bluuebunny/crossref_metadata_2025 huggingface dataset:
Heparin‐Induced Structural Modifications and Oxidative Cleavage of Human Serum Albumin in the Absence and Presence of Glucose
Authors: Finotti Paola , Pagetta Andrea
Published: July 2004

Both unfractionated and fractionated, low‐molecular‐mass heparins were tested on human serum albumin in the absence and presence of glucose at concentrations similar to those frequently found in diabetic hyperglycaemic patients, to ascertain whether heparin and glucose interfered with each other in affecting the conformation of albumin. Reproducible results were obtained with both heparins when used at equal masses, but not when used at equal molar concentrations, suggesting a crucial role of the amount of the saccharide units in determining the observed effects. Spectroscopic studies showed that the binding sites of glucose and heparin on albumin do not overlap and that changes in protein structure depend on complex and mutual interference of glucose and heparin with the protein, although the effects of heparin in modifying the chromophore environment and increasing the ordered structure of the protein also prevailed in the presence of glucose.

Heparin binding to albumin rapidly gave rise to oxidative reactions, which were responsible for the increase in the carbonyl content of the protein together with its higher susceptibility to tryptic digestion. Glucose enhanced and prolonged the production of heparin‐induced oxidants. Oxidation caused peptide bond cleavage at Lys323 in the primary structure of albumin, yielding two large fragments of 27.5 kDa and 35 kDa which aggregated to form disulphide‐linked homodimers visible in SDS/PAGE as two new bands of 54 kDa and 74 kDa, respectively. This was accompanied with a reduction in Val, Glu, and Gly residues, only partially counterbalanced by an increase in Thr and Ser residues. While only a small percentage of albumin molecules underwent fragmentation in the presence of heparin with glucose, albumin turned out to display in an even higher proportion structural modifications consistent with a higher degree of ordered structure. The mechanism(s) underlying this heparin‐driven effect and possible physio‐pathological implications in vivo are discussed.
The Bioactive Conformation of Neuropeptide Y Analogues at the Human Y2‐Receptor
Authors: Rist Beate , Ingenhoven Nikolaus , Scapozza Leonardo , Schnorrenberg Gerd , Gaida Wolfram , Wieland Heike A. , Beck‐Sickinger Annette G.
Published: July 2004

Several attempts to investigate the bioactive conformation of neuropeptide Y have been made so far. As cyclic peptides are much more rigid than linear ones, we decided to synthesise cyclic analogues of the C‐terminal dodekapeptide amide neuropeptide Y Ac‐25‐36. Cyclisation was performed by side chain lactamisation of ornithine or lysine and glutamic or aspartic acid. The affinity of the 19 peptides ranged from K1 0.6 nM to greater than 10000 nM. We found that the size, position, orientation, configuration, and the location of the cycle plays an important role for receptor recognition. Circular dichroic studies have been performed to characterise the secondary structure of each peptide. Receptor binding studies were carried out on human neuroblastoma cell lines SK‐N‐MC (Y1) and SMS‐KAN (Y2), and on rabbit kidney membranes (Y2).

The pharmacological and spectral data showed that the α‐helix content was not the predominant factor for high Y1‐receptor affinity. Instead, the location and the size of the hydrophobic lactam bridge, and the conserved C‐terminal tetrapeptide (Arg‐Glu‐Arg‐Tyr) seemed to be the main parameters.

Using molecular dynamics, the structures of four cyclic peptides (i,i+4) have been investigated and compared with the previously published NMR structure of one of the cyclic peptide analogues. Significant differences have been found in the overall three‐dimensional fold of the peptides. The distances between the N‐ and the C‐terminus allow discrimination between peptides with high binding affinity and those with low binding affinity, because of the correlation that was found with the measured affinity. Thus, this study suggests that a turn‐like structure and the orientation of the C‐terminus towards the N‐terminus play major roles for high affinity binding of cyclic dodecapeptides to the Y2‐receptor.

None of the cyclic segments exhibits significant affinity to the Y1‐receptor. Thus, these results support the hypothesis of a discontinuous binding site of neuropeptide Y at the Y1‐receptor.
Mutational Analysis of Domain II of Flavonol 3‐Sulfotransferase
Authors: Marsolais Frédéric , Varin Luc
Published: July 2004

The flavonol 3‐ and 4′‐sulfotransferases (ST) from Flaveria chloraefolia catalyze the transfer of the sulfonate group from 3′‐phosphoadenosine 5′‐phosphosulfate (PAdoPS) to position 3 of flavonol agly‐cones and position 4′ of flavonol 3‐sulfates. We identified previously a protein segment, designated domain 11, that contains all the determinants responsible for the specificity of these enzymes. Within domain 11, at least five amino acids specific to the 4′‐ST that could bind the sulfate group of quercetin 3‐sulfate were identified. In this study, these amino acid residues were introduced at equivalent positions in the flavonol 3‐ST sequence by site‐directed mutagenesis of the cloned cDNA. No reversal of the substrate specificity was observed after the individual mutations. However, mutation of Leu95 to Tyr had different effects on the kinetic constants depending on the substitution pattern of the flavonoid B ring, suggesting that the tyrosine side chain may be in direct contact with this part of the molecule. The function of conserved amino acids present in domain II was also investigated. Unconservative mutations at Lys134, Tyr137 and Tyr150 resulted in protein instability in solution, suggesting that these residues might be important for the structural stability of the enzyme. Replacement of Arg140 with Lys or Ser had no effect on protein stability, but resulted in a strong reduction in specific activity. The results of photoaffinity‐labeling experiments with PAdoP[35S]S suggest that this residue is required to bind the cosubstrate. In addition, the reduced affinity of [Ser140]ST for 3′‐phosphoadenosine 5′‐phosphate (PAdoP)‐agarose indicates that Arg140 is also involved in binding the coproduct. Replacement of His118 with Glu or Ala resulted in a strong reduction in catalytic activity. However, [Lys118]ST retained a significant amount of catalytic activity. The results of photoaffinity‐labeling experiments with PAdoP[35S]S and affinity chromatography on PAdoP‐agarose suggest that His118 might be involved in catalysis in the flavonol 3‐ST.
The Adenylation Domain of Tyrocidine Synthetase 1
Authors: Dieckmann Ralf , Pavela‐Vrancic Maja , Pfeifer Eva , Von Döhren Hans , Kleinkauf Horst
Published: July 2004

