bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure–function relationships in Drosophila melanogaster alcohol dehydrogenase allozymes ADHS, ADHF and ADHUF, and distantly related forms
Authors: Benach Jordi , Atrian Sílvia , Fibla Joan , Gonzàlez‐Duarte Roser , Ladenstein Rudolf
Published: March 2003

Drosophila melanogaster alcohol dehydrogenase (ADH), a paradigm for gene–enzyme molecular evolution and natural selection studies, presents three main alleloforms (ADHS, ADHF and ADHUF) differing by one or two substitutions that render different biochemical properties to the allelozymes. A three‐dimensional molecular model of the three allozymes was built by homology modeling using as a template the available crystal structure of the orthologous D. lebanonensis ADH, which shares a sequence identity of 82.2%. Comparison between D. lebanonensis and D. melanogaster structures showed that there is almost no amino‐acid change near the substrate or coenzyme binding sites and that the hydrophobic active site cavity is strictly conserved. Nevertheless, substitutions are not distributed at random in nonconstricted positions, or located in external loops, but they appear clustered mainly in secondary structure elements. From comparisons between D. melanogaster allozymes and with D. simulans, a very closely related species, a model based on changes in the electrostatic potential distribution is presented to explain their differential behavior. The depth of knowledge on Drosophila ADH genetics and kinetics, together with the recently obtained structural information, could provide a better understanding of the mechanisms underlying molecular evolution and population genetics.
The blue‐colored linker polypeptide L55 is a fusion protein of phycobiliproteins in the cyanobacterium Synechocystis sp. strain BO 8402
Authors: Neuschaefer‐Rube Olaf , Westermann Martin , Blüggel Martin , Meyer Helmut E. , Ernst Anneliese
Published: March 2003

The cyanobacterium Synechocystis sp. strain BO 8402, isolated from Lake Constance, lacks phycobilisomes but instead forms inclusion bodies containing remnants of phycobiliproteins. The inclusion bodies are surrounded by a proteinaceous capsule and contain α‐phycocyanin and β‐phycocyanin, the rod linker polypeptide L35RPC and a novel blue‐colored protein L55 with an apparent molecular mass of 55 kDa. An antibody raised against β‐phycocyanin showed a strong cross‐reaction with L55. Mass spectrometry analysis of proteolytic peptides from L55 revealed mass identity to proteolytic peptides derived from L35RPC and β‐phycocyanin. However, analysis of the genome of strain BO 8402 revealed only one cpcBACE operon, encoding the apoproteins of β‐phycocyanin and α‐phycocyanin, L35RPC and a subunit of the phycocyanin α subunit phycocyanobilin lyase, respectively. The gene structure, sequence and transcription of these genes were identical to that of a revertant strain, Synechocystis sp. strain BO 9201, which formed phycobilisomes and did not express L55. Based on these observations, we concluded that L55 did not derive from a particular gene or from a special form of mRNA‐processing. We propose that L55 is formed by post‐translational fusion of L35RPC and β‐phycocyanin. Cross‐linking may stabilize the formation of the large paracrystalline phycocyanin aggregates unique to Synechocystis sp. strain BO 8402.
Expression and characterization of recombinant human eosinophil peroxidase
Authors: Romano Maurizio , Baralle Francisco E. , Patriarca Pierluigi
Published: March 2003

Hereditary eosinophil peroxidase deficiency is a genetic abnormality characterized by a decrease or absence of peroxidase activity and a reduction of the granule matrix volume. Recently, we identified two mutations associated with eosinophil peroxidase deficiency in a subject and his siblings, i.e. a base insertion causing the appearance of a premature stop codon and a base transition causing the replacement of an Arg at codon 286 with a His (R286H). In this article we report the stable expression of both the recombinant wild‐type and the R286H eosinophil peroxidase precursor in the K‐562 cell line, and the effects of the R286H substitution on the structure and function of the eosinophil peroxidase precursor. Heme group incorporation into both the recombinant wild‐type and the recombinant R286H eosinophil peroxidase precursor was comparable, as was the stability of both proteins. Instead, the recombinant R286H eosinophil peroxidase precursor exhibited marked alterations of the catalytic properties and an increased sensitivity to four peroxidase inhibitors with respect to both the recombinant wild‐type eosinophil peroxidase precursor and the native enzyme. In addition, the recombinant wild‐type, but not the R286H, eosinophil peroxidase precursor was immunoprecipitated by two anti‐(eosinophil peroxidase) mAbs. Altogether, our results suggest a protein misfolding of the R286H eosinophil peroxidase precursor which might account for its altered catalytic properties and the absence of expression of some epitopes.
A novel transmembrane serine/threonine protein kinase gene from a rifamycin SV‐producing Amycolatopsis mediterranei U32
Authors: Zhang Weiwen , Li Lei , Jiang Weihong , Zhao Guoping , Yang Yunliu , Chiao Juishen
Published: March 2003

