bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of Stereoisomers and Isoforms of a Tryptic Heptapeptide Fragment of Human Growth Hormone and Analysis by Reverse‐Phase HPLC and Capillary Electrophoresis
Authors: Vinther Anders , Holm Arne , Høeg‐Jensen Thomas , Jespersen Anne M. , Klausen Niels K. , Christensen Thorkild , Sørensen Hans H.
Published: August 2004

The amino acid sequence Asn‐Gly has at pH 7 a tendency to induce deamidation of asparagine to aspartic acid via the formation of a cyclic imide. This imide opens up to yield Asp‐Gly or the isoaspartic acid (isoAsp) form, isoAsp‐Gly. Both isomers may be found in their l‐form or d‐form. Like Asn‐Gly, the sequence Asp‐Gly has a tendency for isomerization and racemization via the formation of a cyclic imide intermediate.

When human growth hormone is digested with trypsin, one of the fragments is a heptapeptide (amino acid residues 128–134) containing the amino acid sequence Asp‐Gly (amino acid residues 130 and 131). This heptapeptide, as well as stereoisomers and isoforms where l‐Asp was replaced by d‐Asp, l‐isoAsp, d‐isoAsp or the l‐cyclic imide, respectively, has been synthesized and used as a standard to achieve separation of the five forms by capillary electrophoresis and by reverse‐phase HPLC.

Capillary electrophoresis analysis was performed in uncoated capillaries by the use of aspartic acid/cyclodextrin buffers at low pH. The elution order of the aspartic‐acid‐containing heptapeptides was d‐Asp, asp, l‐isoAsp, d‐isoAsp and l‐cyclic imide. Reverse‐phase HPLC analysis was performed on a C18 column by the use of a shallow acetonitrile gradient in trifluoroacetic acid/water. The elution order was d‐isoasp, l‐isoAsp, l‐asp, d‐Asp and l‐cyclic imide.

Human growth hormone samples were degraded by incubation at high temperature and analyzed for their potential content of isomerization and racemization products. Only l‐forms of aspartic acid and isoaspartic acid of the heptapeptide fragment were found.
Motifs Resembling Hepatocyte Nuclear Factor 1 and Activator Protein 3 Mediate the Tissue Specificity of the Human Plasminogen Gene
Authors: Meroni Germana , Buraggi Giorgio , Mantovani Roberto , Taramelli Roberto
Published: August 2004

Plasminogen is one of the key elements in the fibrinolytic process. Like most of the gene products that participate in such reactions and which interact with plasminogen, the site of its synthesis is mainly confined to the hepatocyte. Plasminogen RNA has additionally been detected in kidney and very low amounts also in testes. Deletional analysis has indicated that two 5′ sequences located within 2.5 kb of the first ATG are responsible for the transcriptional activation and the tissue specificity of the expression of the gene. By DNase protection and gel mobility shift assays with HepG2 nuclear extracts, the two sequences were localized and found to be the recognition sites for the widely known hepatocyte nuclear factor 1 (HNF‐1) a trans‐acting factor, and a nuclear factor like activator protein 3 (AP‐3). The first one lies in a rather unusual position, i.e. within the 5′‐untranslated region. The latter is located further upstream in a region between –2200 and –2100 from the plasminogen mRNA cap site. Moreover, site‐directed mutagenesis coupled by functional experiments in HepG2 cells has demonstrated a synergism between these two positively acting elements in controlling the transcription of the human plasminogen gene.
CTBP1/RBP1, a Saccharomyces Cerevisiae Protein which Binds to T‐Rich Single‐Stranded DNA Containing the 11‐bp Core Sequence of Autonomously Replicating Sequence, is a Poly(Deoxypyrimidine)‐Binding Protein
Authors: Ikeda Masato , Arai Ken‐ichi , Masai Hisao
Published: August 2004

South‐Western screening of a glutathione‐S ‐transferase fusion protein brary constructed from the yeast Saccharomyces cerevisiae genomic DNA lead to isolation of core T‐rich‐strand‐binding protein (CTBP) clones that bound to single‐stranded DNA containing the T‐rich‐strand of the 11‐bp core sequence of autonomously repcating sequences. One of these clones, CTBP1, contains a portion of previously described RBP1 which is an RNA‐binding and single‐stranded DNA‐binding protein of S. cerevisiae. GST‐CTBP1 as well as the full‐length fusion protein with RBP1 (GST‐RBP1) bind exclusively to the T‐rich strand of the core sequence with an apparent dissociation constant of 5 nM, but not to the A‐rich strand or double strand of the same sequence. Mutations within the core which reduce the number of T or C residues decrease the affinity of this protein. In keeping with this, binding of GST‐CTBP1 to the core sequence is efficiently competed by poly(dT), poly(dT‐dC) or poly(dC), but not by poly(dA) or poly(dG) to significant extents. Among polyribonucleic acids, GST‐CTBP1 binds to poly(U) and poly(I) with greatest affinity, whereas GST‐RBP1 binds to RNA in a rather non‐specific manner. In no cases was affinity for RNA greater than that for DNA. Our results indicate that CTBP1/RBP1 is a polydeoxypyri‐midtne‐binding protein of S. cerevisiae. CTBP1 contains two sets of an RNA‐recognition motif (RRM) and a glutamine stretch. The binding affinity of the N‐terminal or C‐terminal set containing one RRM and one glutamine stretch is nearly two orders of magnitude lower than that of the wild‐type CTBP1 containing both sets. The isolated N‐terminal or C‐terminal RRM alone (RRM1 and RRM2, respectively) is sufficient for binding nucleic acids with the binding specificity similar to that of the wild‐type RRM, although the binding affinity of the isolated RRM2 is nearly two orders of magnitude lower than that of RRM1. Our results indicate that the two RRMs present in CTBP1/RBP1 have differential binding affinities and that the high affinity of RRM for polydeoxypyrimidine results from synergy between two lower‐affinity RRMs.
Effects of Major‐Histocompatibility‐Complex‐Encoded Subunits on the Peptidase and Proteolytic Activities of Human 20S Proteasomes
Authors: Ehring Bettina , Meyer Thomas H. , Eckerskorn Christoph , Lottspeich Friedrich , Tampé Robert
Published: August 2004

