bluuebunny/crossref_metadata_2025 huggingface dataset:
Urate oxidase is imported into peroxisomes recognizing the C‐terminal SKL motif of proteins
Authors: MIURA Satoshi , ODA Toshiaki , FUNAI Tsuneyoshi , ITO Masaki , OKADA Yoshiie , ICHIYAMA Arata
Published: March 2005

Rat liver urate oxidase synthesized from cDNA through coupled transcription and translation was incubated at 26°C for 60 min with purified peroxisomes from rat liver. Urate oxidase was efficiently imported into the peroxisomes, as determined by resistance to externally added proteinase K. The amount of imported urate oxidase increased with time and the import was temperature dependent. A synthetic peptide composed of the C‐terminal 10 amino acid residues of acyl‐CoA oxidase (the C‐terminal tripeptide is Ser‐Lys‐Leu) inhibited the import of urate oxidase, whereas other peptides, in which the C‐terminal Ser‐Lys‐Leu (SKL) sequence was deleted or mutated, were not effective. Two mutant urate oxidase proteins in which the C‐terminal Ser‐Arg‐Leu (SRL) sequence was deleted or mutated to Ser‐Glu‐Leu (SEL) were not imported into peroxisomes. With substitution of a lysine residue for arginine in the SRL tripeptide at the C‐terminus the import activity was retained. These results show that urate oxidase is imported into peroxisomes via a common pathway with acyl‐CoA oxidase, and that the C‐terminal SRL sequence functions as a peroxisomal‐targeting signal.
Mast cell proteinases activate precursor forms of collagenase and stromelysin, but not of gelatinases A and B
Authors: LEES Martyn , TAYLOR David J. , WOOLLEY David E.
Published: March 2005

Mast cell activation in vivo is often associated with areas of oedema and connective‐tissue degradation. Tryptase and chymase are the major serine proteinases released by mast cells, but they appear to have little activity on most components of the extracellular matrix. The matrix metalloproteinases (MMP) are purported to degrade almost all connective tissue elements and are secreted by cells in the form of inactive precursors. Since the mechanisms of MMP activation in vivo are poorly understood we have examined the potential of mast cell proteinases to activate the precursor forms of human collagenase (MMP‐1), stromelysin (MMP‐3), gelatinase A (MMP‐2) and gelatinase B (MMP‐9).

Mast cell proteinases prepared from purified dog mastocytoma cells were shown to process and activate purified precursor forms of both MMP‐1 and MMP‐3. Using antipain and chymostatin, inhibitors for tryptase and chymase, respectively, it was demonstrated that both pMMP‐1 and pMMP‐3 were effectively processed and activated by the chymase component. By contrast, tryptase activated only pMMP‐3. The mast cell proteinases were unable to process or activate purified precursor forms of MMP‐2 and MMP‐9. However, MMP‐3 previously activated by mast cell proteinases was shown to activate pMMP‐9, but not pMMP‐2. Since we have no evidence that mast cells express these four metalloenzymes, the release of mast cell serine proteinases following activation/degranulation could contribute to local metalloproteinase activation and subsequent matrix degradation.
Involvement of Src‐homology/collagen (SHC) proteins in signaling through the insulin receptor and the insulin‐like‐growth‐factor‐I‐receptor
Authors: GIORGETTI Sophie , PELICCI Pier Giuseppe , PELICCI Guilana , VAN OBBERGHEN Emmanuel
Published: March 2005

Src homology/collagen (SHC) proteins are thought to participate in signaling through both receptor tyrosine kinases, such as the insulin receptor and the EGF (epidermal growth factor) receptor, and cytoplasmic tyrosine kinases, such as v‐src and v‐fps. Here we approached the insulin‐induced and the insulin‐like‐growth‐factor‐I‐induced (IGF‐I‐induced) phosphorylation of SHC proteins, and the possible role of these proteins in insulin and IGF‐I signaling. First, we showed that SHC proteins are phosphorylated on tyrosine residues upon insulin and IGF‐I treatment of fibroblasts transfected with a SHC cDNA construct. More important, ligand‐activated insulin and IGF‐I receptors phosphorylate SHC proteins in vitro, indicating that SHC proteins could be direct substrates for insulin and IGF‐I receptors. Further, insulin or IGF‐I treatment of SHC‐transfected fibroblasts leads to immunoprecipitation of SHC proteins with insulin‐receptor substrate 1 (IRS‐1).

We next looked at the possible effect of SHC proteins on biological responses in SHC‐transfected fibroblasts. We found that the expression of exogenous SHC proteins results in an increased basal MEK (MAPK/ERK‐activating kinase) activity. Further, neither the basal nor the insulin‐induced or IGF‐I‐induced PtdIns‐3‐kinase activity were modified by expression of exogenous SHC proteins. These results illustrate that SHC proteins are implicated in the MAP(mitogen‐activated protein)‐kinase pathway, but not in that of PtdIns‐3‐kinase. Finally, we show that SHC‐transfected cells, unlike control cells, are able to advance into the early phases of the cell cycle, and are more sensitive to the growth‐promoting effect of insulin.

