bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcription‐dependent neuronal plasticity
Authors: Bading Hilmar
Published: March 2003

In neurons, calcium ions control gene transcription induced by synaptic activity. The states and histories of neuronal activity are represented by a calcium code that comprises the site of calcium entry, and the amplitude, duration and spatial properties of signal‐evoked calcium transients. The calcium code is used to transform specific firing patterns into qualitatively and quantitatively distinct transcriptional responses. The following hypothesis is proposed: electrical activity causes long‐lasting, transcription‐dependent changes in neuronal functions when synaptically evoked calcium transients associated with the stimulation propagate to the nucleus; gene transcription activated by dendritic calcium signals only is insufficient to consolidate functional alterations long‐term. Similar to enduring increases in synaptic efficacy, nuclear calcium transients are induced by high‐frequency firing patterns or by weak synaptic inputs coinciding with backpropagating dendritic action potentials. Nuclear calcium stimulates CREB‐mediated transcription and, through inducing the activity of the transcriptional coactivator CREB‐binding protein (CBP), may modulate the expression of numerous genes including neurotransmitter receptors and scaffolding proteins. Increases in the transcription rate of target genes are predicted to be transient and in many cases small, however, they collectively contribute to the maintenance of changes in synaptic efficacy. Nuclear calcium may be the common regulator of diverse transcription‐dependent forms of neuronal plasticity.
Oct‐1 specifically binds the UEF4 Site of the human AP1‐regulated urokinase enhancer
Authors: Palazzolo Martina , Berthelsen Jens , De Cesare Dario , Blasi Francesco
Published: March 2003

The inducible urokinase enhancer contains three essential elements: a combined PEA3/AP1 and a downstream AP1 site, separated by a 74‐bp DNA region called COM (cooperation mediator), that is required for the synergism between the three sites. The 5′ half of COM (uCOM) forms four retarded complexes with HeLa or HepG2 nuclear proteins (UEF1–4). We now demonstrate that the UEF4 complex is the transcription factor Oct‐1. Because of functional redundancy of the UEF sites, single mutations in UEF4 have no phenotype; we have changed UEF4 from a low to a high affinity binding site for Oct‐1. In vitro, this mutation increases the DNA binding of Oct‐1 and disturbs the binding of the Prep–Pbx complexes to the nearby UEF3 site. In vivo, this mutation reduces the basal transcriptional activity of the urokinase enhancer, while not affecting its phorbol ester inducibility. This is in keeping with the effect of the deletions of the COM region, which result in an increase in the basal level and, as a consequence, in the loss of 4β‐phorbol 12‐myristate 13‐acetate inducibility. Oct‐1 therefore is not involved in the inducibility of the urokinase enhancer but only in determining its basal activity level.
Expression of the gene and processed pseudogenes encoding the human and rabbit translationally controlled tumour protein (TCTP)
Authors: Thiele Holger , Berger Mario , Skalweit Angela , Thiele Bernd‐Joachim
Published: March 2003

In humans and rabbits, the TPT1 gene encoding the translationally controlled tumour protein TCTP generates two mRNAs (TCTP mRNA1 and TCTP mRNA2) which differ in the length of their 3′ untranslated regions. The distribution of these mRNAs was investigated in 10 rabbit and 50 human tissues. They were transcribed in all tissues investigated, but differed considerably in their quantity and ratio of expression. This indicates an extensive transcriptional control and involvement of tissue‐specific factors. In the rabbit genome numerous processed, intronless pseudogenes were detected. Four, corresponding to both types of mRNAs, were sequenced and analysed in detail; all displayed only few mutations and were either preserved completely in the original amino acid sequence of the intron containing gene, or contained only minor mutations in the coding region which did not interrupt the open reading frame. In the mRNA population of rabbit reticulocytes two additional TCTP RNAs of the TCTP mRNA2 type were detected, which have the characteristics of pseudogene transcripts. Pseudogene transcription was supported further by CAT reporter gene assays showing substantial promoter activity of 5′‐flanking regions of two TPT1 pseudogenes.
A fluorescence‐quenched chitopentaose for the study of endo‐chitinases and chitobiosidases
Authors: Cottaz Sylvain , Brasme Bernard , Driguez Hugues
Published: March 2003