The proteasome is responsible for the non‐lysosomal degradation of misfolded, transient, or ubiquitintagged proteins. This fact and the identification of two major‐histocompatibility‐complex‐(MHC)‐encoded proteasomal subunits, LMP2/7, suggest an important role of the proteasome in antigen processing. Using purified 20s proteasomes from a wild‐type and a LMP2/7‐deletion T lymphoblastoid cell line, we analyzed the effect of LMP2/7 on the peptidase and proteolytic activities of the complex in the context of various purification and activation methods. The incorporation of LMP2/7 alters the peptidase activity against fluorogenic substrates, but these effects are not reflected in the time‐dependent degradation pattern of oxidized insulin B chain or of peptide epitopes of an antigenic protein. No effect of LMP2/7 on the degradation pattern of these substrates was observed by either reverse‐phase chromatography, pool sequencing, or mass spectrometry. The 20s proteasome can cleave insulin B chain at nearly every position, showing that the P1 position alone does not determine the cleavage sites. The maximum of the length distribution of the end products, makes these ideal candidates for MHC display; yet we find that a natural epitope derived from human histone H3 is further degraded by 20s proteasomes. Alanine scans and substitutions with related amino acids of this epitope indicate that, as in insulin B chain, the cleavage sites are not determined by the P1 position alone.
Reduced B Subunit of Heat‐Labile Enterotoxin Associates with Membranes In vivo
Authors: Hardy Simon J. S. , Hedges Peter A.
Published: August 2004

The B subunit of heat‐labile enterotoxin, a periplasmic protein of Escherichia coli has an internal disulfide bond that forms after the protein has been exported. The presence of 2.5 mM dithiothreitol in the medium prevents the formation of the disulfide bond and this causes the protein to rapidly bind to membranes, preferentially but not exclusively to the cytoplasmic membrane. The binding is irreversible in vivo but chaotropic agents disrupt the association between the non‐native B subunit and the membranes in vitro. The fact that the reduced B subunit binds to both the cytoplasmic and outer membranes that enclose the periplasm suggests that it is exported normally to the periplasm and then, because it is unable to form its native structure, adsorbs to membranes in the vicinity. This is confirmed by the finding that when synthesised by spheroplasts, in which the outer membrane is disrupted, the majority of reduced B subunit, which is not now confined in the periplasm, is exported to the medium and is not associated with membranes.
O‐Glycosylated Species of Natural Human Tumor‐Necrosis Factor‐α
Authors: Takakura‐Yamamoto Rohko , Yamamoto Shigeto , Fukuda Shigeharu , Kurimoto Masashi
Published: August 2004

Tumor‐necrosis factor‐α, produced by human B‐cell lymphoblastoid cell line BALL‐1, was expressed as four protein bands on SDS/PAGE analysis. It may have been glycosylated, based on the fact that the heavier two of the four bands disappeared after neuraminidase treatment. Sugar composition analyses revealed that the tumor necrosis factor‐α contained galactose, N‐acetylgalactosamine and N‐acetylneuraminic acid as sugar components. To prepare sugar chains, tumor necrosis factor‐α was treated with alkaline borodeuteride and the oligosaccharide‐alditols liberated were fractionated by gel‐filtration chromatography on a Bio‐Gel P‐4 column, followed by normal‐phase HPLC. Three oligosaccharide‐alditols were obtained, and the structures of two of them were identified by methylation analysis and exoglycosidase digestion. The structures of these oligosaccharide‐alditols were Galβ1–3(NeuAcα2–6)GalNAcol and Galβ1–3GalNcol (GalNAcol, N‐acetylgalactosaminitol). The structure of the remaining oligosaccharide‐alditol was determined to be NeuAcα2–3Gal1–3GalNAcol by composition and methylation analyses. About 20% of tumor necrosis factor‐α was found to be O‐glycosylated, based on the results of the sugar composition and structure analyses. An amino acid sequence analysis of the glycosylated peptides was performed after Staphylococcus aureus V8 protease digestion of tumor necrosis factor‐α had been completed, and it was proved that the O‐glycosylation site of tumor necrosis factor‐α was Ser 4.
Three Separate Proteins Constitute the Magnesium Chelatase of Rhodobacter Sphaeroides
Authors: Willows Robert D. , Gibson Lucien C. D. , Kanangara C. Gamini , Hunter C. Neil , von Wettstein Diter
Published: August 2004

The insertion of magnesium into protoporphyrin IX is the first step unique to chlorophyll production and is catalyzed by magnesium chelatase. The Rhodobacter sphaeroides genes, bchl and bchD together, and bchH alone, were cloned and expressed with the pET3a vector in Escherichia coli strain BL21(DE3). The 40‐kDa BchI protein was synthesized in greater abundance compared to the 70‐kDa BchD protein when both were expressed together from the same plasmid. The production of large amounts of the 140‐kDa BchH protein in E. coli was accompanied by an accumulation of protoporphyrin IX. The accumulated protoporphyrin IX was bound specifically to BchH in an approximate molar ratio of 1:1. All three recombinant proteins were soluble; BchH was monomeric, BchI was dimeric, while BchD appeared to be polymeric with a molecular mass of approximately 550 kDa. The BchH and BchI proteins were purified to apparent homogeneity while BchD was separated from BchI and partially purified. Magnesium was inserted into protoporphyrin IX and deuteroporphyrin by combining these three proteins in the presence of ATP. One monomer of BchH to one dimer of BchI gave the optimal magnesium chelatase activity and the activity was dependent on the amount of partially purified BchD added to the assay at the optimum BchH:BchI ratio. The reaction was dissected into two parts with an activation step requiring BchI, BchD, and Mg2+‐ATP, and a metal‐insertion step which in addition requires Mg2+, protoporphyrin IX, and BchH. The stoichiometric binding of protoporphyrin IX to BchH in vitro is direct evidence for BchH carrying out such a role in vivo whereas the other two proteins are involved in ATP activation and magnesium insertion.
The Association of Focal Adhesion Kinase with a 200‐kDa Protein that is Tyrosine Phosphorylated in Response to Platelet‐Derived Growth Factor
Authors: Chen Hong‐Chen , Guan Jun‐Lin
Published: August 2004