In conclusion, SHC proteins are substrates for insulin and IGF‐I receptors, and would appear to function as early post‐receptor signaling components.
Influence of M subunit Thr222 and Trp252 on quinone binding and electron transfer in Rhodobacter sphaeroides reaction centres
Authors: STILZ Hans Ulrich , FINKELE Ulrich , HOLZAPFEL Wolfgang , LAUTERWASSER Christoph , ZINTH Wolfgang , OESTERHELT Dieter
Published: March 2005

M subunit Trp252 is the only amino acid residue which is located between the bacteriopheophytin HA and the quinone QA in the photosynthetic reaction centre of Rhodobacter sphaeroides. Oligodeoxynucleotide‐directed mutagenesis was employed to elucidate the influence of this aromatic amino acid on the electron transfer between these two chromophores. For this, M subunit Trp252 was changed to tyrosine or phenylalanine, and Thr222, which presumably forms a hydrogen bridge to the indole ring of M subunit Trp252, to valine. In all three mutated reaction centres, the electron‐accepting ubiquinone QA is less firmly bound to its binding site than in the wild‐type protein. The electron transfer from the reduced bacteriopheophytin HA− to QA proceeds in the wild‐type and in the mutant ThrM222Val within 220 ps. However, in the mutants TrpM252Tyr and TrpM252Phe the time constants are 600 ps and 900 ps, respectively. This indicates that M subunit Trp252 participates in the binding of QA and reduction of this quinone.
Enzymic O‐sulfation of tyrosine residues in hirudins by sulfotransferase from Eubacterium A‐44
Authors: MURAMATSU Ryo , NUKUI Eriko , SUKESADA Akiko , MISAWA Satoru , KOMATSU Yasuhiko , OKAYAMA Toru , WADA Koichi , MORIKAWA Tadanori , HAYASHI Hideya , KOBASHI Kyoichi
Published: March 2005

The enzymic O‐sulfation of Tyr residues in a recombinant hirudin variant‐1 (rHV‐1) and its analog in which Glu61 and Glu62 were replaced by Tyr, [E61Y, E62Y]rHV‐1, was carried out by use of sulfotransferase isolated from an anaerobic bacterium from the human intestine, Eubacterium A‐44. Although rHV‐1 was not sulfated by this enzyme, the sulfation of [E61Y, E62Y]rHV‐1 was observed, and three kinds of sulfated analog, whose C‐terminal six amino acid residues were ‐PYY(SO3H)YLQ, ‐PYYY(SO3H)LQ, and ‐PYY(SO3H)Y(SO3H)LQ, were obtained. Among the sulfated hirudin analogs tested here, the Tyr62 and Tyr63 bisulfated [E61Y, E62Y]rHV‐1 showed the strongest thrombin inhibition with the inhibition constant (Ki) of 0.0430 pM, followed by the Tyr63 monosulfated analog (Ki= 0.0593 pM) and the Tyr62 monosulfated one (Ki= 0.158 pM). The Tyr63 monosulfated analog and Tyr62 and Tyr63 bisulfated one were more potent inhibitors of thrombin than unsulfated rHV‐1. The increase in affinity caused by sulfation was predominantly due to an increase in the association‐rate constant.
Isolation and refolding of a mutant methionine‐free interleukin‐2‐receptor α chain synthesized as a fusion protein in Escherichia coli
Authors: SEIPELT Irene , ENGELS Joachim W.
Published: March 2005

A soluble domain of the interleukin(IL)‐2 receptor, the α chain synthesized in Escherichia coli, was employed to study expression and refolding of the protein. The results showed that it is possible to obtain biologically active synthetic methionine‐free IL‐2 receptor α chain (synIL‐2Rα) after BrCN cleavage and renaturation of the crude cleavage material, although the α chain is expressed as a deglycosylated, methionine‐free protein. The soluble receptor comprises amino acids 1–219 and forms 5 disulfide bonds in its biologically active state. Biological activity has been analysed by affinity chromatography and ELISA with mutant [Ala125]IL‐2 and monoclonal antibodies as ligands.

Renaturation yield is limited mainly by the high aggregation rate of incorrectly folded protein. Aggregation could be limited by varying the oxidation conditions.

The deletion of a non‐bridging cysteine at position 192 in the synIL‐2Rα did not affect the renaturation yield of the receptor protein. Additionally a cysteine‐free and methionine‐free β‐galactosidase derivative was fused to the soluble synIL‐2Rα derivatives to prevent reoxidation of incorrect disulfide bonds in the crude BrCN‐cleavage material. It is suggested that cysteine impurities from cyanogen‐bromide‐cleaved peptides might interfere seriously with the refolding process of the synthetic IL‐2 receptor α‐subunit.
Expression, purification and functional properties of a soluble form of Bradyrhizobium japonicum TlpA, a thioredoxin‐like protein
Authors: LOFERER Hannes , HENNECKE Hauke
Published: March 2005

The TlpA protein of Bradyrhizobium japonicum was previously identified genetically as a membrane‐anchored, periplasmic thioredoxin‐like protein. Here we describe the heterologous expression in Escherichia coli, subsequent purification and biochemical characterization of TlpA. A soluble form of TlpA, which lacks its N‐terminal membrane anchor, was overexpressed in E. coli and purified by a two‐step procedure. Pure TlpA was shown to be a monomer in solution and was active in reducing the disulfides of insulin and in reactivating reduced, denatured RNaseA. Evidence is presented that two non‐active‐site cysteine residues form an intramolecular disulfide bond, a feature that is not normally found in other prokaryotic thioredoxins.
The sulfoxide of thymosin β4 almost lacks the polymerization‐inhibiting capacity for actin
Authors: HEINTZ Daniela , REICHERT Andreas , MIHELIC‐RAPP Mirna , STOEVA Stanka , VOELTER Wolfgang , FAULSTICH Heinz
Published: March 2005