A new fluorogenic substrate displaying intramolecular fluorescence energy transfer (FRET) has been synthetized from NI,NII,NIII,NIV‐tetra‐acetyl‐chitopentaose. Two molecules, a fluorophore (5‐(2‐aminoethyl) amino‐1‐naphtalene‐sulfonic acid; EDANS) and a quenching group (dimethylaminophenylazophenyl; DAB) were chemically introduced on to the chitopentaose, one at each end. Among eight enzymes tested, only endo‐chitinase and chitobiosidase activities could be specifically assayed by monitoring the variation of fluorescence after enzymatic hydrolysis of this substrate. Chitobiases and N‐acetyl‐β‐glucosaminidases are not active on the compound, the presence of a bulky chromogenic group at the 2 position of the nonreducing end of the subtrate preventing the binding and thus hydrolysis by these two exo‐enzymes. The observation that chitobiosidases are able to hydrolyse a chitooligosaccharide functionalized on both extremities demonstrates the possibility of an endo‐action for this class of chitinases, which are generally classified as exo‐enzymes. This fluorogenic chitooligosaccharide should prove to be very useful for the detection and the convenient assay of chitinolytic activities at nanomolar concentrations.
Enhancement of catalytic efficiency by the combination of site‐specific mutations in a carbonic anhydrase‐related protein
Authors: Elleby Björn , Sjöblom Björn , Tu Chingkuang , Silverman David N. , Lindskog Sven
Published: March 2003

A single mutation, involving the replacement of an arginine residue with histidine to reconstruct a zinc‐binding site, suffices to change a catalytically inactive murine carbonic anhydrase‐related protein (CARP) to an active carbonic anhydrase with a CO2‐hydration turnover number of 1.2 × 104 s−1. Further mutations, leading to a more ‘carbonic anhydrase‐like’ active‐site cavity, results in increased activity. A quintuple mutant having His94, Gln92, Val121, Val143, and Thr200 (human carbonic anhydrase I numbering system) shows kcat = 4 × 104 s−1 and kcat/Km = 2 × 107 m−1·s−1, greatly exceeding the corresponding values for carbonic anhydrase isozyme III and approaching those characterizing carbonic anhydrase I. In addition, a buffer change from 50 mm Taps/NaOH to 50 mm 1,2‐dimethylimidazole/H2SO4 at pH 9 results in a 14‐fold increase in kcat for this quintuple mutant. The CO2‐hydrating activity of a double mutant with His94 and Gln92 shows complex pH‐dependence, but the other mutants investigated behave as if the activity (kcat/Km) is controlled by the basic form of a single group with pKa near 7.7. In a similar way to human carbonic anhydrase II, the buffer behaves formally as a second substrate in a ping‐pong pattern, suggesting that proton transfer between a zinc‐bound water molecule and buffer limits the maximal rate of catalysis in both systems at low buffer concentrations. However, the results of isotope‐exchange kinetic studies suggest that proton shuttling via His64 is insignificant in the CARP mutant in contrast with carbonic anhydrase II. The replacement of Ile residues with Val in positions 121 or 143 results in measurable 4‐nitrophenyl acetate hydrolase activity. The pH–rate profile for this activity has a similar shape to those of carbonic anhydrase I and II. CD spectra of the double mutant with His94 and Gln92 are variable, indicating an equilibrium between a compact form of the protein and a ‘molten globule’‐like form. The introduction of Thr200 seems to stabilize the protein.
Dual nature of the distal histidine residue in the autoxidation reaction of myoglobin and hemoglobin
Authors: Suzuki Tatsuya , Watanabe Yo‐hei , Nagasawa Masashi , Matsuoka Ariki , Shikama Keiji
Published: March 2003

The oxygenated form of myoglobin or hemoglobin is oxidized easily to the ferric met‐form with generation of the superoxide anion. To make clear the possible role(s) of the distal histidine (H64) residue in the reaction, we have carried out detailed pH‐dependence studies of the autoxidation rate, using some typical H64 mutants of sperm whale myoglobin, over the wide range of pH 5–12 in 0.1 m buffer at 25 °C. Each mutation caused a dramatic increase in the autoxidation rate with the trend H64V ≥ H64G ≥ H64L ≫ H64Q > H64 (wild‐type) at pH 7.0, whereas each mutant protein showed a characteristic pH‐profile which is essentially different from that of the wild‐type or native sperm whale MbO2. In particular, all the mutants have lost the acid‐catalyzed process that can play a dominant role in the autoxidation reaction of most mammalian myoglobins or hemoglobins. Kinetic analyses of various types of pH‐profiles lead us to conclude that the distal histidine residue can play a dual role in the nucleophilic displacement of O2− from MbO2 or HbO2 in protic, aqueous solution. One is in a proton‐relay mechanism via its imidazole ring, and the other is in the maximum protection of the FeO2 center against a water molecule or an hydroxyl ion that can enter the heme pocket from the surrounding solvent.
Shifting the NAD/NADP preference in class 3 aldehyde dehydrogenase
Authors: Perozich John , Kuo Ingrid , Wang B. C. , Boesch Josette S. , Lindahl Ronald , Hempel John
Published: March 2003