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase implicated in the signal transduction pathways initiated by integrins. However, we have previously found that platelet‐derived growth factor (PDGF) could stimulate the association of FAK with phosphatidylinositol 3‐kinase in NIH 3T3 cells [Chen, H.‐C. & Guan, J.‐L. (1994) J. Biol. Chem. 269, 31229–31233], suggesting that FAK might participate in some of the cellular effects of the growth factors in modulating cell morphology and migration. In this report, we describe the association of FAK with a 200‐kDa protein (pp200) that is tyrosine phosphorylated in response to PDGF stimulation in NIH 3T3 cells. Although the identity of pp200 is unknown at present, we have excluded the possibilities that it is the PDGF receptor β, tensin, talin, myosin or the guanosine‐triophosphatase activating protein‐associated p190 protein. Furthermore, we found that the tyrosine phosphorylation of FAK‐associated pp200 upon PDGF stimulation is largely independent of cell adhesion or the integrity of the cytoskeleton. Therefore, pp200 and its interactions with FAK may also be involved in growth‐factor‐induced cellular effects such as the modulation of cell adhesion or cell migration via cytoskeleton reorganization or disruption of focal adhesions.
Solid‐state 13C‐NMR of [(3‐13C)Pro]bacteriorhodopsin and [(4‐13C)Pro]bacteriorhodopsin
Authors: Engelhard Martin , Finkler Stefan , Metz Günther , Siebert Friedrich
Published: August 2004

The configuration of an Xaa‐Pro bond can be determined by solid‐state magic‐angle‐sample‐spinning (MASS)‐13C‐NMR spectroscopy since the chemical shifts of Cβand Cγof the proline ring are sensitive to the isomerization state of the preceding peptide bond. (3‐13C)Pro and (4‐13C)Pro have been chemically synthesized; the former by means of an asymmetric synthesis. The 13C‐labeled Pro residues were biosynthetically incorporated into bacteriorhodopsin with a yield of 80%. The solid‐state‐MASS‐13C‐NMR spectra of [(3–13C)Pro]bacteriorhodopsin and [(4‐13C)Pro]bacteriorhodopsin revealed isotropic chemical shifts at 29.8 ppm and 25.5 ppm, respectively. From the chemical‐shift values we conclude that all Xaa‐Pro peptide bonds are in the trans configuration confirming previous results from solution‐NMR studies on solubilized bacteriorhodopsin in organic solvents [Deber, M. C., Sorrell, B. J. & Xu, G.‐Y. (1990) Biochem. Biophys. Res. Commun, 172, 862–869], Inversion‐recovery experiments could differentiate between three classes of Pro residues distinguished by their relaxation time t1. Tentatively, these three distinct groups of Pro residues could be assigned to the helical, the loop, and the C‐terminal parts of the protein. The resonances of the two C‐terminal Pro could be identified by removing the C‐terminus by proteolysis. Although they are separated by only one Glu they occupy different chemical environments and possess different flexibilities. These results indicate that the first part of the C‐terminal tail is constrained. Pro238 marks the position where the tail becomes freely mobile. It is proposed that the C‐terminus is fixed to the membrane via salt bridges between divalent cations and negative charges of the C‐terminus as well as interhelical loops.
Differential Binding of cAMP‐responsive‐element(CRE)‐binding Protein‐1 and Activating Transcription Factor‐2 to a CRE‐Like Element in the Human Tissue‐Type Plasminogen Activator (t‐PA) Gene Promoter Correlates with Opposite Regulation of t‐PA by Phorbol Ester in HT‐1080 and HeLa Cells
Authors: Costa Magdaline , Medcalf Robert L.
Published: August 2004

The human tissue‐type plasminogen activator gene (t‐PA) is induced by the phorbol ester, phorbol 12‐myristate 13‐acetate (PMA), in HeLa cells. Previous studies in transfected HeLa cells identified two cis ‐acting regulatory elements within the t‐PA gene promoter responsible for both constitutive and PMA‐inducible expression. One element differs from the consensus cAMP response element (CRE) by a single nucleotide substitution (referred to in this report as t‐PACRE) and another which bears similarity to the AP‐2 recognition sequence. In HT‐1080 fibrosarcoma cells, t‐PA mRNA levels are expressed at higher constitutive levels and are suppressed by PMA. Nuclear run‐on transcription experiments indicate that PMA‐mediated suppression of t‐PA in these cells is associated with a decrease in t‐PA gene template activity. We designed experiments to determine whether nuclear t‐PACRE or AP‐2‐like binding proteins were differentially expressed in HeLa and HT‐1080 cells and, accordingly, if these could be correlated with the opposite effect of PMA on t‐PA expression. Band shift analyses indicated that the migration profiles of HeLa and HT‐1080 nuclear proteins interacting with the AP‐2‐like site were indistinguishable; however, those produced with the t‐PACRE binding site were qualitatively and quantitatively distinct. The distribution of t‐PACRE binding proteins in these cells was investigated in a supershift assay using specific antibodies against members of the fos/jun and CRE‐binding protein (CREB)/activating transcription factor (ATF) families. In HT‐1080 cells, CREB‐1 was the most prominent t‐PACRE‐binding activity detected and was greatly increased in cells treated with PMA. In contrast, CREB‐1 activity was absent in HeLa cells, but antibodies specific for ATF‐2 produced a marked supershifted complex which was unaffected by PMA treatment. Since CREB‐1 can repress transcription of other target genes (including c‐jun) via association with identical cis ‐acting CRE‐like sequences, we suggest that the mechanism for the transcriptional down‐regulation of t‐PA by PMA in HT‐1080 cells requires CREB‐1 binding to the t‐PACRE while ATF‐2, by associating with the same site, plays a role in PMA‐mediated induction of t‐PA in HeLa cells.
Single‐Step Trypsin Cleavage of a Fusion Protein to Obtain Human Insulin and Its C Peptide
Authors: Jonasson Per , Nilsson Joakim , Samuelsson Elisabet , Moks Tomas , Stårhl Stefan , Uhlén Mathias
Published: August 2004