Thymosin β4 (Tβ4), a peptide of 43 amino acids, binds to actin monomers and inhibits filament formation. In preparations of Tβ4 from bovine lung tissue, the peptide is accompanied by a derivative in which the methionine residue in position 6 is replaced by its sulfoxide. Tβ4 sulfoxide inhibits actin polymerization to an extent approximately 20‐times less than Tβ4. While an equimolar amount of Tβ4 prevented actin polymerization almost completely, polymerization with the corresponding amount of the sulfoxide proceeded in a manner similar to that of pure actin, except for a slight retardation. We showed that the decrease in the inhibitory activity is reflected by a 20‐times lower affinity to actin. Interestingly, under non‐polymerizing conditions, the affinity of Tβ4 sulfoxide for actin is as high as that of Tβ4 (approximately 1 μM). In accordance with this, no differences were found between Tβ4 and the sulfoxide in cross‐linking experiments with the monomer, where both forms of the peptide yielded similar amounts of a 47‐kDa band representing conjugates of actin and β‐thymosin, as proved by Western‐blotting analysis. Likewise, both, Tβ4 and the sulfoxide retarded the exchange of G‐actin‐bound nucleotide to similar extents. Although the sulfoxide is presumably a product of autoxidation, it is attractive to speculate that oxidation of the methionine residue in Tβ4 may represent a regulatory switch for starting filament formation in non‐muscle cells.
Cancellation of the cooperativity of Ca2+ binding to sarcoplasmic reticulum Ca2+‐ATPase by the non‐ionic detergent dodecylmaltoside
Authors: DE FORESTA Béatrice , HENAO Fernando , CHAMPEIL Philippe
Published: March 2005

The perturbation of the kinetics of the sarcoplasmic reticulum (SR) membranous Ca2+‐ATPase cycle by the non‐ionic detergent dodecylmaltoside (DM) has been shown to exhibit specific features which were not observed with the related detergents octa(ethylene glycol) monododecylether and Triton X‐100 [de Foresta, B., Henao, F. & Champeil, P. (1992) Eur. J. Biochem. 209, 1023–1034]. This previous study has been completed here by a detailed analysis of the perturbation by DM of the interaction of Ca2+ with membranous ATPase, both in its unphosphorylated and phosphorylated form. Equilibrium binding measurements, performed at pH 7.5 and 20°C, showed that only one 45Ca2+ was bound with high affinity to the ATPase in the presence of maximally perturbing concentrations of DM, as compared to two 45Ca2+ in the absence of detergent. This binding was also assessed by a small decrease in the tryptophan fluorescence intensity. Binding of a second Ca2+ occurred only with a much lower affinity. In the presence of DM, the pCa dependence of the phosphorylation by [γ‐32P]ATP of the ATPase shifted towards 50‐fold higher Ca2+ concentrations than in its absence. Furthermore, DM completely inhibited the cooperativity of this dependence. This shift strongly suggests that the phosphorylation of DM‐perturbed ATPase requires the binding of this second, low‐affinity Ca2+. In order to assess this, samples of ATPase were intramolecularly cross‐linked with glutaraldehyde. This treatment stabilized the phosphorylated intermediate with occluded Ca2+ [Ross, D. C., Davidson, G. A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613–4621]. Both in the absence and presence of DM, the cross‐linked enzyme occluded close to two Ca2+/phosphorylated molecule. Finally, the pCa dependences of the ATPase hydrolytic activity, measured with two different high‐energy substrates, ATP or p‐nitrophenylphosphate (PNpP), were also found to shift towards higher Ca2+ concentrations in the presence of DM, which was again consistent with a normal coupling ratio, i.e. two bound Ca2+/substrate hydrolyzed.

As compared to other detergents, the maltoside head group of DM might favor a stronger interaction with membranous ATPase, resulting in its high perturbing effect on Ca2+ binding. The loss of cooperativity of Ca2+ binding evidenced here makes DM a useful tool in the analysis of the sequence of events occurring during Ca2+ binding.
Affinity purification and characterization of (2′‐5′)oligo(adenylate)‐dependent RNase from mouse spleen
Authors: BAYARD Bernard , BETTE‐BOBILLO Pascale , ALIAU Serge
Published: March 2005

Murine (2′‐5′)An‐dependent RNase, a key enzyme of the interferon system, was purified from mouse spleen by affinity chromatography to immobilized (2′‐5′)An. Since the ribonuclease has high affinity to (2′‐5′)An, optimal non‐denaturing conditions were obtained to disrupt the (2′‐5′)An‐nuclease complex. Low‐pH buffers in the presence of 0.1% Triton X‐100 removed almost 80% of the enzyme from the (2′‐5′)An‐agarose, preserving its (2′‐5′)An binding activity and RNA cleavage function. Purification was monitored using a classical radiobinding assay, ultraviolet covalent cross‐linking method and denaturing‐renaturing affinity blotting assay. The purified enzyme was a 160‐kDa dimer that migrated with an apparent molecular mass of 78 kDa and was > 80% pure, as assessed by silver‐stained SDS gels. Both a 160‐kDa dimer and 78‐kDa monomer were found in the cellular extract at a 5:1 ratio. Binding of radiolabeled (2′‐5′)An to (2′‐5′)An‐dependent RNase either in crude extract or in purified form reached equilibrium by 5 h at 4°C. 2‐Mercaptoethanol was required to obtain (2′‐5′)An‐binding activity but, interestingly, in the absence of this reducing agent, (2′‐5′)An‐binding activity was initiated by preincubation with poly(U), a synthetic substrate of the nuclease. This new mechanistic feature indicates that interaction of poly(U) with nuclease induced a conformational modification allowing, in a second step, the binding of (2′‐5′)An. Furthermore, when activated by low amounts of (2′‐5′)An, the eluted purified enzyme degraded mRNA but there was still degradation in the absence of (2′‐5′)An. This suggested a loss of regulatory protein(s) during the purification step. Scatchard analysis showed that the purified enzyme had a Kd of 106 pM for (2′‐5′)An, similar to estimates obtained using crude spleen extracts (Kd 112 pM), indicating that the purified nuclease had almost identical (2′‐5′)An‐binding properties to those identified in spleen extracts.
Induction of the polymerization of actin from the actin:thymosin β4 complex by phalloidin, skeletal myosin subfragment 1, chicken intestinal myosin I and free ends of filamentous actin
Authors: BALLWEBER Edda , HANNAPPEL Ewald , NIGGEMEYER Brigitte , MANNHERZ Hans Georg
Published: March 2005