Among pyridine‐nucleotide‐dependent oxidoreductases, the class 3 family of aldehyde dehydrogenases (ALDHs) is unusual in its ability to function with either NAD or NADP. This is all the more surprising because an acidic residue, Glu140, coordinates the adenine ribose 2′ hydroxyl. In many NAD‐dependent dehydrogenases a similarly placed carboxylate is thought to be responsible for exclusion of NADP. The corresponding residue in most (≈ 71%) sequences in the ALDH extended family is also Glu, and most of these are NAD‐specific enzymes. Site‐directed mutagenesis was performed on this residue in rat class 3 ALDH. Our results indicate that this residue contributes to tighter binding of NAD in the native enzyme, but suggest that additional factors must contribute to the ability to utilize NADP. Mutagenesis of an adjacent basic residue (Lys137) indicates that it is even more essential for binding both coenzymes, consistent with its conservation in nearly all ALDHs (> 98%).
Purification and primary structure of a new bovine spermadhesin
Authors: Tedeschi Gabriella , Oungre Emanuela , Mortarino Michele , Negri Armando , Maffeo Giovanni , Ronchi Severino
Published: March 2003

Z13 is a new seminal plasma protein made up of two disulfide‐linked 13‐kDa subunits that was identified in our laboratory by 2D PAGE. In this report we present the purification of Z13 from bovine seminal plasma. In solution, the protein is a nonglycosylated dimer that presents one interchain disulfide bond and does not show heparin‐binding properties. The complete primary structure and the localization of the S–S bridges are reported. The results suggest that Z13 is a new protein of the spermadhesin family whose members are thought to play a prominent role in different aspects of fertilization.
The rat kidney acylase I, characterization and molecular cloning
Authors: Giardina Thierry , Perrier Josette , Puigserver Antoine
Published: March 2003

The soluble acylase I from rat kidney was purified to homogeneity using a five‐step procedure. As the resulting protein was found to have a relative molecular mass of 125 kDa based on size‐exclusion chromatography and 44 kDa based on SDS/PAGE, the native protein was taken to consist of three subunits. The amino‐acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microsequencing (RHEFHALRAGFALDEGLA), was found to be very similar to the corresponding sequence of porcine kidney acylase I. However, as N‐furyl‐acryloyl‐l‐methionine, a synthetic substrate for porcine acylases, was not hydrolyzed by the rat enzyme, it was suggested that the polypeptide chain might differ in other respects from those of the other acylases I. A full length cDNA coding for the rat kidney acylase I was therefore isolated and found to contain a 1224‐bp open reading frame encoding a protein consisting of 408 amino‐acid residues, which corresponded to a calculated molecular mass of 45 847 Da per subunit. The deduced amino‐acid sequence showed 93.6% and 87.2% identity with that of the human liver and porcine kidney, respectively.
Combinatorial mutagenesis and pseudorevertant analysis to characterize regions in loop E of the CP47 protein in Synechocystis sp. PCC 6803
Authors: Tichy Martin , Vermaas Wim
Published: March 2003

Deletion of the I265–F268 and T271–K277 regions in the large lumenally exposed loop of the CP47 protein are known to lead to a loss of photoautotrophic growth. Here, these regions have been investigated by combinatorial mutagenesis and pseudorevertant mapping. No single amino‐acid residue in the I265–F268 region was found to be critical for function, but a large hydrophobic residue at position 267 and preferentially an aromatic residue at position 268 appeared to be required for photoautotrophic growth. Starting from an obligate photoheterotrophic mutant lacking the T271–K277 region, photoautotrophic pseudorevertants were generated with short in‐frame tandem repeats near the site of the original deletion, partially or fully restoring the length of the original protein. These pseudorevertants were sensitive to oxygen indicating that the T271–K277 region may provide PS II stability and/or protection against oxygen‐dependent photoinactivation. Pseudorevertants with much improved photoautotrophic growth were also generated for one of the combinatorial mutants in the I265–F268 region. Surprisingly, the secondary mutations in these pseudorevertants mapped to the ferrochelatase gene. We speculate that the secondary mutation in ferrochelatase gene resulted in altered ferrochelatase activity. Decreased heme (phycobilin) biosynthesis and/or increased chlorophyll biosynthesis could then lead to improved PS II performance of the combinatorial CP47 mutant.
Solubilization, partial purification and photolabeling of the integral membrane protein lysophospholipid:acyl‐CoA acyltransferase (LAT)
Authors: Kerkhoff Claus , Trümbach Bärbel , Gehring Lars , Habben Kai , Schmitz Gerd , Kaever Volkhard
Published: March 2003