The kinetics for trypsin cleavage of different fusion proteins, consisting of human proinsulin and two IgG‐binding domains (ZZ), were investigated. To achieve simultaneous removal of the fusion tag and processing of proinsulin to insulin and free C peptide, three versions of the ZZ‐proinsulin fusion protein were generated, having different trypsin‐sensitive cleavage sites, Arg, Lys‐Arg or Lys. The ZZ‐proinsulin fusion proteins which accumulated as inclusion bodies in Escherichia coli cells were solubilized, refolded and purified by IgG affinity chromatography. The yield of ZZ‐proinsulin monomers exceeded 90%. The kinetics for the trypsin cleavage revealed unexpected differences when comparing the three linkers and it was found that the single arginine linker was most efficiently processed. Characterization of the cleavage products by reverse‐phase chromatography, mass spectrometry and N‐terminal sequencing verified that human insulin and C peptide were generated. The results demonstrate that high yields of native insulin, C peptide and affinity tag can be achieved by simultaneous cleavage of a fusion protein at three different trypsin‐sensitive sites in a single step. The implications for production and recovery of various recombinant proteins are discussed.
Structure of the B‐Myb DNA‐binding Domain in Solution and Evidence for Multiple Conformations in the Region of Repeat‐2 Involved in DNA Binding
Authors: Carr Mark D. , Wollborn Ute , McIntosh Pauline B. , Frenkiel Tom A. , McCormick John E. , Bauer Christopher J. , Klempnauer Karl‐Heinz , Feeney James
Published: August 2004

A range of double and triple resonance heteronuclear NMR experiments has been used to obtain nearly complete sequence‐specific 15N, 13C and 1H resonance assignments for a 110‐residue protein corresponding to the B‐Myb DNA‐binding domain (B‐MybR2R3) and to determine its secondary structure in solution. The protein was found to contain two stable helices in repeat‐2 (R2) and Three in repeat‐3 (R3L involving residues K12–K24 (R2‐1), W30‐H36 (K2‐2), E64–V76 (K3‐1), W81–L7 (R3‐2) and D93–K105 (R3‐3). In addition, the chemical shift and nuclear Overhauser effect data suggest that amino acids Q44–W49 near the C‐terminus of R2 form an unstable or nascent helix, which could be stabilised on binding to a specific DNA target site. The two N‐terminal helices in R2 and R3 occupy essentially identical positions in the two domains, consistent with the high level of sequence similarity between these regions. In contrast, the C‐terminal region forming the third helix in R3 shows low sequence similarity with R2, accounting for the differences in secondary structure. In the case of B‐MybR2R3, there is clear chemical shift and line‐broadening evidence for the existence of multiple conformations in the C‐terminal region of R2, which is believed to form one half of the DNA‐binding site. We propose that conformational instability of part of the DNA‐binding motif is a way of increasing the specificity of Myb proteins for a relatively short (6‐bp) DNA target site by reducing their affinity for lion‐specific 13NA sequences compared to specific sites.
Requirements for Maturation of Bradyrhizobium japonicum Cytochrome c550 in Escherichia coli
Authors: Thöny‐Meyer Linda , Künzler Peter , Hennecke Hauke
Published: August 2004

Various forms of Bradyrhizobioum japonicum cytochrome c550, (the cycA gene product) were overexpressed in Escherichia coli cells grown under different conditions. Antibodies directed against a synthetic cytochrome c550 peptide were used as tools to detect both, apoprotein and holoprotein. Complete maturation of the apoprotein into its holo from with haem covalently bound to the polypeptide was observed only under anaerobic growth conditions and in E. coli K12 derivatives, whereas there binding did not occur in the E. coli EL21 host. When maturation was complete, holocytochrorme c550 was found exclusively in the periplasmic fraction. A cycA ‐expressing plasmid construct lacking the genetic information for the signal sequence produced apoprotein that wits rapidly degraded without further maturation. Mutations in the haem‐binding site resulted in products that were translocated through the cytoplasmic membrane, but apparently became degraded. Our results support the view that attachment of haem to the apoprotein is not a prerequisite for cleavage of the signal sequence and occurs on the periplasmic side of the membrane, subsequent to translocation of the apoprotein precursor.
ATP Synthesis by the F0F1 ATP Synthase from Thermophilic Bacillus PS3 Reconstituted into Liposomes with Bacteriorhodopsin
Authors: Pitard Bruno , Richard Peter , Duñarach Mireia , Girault Guy , Rigaiud Jean‐Louis
Published: August 2004

Optimal conditions for the reconstitution of bacteriorhodopsin and H+‐transporting ATP synthase from thermophilic Bacillus PS3 (TF0F1) were determined. Phosphatidylcholine/phosphatidic acid liposomes prepared by reverse‐phase evaporation were treated with various amounts of Triton X‐100, octyl glucoside, octaethylcne glycol n‐dodecylether, sodium cholate or sodium deoxycholate and the incorporation of proteins by these detergents was studied at each step of the solubilization process. After removal of detergent by means of SM‐2 Bio‐Beads, the light‐driven ATP synthase activities of the resulting proteoliposomes were analyzed at 40°C. The nature of the detergent used for reconstitution wits important for determining the mechanism of protein insertions. The most efficient reconstitutions were obtained with octyl glucoside or Triton X‐100 by insertion of the proteins into detergent‐saturated liposomes.