Thymosin β4 is able to form 1:1 complexes with monomeric (G) actin, thereby stabilizing the intracellular pool of unpolymerized actin. We have searched for factors that are able to induce the polymerization of actin from the actin:thymosin β4 complex. Phalloidin, subfragment 1 isolated from rabbit skeletal muscle myosin and chicken intestinal myosin I are demonstrated to be able to polymerize the actin from this complex in the presence of 1 mM MgCl2. Polymerization of actin was verified by the DNase I inhibition assay, by cosedimentation and from the fluorescence increase of pyrene‐labelled actin. Actin filaments formed under the influence of subfragment 1 or phalloidin were visualized under the electron microscope after negative staining. Polymerization of skeletal muscle actin from the complex with thymosin β4 by phalloidin is accompanied by the hydrolysis of the actin‐bound ATP to ADP. Polymerization was also induced by sonicated F‐actin which possessed a high concentration of free filament ends. F‐actin was severed by 0.01 M human cytoplasmic gelsolin, which is known to possess blocked + ends. Free, slowly growing – ends were unable to induce polymerization of actin from the thymosin β4 complex. However, when gelsolin on its own or in complex with two actin molecules was added to actin:thymosin β4 under nucleating conditions, it was found to be able to promote actin repolymerization provided that its concentration was close to the dissociation constant (Kd) of actin:thymosin β4. This Kd was found to be 0.4 μM in the presence of 1 mM MgCl2 and the absence of KCl and, thus, close to the critical concentration of actin polymerization under these conditions. The source of actin did not influence its polymerization from the thymosin β4 complex; rabbit skeletal muscle, actin and porcine brain actin were polymerized with equal efficiency from their complexes with thymosin β4 by both phalloidin and myosin subfragment 1. Skeletal muscle, but not cytoplasmic actin, was found to be also polymerized in the presence of increased CaCl2 concentrations to values above 1 mM.
Activation of the protein tyrosine kinase tyk2 by interferon α/β
Authors: BARBIERI Giovanna , VELAZQUEZ Laura , SCROBOGNA Marina , FELLOUS Marc , PELLEGRINI Sandra
Published: March 2005

We previously demonstrated that the gene tyk2 rescues the phenotype of a human mutant cell line unresponsive to α (IFN) and partially responsive to IFN‐β. Here, we describe functional complementation of the mutant cells with the corresponding cDNA. To characterize the putative non‐receptor protein tyrosine kinase encoded by the gene tyk2 and begin to understand its functioning, we have raised polyclonal antibodies against a segment of the protein. Using these, we have identified tyk2 as a 134‐kDa protein which is rapidly and transiently phosphorylated on tyrosine in response to IFN‐α/β and possesses an inducible kinase activity when tested in vitro. IFN‐γ has no effect on the phosphorylation state of the protein. In agreement with previous genetic evidence, these results assign a role to tyk2 in the IFN‐α/β signalling pathway and not in the IFN‐γ pathway. Fractionation of cell lysates have helped to localize the bulk of the protein in the cytoplasm, with a minor fraction associated with the cell membrane. Both protein pools undergo activation upon short‐term IFN treatment of intact cells. Through the study of the effect of pervanadate on the phosphorylation level and the activity of tyk2, we conclude that activation of tyk2 by IFN‐α does not require an intermediate regulatory tyrosine phosphatase.
The ribosomal protein S8 from Thermus thermophilus VK1
Authors: VYSOTSKAYA Valentina , TISCHENKO Svetlana , GARBER Maria , KERN Daniel , MOUGEL Marylène , EHRESMANN Chantal , EHRESMANN Bernard
Published: March 2005

The gene of the ribosomal protein S8 from Thermus thermophilus VK1 has been isolated from a genomic library by hybridization of an oligonucleotide coding for the N‐terminal amino acid sequence of the protein, amplified by PCR and sequenced. Nucleotide sequence reveals an open reading frame coding for a protein of 138 amino acid residues (Mr 15839). The codon usage shows that 94% of the codons possess G or C in the third position, and agrees with the preferential usage of codons of high G+C content in the bacteria of the genus Thermus. The amino acid sequence of the protein shows 48% identity with the protein from Escherichia coli. Ribosomal protein S8 from T. thermophilus has been expressed in E. coli under the control of the T7 promoter and purified to homogeneity by heat treatment of the extract followed by cation‐exchange chromatography. Conditions were defined in which T. thermophilus protein S8 binds specifically an homologous 16S rRNA fragment containing the putative S8 binding site with an apparent association constant of 5 × 107 M−1. The overexpressed protein binds the rRNA with the same affinity as that extracted from T. thermophilus, indicating that the thermophilic protein is correctly folded in E. coli. The specificity of this binding is dependent on the ionic strength. The protein S8 from T. thermophilus recognizes the E. coli rRNA binding site as efficiently as the S8 protein from E. coli. This result agrees with sequence comparisons of the S8 binding site on the small subunit rRNA from E. coli and from T. thermophilus, showing strong similarities in the regions involved in the interaction. It suggests that the structural features responsible for the recognition are conserved in the mesophilic and thermophilic eubacteria, despite structural pecularities in the thermophilic partners conferring thermo‐stability.
Proteolytic removal of the C‐terminal transmembrane region of cytochrome f during extraction from turnip and charlock leaves generates a water‐soluble monomeric form of the protein
Authors: GRAY John C. , ROCHFORD Rebecca J. , PACKMAN Leonard C.
Published: March 2005