In the present study, we defined experimental conditions that allowed the extraction of the integral membrane protein lysophospholipid:acyl‐CoA acyltransferase (LAT, EC 2.3.1.23) from membranes while maintaining the full enzyme activity using the nonionic detergent n‐octyl glucopyranoside (OGP) and solutions of high ionic strength. We found that the optimal OGP concentration depended on the ionic strength of the solubilization buffer. Fluorescence measurements with 1,6‐diphenyl‐1,3,5‐hexatriene indicated that the critical micellar concentration (CMC) of OGP decreased with increasing salt concentrations. Analogous studies revealed that the zwitterionic detergent Chaps was ineffective in extracting LAT from membranes in the absence of salt, whereas its solubilization efficiency increased with increasing salt concentrations. Detailed lipid analysis of the different protein/lipid/detergent mixed micelles showed that the protein/lipid/OGP mixed micelles were relatively enriched with sphingomyelin (SPM) compared to protein/lipid/Chaps mixed micelles, indicating that the differences in the solubilization efficiency may be due to the ability to extract more SPM from membranes. When the protein/lipid/OGP mixed micelles were dissociated into protein/detergent and lipid/detergent complexes by the addition of increasing Chaps concentrations, one‐tenth of the LAT enzyme activity was preserved making the enzyme accessible to protein purification. Analysis by native PAGE revealed that in the presence of excess Chaps a high molecular mass protein complex migrated into the gel which could be photolabeled by 125I‐labelled‐18‐(4′‐azido‐2′‐hydroxybenzoylamino)‐oleyl‐CoA. This fatty acid analogue has been shown to be a competitive inhibitor of LAT enzyme activity in the dark, and an irreversible inhibitor after photolysis. Therefore, this protein complex is assumed to contain the LAT enzyme.
Effect of amino‐acid substitutions on Alzheimer's amyloid‐β peptide–glycosaminoglycan interactions
Authors: McLaurin JoAnne , Fraser Paul E.
Published: March 2003

One of the major clinical features of Alzheimer's disease is the presence of extracellular amyloid plaques that are associated with glycosaminoglycan‐containing proteoglycans. It has been proposed that proteoglycans and glycosaminoglycans facilitate amyloid fibril formation and/or stabilize these aggregates. Characterization of proteoglycan–protein interactions has suggested that basic amino acids in a specific conformation are necessary for glycosaminoglycan binding. Amyloid‐β peptide (Aβ) has a cluster of basic amino acids at the N‐terminus (residues 13–16, His‐His‐Gln‐Lys), which are considered critical for glycosaminoglycan interactions. To understand the molecular recognition of glycosaminoglycans by Aβ, we have examined a series of synthetic peptides with systematic alanine substitutions. These include: His13→Ala, His14→Ala, Lys16→Ala, His13His14Lys16→Ala and Arg5His6→Ala. Alanine substitutions result in differences in both the secondary and fibrous structure of Aβ1–28 as determined by circular dichroism spectroscopy and electron microscopy. The results demonstrate that the His‐His‐Gln‐Lys region of Aβ, and in particular His13, is an important structural domain, as Ala substitution produces a dysfunctional folding mutant. Interaction of the substituted peptides with heparin and chondroitin sulfate glycosaminoglycans demonstrate that although electrostatic interactions contribute to binding, nonionic interactions such as hydrogen bonding and van der Waals packing play a role in glycosaminoglycan‐induced Aβ folding and aggregation.
Topology and proximity relationships of yeast mitochondrial ATP synthase subunit 8 determined by unique introduced cysteine residues
Authors: Stephens Andrew N. , Roucou Xavier , Artika I. Made , Devenish Rodney J. , Nagley Phillip
Published: March 2003

We have used site‐directed chemical labelling to demonstrate the membrane topology and to identify neighbouring subunits of subunit 8 (Y8) in yeast mitochondrial ATP synthase (mtATPase). Unique cysteine residues were introduced at the N or C‐terminus of Y8 by site‐directed mutagenesis. Expression and targeting to mitochondria in vivo of each of these variants in a yeast Y8 null mutant was able to restore activity to an otherwise nonfunctional ATP synthase complex. The position of each introduced cysteine relative to the inner mitochondrial membrane was probed with thiol‐specific nonpermeant and permeant reagents in both intact and lysed mitochondria. The data indicate that the N‐terminus of Y8 is located in the intermembrane space of mitochondria whereas the C‐terminus is located within the mitochondrial matrix. The proximity of Y8 to other proteins of mtATPase was tested using heterobifunctional cross‐linking reagents, each with one thiol‐specific reactive group and one nonspecific, photoactivatible reactive group. These experiments revealed the proximity of the C‐terminal domain of Y8 to subunits d and f, and that of the N‐terminal domain to subunit f. It is concluded that Y8 possesses a single transmembrane domain which extends across the inner membrane of intact mitochondria. As subunit d is a likely component of the stator stalk of mitochondrial ATP synthase, we propose, on the basis of the observed cross‐links, that Y8 may also be part of the stator stalk.
Enzymes of hydrogen metabolism in Pyrococcus furiosus
Authors: Silva Pedro J. , van den Ban Eyke C. D. , Wassink Hans , Haaker Huub , de Castro Baltazar , Robb Frank T. , Hagen Wilfred R.
Published: March 2003