The conditions for reconstitution were further optimized with regard to functional coupling between bacteriorhodopsin and TF0F1. It was demonstrated that one of the main factors limiting the production of efficient reconstituted proteoliposomes was related to activation of the highly stable TF0F1. Activation was accomplished by total solubilization of phospholipids and proteins in a Triton X‐100/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold slii1iulation of the ATP synthase activities. Final ATP synthase activities depended greatly on the lipid/bacteriorhodopsin and the lipid/TF0F1 ratios as well as on the phospholipid used. In particular, light‐driven ATP synthesis depended upon the presence of negatively charged phospholipids. Cholesterol was found to induce a fourfold increase in ATP synthase activity with a concomitant 65 % decrease in the Km for ADP, suggesting that sterols can modulate catalytic events mediated by F1.

Preparations obtained by this step‐by‐step reconstitution procedure displayed activities up to 20‐fold higher (500–800 nmol ATP. min‐1 mg TF0F1−1 in the presence of cholesterol) than the maximal values reported in the literature for light‐driven ATP synthesis by TF0F1 measured under similar conditions. This study also allowed rationalization of the different parameters involved in reconstitution experiments and the present simple method is shown to be of general use for preparation of efficient proteoliposomes containing bacteriorhodopsin and choloroplast or mitochondrial F0F1‐type ATP synthases.
NMR Studies of the Mode of Binding of Corepressors and Inducers to Escherichia coli Trp Repressor
Authors: Ramesh Vasudevan , Syed Shabih E. H. , Frederick Ronnie O. , Sutcliffe Michael J. , Barnes Maria , Roberts Gordon C. K.
Published: August 2004

The binding of the corepressors tryptophan and 5‐methyltryptophan and of the inducers 3‐indolepropionate, 3‐indoleacrylate and 5‐methylindole to the Escherichia coli trp repressor have been studied by 1H‐NMR spectroscopy. Identification of the resonances of the protons of bound ligands and their NOEs to protons of the protein (measured as transferred NOE) was greatly facilitated by the use of samples of the protein in which the hydrogens of all residues except alanine, isoleucine and threonine were replaced by deuterium. Chemical‐shift changes of protein‐backbone resonances and side‐chain‐amide resonances on ligand binding were measured with generally or selectively 15N‐labelled protein. The patterns of changes in the chemical shifts of protein resonances and, particularly, ligand resonances distinguish the corepressors from the inducers, indicating, in agreement with earlier work, that corepressors and inducers bind to the protein in different ways. The NOEs observed for the bound ligands have been used to determine the position of the ligands in the crystallographically determined binding site, by means of a simulated‐annealing molecular‐dynamics protocol. The structures obtained show that the orientation in the binding site of the indole rings of tryptophan and 5‐mcthyltryptophan and of 3‐intlolepropionate and 3‐indoleacrylate differ by approximately 180° in solution (in agreement with the crystallographic data for complexes of the trp repressor with tryptophan or with 3‐indolepropionate). The value and limitations of calculating ligand positions based on transferred NOE are discussed.
Purification of a 60‐kDa Protein from Chicken Liver Associated with the Internal Nuclear Matrix and Closely Related to Carboxylesterases
Authors: Altieri Fabio , Maras Bruno , Ferraro Anna , Turano Carlo
Published: August 2004

A 60‐kDa protein was purified from chicken liver internal nuclear matrix and its nuclear localization was confirmed by immunofluorescence analysis. Structural information acquired from sequence analysis of the intact protein and of fragments obtained from enzymatic and chemical cleavages strongly suggests that it belongs to the carboxylesterases family, even if with some very peculiar features. The N‐terminal sequence of the 60‐kDa protein is completely different from the other carboxylesterases, but is similar to a region that is normally internal to all mammalian esterase sequences and localized after the serine residue at the active site. This suggests that the protein may be derived from a gene duplication and/or rearrangement. Since the 60‐kDa protein shows a low esterase activity of about 0.2 μmol · min−1· mg−1using either p‐nitrophenyl acetate or p‐nitrophenyl butyrate as substrates, it is not possible to rule out that the protein shares only a sequence similarity with carboxylesterases and is not a true esterase. Otherwise it could be an esterase which has developed different properties, i.e. a special substrate specificity, the requirement of additional factors or a different stability in solution. In the latter case, this protein could be related to the physiological control of hydrolysis of exogenous and endogenous esters which can act on nuclear functions and/or metabolism.
The Defective Secretion of a Naturally Occurring α‐1‐Antichymotrypsin Variant with a Frameshift Mutation
Authors: Tsuda Michio , Sei Yukari , Ohkubo Tomoichi , Yamamura Masaichi , Kamiguchi Hiroshi , Akatsuka Akira , Tsuda Toshio , Tachikawa Hiroshi , Yamamoto Masahiro , Shinohara Yukito
Published: August 2004