Water‐soluble, monomeric cytochrome f purified from leaves of turnip (Brassica rapa) and charlock (Sinapis arvensis) is approximately 3 kDa smaller than the protein in chloroplast thylakoid membranes determined by SDS/PAGE. Sequencing the N‐terminal and C‐terminal regions of the monomeric protein, by automated Edman degradation and carboxypeptidase P digestion, suggested the loss of 33 amino acid residues at the C‐terminus by comparison to sequences of cytochrome f from other higher plants. This was confirmed by the isolation and nucleotide sequencing of the turnip petA gene and by determination of the molecular mass of the monomeric turnip protein by electrospray mass spectrometry. The turnip petA gene encodes a protein of 320 amino acid residues consisting of a presequence of 35 amino acid residues and a mature protein of 285 amino acid residues. The molecular mass of the monomeric turnip protein was 28160.2 ± 5.4 Da, indicating cleavage after Gln252 of the mature protein. Electrospray mass spectrometry of the monomeric charlock protein indicated the presence of two main forms with molecular masses of 28135.1 ± 5.5 Da and 27750.7 ± 4.3 Da corresponding to cleavage after Gln252 and Leu249, respectively. Cleavage in this region of the cytochrome f polypeptide during extraction with butanone removes the single transmembrane span of the protein and liberates the water‐soluble globular domain of cytochrome f.
Activation of thrombin‐inactivated single‐chain urokinase‐type plasminogen activator by dipeptidyl peptidase I (cathepsin C)
Authors: NAULAND Uwe , RIJKEN Dingeman C.
Published: March 2005

Single‐chain urokinase‐type plasminogen activator (scu‐PA) is inactivated by thrombin, which cleaves the peptide bond between Arg156 and Phe157. In a search for potential activators of thrombin‐cleaved two‐chain urokinase‐type plasminogen activator (tcu‐PA/T), we found that the lysosomal aminopeptidase dipeptidyl‐peptidase I or cathepsin C efficiently activates tcu‐PA/T. Cathepsin C was as active towards tcu‐PA/T as the bacterial proteinase thermolysin and about 300‐times more active than plasmin. The activation by cathepsin C proceeded in a concentration‐dependent and time‐dependent manner with a pH optimum between 5 and 7. Furthermore, the effect of cathepsin C was inhibited by cystatin and stimulated by cysteine, typical for the action of a thiol proteinase. As no degradation of the tcu‐PA/T molecule by cathepsin C was visible on SDS/PAGE, we suggest that activation of tcu‐PA/T occurs by cleavage between Lys158‐Ile159 and removal of the two N‐terminal amino acid residues (Phe157‐Lys158) of the B chain of tcu‐PA/T. We conclude that both thrombin and dipeptidyl‐peptidases like cathepsin C might play a regulatory role in the plasminogen‐plasmin system by inactivating scu‐PA and activating tcu‐PA/T, respectively.
Antibodies against subunits of F0 sector of ATP synthase from Saccharomyces cerevisiae
Authors: GRANDIER‐VAZEILLE Xavier , OUHABI Rachid , GUÉRIN Martine
Published: March 2005

Polyclonal antibodies against the three purified proteolipids of the F0 sector [subunit 6 (Su6), subunit 8 (Su8), subunit 9 (Su9)] and against the β subunit (F1) of ATP synthase were raised in rabbits. All antisera showed ELISA reactivities with F0F1‐ATPase. Antisera used to immunoblot partially purified ATP synthase labeled a single band migrating with the same molecular mass as that of the purified protein. Mitochondria were incubated with IgG of each antiserum and oxidative phosphorylation was measured. Anti‐Su6 IgG, as anti‐Suβ IgG, was without effect whereas anti‐Su9 IgG decreased both respiration and ATP‐synthesis rates, resulting in a decrease of ATP/O. In contrast, anti‐Su8 IgG enhanced respiratory control and stimulated the ATP‐synthesis rate, resulting in an increase of ATP/O. In the same manner, anti‐Su9 IgG inhibited ATP hydrolysis whereas anti‐Su8 IgG stimulated this activity. Antimycin titration of phosphorylation and respiration rates demonstrated that anti‐Su9 IgG decreased the H+/ATP ratio and promoted a H+ leak, whereas anti‐Su8 IgG increased H+/ATP without modification of the proton permeability. Anti‐Su9 IgG decreased proton‐motive force whereas anti‐Su8 IgG did not. It is proposed that both antibodies promoted opposite mechanistic changes of the H+/ATP stoichiometry of the ATP synthase, and that in vivo Su8 could have a negative regulatory role in the oxidative phosphorylation process.
Transcriptional control of the human biliary glycoprotein gene, a CEA gene family member down‐regulated in colorectal carcinomas
Authors: HAUCK Wendy , NÉDELLEC Patrick , TURBIDE Claire , STANNERS Clifford P. , BARNETT Thomas R. , BEAUCHEMIN Nicole
Published: March 2005

Biliary glycoprotein (BGP) isoantigens are derived by alternative splicing from a single gene and are the human homologs of rat C‐CAM and the mouse Bgp species. These glycoproteins represent a family of cell‐adhesion molecules. The mouse Bgp isoforms also act as receptors for the hepatitis viral capsid‐protein. BGP is a member of the carcinoembryonic antigen (CEA) gene family, which belongs to the immunoglobulin supergene family, yet it displays restricted expression patterns and unique functions. Since the loss or reduced expression of BGP is associated with human colorectal carcinomas, the elements in its upstream regulatory region were analyzed. A cluster of transcriptional initiation sites and the minimal promoter, located within 150 bp upstream of the major transcriptional start site, were active in human colon carcinoma and hepatoma cells. Unlike the CEA gene, BGP gene transcription was not modulated by a silencer region; repetitive elements in the BGP upstream region were not involved in activation or repression. Footprinting experiments identified two cis‐acting elements and mobility‐shift assays demonstrated that these elements bound several transcription factors, among them, USF, HNF‐4 and an AP‐2‐like factor. In cotransfection experiments, both the USF and HNF‐4 transcription factors transactivate the BGP gene promoter and compete for the same regulatory element. The Sp1 transcription factor, shown to be involved in CEA gene transcriptional regulation, does not bind to the BGP gene promoter. We, therefore, propose that the relative distributions and interactions of these transcription factors mediate distinct transcriptional regulation of the BGP gene in colon and liver; this regulation could be distorted during the oncogenic process.
Carbamoyl phosphate and ureagenesis are not involved in amino‐acid‐stimulated glycogenesis
Authors: GUSTAFSON Lori A. , ROMP Nancy , VAN WOERKOM George M. , MEIJER Alfred J.
Published: March 2005

Amino acids are known to stimulate glycogen synthesis via an increase in cell volume [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990)J. Biol. Chem. 265, 955–959]. It has recently been postulated, however, that carbamoyl phosphate, an intermediate of ureagenesis, can function as a substrate for glucose phosphorylation via carbamoyl‐phosphate:glucose phosphotransferase activity of the glucose‐6‐phosphatase system. This hypothesis would account for the stimulation of glycogenesis by amino acids such as glutamine and proline [Bode, A. M. & Nordlie, R. C. (1993)J. Biol. Chem. 268, 16298–16301].