Sequence analysis of peptide synthetases revealed extensive structure similarity with firefly luciferase, whose crystal structure has recently become available, providing evidence for the localization of the active site at the interface between two subdomains separated by a distorted linker region [Conti, E., Franks, N. P. & Brick, P. (1996) Structure 4, 287–2981. The functional importance of two flexible loops, corresponding to the linker region of firefly luciferase and the highly conserved (S/T)GT(T/S)GXPKG core sequence, has been studied in view of the proposed conformational changes by the use of mutant analysis, limited proteolysis and chemical modification of tyrocidine synthetase 1. Substitution of the highly conserved Arg416, residing in the loop separating the subdomains of the adenylation domain, resulted in profound loss of activity. Limited proteolysis of the mutant suggested significant structural changes as manifested by lack of protection to degradation in the presence of substrates, revealing a probable disturbance of the induced‐fit mechanism regulating the transformation from an open to a closed conformation. Mutants, obtained by replacement of the conserved Lys186 from the (S/T)GT(T/S)GXPKG core sequence, displayed only minor differences in substrate‐binding affinity despite significant reduction of catalytic efficiency. Residue Lys186 appears to play an important role in either stabilization of the bound substrate through charge‐charge‐interactions, and/or fixing of the loop for maintainance of the active‐site conformation.
All‐Transglycolytic Synthesis and Characterization of sialyl(α2–3)galactosyl(β1–4)xylosyl‐p‐nitrophenyl(β1–, an Oligosaccharide Derivative Related to Glycosaminoglycan Biosynthesis
Authors: Vetere Amedeo , Ferro Salvatore , Bosco Marco , Cescutti Paola , Paoletti Sergio
Published: July 2004

β‐d‐Xylopyranosides, such as p‐nitrophenyl‐β‐d‐xylopyranoside (Xyl‐Np) or 4‐methylumbelliferyl‐β‐d‐xylopyranoside (Xyl‐MeUmb), when added to the culture medium of human skin fibroblasts have previously been shown to produce some Np‐ or MeUmb‐oligosaccharides related to the regulation of glycosaminoglycan biosynthesis. Among these oligosaccharide derivatives, we synthesized the trisaccha‐ride derivative NeuAc(α2–3)Gal(β1–4)Xyl‐Np(β1– as a potential inhibitor of human skin fibroblast glycosaminoglycan biosynthesis. This synthesis was achieved by sequential use of transglycosylating activities of Escherichia coliβ‐galactosidase and Tvpanosornu cruzi trans‐sialidase. The structure of the oligosaccharide obtained was determined by HPLC, ion‐spray mass spectrometry, and NMR.
The Subunit F of Mitochondrial Yeast ATP Synthase
Authors: Spannagel Christelle , Vaillier Jacques , Arselin Geneviève , Graves Pierre‐Vincent , Velours Jean
Published: July 2004

The subunit f of the yeast F1 F0 ATP synthase has been isolated from the purified enzyme. Amino acid composition, protein and peptide sequencing were performed. The data are in agreement with the sequence of the predicted product of the gene D9481.21 identified on the Saccharomyces cerevisiae chromosome IV. A 303‐bp open reading frame encoding a 101‐amino acid polypeptide is described. The deduced amino acid sequence from the ATP 17 gene is 6 amino acids longer than the mature protein, which displays a molecular mass of 10567 Da. The protein is basic with a short hydrophobic segment located in the C‐terminal part of the subunit. Subunit f remained associated with other F0 subunits upon sodium bromide treatment of the whole enzyme. A null mutant was constructed. The disrupted strain was unable to grow on glycerol medium and the mutation was recessive; rho− cells arose spontaneously. The null mutant mitochondria were devoid of oligomycin‐sensitive ATPase, but still contained an active F1 while the subunits f, 6 and 8 were absent.
1H‐NMR‐Derived Secondary Structure and Overall Fold of a Natural Anatoxin from the Scorpion Androctonus Australis Hector
Authors: Blanc Eric , Hassani Oussama , Meunier Sylvie , Mansuelle Pascal , Sampieri Francois , Rochat Hervé , Darbon Hervé
Published: July 2004

The venom of the scorpion Androctonus australis hector contains several protein neurotoxins of which structure and structure/activity relationships have been extensively studied. It also contains polypeptides such as Aah STR1, which are not toxic, while having highly similar sequences to fully active toxins. We have determined the solution structure of Aah STR1 by use of conventional two‐dimensional NMR techniques followed by distance‐geometry and energy minimization. We have demonstrated that, despite its lack of toxicity, Aah STR1 is structurally highly related to anti‐mammal scorpion toxins specific for Na′ channels. The calculated structure is composed of a short α‐helix (residues 26–33) connected by a tight turn to a three‐stranded antiparallel β‐sheet (sequences 3–6, 38–41 and 44–48). This β‐sheet is right‐handed twisted as usual for such secondary structures. The β‐turn connecting the strands 38–41 and 44–48 belongs to type II′. The overall fold of Aah STR1 is typical of β‐type scorpion toxins. This is, however, the first example of such a fold in Old World scorpion toxins. Either the absence of a basic residue in position 63 or the high mobility of loops, compared to active β‐type neurotoxins, may explain the lack of activity of this protein.
Characterization of Two F‐Actin‐Binding and Oligornerization Sites in the Cell‐Contact Protein Vinculin
Authors: Hüttelmaier Stefan , Bubeck Peter , Rüdiger Manfred , Jockusch Brigitte M.
Published: July 2004