A newly found variant α‐1‐antichymotrypsin (ACT), ACT Tsehara‐2, has a deletion of two bases (AA) at codon 391 near the carboxyl terminus. This frameshift mutation caused a change in the amino acid sequence and generated 10 extra amino acids (408 amino acids total) [Tsuda, M., Sei, Y., Matsumoto, M., Kamiguchi, H., Yamamoto, Y., Shinohara, Y., Igarashi, T., & Yamamura, M. (1992) Hum. Genet. 91, 467–468]. The serum ACT levels in three unrelated heterozygotes with this mutant ACT gene were 37%, 49% and 54% that of the normal individuals. To examine the reduced serum levels, the normal ACT and the mutant ACT created by site‐directed mutagenesis were transfected into COS‐7 cells for comparison. The value for the retention rate (intracellular ACT/total ACT) was apparently higher in the cells expressing mutant ACT Isehonr‐2 than those bearing the normal gene. In the pulse‐chase experiments, the secretion of the synthesized mutant ACT into the medium was riot observed, whereas the normal ACT was mostly secreted as a 64‐kDa form. The endoglycosidase H digestion and an electron microscopic analysis indicated that the retained mutant ACT was present in the endoplasmic reticulum. These results provide the biochemical basis for the decreased serum ACT level of individuals with ACT Isehara‐2, and suggest the importance of the carboxyl‐terminal region for its secretion.
Enzymic Characterization of Fission Yeast Farnesyl Transferase
Authors: Danjoh Inaho , Fujiyama Asao
Published: August 2004

The enzyme farnesyl transferase (FTase) catalyzes the posttranslational modification of Ras and other Ras family proteins with a C15 farnesyl group. The target proteins have a consensus‐CAAX motif (X, any amino acid except leucine) at the C‐terminus. Since proteins that have leucine as the C‐terminal amino acid X are modified with a C20 geranylgeranyl group, it is thought that the C‐terminal leucine is the signal (‐CAAL motif) for selection of isoprenoid molecules. Here, we report the presence of multiple FTase activities in the fission yeast Schizosaccharomyces pombe, each seeming to correspond to a particular protein known to be modified by the farnesyl group in vivo. Using enzymic activities specific to S. pombe Ras1, we found similar affinities for FTases in the wild‐type (EVSTKCCVIC) and mutant Ras1 peptide, in which the C‐terminal amino acid is replaced by leucine (EVSTKCCVIL). These results suggest that recognition and selection of the correct isoprenoid group by the FTases require other amino acid sequences of the target protein in addition to the C‐terminal‐CAAX motif.
CMP–3‐Deoxy‐d‐Glycerol‐d‐Galacto‐Nonulosonic Acid (CMP‐Kdn) Synthetase
Authors: Terada Takaho , Kitajima Ken , Inoue Sadako , Koppert Klaus , Brossmer Reinhard , Inoue Yasuo
Published: August 2004

In this report we present kinetic data of the activation reaction of several synthetic 3‐deoxy‐d‐glycero‐d‐galacto‐nonulosonic acid (Kdn) and N‐acetylneuraminic acid (Neu5Ac) analogues catalyzed by the rainbow trout testis CMP‐Kdn synthetase. This enzyme showed broad substrate specificity in terms of substitutions at C4 or C5 position of Kdn and Neu5Ac. In contrast, calf brain CMP‐N‐acylneuraminic acid synthetase had narrow substrate specificity, being active only on various N‐acyl analogues of Neu5Ac and only slightly active on Kdn derivatives.

Usefulness of the trout testis enzyme for synthesis of various CMP‐sialate analogues, which could be donor substrates for sialyltransferases, was demonstrated.
Determinants in the Presequence of Cytochrome b2 for Import into Mitochondria and for Proteolytic Processing
Authors: Klaus Christian , Guiard Bernard , Neupert Walter , Brunner Michael
Published: August 2004

Determinants in a mitochondrial targeting signal for import and processing were analyzed by introducing deletions into the presequence of cytochrome b2. The matrix targeting signal and the signal recognized by the mitochondrial processing peptidase were found to be separate. The signal for import into the matrix is located at the N‐terminus within a stretch of 20 amino acid residues that has the potential to form a positively charged, amphipathic α‐helix. The mitochondrial processing peptidase cleaves after residue 31 and recognizes a short sequence motif around the scissile bond. In the context of a presequence, the cleavage site is accessible for the processing peptidase. At a different location or in a different context, the cleavage site motif is still specifically recognized but processed with lower efficiency. The matrix targeting signal may help to present the cleavage site motif to the mitochondrial processing peptidase.
Properties of the Recombinant β Subunit of Glutamate Synthase
Authors: Vanoni Maria A. , Verzotti Enrico , Zanetti Giuliana , Curti Bruno
Published: August 2004

Glutamate synthase is a complex iron‐sulfur flavoprotein containing one molecule each of FAD and FMN and three distinct iron‐sulfur centers/αβ protomer. Production of the β subunit was observed in total extracts of Escherichia coli BL21 (DE) cells harbouring a pT7–7 derivative carrying gltD, the gene encoding the Azospirillum brasilense glutamate synthase β subunit. The protein was soluble, and the identity of the purified protein with the Azospirillum glutamate synthase β subunit was confirmed by N‐terminal sequence analysis. The kinetic and spectroscopic characterization of the glutamate synthase β subunit confirmed that it contains the NADPH binding site, but, in contrast with earlier proposals that assigned both FAD and FMN binding sites to the α subunit of glutamate synthase, the β subunit was shown to contain stoichiometric amounts of FAD. No iron‐sulfur centers were detected by EPR spectroscopy measurements of the recombinant β subunit. Under steady‐state conditions, the glutamate synthase β subunit can catalyze the NADPH‐dependent reduction of several synthetic electron acceptors but no glutamate synthase or glutamate dehydrogenase reactions in either direction. These results are in agreement with previous data from our laboratory and, together with the absence of amino acid sequence similarity between glutamate synthase β subunit and glutamate dehydrogenases, are against the hypothesis that glutamate synthase is evolutionarily derived from β the association of an ancestral glutamate dehydrogenase (the β subunit) and an amidotransferase (the α subunit). The protein‐bound FAD is reduced by NADPH at a rate much faster than turnover with synthetic electron acceptors, leading to formation of a stable reduced flavin‐NADP+ charge‐transfer complex. The rate of reduction of the bound FAD by NADPH is also similar to the rate at which one of the flavins is reduced in the native glutamate synthase, as measured in a stopped‐flow spectrophotometer under pre‐steady‐state conditions. The ability of FAD bound to the β subunit of glutamate synthase to react with NADPH and the lack of reactivity with sulfite lead us to conclude that FAD is Flavin 1 of glutamate synthase [Vanoni, M. A., Edmondson, D. E., Zanetti, G. & Curti, B. (1992) Biochemistry 31, 4613–46237.
Influence of Expression System on Chromophore Binding and Preservation of Spectral Properties in Recombinant Phytochrome A
Authors: Gärtner Wolfgang , Hill Christiane , Worm Karl , Braslavsky Silvia E. , Schaffner Kurt
Published: August 2004