To further examine the role carbamoyl phosphate may play in glycogenesis, isolated hepatocytes were incubated under a variety of conditions to manipulate ureagenesis, glycogenesis and carbamoyl‐phosphate levels. Our data indicate that carbamoyl‐phosphate levels do not correlate with amino‐acid‐stimulated glycogenesis and that ureagenesis and glycogenesis are not competing metabolic pathways.
Calcium inhibition of the activation of protein C by thrombin Role of the P3 and P3′ residues
Authors: REZAIE Alireza R. , ESMON Charles T.
Published: March 2005

Protein C, a precursor to a natural plasma anticoagulant, and the platelet thrombin receptor, involved in cell activation, both require proteolytic cleavage to be activated. In humans, the sequences adjacent to the scissile bond of protein C, DPR/LID and the thrombin receptor DPR/SFL are similar. Previous studies with Asp→Gly mutants indicated that both the P3 and the P3′ Aspresidues make either peptides or protein C a poor substrate for free thrombin, but thrombin interaction with thrombomodulin overcomes these inhibitory interactions. Similar mechanisms are probably operative in the thrombin receptor. In rodents, the P3 Asp residue of the human thrombin receptor is replaced by Asn and in protein C, the P3′; residue is Asn. To determine the functional significance of these Asp→Asn substitutions, the Asp in the P3 or P3′ position of human protein C was changed to Asn. The resultant mutants were still resistant to activation by thrombin, and still required Ca2+ for activation by thrombin‐thrombomodulin complex. We conclude that, unlike activation of the Asp→Gly mutants by thrombin, activation of the P3 and P3′ Asp→Asn mutants is still potently inhibited by physiological Ca2+. Furthermore, even though the charge has been deleted, thrombomodulin acceleration is retained.
Differential inhibitory effects of GMP‐2′,3′‐dialdehyde on human and schistosomal hypoxanthine–guanine phosphoribosyltransferases
Authors: KANAANEH Jamil , CRAIG Sydney P. , WANG Ching C.
Published: March 2005

The hypoxanthine–guanine phosphoribosyltransferase (HGPRTase) of human and the parasitic trematode, Schistosoma mansoni, were expressed at high levels in transformed Escherichia coli in their native forms. Guanosine 2′,3′‐dialdehyde 5′‐phosphate (ox‐GMP) was shown to bind irreversibly to both enzymes in a time‐dependent manner. This binding was stabilized by sodium borohydride reduction, suggesting that a Schiff's base is formed between the dialdehyde groups of ox‐GMP and the amino group of a lysine residue in the enzymes. This linkage formation applies also to inosine 2′,3′‐dialdehyde 5′‐phosphate but not to adenosine 2′,3′‐dialdehyde 5′‐phosphate. GMP was found to be protective against ox‐GMP inactivation and [3H]ox‐GMP labeling of both HGPRTases. 5‐Phosphoribosyl‐1‐diphosphate (PRibPP) also protects human HGPRTase against the ox‐GMP inactivation and [3H]ox‐GMP labeling but provides virtually no protection against the ox‐GMP inactivation and labeling of the schistosomal enzyme, even though PRibPP binds to the latter with a threefold higher affinity. These results imply that PRibPP and ox‐GMP compete with each other for binding to the human HGPRTase but not for binding to the schistosomal enzyme. This discrepancy could be exploited for the purpose of designing selective inhibitors of the schistosomal HGPRTase. Guanosine 2′,3′‐dialdehyde (ox‐guanosine) is nearly as active as ox‐GMP in inhibiting schistosomal HGPRTase but much less potent in inhibiting human HGPRTase, suggesting that ox‐guanosine and ox‐GMP may bind equally well to the parasite enzyme. PRibPP can protect human but not schistosomal HGPRTase against the inactivation by ox‐guanosine. Therefore, ox‐GMP and ox‐guanosine must be forming Schiff's bases with the same amino acid residues in each of the two HGPRTases.
Carbodiimide modification enhances activity of pig pancreatic phospholipase A2
Authors: FERREIRA João Paulo M. , SASISEKHARAN Ram , LOUIE Otway , LANGER Robert
Published: March 2005

Pig phospholipase A2, pig iso‐phospholipase A2 and bovine pancreatic phospholipase A2 were reacted in solution with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide, in the presence of N‐hydroxysulfosuccinimide, at pH 7. The influence of micellar protectants was analyzed. In the presence of n‐hexadecylphosphocholine, the losses of activity in micellar diheptanoyl‐lecithin were 80, 35, and 10% in bovine phospholipase A2, pig iso‐phospholipase A2, and pig phospholipase A2, respectively. With 1‐oleoylglycerophosphocholine, the bovine enzyme lost 40% activity, but the pig enzyme was activated sevenfold. The modified pig enzyme showed pre‐micellar activation on monomeric diheptanoyl‐lecithin, and either reduced or increased activities on mixed micelles of bile salt with egg phosphatidylcholine, depending on the composition of the micelles. This activation is consistent with previous protein‐engineering studies of pig pancreatic phospholipase A2. In this study, we present new information concerning the specificity and interfacial recognition behaviour of this enzyme in relation to this activation.
Protease‐catalyzed conversion of insulin‐like growth factor‐1 and interleukin‐6 into high‐molecular‐mass species through the sequential action of hematopoietic surface‐associated cathepsin G and γ‐glutamyl transpeptidase‐related activities
Authors: LAOUAR Amale , WIETZERBIN Juana , BAUVOIS Brigitte
Published: March 2005