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14‐subunit transmembrane complex associated with a Ni–Fe hydrogenase. Ten ORFs (mbhA–I and mbhM) encode hydrophobic, membrane‐spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni–Fe hydrogenase, however, the small subunit can coordinate only a single iron‐sulfur cluster, corresponding to the proximal [4Fe–4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe–4S] protein. With P. furiosus soluble [4Fe–4S] ferredoxin as the electron donor the membranes produce H2, and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane‐bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H2 evolution to H2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron‐sulfur clusters with Em ≈ −0.33 V. The genome contains a second putative operon, mbxABCDFGHH’MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni–Fe‐cluster in the large hydrogenase subunit. Kinetic studies of membrane‐bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H2) connected by devices for transduction, transfer, recovery and safety‐valving of energy.
Specificities of functionally expressed chalcone and acridone synthases from Ruta graveolens
Authors: Springob Karin , Lukačin Richard , Ernwein Claudia , Gröning Inga , Matern Ulrich
Published: March 2003

The common rue, Ruta graveolens L., expresses two types of closely related polyketide synthases that condense three malonyl‐CoAs with N‐methylanthraniloyl‐CoA or 4‐coumaroyl‐CoA to produce acridone alkaloids and flavonoid pigments, respectively. Two acridone synthase cDNAs (ACS1 and ACS2) have been cloned from Ruta cell cultures, and we report now the cloning of three chalcone synthase cDNAs (CHS1 to CHS3) from immature Ruta flowers. The coding regions of these three cDNAs differ only marginally, and the translated polypeptides show about 90% identity with the CHSs from Citrus sinensis but less than 75% with the Ruta endogeneous ACSs. CHS1 was functionally expressed in Eschericha coli and its substrate specificity compared with those of the recombinant ACS1 and ACS2. 4‐Coumaroyl‐CoA was the preferred starter substrate for CHS1, but cinnamoyl‐CoA and caffeoyl‐CoA were also turned over at significant rates. However, N‐methylanthraniloyl‐CoA was not accepted. In contrast, highly active preparations of recombinant ACS1 or ACS2 showed low, albeit significant, CHS side activities with 4‐coumaroyl‐CoA, which on average reached 16% (ACS1) and 12% (ACS2) of the maximal activity determined with N‐methylanthraniloyl‐CoA as the starter substrate, while the conversion of cinnamoyl‐CoA was negligible with both ACSs. The condensation mechanism of the acridone ring system differs from that of chalcone/flavanone formation. Nevertheless, our results suggest that very minor changes in the sequences of Ruta CHS genes are sufficient to also accommodate the formation of acridone alkaloids, which will be investigated further by site‐directed mutagenesis.
Conversion of the central [4Fe–4S] cluster into a [3Fe–4S] cluster leads to reduced hydrogen‐uptake activity of the F420‐reducing hydrogenase of Methanococcus voltae
Authors: Bingemann Ruth , Klein Albrecht
Published: March 2003

As in many other hydrogenases, the small subunit of the F420‐reducing hydrogenase of Methanococcus voltae contains three iron‐sulfur clusters. The arrangement of the three [4Fe–4S] clusters corresponds to the arrangement of [Fe‐S] clusters in the [NiFeSe] hydrogenase of Desulfomicrobium baculatum. Many other hydrogenases contain two [4Fe–4S] clusters and one [3Fe–4S] cluster with a relatively high redox potential, which is located in the central position between a proximal and a distal [4Fe–4S] cluster. We have investigated the role of the central [4Fe–4S] cluster in M. voltae with regard to its effect on the enzyme activity and its spectroscopic properties. Using site‐directed mutagenesis, we constructed a strain in which one cysteine ligand of the central [4Fe–4S] cluster was replaced by proline. The mutant protein was purified, and the [4Fe–4S] to [3Fe–4S] cluster conversion was confirmed by EPR spectroscopy. The conversion resulted in an increase in the redox potential of the [3Fe–4S] cluster by about 400 mV. The [NiFe] active site was not affected significantly by the mutation as assessed by the unchanged Ni EPR spectrum. The specific activity of the mutated enzyme did not show any significant differences with the artificial electron acceptor benzyl viologen, but its specific activity with the natural electron acceptor F420 decreased tenfold.
Redox potentials and their pH dependence of D‐amino‐acid oxidase of Rhodotorula gracilis and Trigonopsis variabilis
Authors: Pollegioni Loredano , Porrini Davide , Molla Gianluca , Pilone Mirella S.
Published: March 2003

