviolet–visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5′‐phosphate that was not observed when HemT was incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates. Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross‐react with mouse ALAS, indicating that all of the proteins have conserved epitopes.
Orientation of GTP and ADP within their respective binding sites in glutamate dehydrogenase
Authors: Rajagopalan Krishnan , Watt David S. , Haley Boyd E.
Published: March 2003

Previous studies have identified the guanine and adenine binding domains of the GTP and ADP binding sites of GDH. In this study the peptide sequences within or near to the terminal phosphate‐binding domains of the GTP and ADP binding sites of bovine liver glutamate dehydrogenase (GDH) were identified using photoaffinity labeling with the benzophenone nucleotide derivatives, [γ‐32P]GTPγBP and [γ‐32P]ATPγBP. Without activating light, GTPγBP exhibited inhibiting effects on the GDH reaction similar to GTP; ATPγBP, as expected, produced activating effects similar to those of ADP. Photoinsertion into GDH by both probes exhibited saturation effects in agreement with the respective kinetic effects. Specificity of labeling was supported by specific and effective reduction of photoinsertion of [γ‐32P]GTPγBP and [γ‐32P]ATPγBP into GDH by GTP and ADP, respectively. Using a combination of immobilized Fe3+‐chelate affinity chromatography and reversed‐phase HPLC, photolabeled peptides located within or near the phosphate‐binding domains of the GTP and ADP sites were isolated. Sequence analysis showed that GTPγBP primarily modified a peptide near the middle of the GDH sequence, Asn135–Lys143 and Glu290–Lys295. However, ATPγBP modified a single peptide corresponding to the sequence Met411–Arg419 near the C‐terminal domain. Using these results and the data from the previously identified base‐binding domain peptides the orientation of GTP and ADP within their respective binding sites in the catalytic cleft of GDH is proposed and explained on the basis of a proposed three‐dimensional schematic model structure derived from the bacterial enzyme.
Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines
Authors: Steinmetzer Torsten , Renatus Martin , Künzel Sebastian , Eichinger Andreas , Bode Wolfram , Wikström Peter , Hauptmann Jörg , Stürzebecher Jörg
Published: March 2003

Two bivalent thrombin inhibitors were synthesized, which consist of a benzamidine‐based active‐site‐blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments. BZA‐1 hirulog contains an Nα‐(2‐naphthylsulfonyl)‐S‐3‐amidinophenylalanyl‐isonipecotic acid residue connected via the carboxyl group to the linker segment. The active‐site‐directed moiety of BZA‐2 hirulog [Nα‐(2‐naphthylsulfonyl‐glutamyl)‐R‐4‐amidinophenylalanyl‐piperidide] was coupled to the linker via the side chain of the glutamic acid. Both BZA‐hirulogs contain almost identical linker‐exo site inhibitor parts, except for the substitution of a glycine as the first linker residue in BZA‐1 hirulog by a γ‐amino butyric acid in BZA‐2 hirulog, thus increasing flexibility and linker length by two additional atoms. BZA‐1 hirulog showed moderate potency (Ki = 0.50 ± 0.14 nm), while BZA‐2 hirulog was characterized as a slow, tight binding inhibitor of thrombin (Ki = 0.29 ± 0.08 pm). The stability in human plasma of both analogs was strongly improved compared with hirulog‐1. For BZA‐2 hirulog a significantly reduced plasma clearance was observed after intravenous injection in rats compared with BZA‐1 hirulog and hirulog‐1. The X‐ray structure of the BZA‐2 hirulog in complex with human α‐thrombin was solved and confirmed the expected bivalent binding mode.
Cloning, sequencing and heterologous expression of pyrogallol‐phloroglucinol transhydroxylase from Pelobacter acidigallici
Authors: Baas Dietmar , Rétey Jànos
Published: March 2003

A genomic λ‐library of Pelobacter acidigallici has been established. Proteolytic digestion of homogeneous pyrogallol‐phloroglucinol transhydroxylase from the same microorganism afforded polypeptide fragments whose N‐terminal sequences allowed the generation of oligonucleotide primers. Together with primers deduced from the known N‐terminal sequences of the two intact subunits these were used in PCR experiments to obtain three amplificates. Screening the λ‐library with the three amplificates led eventually to clones containing the whole gene coding for the transhydroxylase.

 Sequencing the gene revealed two open reading frames coding for 875 and 275 amino acids which correspond to the α‐ and β‐subunits of THL, respectively. The two subunits are separated by a 48‐bp noncoding region. Comparison of the sequence with those of other molybdopterin cofactor (MoCo)‐enzymes places THL in the dimethylsulfoxide reductase family. Possible contact sites to the MoCo and to the iron‐sulphur clusters were spotted.

 Using the expression vectors pQE 30 and pT 7‐7 three constructs harbouring the THL gene were created. One of them carried a His6‐tag at the N‐terminus of the α‐subunit, another at the C‐terminus of the β‐subunit. Immunoblot analysis showed high expression of THL, but the inclusion bodies could not be refolded to active enzyme.