Interleukin‐6 (IL‐6) and insulin‐like growth‐factor‐1 (IGF‐1) are cytokines produced by a variety of cells that act on a wide range of tissues, influencing cell growth and differentiation. Purified plasma membranes from human U937 monoblastic cells produced in vitro dimeric species of IL‐6‐and IGF‐1‐derived peptides through the sequential actions of surface‐associated enzymes cathepsin G and transpeptidase activities. Cathepsin G degraded native unglycosylated IL‐6 and IGF‐1 molecules into 8‐kDa and 7‐kDa peptides respectively. Subsequent dimerisation of these intermediate forms into 16‐kDa IL‐6‐ and 14‐kDa IGF‐1‐derived peptides was inhibited by acivicin and glutathione which are specific inhibitors of the standard cell‐surface γ‐glutamyl transpeptidase (γ‐GT). However U937 plasma membranes, cleared of γ‐GT activity by immunoprecipitation with anti‐γ‐GT and adsorption on protein‐G‐Sepharose, were still able to convert the intermediate forms of IL‐6 and IGF‐1 into dimers. Together, these observations indicate that the transpeptidase involved in the formation of the dimeric species of IL‐6 and IGF‐1 was related to, but distinct from, standard cell‐surface γ‐GT. Cells of all hematopoietic lineages expressed γ‐GT‐related activity. In contrast to the 16‐kDa IL‐6‐derived peptide that did not retain growth‐stimulating activity, the 14‐kDa IGF‐1 peptide was at least equipotent with native IGF‐1 in the BALB/c 3T3 fibroblast DNA synthesis response. The N/O‐glycosylated IL‐6 was clearly as sensitive to cathepsin‐G‐ and γ‐GT‐related activities as the unglycosylated IL‐6 from Escherichia coli, thus indicating that the sugar chains did not protect the cleavage sites of the two proteases on the IL‐6 molecule. Our in vitro findings raise the possibility that similar proteases participate in the regulation of the catabolism of IL‐6 and IGF‐1 in vivo.
Phosphorylation of synthetic fragments of inhibitor‐2 of protein phosphatase‐1 by casein kinase‐1 and ‐2
Authors: MARIN Oriano , MEGGIO Flavio , SARNO Stefania , ANDRETTA Monica , PINNA Lorenzo A.
Published: March 2005

The major phosphorylation site for both casein kinase‐2 (CK2) and casein kinase‐1 (CK1) in protein phosphatase‐1 (PP‐1) inhibitor‐2 (I‐2) is Ser86. Minor phosphorylation sites affected by either CK2 or CK1 are Ser120/Ser121 and Ser174, respectively. A synthetic peptide of 25 amino acids encompassing residues 67–93 of I‐2 is phosphorylated by either CK2 or CK1 at its seryl residue corresponding to Ser86 with higher Vmax and Km values similar to those of the intact protein (9 vs 7.2 μM and 14.2 vs 5.3 μM with CK2 and CK1, respectively). No detectable phosphorylation of this peptide which also includes the glycogen synthase kinase‐3 (GSK‐3) site (Thr72), could be observed with either GSK‐3 or p34cdc2 kinase whether or not its seryl residue equivalent to Ser86 had been previously phosphorylated by CK2. Shorter derivatives of I‐2(67–93), encompassing residues 72–93 and 78–93, are also readily phosphorylated by both CK1 and CK2, with phosphorylation efficiencies similar to those of the parent peptide.

A synthetic heptadecapeptide reproducing the phosphoacceptor site around Ser120/Ser121 is phosphorylated by CK2, but not to any detectable extent by CK1, with a Km value fivefold higher than that of the corresponding pentadecapeptide including Ser86 (78–93).

A synthetic pentadecapeptide (166–180) reproducing the phosphoacceptor site around Ser174 is phosphorylated by CK1 less efficiently than the pentadecapeptide including its main phosphorylation site (78–93) (Km 280 μM vs 33 μM). This peptide is readily phosphorylated by CK2 as well, although it lacks the canonical consensus sequence for CK2 and its Ser174 is almost unaffected by CK2 in intact I‐2.

These data provide the clear‐cut demonstration that the consensus sequence with N‐terminal prephosphorylated residue(s), SerP/ThrP‐Xaa‐Xaa‐Ser/Thr, [Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W. & Roach, P. J. (1990) J. Biol. Chem. 265, 14264–14269; Meggio, F., Perich, J. W., Reynolds, E. C. & Pinna, L. A. (1991) FEBS Lett. 283, 303–306] is not always required to achieve efficient and high‐affinity phosphorylation by CK1. They also show that the specificity determinants for I‐2 phosphorylation by either CK2 or CK1, but not by GSK3, are entirely grounded on local structural features of the phosphoacceptor site, being only marginally affected by the overall structure of I‐2.
Interaction of Serratia marcescens hemolysin (ShlA) with artificial and erythrocyte membranes
Authors: SCHÖNHERR Roland , HILGER Martina , BROER Stefan , BENZ Roland , BRAUN Volkmar
Published: March 2005