The redox potentials and pH characteristics of d‐amino‐acid oxidase (EC 1.4.3.3; DAAO) from the yeast Rhodotorula gracilis and Trigonopsis variabilis were measured in the pH range 6.5–8.5 at 15 °C. In the free enzyme form, the anionic red semiquinone is quantitatively formed in both DAAOs, indicating that a two single‐electron transfer mechanism is active. The semiquinone species is also thermodynamically stable, as indicated by the large separation of the single‐electron transfer potentials. The first electron potential is pH‐independent, while the second electron transfer is pH‐dependent exhibiting a ≈ −60 mV/pH unit slope, consistent with a one‐electron/one‐proton transfer. In the presence of the substrate analogue benzoate, the two‐electron transfer is the thermodynamically favoured process for both DAAOs, with only a quantitative difference in the stabilization of the anionic semiquinone. Clearly binding of the substrate (or substrate analogue) modulates the redox properties of the two enzymes. In both cases, in the presence and absence of benzoate, the slope of Em vs. pH (−30 mV/pH unit) corresponds to an overall two‐electron/one‐proton transfer in the reduction to yield the anionic reduced flavin. This behaviour is similar to that reported for DAAO from pig kidney. The differences in potentials and the stability of the semiquinone intermediate measured for the three DAAOs probably stem from different isoalloxazine environments. In the case of R. gracilis DAAO, the low stability of the semiquinone form in the DAAO–benzoate complex can be explained by the shift in position of the side chain of Arg285 following substrate analogue binding.
Protein disulfide isomerase‐mediated cell‐free assembly of recombinant interleukin‐12 p40 homodimers
Authors: Martens Erik , Alloza Iraide , Scott Christopher J. , Billiau Alfons , Vandenbroeck Koen
Published: March 2003

Interleukin‐12 (IL‐12) is a heterodimeric cytokine composed of two subunits, p35 and p40. The disulfide‐linked homodimer (p40)2 has been shown to be a potent IL‐12 antagonist. In the present study, the p40 subunit was refolded from Escherichia coli inclusion bodies. Formation of (p40)2 was greatly increased in a redox buffer containing reduced and oxidized glutathione, but was not significantly affected by the cosolvents urea, GdnHCl or Chaps. While protein disulfide isomerase (PDI), GroEL/ES or DnaK/J/GrpE suppressed aggregation during refolding of p40, only DnaK/J/GrpE and PDI enhanced p40 dimerization. Oxidative assembly of p40 into (p40)2 by PDI, but not suppression of aggregation, was strongly dependent on inclusion of BSA in the refolding buffer. It is concluded that both chaperone‐like and disulfide isomerase effects are essential for correct folding of p40 into dimers.
Cloning, expression and characterization of a cDNA (6A8) encoding a novel human α‐mannosidase
Authors: Li Bo , Wang Zhaung‐Zhi , Ma Feng‐Rong , Shi Geng‐Xian , Zhang Li‐Xin , Zeng Xuan , Liu Yin , Zhao Fang‐Tao , Zhu Li‐Ping
Published: March 2003

A 3300‐bp cDNA (6A8) has been isolated from a human tonsil cell λgt11 cDNA library (GenBank accession number: AF044414). The 6A8 gene is localized on human chromosome 13q31–32. Its cDNA has an open reading frame from position 57 bp to 3243 bp, encoding a 1062 amino‐acid polypeptide. The sequence of the polypeptide has 89% identity to rat liver ER α‐mannosidase. Homogenates of COS‐7 cells transfected with 6A8 cDNA showed an enhanced enzymatic activity with p‐nitro‐phenyl‐α‐D‐mannopyranoside, which was not inhibited by swainsonine. These data suggest that 6A8 α‐mannosidase belongs to the class II α‐mannosidase. Western blot analysis showed a band for 6A8 cDNA encoded protein of ≈120 kDa. Northern blot analysis revealed two 6A8 mRNA transcripts with different tissue distribution. Enhanced concanavalin A (ConA) binding to CNE‐2L2 cells transfected with a reverse 6A8 DNA was observed, indicating that the 6A8 protein is an important cellular α‐mannosidase.
Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N‐oxide on the self‐association of muscle glycogen phosphorylase b
Authors: Chebotareva Natalia A. , Harding Stephen E. , Winzor Donald J.
Published: March 2003