Genomic DNA sequencing in the vicinity of methylmalonyl‐CoA mutase gene (mutAB) from a rifamycin SV‐producing Amycolatopsis mediterranei U32 allowed us to clone, sequence, and identify a gene encoding a novel serine/threonine protein kinase (amk). The sequence contains a complete ORF of 1821 base pairs encoding a predicted protein of 606 amino acids in length. The N‐terminal domain of the protein shows significant homology to the catalytic domain of other protein kinases from both prokaryotic and eukaryotic sources. It also contains all the structural features that are highly conserved in active protein kinases, including the Gly‐X‐Gly‐X‐X‐Gly motif of ATP‐binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein kinase. This protein kinase gene was expressed in Escherichia coli and was shown to have the ability of autophosphorylation. The autophosphorylated site was found to be the threonine at position 164 by labeled phosphoaminoacid analysis and site‐directed mutagenesis. The C‐terminal half of protein kinase was found to contain strong transmembrane structures by PhoA fusion protein analysis, suggesting that Amk protein kinase is a transmembrane protein. A Southern hybridization experiment showed that this type of protein kinase is distributed ubiquitously and might play significant physiological roles in the various species of streptomycetes. However, overexpression of amk gene in Streptomyces cinnamonensis showed no effect on methylmalonyl‐CoA mutase activity, monensin production and the hyphae morphology. Although its biological role is still unknown, Amk protein kinase is the first transmembrane serine/threonine protein kinase described for genus Amycolatopsis.
NMR investigations of protein–carbohydrate interactions
Authors: Espinosa Juan Felix , Asensio Juan Luis , García Jose Luis , Laynez José , Bruix Marta , Wright Christine , Siebert Hans‐Christian , Gabius Hans‐Joachim , Cañada Francisco Javier , Jiménez‐Barbero Jesus
Published: March 2003

The specific interaction of the isolated B domain of wheat germ agglutinin (WGA‐B) with N,N′,N″‐triacetylchitotriose has been analyzed by 1H‐NMR spectroscopy. The association constants for the binding of WGA‐B to this trisaccharide have been determined from both 1H‐NMR titration experiments and microcalorimetry methods. Entropy and enthalpy of binding have been obtained. The driving force for the binding process is provided by a negative ΔH which is partially compensated by negative ΔS. These negative signs indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. NOESY NMR experiments in water solution provided 327 protein proton‐proton distance constraints. All the experimental constraints were used in a refinement protocol including restrained molecular dynamics in order to determine the refined solution conformation of this protein/carbohydrate complex. With regard to the NMR structure of the free protein, no important changes in the protein NOEs were observed, indicating that carbohydrate‐induced conformational changes are small. The average backbone rmsd of the 35 refined structures was 1.05 Å, while the heavy atom rmsd was 2.10 Å. Focusing on the bound ligand, two different orientations of the trisaccharide within WGA‐B binding site are possible. It can be deduced that both hydrogen bonds and van der Waals contacts confer stability to both complexes. A comparison of the three‐dimensional structure of WGA‐B in solution to that reported in the solid state and to those deduced for hevein and pseudohevein in solution has also been performed.
α‐Crystallin prevents irreversible protein denaturation and acts cooperatively with other heat‐shock proteins to renature the stabilized partially denatured protein in an ATP‐dependent manner
Authors: Wang Keyang , Spector Abraham
Published: March 2003

α‐Crystallin, a major lens protein of ≈ 800 kDa with subunits of ≈ 20 kDa has previously been shown to act as a chaperone protecting other proteins from stress‐induced aggregation. Here it is demonstrated that α‐crystallin can bind to partially denatured enzymes at 42–43 °C and prevent their irreversible aggregation, but cannot prevent loss of enzyme activity. However, the α‐crystallin‐bound enzymes regain activity on interaction with other chaperones. The data indicate that the re‐activated enzymes are no longer associated with the α‐crystallin, and ATP is required for re‐activation. When inactive luciferase bound to α‐crystallin was treated with reticulocyte lysate, a rich source of chaperones, up to 60% of the original luciferase activity could be recovered. Somewhat less re‐activation was observed when the α‐crystallin‐bound enzyme was treated with heat‐shock protein (HSP)70, HSP40, HSP60 and an ATP‐generating system. Similar results were also obtained with citrate synthase. The overall results suggest that α‐crystallin acts to stabilize denaturing proteins so that they can later be re‐activated by other chaperones.
Expression of silicatein and collagen genes in the marine sponge Suberites domuncula is controlled by silicate and myotrophin
Authors: Krasko Anatoli , Lorenz Bernd , Batel Renato , Schröder Heinz C. , Müller Isabel M. , Müller Werner E. G.
Published: March 2003

The major skeletal elements in the (Porifera) sponges, are spicules formed from inorganic material. The spicules in the Demospongiae class are composed of hydrated, amorphous silica. Recently an enzyme, silicatein, which polymerizes alkoxide substrates to silica was described from the sponge Tethya aurantia. In the present study the cDNA encoding silicatein was isolated from the sponge Suberites domuncula. The deduced polypeptide comprises 331 amino acids and has a calculated size of Mr 36 306. This cDNA was used as a probe to study the potential role of silicate on the expression of the silicatein gene. For these studies, primmorphs, a special form of aggregates composed of proliferating cells, have been used. It was found that after increasing the concentration of soluble silicate in the seawater medium from around 1 µm to approximately 60 µm, this gene is strongly upregulated. Without additional silicate only a very weak expression could be measured. Because silica as well as collagen are required for the formation of spicules, the expression of the gene encoding collagen was measured in parallel. It was also found that the level of transcripts for collagen strongly increases in the presence of 60 µm soluble silicate. In addition, it is demonstrated that the expression of collagen is also upregulated in those primmorphs which were treated with recombinant myotrophin obtained from the same sponge. Myotrophin, however, had no effect on the expression of silicatein. From these data we conclude that silicate influences the expression of the enzyme silicatein and also the expression of collagen, (via the mediator myotrophin).
Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics
Authors: Sich Christian , Improta Sabina , Cowley David John , Guenet Chantal , Merly Jean‐Pierre , Teufel Michael , Saudek Vladimir
Published: March 2003