Pore formation by hemolysin (ShlA) of Serratia marcescens was studied in erythrocytes and in artificial lipid bilayer membranes. The results with erythrocytes demonstrated that hemolysin pores varied in size. In erythrocyte membranes with reduced fluidity (0°C), the toxin formed small pores with diameter 1–1.5 nm. In fluid membranes (above 20°C), hemolysin pores with larger diameters (approximately 2.5–3.0 nm) were observed, which may be caused by association of ShlA monomers into oligomers. Comparison of the channels formed by Staphylococcus aureusα‐toxin with channels formed by ShlA indicated a slightly smaller pore diameter of ShlA pores. Analysis of ShlA in artificial lipid bilayers showed the formation of pores with a broad distribution of single channel conductances, suggesting variable sizes of the ShlA pore. The lower limit for the pore diameter was approximately 1.0 nm. The ShlA pores did not exhibit pronounced ion selectivity nor voltage dependence, supporting the presence of a large water‐filled pore.
Ligand‐binding properties and N‐glycosylation of α1 subunit of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate(AMPA)‐selective glutamate receptor channel expressed in a baculovirus system
Authors: KAWAMOTO Susumu , HATTORI Satoshi , OIJI Izumi , HAMAJIMA Kenji , MISHINA Masayoshi , OKUDA Kenji
Published: March 2005

The α1 subunit of the mouse α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate(AMPA)‐selective glutamate receptor channel has been expressed in insect Spodoptera frugiperda cells using a baculovirus system. The recombinant receptor proteins were identified by immunocytochemical detection, Western‐blot analysis, and [35S]methionine/[35S]cysteine metabolic labeling experiments. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of approximately 104 kDa and a minor species of approximately 100 kDa, correspond to glycosylated and non‐N‐glycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. The lack of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate‐binding activity of non‐N‐glycosylated glutamate receptor expressed in the presence of tunicamycin suggested that N‐glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. Scatchard analysis of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate binding showed a single binding site with Kd 30 nM and a Bmax value of 2.6 × 105 binding sites/cell or 1.5 pmol/mg protein in the total particulate fraction. Among the compounds tested in the competition studies, β‐(3,5‐dioxo‐1,2,4‐oxadiazolidin‐2‐yl)‐l‐alanine (quisqualate) was the most potent inhibitor of the 3H‐labeled α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate binding (IC50= 30 nM), followed in decreasing order by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate, l‐glutamate, 6,7‐dinitroquinoxaline‐2,3‐dione, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, and 2‐carboxy‐4‐(1‐methylethenyl)‐3‐pyrrolidineacetate (kainate). Thus, in this study we present detailed analysis of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate‐binding activity of the homomeric (single subunit) glutamate receptor channel of mouse α1 subunit and discuss possible roles of N‐glycosylation of the glutamate receptor channel α1 subunit.
Characterization of baculovirus recombinant wild‐type p53
Authors: DELPHIN Christian , CAHEN Pierre , LAWRENCE Jean Jacques , BAUDIER Jacques
Published: March 2005

A high‐yield, rapid and non‐denaturing purification protocol for baculovirus recombinant wild‐type p53 is described. Gel‐filtration chromatography and chemical cross‐linking experiments indicated that purified p53 assembles into multimeric forms ranging from tetramer to higher oligomers. A gel‐mobility‐shift assay and protein‐DNA cross‐linking studies demonstrated that purified baculovirus recombinant p53 binds to consensus DNA target as a dimer but that additional p53 molecules may then associate with the preformed p53‐dimer–DNA complexes to form larger p53 DNA complexes. These observations suggest that the p53 tetramers and higher oligomers that form the minimal p53 association in solution dissociate upon DNA binding to form p53 dimer‐DNA complexes. Binding of the mAB PAb 421 to the oligomerization‐promoting domain on p53 stimulated sequentially formation of both p53‐dimer–DNA and larger p53–DNA complexes. This observation suggests that factors may exist in vivo that could participate in the formation and the stabilization of the various p53‐DNA complexes. Further characterization of the purified p53 revealed that the protein possesses highly reactive cysteine residues. We show that intrachain disulfide bonds form within the purified p53 molecules during storage in the absence of reducing agent. Zn2+ binding to p53 protect sulfhydryl groups from oxidation. Cysteine oxidation by intramolecular disulfide‐bond formation did not modify the wild‐type immunoreactive phenotype of the p53 protein but totally inhibited its DNA‐binding activities. The oxidation of the p53 cysteine residues was also observed for nuclear p53 in baculovirus‐infected insect cells. The redox status of the nuclear p53 regulates its DNA‐binding activity in vitro confirming the essential role of the reduced state of cysteine residues in p53 for detectable DNA‐binding activity.
Rack‐induced bonding in blue‐copper proteins
Authors: MALMSTRÖM Bo G.
Published: March 2005

The unique spectroscopic properties of blue‐copper centers, i.e. the strong charge‐transfer band at approximately 600 nm and the narrow hyperfine coupling in the EPR spectrum, are reviewed. The concept of rack‐induced bonding is summarized. The tertiary structure of the protein creates a preformed chelating site with very little flexibility, the geometry of which is in conflict with that preferred by Cu2+. The structure of the metal site in azurin is discussed. It is shown that the three strong ligands, one thiolate S and two imidazole N, are in a configuration intermediate between those preferred by Cu2+ and Cu+. It is emphasized that cysteine is an obligatory component of a blue site, whereas the weak interaction with a methionine S is not necessary. The minimum rack energy is estimated to be 70 kJ · mol−1. It is pointed out that the high reduction potentials of blue‐copper centers are a result of the protein‐forced ligand‐field‐destabilized site structure. It is suggested that the potentials are tuned by variations in π back bonding, and this is supported by a linear increase in ΔLF (ligand field) with decreasing electron‐transfer enthalpy. Site‐directed mutagenesis has shown that large hydrophobic residues in the site increase the potential, whereas negative groups or water decrease it. It is also shown that the fine‐tuning of the properties of the metal site by rack‐induced bonding can alter the electron‐transfer reorganization energy. Kinetic results with azurin mutants support a through‐bond tunneling mechanism for intramolecular electron transfer in proteins. Finally, it is pointed out that the concept of rack‐induced bonding is a universal principle of macromolecular structure/function relationships, which should be applied also to other systems.