Vinculin, a structural protein of animal cells, is critically involved in the assembly of microfilament/plasma membrane junctions at cell contacts. To understand its role in organizing the distal portions of microfilaments into specific, morphologically distinct structures at these sites in more detail, we characterized its interaction with filamentous actin and with itself by means of in vitro assays. Using recombinant proteins comprising different parts of the vinculin tail fused to the maltose‐binding protein of Escherichia coli, we show in sedimentation assays that this part of vinculin harbors two discrete sites that can bind to actin independently. They reside within amino acid residues 893–985 and 1016–1066 of the 1066‐residue polypeptide chain. However, both sites are necessary to cross‐link or bundle actin filaments, as demonstrated by low shear viscometry. Crosslinking and bundling are alternatives determined by the molar ratio of fusion protein to F‐actin. Both actin‐binding sequences are capable of oligomer formation, as shown in chemical‐cross‐linking and dot‐overlay assays. These data allow us to propose a possible role for vinculin in organizing the distal ends of microfilaments at the plasma membrane into the point‐like structure characteristic for cell‐matrix contacts.
Selective Stimulation of a CAMP‐Specific Phosphodiesterase (PDE4A5) Isoform by Phosphatidic Acid Molecular Species Endogenously Formed in Rat Thymocytes
Authors: El Bawab Samer , Macovschi Olga , Sette Claudio , Conti Marco , Lagarde Michel , Nemoz Georges , Prigent Annie‐France
Published: July 2004

We have previously reported that concanavalin A (ConA) stimulation of rat thymocytes induces an increase in the cellular phosphatidic acid mass as well as a change in its fatty acid composition. An increase in phosphodiesterase (PDE) activity, mostly due to CAMP‐specific (PDE4) isoforms, has also been observed in thymocytes stimulated by ConA. Furthermore, phosphatidic acid was able to stimulate PDE4 activity in vitro. In the present study, cAMP levels have been shown to decrease upon ConA stimulation of thymocytes. Decreasing phosphatidic acid level using diacylglycerol kinase inhibitors induced a parallel decrease of the ConA‐stimulated CAMP‐specific PDE activity in these cells. Analyses of phosphatidic acid molecular species in cells stimulated for 5 min by ConA revealed a significant increase in l‐stearoyl‐2‐arachidonoyl‐sn‐glycerol‐3‐phosphate and a relative decrease in the other molecular species of phosphatidic acid, mainly species containing palmitate. On the other hand, phosphatidic acid extracted from ConA‐stimulated cells activated more efficiently the recombinant PDE4A5 isoform in vitro, as compared to phosphatidic acid extracted from unstimulated cells. In addition, phosphatidic acid species containing unsaturated fatty acids were stimulatory, while those containing two saturated fatty acids had only a marginal effect on the enzyme activity. Taken together, these data suggest that the mitogenic stimulation of thymocytes is accompanied by the synthesis of peculiar phosphatidic acid molecular species able to activate a PDE4 isoform. This activation might be of physiological relevance since cAMP is a major negative effector of the mitogenic response.
The Regulatory Functions of the γ and ɛ Subunits from Chloroplast CF1 are Transferred to the Core Complex, α3β3, from Thermophilic Bacterial F1
Authors: Hisabori Toru , Kato Yasuyuki , Motohashi Ken , Kroth‐Pancic Peter , Strotmann Heinrich , Amano Toyoki
Published: July 2004

The expression plasmids for the subunit γ (γc) and the subunit ɛ (ɛc) of chloroplast coupling factor (CF1) from spinach were constructed, and the desired proteins were expressed in Escherichia coli. Both expressed subunits were obtained as inclusion bodies. When recombinant γc was mixed with recombinant α and β subunits of F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (α3β3γc) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCI2, which induces formation of a disulfide bond between two cysteine residues in γc. Hence, this complex has similar modulation characteristics as CF1.

The effects of recombinant ɛc, and ɛ subunit from TF1 (ɛ1) on α3β3γc were also investigated. ɛc, strongly inhibited the ATP hydrolysis activity of chimeric α3β3γc complex but ɛ1 did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium. The addition of ɛc, or ɛ1, to the α3β3γt complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity.

These results suggest that (a) the specific regulatory function of γc can be transferred to the bacterial subunit complex; (b) the interaction between the γc subunit and ɛc, strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the α3β3 core.
ADP‐Binding and ATP‐Binding Sites in Native and Proteinase‐K‐Digested Creative Kinase, Probed by Reaction‐Induced Difference Infrared Spectroscopy
Authors: Raimbault Cyrille , Clottes Eric , Leydier Chantal , Vial Christian , Buchet René
Published: July 2004

Conformational changes induced by nucleotide binding to native creatine kinase (CK) from rabbit muscle and to proteinase‐K‐digested (nicked) CK, were investigated by infrared spectroscopy. Photochemical release of ATP from ATP[Et(PhNO2)] in the presence of creatine and native CK produced reaction‐induced difference infrared spectra (RIDS) of CK related to structural changes of the enzyme that paralleled the reversible phosphoryl transfer from ATP to creatine. Similarly the photochemical release of ADP from ADP[Et(PhNO2)] in the presence of phosphocreatine and native CK allowed us to follow the backward reaction and its corresponding RIDS. Infrared spectra of native CK indicated that carboxylate groups of Asp or Glu, and some carbonyl groups of the peptide backbone are involved in the enzymatic reaction. Native and proteinase nicked CK have similar Stokes' radii, tryptophan fluorescence, fluorescence fraction accessible to iodide, and far‐ultraviolet CD spectra, indicating that native and modified enzymes have the same quaternary structures. However, infrared data showed that the binding site of the γ‐phosphate group of the nucleotide was affected in nicked CK compared with that of the native CK. Furthermore, the infrared absorptions associated with ionized carboxylate groups of Asp or Glu amino acid residues were different in nicked CK and in native CK.
Structural and Functional Consequences of Mutations within the Hydrophobic Cores of the HMG1‐Box Domain of the Chironomus High‐Mobility‐Group Protein 1a
Authors: Wiśniewski Jacek R. , Hessler Katja , Claus Peter , Zechei Kasper
Published: July 2004