The structure of a recently reported neurotrophic ligand, 3‐(3‐pyridyl)‐1‐propyl(2S)‐1‐(3,3‐dimethyl‐1,2‐dioxopentyl)‐2‐pyrrolidinecarboxylate, in complex with FKBP12 was determined using heteronuclear NMR spectroscopy. The inhibitor exhibits a binding mode analogous to that observed for the macrocycle FK506, used widely as an immunosuppressant, with the prolyl ring replacing the pipecolyl moiety and the amide bond in atransconformation. However, fewer favourable protein–ligand interactions are detected in the structure of the complex, suggesting weaker binding compared with the immunosuppressant drug. Indeed, a micromolar dissociation constant was estimated from the NMR ligand titration profile, in contrast to the previously published nanomolar inhibition activity. Although the inhibitor possesses a remarkable structural simplicity with respect to FK506,15N relaxation studies show that it induces similar effects on the protein dynamics, stabilizing the conformation of solvent‐exposed residues which are important for mediating the interaction of immunophilin/ligand complexes with molecular targets and potentially for the transmission of the neurotrophic action of FKBP12 inhibitors.
Independent pathways leading to apoptotic cell death, oxidative burst and defense gene expression in response to elicitin in tobacco cell suspension culture
Authors: Sasabe Michiko , Takeuchi Kasumi , Kamoun Sophien , Ichinose Yuki , Govers Francine , Toyoda Kazuhiro , Shiraishi Tomonori , Yamada Tetsuji
Published: March 2003

We characterized pharmacologically the hypersensitive cell death of tobacco BY‐2 cells that followed treatments with Escherichia coli preparations of INF1, the major secreted elicitin of the late blight pathogen Phytophthora infestans. INF1 elicitin treatments resulted in fragmentation and 180 bp laddering of tobacco DNA as early as 3 h post‐treatment. INF1 elicitin also induced rapid accumulation of H2O2 typical of oxidative burst, and the expression of defense genes such as phenylalanine ammonia‐lyase (PAL) gene at 1 h and 3 h after elicitin treatment, respectively. To investigate the involvement of the oxidative burst and/or the expression of defense genes in the signal transduction pathways leading to hypersensitive cell death, we analyzed the effect of several chemical inhibitors of signal transduction pathways on the various responses. The results indicated that (a) the cell death required serine proteases, Ca2+ and protein kinases, (b) the oxidative burst was involved in Ca2+ and protein kinase mediated pathways, but elicitin‐induced AOS was neither necessary nor sufficient for cell death and PAL gene expression, and (c) the signaling pathway of PAL gene expression required protein kinases. These results suggest that the three signal transduction pathways leading to cell death, oxidative burst and expression of defense genes branch in the early stages that follow elicitin recognition by tobacco cells.
Oxidation by mushroom tyrosinase of monophenols generating slightly unstable o‐quinones
Authors: Fenoll Lorena G. , Rodríguez‐López José N. , García‐Sevilla Francisco , Tudela José , García‐Ruiz Pedro Antonio , Varón Ramón , García‐Cánovas Francisco
Published: March 2003

Tyrosinase can act on monophenols because of the mixture of mettyrosinase (Em) and oxytyrosinase (Eox) that exists in the native form of the enzyme. The latter form is active on monophenols although the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex as the products of the enzymatic reaction, the o‐quinones, are unstable and continue evolving to generate o‐diphenols in the medium. In the case of substrates such as 4‐methoxyphenol, 4‐ethoxyphenol and 4‐tert‐butylphenol, tyrosinase generates o‐quinones which become unstable with small constants of approximately < 10−3 s−1. The system evolves from an initial steady state, reached when t→0, through a transition state towards a final steady state, which is never reached because the substrate is largely consumed. The mechanisms proposed to explain the enzyme's action can be differentiated by the kinetics of the first steady state. The results suggest that tyrosinase hydroxylates monophenols to o‐diphenols, generating an intermediate Em‐diphenol in the process, which may oxidize the o‐diphenol or release it directly into the medium. In the case of o‐quinone formation, its slow instability generates o‐diphenol which activates the enzymatic system yielding parabolic time recordings.
Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii
Authors: Costello Colleen A. , Payson Robert A. , Menke Michael A. , Larson Jeffrey L. , Brown Keith A. , Tanner Joseph E. , Kaiser Raymond E. , Hershberger Charles L. , Zmijewski Milton J.
Published: March 2003

A novel ketoreductase isolated from Zygosaccharomyces rouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8‐kDa ketoreductase was purified more than 300‐fold to > 95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4‐methylenedioxyphenyl acetone of 2.9 mm and a Km for NADPH of 23.5 µm. The enzyme is able to effectively reduce α‐ketolactones, α‐ketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0‐kb ketoreductase gene was cloned and sequenced from a Z. rouxii cDNA library using a degenerate primer to the N‐terminal sequence of the purified protein. Furthermore, it was expressed in both Escherichia coli and Pichia pastoris and shown to be active. Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class.
Structural analysis of the lipopolysaccharide from Chlamydophila psittaci strain 6BC
Authors: Rund Sabine , Lindner Buko , Brade Helmut , Holst Otto
Published: March 2003