N‐Terminal deletion mutants of the plant photoreceptor phytochrome, additionally truncated at two different positions at their C‐terminal ends, were expressed both in Escherichia coli and in yeast (Pichia pastoris) and converted into chromoproteins upon chromophore incorporation. The start and end positions of the cDNA employed (phyA from oat) mimic the positions of tryptic cleavage (deletion of the first 64 amino acids, and stop codons after amino acid positions 425 or 595, generating 39‐kDa and 59‐kDa peptides, respectively). The absorption properties and photochromicity upon red/far‐red irradiation of these mutants were compared with their tryptic counterparts derived from native oat phytochrome and with recombinant products possessing intact N‐termini, but C‐terminal positions identical to those of the corresponding tryptic fragments (45‐kDa and 65‐kDa peptides). All recombinant 65‐kDa and 59‐kDa peptides bound the chromophore after expression and showed the appropriate absorption spectra of the Pr and the Pfr forms. The smaller chromopeptides (45‐kDa and 39‐kDa) behaved differently depending on the expression system employed. E. coli‐derived peptides exhibited a phytochrome‐like difference spectrum only when the intact N‐terminus was present (45‐kDa product). The recombinant 39‐kDa pep‐tide from E. coli was incapable of chromophore binding whereas the identical peptide sequence expressed by P pastoris formed a chromoprotein with phycocyanobilin. This recombinant phytochrome fragment exhibited a difference spectrum (Pr–Ppr) with an even larger Pfr absorption band than the comparable tryptic 39‐kDa fragment. Selectivity of chromophore incorporation and spectral properties suggest that interactions between protein domains of phytochrome control the protein folding and the Pr/Pfr absorption characteristics. Evidently, trypsin digestion down to the 39‐kDa fragment affects protein conformation also in terms of Pfr conservation.
Insulin‐Induced GLUT4 Recycling in Rat Adipose Cells by a Pathway Insensitive to Brefeldin A
Authors: Kono‐Sugita Eri , Satoh Shinobu , Suzuki Yoichi , Egawa Masato , Udaka Naoko , Ito Takaaki , Sekihara Hisahiko
Published: August 2004

Brefeldin A induces a rapid redistribution of coat proteins associated with clathrin‐coated vesicles that bud from the trans‐Golgi network but not of those associated with vesicles derived from the plasma membrane. Its effect on glucose transport and the subcellular distribution of the glucose transporter GLUT4 were therefore investigated in the absence or in the presence of insulin in order to characterize the mechanism of insulin‐induced GLUT4 recycling. Exposure of rat adipose cells to brefeldin A in the absence of insulin resulted in a slight increase both in 3‐O‐methy‐d‐glucose transport and in the amount of GLUT4 in the plasma membrane as determined by immunoblot analysis. In contrast, it had no effect on the insulin‐induced increase in 3‐O‐methyl‐glucose transport and plasma‐membrane‐associated GLUT4. Immunohistochemical analysis revealed clathrin immunoreactivity in the juxtanuclear region and in the cell periphery, and the staining in the perinuclear region decreased after exposure of cells to 35 μM brefeldin A. However, it had no effect on the basal pattern of GLUT4 staining or on the insulin‐induced redistribution of GLUT4 staining to the cell periphery. These results suggest that basal recycling of GLUT4 responsible for maintaining the intracellular location of the transporter may include a membrane budding step sensitive to brefeldin A, possibly associated with clathrin‐coated vesicles, but that the insulin‐stimulated GLUT4 recycling pathway is mediated predominantly by a brefeldin A‐insensitive mechanism.
Structure of the O‐Specific Polysaccharide of an Aeromonas Trota Strain Cross‐Reactive with Vibrio Cholerae O139 Bengal
Authors: Knirel Yuriy A. , Senchenkova Sof'ya N. , Jansson Per‐Erik , Weintraub Andrej , Ansaruzzaman Mohammed , Albert M. John
Published: August 2004

The O‐specific polysaccharide of an Aeromonas trota strain was isolated by hydrolysis of the lipopoly‐saccharide at pH 4.5 followed by gel‐permeation chromatography and found to consist of hexasaccharide repeating units containing D‐galactose, L‐rhamnose, 3,6‐dideoxy‐L‐xylo ‐hexose (colitose, Col), 2‐acet‐amido‐2‐deoxy‐D‐glucose and 2‐acetamido‐2‐deoxy‐D‐galactose in the ratios 1:1:2:1:1. Partial hydrolysis of the polysaccharide with 48% hydrofluoric acid resulted in selective removal of colitose to give a modified polysaccharide containing the other four sugar constituents. On the basis of methylation analysis and NMR spectroscopic studies of the initial and modified, colitose‐free polysaccharide, it was concluded that the repeating unit of the O‐specific polysaccharide has the following structure

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The known cross‐reactivity between the strain studied and Vibrio cholerae 0139 Bengal is substantiated by the presence of a common colitose‐containing epitope shared by the O‐specific polysaccharide of A. trota and the capsular polysaccharide of V. cholerae, which is thought to carry determinants of O‐specificity.
Association of the Cytoplasmic Domain of Intercellular‐Adhesion Molecule‐1 with Glyceraldehyde‐3‐Phosphate Dehydrogenase and β‐Tubulin
Authors: Federici Christian , Camoin Luc , Hattab Maurice , Strosberg Arthur Donny , Couraud Pierre‐Olivier
Published: August 2004