The high‐mobility‐group protein 1 box domain (HMGI‐BD) is a structural element found in several DNA‐binding proteins in eukaryotic cells. Its structure is dominated by three α‐helices. The spatial arrangement of these helices into an L‐shaped molecule is maintained by a number of apolar residues organized into a main and a secondary hydrophobic core. To analyze the significance of these residues for proper folding, conformational stability, and ability to bind and bend DNA, we have mutated the highly conserved Trpl4 of the Chironomus HMG1 a protein and have synthesized a series of N‐terminally truncated forms. The observed alterations in DNA‐binding and DNA‐bending characteristics were correlated with structural consequences, as revealed by CD spectroscopy, limited trypsin digestion, and transverse urea gradient gel electrophoresis. Mutation of the Trp14 residue (Chironomus [W14A]HMG1a) and deletion of the seven N‐terminal residues, respectively, which are members of the main and the secondary core of Chironomus HMG1a, both resulted in a substantial unfolding of the protein. Unexpectedly, these mutants still retained their ability to bind and bend DNA. Conformational analysis of wild‐type cHMG1a and [W14A]cHMG1a showed that the proteins unfold at 2‐4 M urea. In contrast, their DNA complexes persisted even at 6–8 M of the denaturant. Multiple contacts between the HMG1‐BD and the DNA are probably responsible for the unusual stability of the complexes.
The Surface‐Dependent Autoactivation Mechanism of Factor XII
Authors: Røjkjær Rasmus , Schousboe Inger
Published: July 2004

The dependency of concentrations of Zn2+ and the negatively charged surfaces, phosphatidylinositol phosphate (PtdInsP), sulfatide and dextran sulfate, on the autoactivation of human factor XII, has been studied. While the autoactivation induced by sulfatide, and low concentrations of dextran sulfate, was unaffected by the presence of Zn2+, that induced by PtdInsP and higher concentrations of dextran sulfate was completely dependent on Zn2+: the excess of Zn2+ needed to induce maximal activity with PtdInsP was 12‐fold the concentration of factor XII, while with dextran sulfate it was 40‐fold. Determination of the Zn2+‐binding properties of factor XII revealed that a total of four zinc ions could bind to each factor XII molecule. The first bound zinc ions (Kd 0.1 μM) induced an increase in the intrinsic tryptophan fluorescence of factor XII, while further titration up to a 40‐fold surplus resulted in a quenching of the fluorescence. Binding of the zinc ions that caused the quenching had an average Kd of approximately 1 μM, independent of whether it was determined from the fluorescence changes or by equilibrium filtration. Low concentrations of both sulfatide and PtdInsP induced a fluorescence increase similar to that at low concentrations of Zn2+ but, in contrast to sulfatide, higher concentrations of PtdInsP did not induce a quenching in fluorescence. As the Zn2+‐independent activating surface (sulfatide) induced quenching in the fluorescence intensity, while the Zn2+‐dependent activating surface (PtdInsP) did not, the quenching, whether it was caused by sulfatide or zinc ions, was assigned to a change in the conformation which resulted in a molecular structure of factor XII that could be autoactivated. Association of factor XII in this conformation on the activating surface was suggested to be responsible for the autoactivation.
Theoretical Approaches to the Evolutionary Optimization of Glycolysis
Authors: Helnrlch Reinhart , Montero Francisco , Klipp Edda , Waddell Thomas G. , Meléndez‐Hevia Enrique
Published: July 2004

It is analyzed whether the structural design of contemporary glycolysis can be explained theoretically on the basis of optimization principles originating from natural selection during evolution. Particular attention is paid to the problem of how the kinetic and thermodynamic properties of the glycolytic pathway are related to its stoichiometry with respect to the number and location of ATP‐coupling sites. The mathematical analysis of a minimal model of unbranched energy‐converting pathways shows that the requirement of high ATP‐production rate favours a structural design that includes not only ATP‐producing reactions (P‐sites) but also ATP‐consuming reactions (C‐sites). It is demonstrated that, at fixed overall thermodynamic properties of a chain, the ATP‐production rate may be enhanced by kinetic optimization. The ATP‐production rate is increased if the C‐sites are concentrated at the beginning and all the P‐sites at the end of the pathway. An optimum is attained, which is characterized by numbers of coupling sites corresponding to those found in glycolysis. Various extensions of the minimal model are considered, which allow the effects of internal feedback‐regulations, variable enzyme concentrations, and the symmetric branching of glycolysis at the aldolase step to be considered.
Processing of Thionin Precursors in Barley Leaves by a Vacuolar Proteinase
Authors: Romero Alicia , Alamillo Jocefa M. , García‐Olmedo Francisco
Published: July 2004