The lipopolysaccaride of Chlamydophila psittaci 6BC was isolated from tissue culture‐grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. Compositional analyses indicated the presence of 3‐deoxy‐
dmanno‐oct‐2‐ulosonic acid, GlcN, organic bound phosphate and fatty acids in a molar ratio of ≈ 3.3 : 2 : 1.8 : 4.6. Deacylated lipopolysaccharide was obtained after successive microscale treatment with hydrazine and potassium hydroxide, and was then separated by high performance anion‐exchange chromatography into two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as α‐Kdo‐(2→8)‐α‐Kdo‐(2→4)‐α‐Kdo‐(2→6)‐β‐
d‐GlcpN‐(1→6)‐α‐
d‐GlcpN 1,4′‐bisphosphate and α‐Kdo‐(2→4)‐[α‐Kdo‐(2→8)]‐α‐Kdo‐(2→4)‐α‐Kdo‐(2→6)‐β‐
d‐GlcpN‐(1→6)‐α‐
d‐GlcpN 1,4′‐bisphosphate. The distribution of fatty acids in lipid A was determined by compositional analyses and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry experiments on lipid A and de‐O‐acylated lipid A. It was shown that the carbohydrate backbone of lipid A is replaced by a complex mixture of fatty acids, including long‐chain and branched (R)‐configured 3‐hydroxy fatty acids, the latter being exclusively present in an amide linkage.
Cytoprotective antioxidant function of tyrosine and tryptophan residues in transmembrane proteins
Authors: Moosmann Bernd , Behl Christian
Published: March 2003

The transmembrane domains of integral membrane proteins show an astounding accumulation of tyrosine and tryptophan residues, especially in the region of the highest lipid density. We found that these residues perform vital antioxidant functions inside lipid bilayers and protect cells from oxidative destruction. First, tyrosine‐ and tryptophan‐containing peptides representing stretches from the transmembrane domains of different integral membrane proteins, including presenilin and the cystic fibrosis transmembrane conductance regulator, prevent oxidative lysis in clonal and primary cells. Second, long‐chain acylated tyrosine and tryptophan, but not phenylalanine or short‐chain acylated derivatives, are potent inhibitors of lipid peroxidation and oxidative cell death. The antioxidant functions of tyrosine and tryptophan may provide a specific explanation for (a) their unique transmembrane distribution pattern and (b) the high vulnerabiliy of low‐protein neuronal membranes to oxidative stress, as seen in neurodegenerative disorders.
Characterization of acid‐induced unfolding intermediates of glucose/xylose isomerase
Authors: Pawar S. A. , Deshpande V. V.
Published: March 2003

Acid‐induced unfolding of the tetrameric glucose/xylose isomerase (GXI) from Streptomyces sp. NCIM 2730 has been investigated using intrinsic fluorescence, fluorescence quenching, second derivative spectroscopy, hydrophobic dye (1‐anilino‐8‐naphthalene‐sulfonate) binding and CD techniques. The pH dependence of tryptophanyl fluorescence of GXI at different temperatures indicated the presence of two stable intermediates at pH 5.0 and pH 3.0. The pH 3.2 intermediate was a dimer and exhibited molten globule‐like characteristics, such as the presence of native‐like secondary structure, loss of tertiary structure, increased exposure of hydrophobic pockets, altered microenvironment of tyrosine residues and increased accessibility to quenching by acrylamide. Fluorescence and CD studies on GXI at pH 5.0 suggested the involvement of a partially folded intermediate state in the native to molten globule state transition. The partially folded intermediate state retained considerable secondary and tertiary structure compared to the molten globule state. This state was characterized by its hydrophobic dye binding capacity, which is smaller than the molten globule state, but was greater than that of the native state. This state shared the dimeric status of the molten globule state but was prone to aggregate formation as evident by the Rayleigh light scattering studies. Based on these results, the unfolding pathway of GXI can be illustrated as: N→PFI→MG→U; where N is the native state at pH 7.5; PFI is the partially folded intermediate state at pH 5.0; MG is the molten globule state at pH 3.2 and U is the monomeric unfolded state of GXI obtained in the presence of 6 m GdnHCl. Our results demonstrate the existence of a partially folded state and molten globule state on the unfolding pathway of a multimeric α/β barrel protein.
Identification of novel prostate‐specific antigen‐binding peptides modulating its enzyme activity
Authors: Wu Ping , Leinonen Jari , Koivunen Erkki , Lankinen Hilkka , Stenman Ulf‐Håkan
Published: March 2003

Prostate‐specific antigen (PSA) is a serine protease with highly prostate‐specific expression. Measurement of PSA in serum is widely used for diagnosis and monitoring of prostate cancer. PSA dissolves the seminal gel forming after ejaculation. It has been suggested to mediate invasion and metastasis of prostate cancer but also to exert antiangiogenic activity. We have identified peptides specific for PSA by screening cyclic phage display peptide libraries. PSA‐binding peptides were isolated from four different libraries and produced as a fusion protein with glutathione S‐transferase (GST). The phage and fusion proteins were shown to bind to PSA specifically as indicated by lack of binding to other serine proteinases. A peptide with four cysteines showed the highest affinity for PSA. Zn2+, an inhibitor of PSA activity, increased the affinity of the peptides to PSA. The binding specificity was characterized by cross‐inhibition using monoclonal anti‐PSA antibodies of known epitope specificities. The peptides bound to the same region as mAbs specific for free PSA indicating that they bind close to the active site of the enzyme. The peptides enhanced the enzyme activity of PSA against a chromogenic substrate. These results show that peptides binding to PSA and modulating its enzyme activity can be developed by phage display technique. The peptides have the potential to be used for identification of PSA variants and for imaging and targeting of prostatic tumors.
The assembly factor P17 from bacteriophage PRD1 interacts with positively charged lipid membranes
Authors: Holopainen Juha M. , Säily Matti , Caldentey Javier , Kinnunen Paavo K. J.
Published: March 2003