The suitability of sedimentation equilibrium for characterizing the self‐association of muscle glycogen phosphorylase b has been reappraised. Whereas sedimentation equilibrium distributions for phosphorylase b in 40 mm Hepes buffer (pH 6.8) supplemented with 1 mm AMP signify a lack of chemical equilibrium attainment, those in buffer supplemented additionally with potassium sulfate conform with the requirements of a dimerizing system in chemical as well as sedimentation equilibrium. Because the rate of attainment of chemical equilibrium under the former conditions is sufficiently slow to allow resolution of the dimeric and tetrameric enzyme species by sedimentation velocity, this procedure has been used to examine the effects of thermodynamic nonideality arising from molecular crowding by trimethylamine N‐oxide on the self‐association behaviour of phosphorylase b. In those terms the marginally enhanced extent of phosphorylase b self‐association observed in the presence of high concentrations of the cosolute is taken to imply that the effects of thermodynamic nonideality on the dimer–tetramer equilibrium are being countered by those displacing the T ⇄ R isomerization equilibrium for dimer towards the smaller, nonassociating T state. Because the R state is the enzymically active form, an inhibitory effect is the predicted consequence of molecular crowding by high concentrations of unrelated solutes. Thermodynamic nonideality thus provides an alternative explanation for the inhibitory effects of high concentrations of glycerol, sucrose and ethylene glycol on phosphorylase b activity, phenomena that have been attributed to extremely weak interaction of these cryoprotectants with the T state of the enzyme.
Proteolytic degradation of ribonuclease A in the pretransition region of thermally and urea‐induced unfolding
Authors: Arnold Ulrich , Ulbrich‐Hofmann Renate
Published: March 2003

The method of limited proteolysis has proven to be appropriate for the determination of unfolding rate constants (kU) of ribonuclease A in the transition region of thermal denaturation [Arnold, U. & Ulbrich‐Hofmann, R. (1997) Biochemistry36, 2166–2172]. The aim of the present paper was to extend this procedure to the pretransition region of thermally and urea‐induced denaturation where spectroscopic methods do not allow direct measurement of kU. The results show that the approach can be applied successfully to denaturing (free energy of unfolding ΔG < 10 kJ·mol−1) and to marginally native conditions (ΔG = 10–25 kJ·mol−1). Under moderately (ΔG = 25–30 kJ·mol−1) and strongly native conditions (ΔG > 30 kJ·mol−1), however, the determination of kU was not possible in this way as the proteolytic degradation of ribonuclease A by thermolysin or trypsin was no longer determined by global unfolding. Here, proteolysis proceeds via the native RNase A. In the presence of low concentrations of urea, the rate constants of proteolysis were, surprisingly, smaller than in the absence of urea. As the protease activity has been taken into account, this result points to a local stabilization of the RNase A molecule.
A folding variant of human α‐lactalbumin induces mitochondrial permeability transition in isolated mitochondria
Authors: Köhler Camilla , Gogvadze Vladimir , Håkansson Anders , Svanborg Catharina , Orrenius Sten , Zhivotovsky Boris
Published: March 2003

A human milk fraction containing multimeric α‐lactalbumin (MAL) is able to kill cells via apoptosis. MAL is a protein complex of a folding variant of α‐lactalbumin and lipids. Previous results have shown that upon treatment of transformed cells, MAL localizes to the mitochondria and cytochrome c is released into the cytosol. This is followed by activation of the caspase cascade. In this study, we further investigated the involvement of mitochondria in apoptosis induced by the folding variant of α‐lactalbumin. Addition of MAL to isolated rat liver mitochondria induced a loss of the mitochondrial membrane potential (ΔΨm), mitochondrial swelling and the release of cytochrome c. These changes were Ca2+‐dependent and were prevented by cyclosporin A, an inhibitor of mitochondrial permeability transition. MAL also increased the rate of state 4 respiration in isolated mitochondria by exerting an uncoupling effect. This effect was due to the presence of fatty acids in the MAL complex because it was abolished completely by BSA. BSA delayed, but failed to prevent, mitochondrial swelling as well as dissipation of ΔΨm, indicating that the fatty acid content of MAL facilitated, rather than caused, these effects. Similar results were obtained with HAMLET (human α‐lactalbumin made lethal to tumour cells), which is native α‐lactalbumin converted in vitro to the apoptosis‐inducing folding variant of the protein in complex with oleic acid. Our findings demonstrate that a folding variant of α‐lactalbumin induces mitochondrial permeability transition with subsequent cytochrome c release, which in transformed cells may lead to activation of the caspase cascade and apoptotic death.
Isolation and structural characterization of an R‐form lipopolysaccharide from Yersinia enterocolitica serotype O:8
Authors: Oertelt Clemens , Lindner Buko , Skurnik Mikael , Holst Otto
Published: March 2003