Thionins are synthesized as precursors with a signal peptide and a long C‐terminal acidic peptide that is post‐translationally processed. A fusion protein including the maltose‐binding protein from Eschrrichia coli (MalE), thionin DG3 froin barley leaves, and its acidic C‐terminal peptide has been used to obtain antibodies that recognize both domains of the precursor. In barley leaf sections. mature thionins accuinulated in the vacuolar content, while the acidic peptide was not detected in any cell fraction. Brefeldin A and inonensin inhibited processing of the precursor but its export from the microsomal fraction was not inhibited. Both purified vacuoles aiid an acid (pH 5.5) extract from leaves processed the fusion protein into a MalE‐thionin and an acidic peptide fragment. A 70‐kDa proteinase that effected this cleavage was purified froin the acid extract. Processing of the fusion protein by both lysed vacuoles and the purified proteinase was inhibited by Zn2+ and by Cu2+, but not by inhibitors of the previously described vacuolar processing thiol or aspartic proteinases. In vivo processing of the thionin precursor in leaf sections was also inhibited by Zn+, and Cu2+, Variants of the fusion protein with altered processing sites that represented thme of thionin precursors from different taxa were readily processed by the proteinase, whereas changing the polarity of either the C‐terminal or N‐terminal residues of the processing site prevented cleavage by the proteinase.
Identification and Structure of Activated‐Platelet Protein‐1, a Protein with RNA‐Binding Domain Motifs that is Expressed by Activated Platelets
Authors: Houng Aiilyan K. , Maggini Laura , Clement Chris Y. , Reed Guy L.
Published: July 2004

Beyond their critical role in thrombosis, platelets perform important functions in vascular remodeling, inflammation, and wound repair. Many of these functions are executed by molecules expressed by activated platelets. A novel molecule, activated‐platelet protein‐1 (APP‐1), was identified by a monoclonal antibody against activated rabbit platelets. When platelets were stimulated by thrombin, A23187 or ADP, APP‐I was expressed on the platelet surface. APP‐1 was also detected in whole cell lysates of platelets, but not on the external surfaces of resting platelets. With maximal activation by thrombin, 15900±2800 molecules APP‐1 were expressed/platelet. A 2.3‐kb cDNA fragment containing a partial coding sequence for APP‐1 was isolated from a rabbit bone marrow library by expression cloning with the anti‐APP‐l monoclonal antibody. When expressed as a recombinant fusion protein in bacteria, APP‐1 bound specifically to poly(A)‐Sepharose. The full‐length cDNA coding for human APP‐1, obtained by DNA hybridization techniques, showed 98.7% amino acid sequence identity with the rabbit protein. Northern analysis with human APP‐1 identified a 3.7‐kb mRNA transcript in megakaryocytic lines that express transcripts for platelet proteins. Human APP‐1 has four ribonucleotide binding domains with ribonucleoprotein 1 and 2 motifs. By virtue of its ribonucleotide binding domains, APP‐1 is structurally related to polyadenylate‐binding protein, which regulates translation initiation and polyadenylate shortening, and to nucleolysin, a specific effector molecule found in the granules of cytotoxic T lymphocytes.
Active Efflux of the Free Acid form of the Fluorescent Dye 2′,7′‐Bis(2‐Carboxyethyl)‐5(6)‐Carboxyfluorescein in Multidrug‐Resistance‐Protein‐Overexpressing Murine and Human Leukemia Cells
Authors: Draper Michael P. , Martell Robin L. , Levy Stuart B.
Published: July 2004

Murine and human cell lines overexpressing the multidrug‐resistance protein (MRP) showed a marked decreased accumulation of the fluorescent dye 2′,7′‐bis(2‐carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF). In contrast, less altered accumulation was seen in the P‐glycoprotein(P‐gp)‐overexpressing cell lines. The decreased drug accumulation was reversed by the energy inhibitors sodium azideI2‐deoxyglu‐cose and by the vinca alkaloid, vincristine, but not by the chemotherapeutic agents, etoposide and adriamycin. Decreased accumulation was linked to active efflux of the hydrophilic free acid form of BCECF from the MRP‐overexpressing cell lines, indicating that dye extrusion occurs after the dye ester has been converted to the free acid form in the cytoplasm. The finding suggests that MRP mediates removal of substrates from a cytoplasmic location. Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, decreased the vincristine and etoposide resistance displayed by the MRP‐expressing murine cell lines, but did not affect the accumulation of BCECF. Thus, while glutathione may be involved in MRP‐mediated resistance to some chemotherapeutic agents, it is not necessary for efflux of substrates such as BCECF.
The Phosphatidylinositol 3‐Kinase Inhibitors Wortmannin and LY294002 Inhibit Autophagy in Isolated Rat Hepatocytes
Authors: Blommaart Edward F. C. , Krause Ulrike , Schellens Jacques P. M. , Vreeling‐Sindelárová Heleen , Meijer Alfred J.
Published: July 2004

Recent studies indicate that phosphatidylinositol 3‐kinase is essential in the regulation of many processes dependent on membrane flow. Autophagy is a complex pathway in which cell material, including proteins, can be degraded. Membrane flow plays a pivotal role in this process. To find out whether phosphatidylinositol 3‐kinase is also required for autophagy, we tested the effects on autophagy of two structurally unrelated phosphatidylinositol 3‐kinase inhibitors, wortmannin and 2‐(4‐morpholinyl)‐8‐phe‐nylchromone (LY294002).

The addition of low concentrations of each of these inhibitors to incubations of hepatocytes in the absence of amino acids resulted in a strong inhibition of proteolysis. The antiproteolytic effect of wortmannin (IC50, 30 nM) and LY294002 (IC50 10 μM) was accompanied by inhibition of autophagic sequestration and not by an increase in lysosomal pH or a decrease in intracellular ATP. No further inhibition of proteolysis by the two compounds was observed when autophagy was already maximally inhibited by high concentrations of amino acids.

3‐Methyladenine, which is commonly used as a specific inhibitor of autophagic sequestration, was an inhibitor of phosphatidylinositol 3‐kinase, thus providing a target for its action.