The interactions of the assembly factor P17 of bacteriophage PRD1 with liposomes were investigated by static light scattering, fluorescence spectroscopy, and differential scanning calorimetry. Our data show that P17 binds to positively charged large unilamellar vesicles composed of the zwitterionic 1‐palmitoyl‐2‐oleoyl‐phosphatidylcholine and sphingosine, whereas only a weak interaction is evident for 1‐palmitoyl‐2‐oleoyl‐phosphatidylcholine vesicles. P17 does not bind to negatively charged membranes composed of 1‐palmitoyl‐2‐oleoyl‐phosphatidylglycerol and 1‐palmitoyl‐2‐oleoyl‐phosphatidylcholine. Our differential scanning calorimetry results reveal that P17 slightly perturbs the phase behaviour of neutral phosphatidylcholine and negatively charged multilamellar vesicles. In contrast, the phase transition temperature of positively charged dimyristoylphosphatidylcholine/sphingosine multilamellar vesicles (molar ratio 9 : 1, respectively) is increased by approximately 2.4 °C and the half width of the enthalpy peak broadened from 1.9 to 5.6 °C in the presence of P17 (protein : lipid molar ratio 1 : 47). Moreover, the enthalpy peak is asymmetrical, suggesting that lipid phase separation is induced by P17. Based on the far‐UV CD spectra, the α‐helicity of P17 increases upon binding to positively charged micelles composed of Triton X‐100 and sphingosine. We propose that P17 can interact with positively charged lipid membranes and that this binding induces a structural change on P17 to a more tightly packed and ordered structure.
Examining the zinc binding site of the amyloid‐β peptide
Authors: Yang Dun‐Sheng , McLaurin JoAnne , Qin Kefeng , Westaway David , Fraser Paul E.
Published: March 2003

The amyloid β‐peptide (Aβ) is a principal component of insoluble amyloid plaques which are characteristic neuropathological features of Alzheimer's disease. Aβ also exists as a normal soluble protein that undergoes a pathogenic transition to an aggregated, fibrous form. This transition can be affected by extraneous proteinaceous and nonproteinaceous elements, such as zinc ions, which may promote aggregation and/or stabilization of the fibrils. Protein chelation of zinc is typically mediated by histidines, cysteines and carboxylates. Previous studies have demonstrated that the Aβ‐Zn2+ binding site is localized within residues 6–28 and that histidines may serve as the principal sites of interaction. To localize key residues within this region, a series of Aβ peptides (residues 1–28) were synthesized that contained systematic His/Ala substitutions. Circular dichroism and electron microscopy were used to monitor the effects of Zn2+ on the peptide β‐sheet conformation and fibril aggregation. Our results indicate that substitution of either His13 or His14 but not His6 eliminates the zinc‐mediated effects. These observations indicate a specific zinc binding site within Aβ that involves these central histidine residues.
Identification of low‐density Triton X‐100‐insoluble plasma membrane microdomains in higher plants
Authors: Peskan Tatjana , Westermann Martin , Oelmüller Ralf
Published: March 2003

Low density Triton X‐100‐insoluble plasma membrane microdomains can be isolated from different mammalian cell types and are proposed to be involved in membrane trafficking, cell morphogenesis and signal transduction. Heterotrimeric G‐proteins and their receptors are often associated with such domains, suggesting that these structures are involved in G‐protein‐coupled signaling. Here we report that detergent‐insoluble plasma membrane microdomains also exist in higher plants and contain about 15% of membrane‐bound heterotrimeric G‐protein beta‐subunit (Gβ). Plasma membrane microdomains were isolated from tobacco leaves. They have low buoyant density relative to the surrounding plasma membrane, and are insoluble in Triton X‐100 at 4 °C. Detergent‐insoluble vesicles were examined by freeze‐fracture electron microscopy. They have sizes in the range 100–400 nm, and often contain aggregated protein complexes. The majority of plasma membrane proteins cannot be detected in the Triton X‐100‐insoluble fraction, while few polypeptides are highly enriched. We identified six proteins with molecular masses of 22, 28, 35, 60, 67 and 94 kDa in detergent‐insoluble fractions that are glycosylphosphatidylinositol (GPI)‐anchored.
Cloning and characterization of the Rainbow trout (Oncorhynchus mykiss) type II interleukin‐1 receptor cDNA
Authors: Sangrador‐Vegas Amaia , Martin Samuel A. M. , O’Dea Paul G. , Smith Terry J.
Published: March 2003

A homologue of mammalian type II interleukin‐1 receptor (IL‐1RII) was isolated from a rainbow trout cDNA library by differential hybridization using a suppression subtractive hybridization generated probe enriched for sequences upregulated after immune stimulation. The trout cDNA has an ORF encoding 441 amino acids, and represents the first piscine IL‐1 receptor described. The predicted amino‐acid sequence has 29 and 26% identity with human and mouse IL‐1RII, respectively. The trout IL‐1 receptor has a domain organization similar to that of mammalian type II receptor, with a short cytoplasmic tail of 24 amino acids. These results suggest that type II receptor is also present in lower vertebrates, and therefore the duplication of an ancestral gene that generated type I and type II IL‐1 receptors occurred prior to the time mammals emerged.
Evidence for an evolutionary conserved role of bone morphogenetic protein growth factors and Phox2 transcription factors during noradrenergic differentiation of sympathetic neurons
Authors: Ernsberger Uwe
Published: March 2003