To elucidate the molecular mechanisms of the transendothelial migration of leukocytes, we attempted to identify the cellular proteins capable of interaction with the cytoplasmic domain of the intercellular adhesion molecule‐1 (ICAM‐1) in a rat brain microvessel endothelial cell line (RBE4 cells). A 27‐amino‐acid synthetic peptide, corresponding to the cytoplasmic domain of rat ICAM‐1, was covalently linked to a Sepharose matrix. Upon affinity chromatography of RBE4 cell cytosol, several ICAM‐1‐interacting proteins were specifically eluted by the soluble peptide. Two of these proteins have been identified by microsequencing as the cytoskeletal protein β‐tubulin and the glycolytic enzyme glyceraldehyde‐3‐phos‐phate dehydrogenase (GraP‐DH). Experiments carried out with purified GraP‐DH or CNBr fragments of GraP‐DH indicated that binding to the ICAM‐1 matrix was mediated by the C‐terminal domain of GraP‐DH, containing the binding site of the cofactor NATL+, and that NATL+ could compete with this binding. Using a series of ICAM‐1 C‐terminal truncated peptides, we could demonstrate that (a) the nitric‐oxide‐induced covalent linkage of NATL+ to GraP‐DH was impaired by these peptides, (b) the glycolytic activity of GraP‐DH was drastically inhibited by a truncated peptide containing the 15 C‐terminal residues, (c) nitric oxide appeared to prevent this inhibition.

Together, our results demonstrate that GraP ‐DH specifically associates with the isolated ICAM‐1 cytoplasmic domain. Since GraP ‐DH is known as a microtubule bundling protein, these findings suggest that, in a cellular environment, GraP‐DH may behave as an Actaptor molecule by linking ICAM‐1 to the microtubule network. The role of nitric oxide in the modulation of this interaction deserves further investigation.
Conserved Regulation of the Hansenula Polymorpha MOX Promoter in Saccharomyces Cerevisiae Reveals Insights in the Transcriptional Activation by Adrlp
Authors: Pereira Goncalo G. , Hollenberg Cornells P.
Published: August 2004

The Hansenula polymorpha MOX gene encodes a peroxisomal enzyme that catalyzes the first step of the highly specialized methanol metabolism. MOX is strongly transcribed in cells growing in methanol and completely repressed in glucose. We show here that the MOX promoter confers a glucose‐repressible expression upon a lacZ reporter gene in Saccharomyces cerevisiae, an unrelated yeast species that lacks the methanol metabolism. Repression was mediated by a 200–bp region of the MOX promoter, termed MOX‐B, and was counteracted by Adr1p, a transcription factor involved in the derepression of S. cerevisiae genes encoding peroxisomal proteins, the class to which MOX belongs. Binding of Adr1p to MOX‐Bwas demonstrated by gel retardation and DNasel‐footprinting, and Adr1p was shown to interact with a DNA region containing only a half of the putative Adr1p consensus binding site. Our findings suggest that Adrlp is a conserved regulator for genes encoding peroxisomal proteins at least in other yeast species, and that its interaction with the DNA is dependent on the promoter context.
Secondary Structural Elements as a Basis for Antibody Recognition in the Immunodominant Region of Human Immunodeficiency Viruses 1 and 2
Authors: Markert Robert L. M. , Ruppach Horst , Gehring Stephan , Dietrich Ursula , Mierke Dale F. , Köck Matthias , Rübsamen‐waigmann Helga , Griesinger Christian
Published: August 2004

Synthetic peptide antigens corresponding to the entire third variable region V3, the principal neutralizing determinant of the human immunodeficiency virus (HIV) envelope glycoprotein of HIV‐1 subtype B (1), HIV‐2 subtype A (5), and HIV‐2 subtype B (7) were synthesized by solid‐phase peptide synthesis (Table 1).1and5were also prepared as their GlcNAc‐glycosylated forms at the natural N‐glycosylation site NXT (positions 6–8; peptides4and6). Additionally, the proposed β‐turn region of 1 (GPGR; positions 15–18) was altered by introducing D‐Ala17 (2) and D‐Pro16 (3). All compounds have been studied by two‐dimensional NMR techniques. Interproton distances and3JNH/Hαcoupling constants derived from NMR data are used as restraints in distance geometry and ENSEMBLE‐Distance and angle‐bound driven dynamics calculations. The simulations led to disordered conformations except for a high propensity of a βII‐turn in the region GPXR (positions 15–18) in1,2, and4. In3(G‐D‐ProGR, positions 15–18), a type βII‐turn was mainly found instead. For peptide7, the consensus sequence of HIV‐2 subtype B, a type βrturn was also found although the primary structure (VSGL; positions 15–18) differs grossly from the HIV‐1 peptide1. With the exception of2, all βIIturns were able to form a canonically opened β‐turn by a 180° rotation of φ(G17). Surprisingly, compounds5and6that are highly similar to7showed no βIItype turn within MSGL (positions 15–18). They form a type βVIII‐turn across the tetrapeptide SGLV (positions 16–19) together with a non‐canonical turn conformation across LMSG (positions 14–17) leading to an S‐conformation.

The reaction of the peptides with HIV‐positive sera from patients infected with different subtypes of HIV‐1 and HIV‐2 was tested in enzyme‐linked immunosorbent assays (ELISA reactions). No HIV‐2 sera reacted with peptide1and no HIV‐1 sera showed reactivity to peptide5. We propose that certain amino acid exchanges within the V3 domain lead to altered conformations of the V3 loop resulting in antibodies that show altered binding properties to the peptide antigens used in the ELISA reactions.