It is proposed that phosphatidylinositol 3‐kinase activity is required for autophagy. 3‐Methyladenine inhibits autophagy by inhibition of this enzyme.
Aspartyl‐tRNA Synthetase from Rat
Authors: Agou Fabrice , Mirande Marc
Published: July 2004

In mammalian cells, nine aminoacyl‐tRNA synthetases, including aspartyl‐tRNA synthetase, are associated within a multienzyme complex. Rat aspartyl‐tRNA synthetase has a N‐terminal polypeptide extension of about 40 amino acid residues which can be removed without impairing its catalytic activity. Earlier, in vivo studies showed that enzymes deprive of this N‐terminal segment behave in vivo as free entities. We designed an experimental in vitro approach, based on the exchange of the complexed endogenous enzyme by free recombinant species, to assess the contribution of that domain in the association of aspartyl‐tRNA synthetase to the complex. A phosphorylation site was introduced at the N‐terminus of rat aspartyl‐tRNA synthetase. The enzyme served as a reporter protein to evaluate the dissociation constants of native and N‐terminal‐truncated species towards the complex. Our data show that a moderate but significant drop in affinity is inferred by the removal of the N‐terminal domain. The results suggest that this domain binds to another component of the complex, but might primarily serve a targeting function absolutely required in vivo for the assembly within the multienzyme structure.
The Interactions with Solvent, Heat Stability, and 13C‐Labelling of Alamethicin, an Ion‐Channel‐Forming Peptide
Authors: Yee Adelinda A. , Marat Kirk , O'neil Joe D. J.
Published: July 2004

The peptide alamethicin was labelled with 13C and 15N by growing the fungus Trichoderma viride in a medium containing [U‐13C]glucose and K15NO3, Spin‐echo difference spectroscopy showed that13C was incorporated to a level of about 50% and15N to about 98%. Incorporation of 13C into the peptide provided residue‐specific probes of the interactions with solvent and heat stability of this ion‐channel‐forming peptide. All of the carbonyl carbons and the α‐carbons of the α‐aminoisobutyric acid [Ala(Me)J residues of alamethicin in methanol were assigned using two‐dimensional and three‐dimensional hetero‐nuclear correlation experiments. Measurements of 1Jc,N revealed hydrogen bonding with solvent at residues 1 and 19 at the ends of the peptide and at Glyl1 in the middle. The data also support the thesis [see Juranic, N., Ilich, P. K. & Macara, S. (1995) J. Am. Chem. Soc. 117, 405–4101 that intramolecular hydrogen bonds in proteins and peptides are weaker than hydrogen bonds to solvent. The sensitivity of alamethicin carbonyl and proton chemical shifts to perturbation by dimethyl sulfoxide correlates well with the calculated solvent accessibilities of the carbonyls in the crystal structures and reveals residues in the middle of the peptide and at the C‐terminus which interact with solvent. Taken together with the 1Jc, measurements, the data support a model in which hydrogen bonding to solvent at the Gly1l/Leu12 amide could provide a site of hydration in the interior of the alamethicin channel structure. The temperature dependencies of the carbonyl chemical shifts support the suggestion that the peptide is flexible in the regions where solvent interacts with the backbone of the peptide. The linear temperature dependence of the carbonyl chemical shifts and molar ellipticity indicate that, due to steric constraints at the Ala(Me) residues, the peptide folding/folding transition is non‐cooperative and that the peptide is remarkably heat stable.
The Fructose Transporter of Bacillus subtilis Encoded by the Lev Operon Backbone Assignment and Secondary Structure of the IIBLev Subunit
Authors: Seip Stephan , Lanz Regina , Gutknecht Regula , Flükiger Karin , Erni Bernhard
Published: July 2004

The fructose transporter of the Bacillus subtilis phosphotransferase system consists of two membrane associated (IIA and IIB) and two transmembrane (IIC and IID) subunits [Martin‐Verstraete, I., Débarbouillé, M., Klier, A. & Rapoport, G. (1990) J. Mol. Biol. 214, 657–671]. It mediates uptake by a mechanism which couples translocation to phosphorylation of the transported solute. The 18‐kDa IIBLev subunit transfers phosphoryl groups from His9 of the IIA subunit to the sugar. The three‐dimensional structure of IIBLev or similar proteins is not known. IIBLev was overexpressed in Escherichia coli and isotopically labelled with 13C/15N in H2O as well as in 70% D2O. 15N‐edited NOESY, 13C‐edited NOESY and 13C,15N triple‐resonance experiments yielded a nearly complete assignment of the 1H, 13C and 15N resonances. Based on qualitative interpretation of NOE, scalar couplings, chemical shift values and amide exchange data, the secondary structure and topology of IIBLev was determined. IIBLev comprises six parallel β‐strands, one antiparallel β‐strand and 5 α‐helices. The order of the major secondary‐structure elements is (βα)5β (strand order 7651423). Assuming that the βαβ‐motives form right‐handed turn structures, helices αA and αB are packed to one face and helices αC, αD and αE to the opposite face of the parallel βsheet. His15 which is transiently phosphorylated during catalysis is located in the loop β1/αA of the topological switch point. The amino terminal (β/α)4 part of IIBLev has the same topology as phosphoglyceromutase (PGM; PDB entry 3pgm). Both proteins catalyze phosphoryltransfer reactions which proceed through phosphohistidine intermediates and they show a similar distribution of invariant residues in the topologically equivalent positions of their active sites. The protein fold of IIBLev has no similarity to any of the known structures of other phosphoenol pyruvate‐dependent‐carbohydrate‐phosphotransferase‐system proteins.
Purification and Characterization of an Endo‐1,3‐β‐Glucanase from Aspergillus fumigatus
Authors: Fontaine Thierry , Hartland Robbert P. , Beauvais Anne , Diaquin Michel , Latge Jean‐Paul
Published: July 2004