The noradrenergic transmitter phenotype in postganglionic sympathetic neurons is induced early during embryonic development in avian and mammalian primary sympathetic ganglia. The simultaneous expression of tyrosine hydroxylase and dopamine β‐hydroxylase, enzymes of the noradrenaline biosynthesis pathway, indicates that different genes contributing to the noradrenergic transmitter phenotype are regulated as a synexpression group. This conclusion is supported by the demonstration of bone morphogenetic protein (BMP) growth factors and Phox2 transcription factors being necessary for the expression of both tyrosine hydroxylase and dopamine β‐hydroxylase in differentiating sympathetic neurons. The close similarity in the expression patterns of the relevant genes as well as in the function of BMPs and Phox2s between avian and mammalian embryos strongly suggests that noradrenergic induction occurs along a conserved signalling pathway in these vertebrate classes.
Characterization of the Co2+ and Ni2+ binding amino‐acid residues of the N‐terminus of human albumin
Authors: Bar‐Or David , Curtis Gerald , Rao Nagaraja , Bampos Nick , Lau Edward
Published: March 2003

Patients suffering from myocardial ischemia reportedly exhibit reduced in vitro binding of exogenous Co2+ to the N‐terminal of human serum albumin (HSA). The purpose of our investigation was to simulate changes in the N‐terminus of HSA that may account for these ischemia‐induced modifications to the cobalt binding site. HPLC, LC‐MS and 1H NMR analyses have shown that the N‐terminal region of HSA Asp‐Ala‐His‐Lys binds the transition metals Co2+ and Ni2+. Synthetic peptides with the first 2–12 amino acids of the HSA sequence demonstrated that the first three amino acids, Asp‐Ala‐His, are essential for strong binding of cobalt. Modification of the N‐terminus peptide of HSA by way of N‐acetylation or the deletion of one or more amino acid resulted in no binding of cobalt. Because the degradation of the susceptible, specific transition metal binding site of HSA may account for the decreased cobalt binding observed during ischemic events, an assay that detects this reduced binding could be useful in the diagnosis of ischemia.
Thermodynamic analysis of saccharide binding to snake gourd (Trichosanthes anguina) seed lectin
Authors: Komath Sneha Sudha , Kenoth Roopa , Swamy Musti J.
Published: March 2003

The interaction of different saccharides with the snake gourd (Trichosanthes anguina) seed lectin (SGSL) was investigated by fluorescence spectroscopy. Binding of 4‐methylumbelliferyl‐β‐d‐galactopyranoside (MeUmbβGal) to SGSL resulted in a significant increase in the fluorescence emission intensity of the sugar at 376 nm, and this change was used to estimate the association constants for the binding interaction. Interestingly, the increase in emission intensity changed with a change in temperature, increasing from 19.2% at 20 °C to 80.2% at 40 °C. At 20 °C the association constant, Ka, for the MeUmbβGal–SGSL interaction was found by fluorescence titration to be 5.8 × 104 m−1. From the temperature dependence of the association constants, the changes in enthalpy (ΔH) and entropy (ΔS) associated with binding of MeUmbβGal to SGSL were estimated to be −80.85 kJ·mol−1 and −184.0 J·mol−1·K−1, respectively. Binding of unlabeled sugars was investigated by monitoring the decrease in fluorescence intensity when they were added to a mixture of SGSL and MeUmbβGal. The Ka values for different sugars were determined at several temperatures, and ΔH and ΔS were determined from the van’t Hoff plots. Enthalpy–entropy compensation was noticed in all cases. The results indicate that saccharide binding to SGSL is enthalpy‐driven and the negative contribution from entropy is, in general, quite high.
Different functional modulation by heterotropic ligands (2,3‐diphosphoglycerate and chlorides) of the two haemoglobins from fallow‐deer (Dama dama)
Authors: Angeletti Mauro , Pucciarelli Stefania , Priori Anna M. , Canofeni Silvia , Barra Donatella , Fioretti Evandro , Coletta Massimo
Published: March 2003

Two haemoglobin components have been identified and purified from fallow‐deer (Dama dama) erythrocytes. They are present in similar amounts and the two tetrameric molecules share the same α chain, while two different β chains are detected in the two components. The β chains differ by 14 residues, even though they both have 145 amino‐acid residues, which account for a molecular mass of 16 023 and 16 064 Da, respectively, while α chain has 141 residues, yielding a molecular mass of 15 142 Da. Compared with human Hb, the N‐terminal region of both β chains shows deletion of Valβ1 and the replacement of Hisβ2 by a methionyl residue, a modification which is common to most ruminant haemoglobins. Although both isolated components show a low intrinsic affinity for oxygen, meaningful differences between the two haemoglobins have been found with respect to the effect of heterotropic effectors, such as 2,3‐diphosphoglycerate and chloride ions. In view of the high sequence homology between the two components, the different effect of heterotropic ligands has been tentatively correlated to possible localized structural variations between β chains of the two haemoglobin components.
Three‐dimensional structure of the histidine‐containing phosphocarrier protein (HPr) from Enterococcus faecalis in solution
Authors: Maurer Till , Döker Rolf , Görler Adrian , Hengstenberg Wolfgang , Kalbitzer Hans R.
Published: March 2003

The histidine‐containing phosphocarrier protein (HPr) transfers a phosphate group between components of the prokaryotic phosphoenolpyruvate‐dependent phosphotransferase system (PTS), which is finally used to phosphorylate the carbohydrate transported by the PTS through the cell membrane. Recently it has also been found to act as an intermediate in the signaling cascade that regulates transcription of genes related to the carbohydrate‐response system. Both functions involve phosphorylation/dephosphorylation reactions, but at different sites. Using multidimensional 1H‐NMR spectroscopy and angular space simulated annealing calculations, we determined the structure of HPr from Enterococcus faecalis in aqueous solution using 1469 distance and 44 angle constraints derived from homonuclear NMR data. It has a similar overall fold to that found in HPrs from other organisms. Four β strands, A, B, C, D, encompassing residues 2–7, 32–37, 40–42 and 60–66, form an antiparallel β sheet lying opposite the two antiparallel α helices, a and c (residues 16–26 and 70–83). A short α helix, b, from residues 47–53 is also observed. The pairwise root mean square displacement for the backbone heavy atoms of the mean of the 16 NMR structures to the crystal structure is 0.164 nm. In contrast with the crystalline state, in which a torsion angle strain in the active‐center loop has been described [Jia, Z., Vandonselaar, M., Quail, J.W. & Delbaere, L.T.J. (1993) Nature (London) 361, 94–97], in the solution structure, the active‐site His15 rests on top of helix a, and the phosphorylation site Nδ1 of the histidine ring is oriented towards the surface, making it easily accessible to the solvent. Back calculation of the 2D NOESY NMR spectra from both the NMR and X‐ray structures shows that the active‐center structure derived by X‐ray crystallography is not compatible with experimental data recorded in solution. The observed torsional strain must either be a crystallization artefact or represents a conformational state that exists only to a small extent in solution.
Analogs of the antimicrobial peptide trichogin having opposite membrane properties
Authors: Epand Raquel F. , Epand Richard M. , Formaggio Fernando , Crisma Marco , Wu Huiyuan , Lehrer Robert I. , Toniolo Claudio
Published: March 2003

Four analogs of the antimicrobial peptide trichogin GA IV were studied. Their sequences are as follows: GT, n‐octanoyl‐Aib‐Gly‐Leu‐Aib‐Gly‐Gly‐Leu‐Aib‐Gly‐Ile‐Leu‐OMe; ST, n‐octanoyl‐Aib‐Ser‐Leu‐Aib‐Ser‐Ser‐Leu‐Aib‐Ser‐Ile‐Leu‐OMe; BT, n‐octanoyl‐Aib‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ile‐Leu‐OMe; and DT, n‐octanoyl‐Aib‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ile‐Leu‐Aib‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ile‐Leu‐OMe. The trichogin GA IV differs from GT only in the nature of the C‐terminal residue, being a 1,2 aminoalcohol (leucinol) in the case of the parent peptide. Compared with GT, ST has an increased amphiphilicity. In contrast, BT has little amphiphilicity being composed only of hydrophobic amino acids. DT is an octanoylated head‐to‐tail dimer of BT. We show that BT and DT lower the bilayer‐to‐hexagonal phase transition temperature (TH) of dipalmitoleoylphosphatidylethanolamine, indicating that the peptides promote negative curvature. These two peptides, composed of only hydrophobic amino acids, have their bulkier groups on one face of the helix, suggesting that they may penetrate membranes at an oblique angle. In contrast, GT and ST, like trichogin itself, increase TH, promoting positive curvature. These peptides have contrasting membrane lytic activities. Whereas DT and BT did not produce leakage of aqueous contents, GT and ST, like trichogin, did cause rapid leakage. The leakage activity with liposomes also correlates with the greater potency of GT and ST, compared with the hydrophobic analogs, in their hemolytic and bacteriostatic action. ST has greater lytic ability than GT in liposomal leakage as well as hemolysis. We also measured the rate of peptide‐promoted lipid mixing as an indication of membrane fusion. BT produced lipid mixing only with large unilamellar vesicles enriched with dioleoylphosphatidylethanolamine; ST did not produce lipid mixing, as its apparent reduction of energy transfer proved to be artifactual. Quasi‐elastic light scattering of large unilamellar vesicles was also carried out after adding ST and BT. Peptide BT, but not ST, was able to aggregate large unilamellar vesicles. Thus, one of the properties of BT that leads to the induction of lipid mixing is that it is able to aggregate vesicles, placing the bilayers in juxtaposition. Thus, the two pairs of peptides, BT and DT vs GT and ST, exhibit contrasting behaviour with respect to a number of membrane biophysical properties. This occurs despite the fact that the chemical structures of the peptides are rather similar. Such distinct behavior is also reflected in their hemolytic and bacteriostatic actions.
Assembly of cytochrome f into the cytochrome bf complex in isolated pea chloroplasts
Authors: Mould Ruth M. , Kapazoglou Aliki , Gray John C.
Published: March 2003

Structural features of cytochrome f necessary for assembly into the cytochrome bf complex were examined in isolated pea chloroplasts following import of 35S‐labelled chimeric precursor proteins, consisting of the presequence of the small subunit of Rubisco fused to the turnip cytochrome f precursor. Assembly was detected by nondenaturing gel electrophoresis of dodecyl maltoside‐solubilized thylakoid membranes. A cytochrome f polypeptide unable to bind haem because of mutagenesis of Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild‐type polypeptide. This indicates that covalent haem binding to cytochrome f is not necessary for assembly of the protein into the cytochrome bf complex. A truncated protein lacking the C‐terminal 33 amino acid residues, including the transmembrane span and the stroma‐exposed region, was translocated across the thylakoid membrane, had a similar stability to wild‐type cytochrome f but was not assembled into the complex. This indicates that the C‐terminal region of cytochrome f is important for assembly into the complex. A mutant cytochrome f unable to bind haem and lacking the C‐terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence of the C‐terminal membrane anchor, haem‐less cytochrome f is recognized by a thylakoid proteolytic system.
Factors affecting the dimerization of the p66 form of HIV‐1 reverse transcriptase
Authors: Cabodevilla Jesús F. , Odriozola Leticia , Santiago Esteban , Martínez‐Irujo Juan J.
Published: March 2003