An endo‐1,3‐β‐glucanase was purified from a cell wall autolysate of Aspergillus fumigatus. This β‐glucanase activity was associated with a glycosylated 74‐kDa protein. Using a sensitive colorimetric assay and a high‐performance anion‐exchange chromatography with a pulsed electrochemical detector for product analysis, it was shown that the endoglucanase hydrolysed exclusively linear 1,3‐β‐glucan chains, had an optimum pH of 7.0 and an optimum temperature of 60°C. A substrate kinetic study gave a Km value of 0.3 mg/ml for soluble (laminarin and laminari‐oligosaccharides) and 1.18 mg/ml for insoluble (curdlan) 1,3‐β‐glucan. Laminar‐oligosaccharide degradation, analysed by HPLC, showed that the endoglucanase bind to the subtrate at several positions and suggested that the active site of the enzyme recognized five glucose units linked by a 1,3‐β bond. The association of the present endo‐1,3‐β‐glucanase with the cell wall of A. fumigatus suggests a putative role for this enzyme during cell‐wall morphogenesis.
Imidase, a Dihydropyrimidinase‐Like Enzyme Involved in the Metabolism of Cyclic Imides
Authors: Ogawa Jun , Soong Chee‐Leong , Honda Michinari , Shlmizu Sakayu
Published: July 2004

Imidase, which preferably hydrolyzed cyclic imides to monoamidated dicarboxylates, was purified to homogeneity from a cell‐free extract of Blastobacter sp. A17p‐4. Cyclic imides are known to be hydrolyzed by mammalian dihydropyrimidinases. However, imidase was quite different from known dihy‐dropyrimidinases in structure and substrate specificity. The enzyme has a relative molecular mass of 105000 and consists of three identical subunits. The purified enzyme showed higher activity and affinity toward cyclic imides, such as succinimide (Km= 0.94 mM; Vmax= 910 μmol · min‐1· mg‐1) glutarimide (Km= 4.5 mM; Vmax= 1000 μmol · min‐1· mg‐1) and maleimide (Km= 0.34 mM; Vmax= 5800 μmol · min‐1· mg‐1), than toward cyclic ureides, which are the substrates of dihydropyrimidinases, such as dihydrouracil and hydantoin. Sulfur‐containing cyclic imides, such as 2,4‐thiazolidinedione and rhodanine, were also hydrolyzed. The enzyme catalyzed the reverse reaction, cyclization, but with much lower activity and affinity. The enzyme was non‐competitively inhibited by succinate, which was found to be a key compound in cyclic‐imide transformation in relation with the tricarboxylic acid cycle in this bacterium, suggesting that the role of imidase is to catalyze the initial step of cyclic‐imide degradation.
Turnover Number of Escherichia coli F0F1 ATP Synthase for ATP Synthesis in Membrane Vesicles
Authors: Etzold Carsten , Deckers‐Hebestreit Gabriele , Altendorf Karlheinz
Published: July 2004

The rate of ATP synthesized by the ATP synthase (F0F1‐ATPase) is limited by the rate of energy production via the respiratory chain, when measured in everted membrane vesicles of an Escherichia coli atp wild‐type strain. After energization of the membranes with NADH, fractional inactivation of F0F1 by the covalent inhibitor N,N′‐dicyclohexylcarbodiimide allowed the rate of ATP synthesis/mol remaining active ATP synthase complexes to increase; the active ATP synthase complexes were calculated using ATP hydrolysis rates as the defining parameter. In addition, variation of the assay temperature revealed an increase of the ATP synthesis rate up to a temperature of 37°C, the optimal growth temperature of E. coli. In parallel, the amount of F0F1 complexes present in membrane vesicles was determined by immnnoquantitation to be 3.3±0.3% of the membrane protein for cells grown in rich medium and 6.6±0.3% for cells grown in minimal medium with glycerol as sole carbon and energy source. Based on these data, a turnover number for ATP synthesis of 270±540 s‐1 could be determined in the presence of 58 active F0F1 complexes. Therefore, these studies demonstrate that the ATP synthase complex of E. coli has, with respect to maximum rates, the same capacity as the corresponding enzymes of eukaryotic organells.
Characterization of Two Vaccinia CD36 Recombinant‐Virus‐Generated Monoclonal Antibodies (10/5, 13/10)
Authors: Daviet Laurent , Craig Alister G. , Mcgregor Lilian , Pinches Robert , Wild T. Fabian , Berendt Anthony R. , Newbold Christopher I. , Mcgregor John L.
Published: July 2004

Extensive evidence is now available to show that the human CD36 antigen is a cellular receptor for thrombospondin, collagen, modified low‐density lipoproteins, and long‐chain fatty acids. Moreover, CD36 functions as one of the receptors that mediates the adhesion of Plasmodium‐fulciparum‐infected erythro‐cytes to microvascular endothelium. In an attempt to identify new functional sites of this surface glycoprotein, anti‐CD36 monoclonal antibodies were prepared using a vaccinia CD36 recombinant virus as a highly efficient immunization vector. In functional studies, one of these antibodies (clone 10/5) strongly inhibited the adhesion of P. filciparum‐infected erythrocytes to purified CD36. This antibody also potentiated ADP‐induced platelet activation. In contrast, a second antibody (clone 13/10) did not affect the cytoadherence of infected erythrocytes or platelet functions. Previous structural work performed on these antibodies has shown that clone 10/5 is directed against an epitope within the CD36 domain 155–183, whereas clone 13/10 interacts with another antigenic determinant defined by amino acids 30‐76 [Daviet, L., Buckland, R., Puente Navazo, M. D. & McGregor, J. L. (1995) Biochem. J. 305, 221‐2241, Taken together, these current studies show that: (a) the methodology of immunization using recombinant vaccinia virus is a powerful tool in the generation of monoclonal antibodies directed against polyimmunogenic membrane glycoproteins such as CD36; (b) the CD36 domain, recognized by clone 1015 but not by 13110, is functionnally important regarding the adhesion of P. falciparum‐infected erythrocyte and CD36‐dependent platelet activation.
The Wheat Poly (A)‐Binding Protein Functionally Complements Pab1 in Yeast
Authors: Le Hanh , Chang Su‐Chih , Tanguay Robert L. , Gallie Daniel R.
Published: July 2004