The association and dissociation of the homodimeric p66/p66 form of HIV‐1 reverse transcriptase were investigated. The effects on the dimerization process of different salt concentrations, pH and the presence of a template/primer and nucleotide substrates were monitored by measuring polymerase activity and analytical size‐exclusion HPLC. At submicromolar concentrations of enzyme and physiological salt concentrations, most of the enzyme exists in the inactive monomeric form. Increasing NaCl concentration from 0.05 to 1 mdecreased the equilibrium dissociation constant from 2.0 to 0.34 µm. Analysis of the kinetics of the dimerization process indicated it followed a two‐step mechanism, with rapid initial association of the two subunits to form an inactive homodimer followed by a slow isomerization step rendering the active enzyme form. The presence of poly(rA)/dT20decreased the equilibrium dissociation constant of the homodimer about 30‐fold, while the addition of 5 µmdTTP had no effect. The kinetics of the process showed that the template/primer favored dimerization by binding to the inactive homodimer and promoting its isomerization to the active form. These results were confirmed by analyzing the reverse reaction, i.e. the dissociation of the enzyme, by dilution in a low‐ionic‐strength buffer. The results suggest that binding of immature HIV‐1 reverse transcriptase to its natural template/primer may be relevant in both the dimerization process and the selection of its natural primer.
Mechanism of porcine pancreatic α‐amylase
Authors: Koukiekolo Roger , Desseaux Véronique , Moreau Yann , Marchis‐Mouren Guy , Santimone Marius
Published: March 2003

The effects of α‐, β‐ and γ‐cyclodextrins on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic α‐amylase (PPA) were investigated. The results of the statistical analysis performed on the kinetic data using the general initial velocity equation of a one‐substrate reaction in the presence of one inhibitor indicate that the type of inhibition involved depends on the substrate used: the inhibition of amylose hydrolysis by α‐, β‐ and γ‐cyclodextrin is of the competitive type, while the inhibition of maltopentaose hydrolysis is of the mixed noncompetitive type. Consistently, the Lineweaver–Burk plots intersect on the vertical axis when amylose is used as the substrate, while in the case of maltopentaose, the intersection occurs at a point located in the second quadrant. The inhibition of the hydrolysis therefore involves only one abortive complex, PPA–cyclodextrin, when amylose is used as the substrate, while two abortive complexes, PPA–cyclodextrin and PPA–maltopentaose–cyclodextrin, are involved with maltopentaose. The mixed noncompetitive inhibition thus shows the existence of one accessory binding site. In any case, only one molecule of inhibitor binds to PPA. In line with these findings, the difference spectra of PPA produced by α‐, β‐ and γ‐cyclodextrin indicate that binding occurs at a tryptophan and a tyrosine residue. The corresponding dissociation constants and the inhibition constants obtained using the kinetic approach are in the same range (1.2–7 mm). The results obtained here on the inhibition of maltopentaose hydrolysis by cyclodextrin are similar to those previously obtained with acarbose as the inhibitor [Alkazaz, M., Desseaux, V., Marchis‐Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100–107], but differ from those obtained with amylose as the substrate and acarbose as inhibitor [Alkazaz, M., Desseaux, V., Marchis‐Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787–796]. It is concluded that the hydrolysis of both long and short chain substrates requires at least one secondary binding site, including a tryptophan residue.
Mutagenesis and modelling of linoleate‐binding to pea seed lipoxygenase
Authors: Hughes Richard K. , Lawson David M. , Hornostaj Andrzej R. , Fairhurst Shirley A. , Casey Rod
Published: March 2003

We have produced a model to define the linoleate–binding pocket of pea 9/13‐lipoxygenase and have validated it by the construction and characterization of eight point mutants. Three of the mutations reduced, to varying degrees, the catalytic centre activity (kcat) of the enzyme with linoleate. In two of the mutants, reductions in turnover were associated with changes in iron‐coordination. Multiple sequence alignments of recombinant plant and mammalian lipoxygenases of known positional specificity, and the results from numerous other mutagenesis and modelling studies, have been combined to discuss the possible role of the mutated residues in pea 9/13‐lipoxygenase catalysis. A new nomenclature for recombinant plant lipoxygenases based on positional specificity has subsequently been proposed. The null‐effect of mutating pea 9/13‐lipoxygenase at the equivalent residue to that which controlled dual positional specificity in cucumber 13/9‐lipoxygenase, strongly suggests that the mechanisms controlling dual positional specificity in pea 9/13‐lipoxygenase and cucumber 13/9‐lipoxygenase are different. This was supported from modelling of another isoform of pea lipoxygenase, pea 13/9‐lipoxygenase. Dual positional specificity in pea lipoxygenases is more likely to be determined by the degree of penetration of the methyl terminus of linoleate and the volume of the linoleate‐binding pocket rather than substrate orientation. A single model for positional specificity, that has proved to be inappropriate for arachidonate‐binding to mammalian 5‐, 12‐ and 15‐lipoxygenases, would appear to be true also for linoleate‐binding to plant 9‐ and 13‐lipoxygenases.