Poly(A)‐binding protein (PAB) binds to the poly(A) tail of most eukaryotic mRNAs and influences its translational efficiency as well as its stability. Although the primary structure of PAB is well conserved in eukaryotes, its functional conservation across species has not been extensively investigated. In order to determine whether PAB from a monocot plant species could function in yeast, a protein characterized as having PAB activity was purified from wheat and a cDNA encoding for PAB was isolated from a wheat seedling expression library. Wheat PAB (72 kDa as estimated by SDSPAGE and a theoretical mass of 70823 Da as determined from the cDNA) was present in multiple isoforms and exhibited binding characteristics similar to that determined for yeast PAB. Comparison of the wheat PAB protein sequence with PABs from yeast and other species revealed that wheat PAB contained the characteristic features of all PABs, including four RNA binding domains each of which contained the conserved RNPI and RNP2 sequence motifs. The wheat PAB cDNA functionally complemented a pab1 mutant in yeast suggesting that, although the amino acid sequence of wheat PAB is only 47% conserved from that of yeast PAB, this monocot protein can function in yeast.
Cooperative Amplification of Templates by Cross‐Hybridization (CATCH)
Authors: Ehricht Ralf , Ellinger Thomas , Mcaskill John S.
Published: July 2004

In vitro amplification systems not only serve as a tool for the processing of DNA, but have also provided important model systems for the investigation of fundamental issues in evolutionary optimization. In this work we present a coupled amplification system based on the self‐sustained sequence replication (3SR), also known as nucleic acid sequence‐based amplification (NASBA), which allows the experimental investigation of evolving molecular cooperation. The 3SR reaction is an isothermal method of nucleic acid amplification and an alternative to PCR. A target nucleic acid sequence can be amplified exponentially in vitro using two enzymes: reverse transcriptase (RT) and a DNA‐dependent RNA poly‐merase (RNAP). A system has been constructed in which amplification of two molecular species is cooperatively coupled. These species are single‐stranded (ss)DNA templates (D1 and D2) of lengths 58 and 68 nucleotides, respectively. Coupling occurs when D1 and D2 anneal to each other via a complementary region (DB and DB') situated at the 3′ end of each template. RT elongates the hybridized templates producing a double‐stranded (ds)DNA of 106 base pairs (bp). This double strand contains two promoters, which are situated on either side of, and directly adjacent to DB, and which are oriented towards each other. These promoters specify two RNA transcripts encompassing, respectively, the D1 and D2 portion of the dsDNA. After hybridization of two primers (P1 and P2) to the transcripts (R1 and R2) and reverse transcription, the ss templates D1 and D2 are regenerated. Amplification cycles of D1 and D2 are coupled cooperatively via the common dsDNA intermediate. Under optimized batch conditions the system shows the expected growth phases: exponential, linear and saturation phase. The enzymes of the 3SR cycle tend to misincorporate nucleotides and to produce abortive products. In future experiments, we intend to use the system for studies of evolutionary processes in spatially distributed systems where new strategies for optimization at the molecular level are possible.
Recombinant [Pheβ63]Hemoglobin Shows Rapid Oxidation of the β Chains and Low‐Affinity, Non‐Cooperative Oxygen Binding to the α Subunits
Authors: Kiger Laurent , Baudin Véronique , Desbois Alain , Pagnier Josée , Kister Jean , Griffon Nathalie , Henry Yann , Poyart Claude , Marden Michael C.
Published: July 2004

We have engineered αβ2 [Phe63]hemoglobin by changing the highly conserved distal histidine of the β chains to a phenylalanine. The mutant tetramer binds four high‐affinity ligands, such as CO or NO, to the ferrous form, or CN to the oxidized iron; however, it binds only two low‐affinity ligands, oxygen and azide. The absorption spectrum of the ferrous deoxy or ferric forms are not normal, displaying an enhanced absorption of the visible band near 560 nm. Half of the autooxidation process, attributed to the mutated β subunits, is over 1000‐fold faster than for Hb A.

The mutant Hb exhibits non‐cooperative binding of two oxygens with an affinity about fivefold lower than those of HbA valency hybrids (αmetβ)2. Functional properties of this mutant Hb resemble those of Hb Saskatoon ([Tyr63]Hb) [Suzuki, T., Hayashi, A., Shimizu, A. & Yamamura, Y. (1966) Biochim. Biophys. Acta 127, 280–2821, Flash‐photolysis experiments also indicate non‐cooperative behaviour: the CO‐recombination kinetics were independent of the fraction dissociated. Furthermore, the amplitude of the CO bimolecular phase was the same for the (αcometβ), valency hybrid or the (αcoβco), form, suggesting mainly geminate CO‐recombination kinetics to the β chains.

EPR and Resonance Raman spectra did not show evidence for a hemichrome, normally considered as a six‐coordinated iron with low‐spin character. The EPR and resonance Raman spectra for the mutated β subunits demonstrate the presence of a high‐spin compound in the ferric and deoxy ferrous forms. In particular, the ferrous mutated β subunits are penta‐coordinated. The abnormal absorption spectra are possibly due to an interaction between the porphyrin and the phenyl ring in the distal position rather than to direct binding to the iron.