The lipopolysaccharide (LPS) of strain 8081‐c‐R2, a spontaneous R‐mutant of Yersinia enterocolitica serotype O:8, was isolated using extraction with phenol/chloroform/light petroleum. Its compositional analysis indicated the presence of d‐GlcN, d‐Glc, lglycerodmanno‐ and dglycerodmanno‐heptose, 3‐deoxy‐dmanno‐oct‐2‐ulosonic acid (Kdo) and phosphate. From deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide, three oligosaccharides (1–3) were isolated using high‐performance anion‐exchange chromatography, the structures of which were determined by compositional analysis and one‐ and two‐dimensional NMR spectroscopy as

in which all sugars are pyranoses, and R and R′ represent β‐d‐Glc (in 1 and 2) and β‐d‐GlcN (in 1 only), respectively. d‐α‐d‐Hep is dglycero‐α‐dmanno‐heptose, l‐α‐d‐Hep is lglycero‐α‐dmanno‐heptose, Kdo is 3‐deoxy‐dmanno‐oct‐2‐ulosonic acid, and P is phosphate. The liberated lipid A was analyzed by compositional analyses and MALDI‐TOF MS. Its β‐d‐GlcN4P‐(1→6)‐α‐d‐GlcN‐1→P backbone is mainly tetra‐acylated with two amide‐ and one ester‐linked (at O3 of the reducing GlcN) (R)‐3‐hydroxytetradecanoic acid residues, and one tetradecanoic acid that is attached to the 3‐OH group of the amide‐linked (R)‐3‐hydroxytetradecanoic acid of the nonreducing GlcN. Additionally, small amounts of tri‐ and hexa‐acylated lipid A species occur.
COUP‐TFI and COUP‐TFII regulate expression of the NHE through a nuclear hormone responsive element with enhancer activity
Authors: Fernandez‐Rachubinski Francoise , Fliegel Larry
Published: March 2003

The chicken ovalbumin upstream promoter‐transcription factors (COUP‐TFs) are orphan receptors involved in regulation of embryonic development and neuronal cell fate determination. We identified a target of COUP‐TF involved in cell proliferation and cell differentiation. Using reporter assays, footprint analysis, and electrophoretic mobility shift assays, we showed that a nuclear hormone‐responsive element located at −841/−800 nt of the mouse Na+/H+ exchanger (NHE) promoter binds COUP‐TF with enhancer activity. Mutation at −829/−824 nt (and secondarily at −837/−833) prevents COUP binding and activation of the NHE promoter. In vivo expression of COUP isoforms in NIH 3T3 or CV1 cells transactivates from the nuclear hormone‐responsive element and from the entire NHE1 promoter. Transactivation is greater for COUP‐TFII, is increased for either COUP isoform by the presence of high serum concentrations, and is greatly reduced by mutations preventing COUP binding. In vivo COUP expression in NIH 3T3 cells results in increased synthesis of NHE. Expression of COUP‐TFII induced by either retinoic acid or dimethyl sulfoxide in differentiating P19 cells increases NHE expression. The results show that COUP‐TF regulates expression of the NHE and provide a mechanism that may be important in physiological and pathological situations linked to its upregulation.
Histamine prevents polyamine accumulation in mouse C57.1 mast cell cultures
Authors: Fajardo Ignacio , Urdiales José L. , Paz José C. , Chavarría Teresa , Sánchez‐Jiménez Francisca , Medina Miguel A.
Published: March 2003

The effects of histamine on polyamine uptake and metabolism was studied in a mouse mast cell line (C57.1), as a cell model in which both biogenic amines are important for maintaining cell function and viability. Results obtained after incubations with exogenous histamine indicated that histamine prevents polyamine accumulation by affecting polyamine uptake. A plasma membrane transport system for polyamines has been also studied in mast cells. It seems to be a Na+‐dependent uptake with high affinity for both spermine and spermidine and lower affinity for putrescine and agmatine. Polyamine uptake was reduced in both cells treated with exogenous histamine and histamine‐preloaded cells. However, ornithine decarboxylase activity and cell proliferation were not affected by histamine. Incubation with histamine enhanced the spermidine/spermine acetyl transferase induction caused by N1‐ethyl‐N11‐[(cyclopropyl)methyl]‐4,8‐diazaundecane, suggesting that polyamine acetylation could be another mechanism by which histamine prevents polyamine accumulation in C57.1 mast cells.
Inhibitory sequences in the N‐terminus of the double‐stranded‐RNA‐dependent protein kinase, PKR, are important for regulating phosphorylation of eukaryotic initiation factor 2α (eIF2α)
Authors: Vattem Krishna M. , Staschke Kirk A. , Zhu Shuhao , Wek Ronald C.
Published: March 2003

During viral infection, phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) by the interferon‐induced RNA‐dependent protein kinase, PKR, leads to inhibition of translation initiation and viral proliferation. Activation of PKR is mediated by association of virally encoded double‐stranded RNAs (dsRNAs) with two dsRNA binding domains (dsRBDs) located in the N‐terminus of PKR. To better understand the molecular mechanisms regulating PKR, we characterized the activities of wild‐type and mutant versions of human PKR expressed and purified from yeast. The catalytic rate of eIF2α phosphorylation by our purified PKR was increased in response to dsRNA, but not single‐stranded RNA or DNA, consistent with the properties previously described for PKR purified from mammalian sources. While both dsRBD1 and dsRBD2 were required for activation of PKR by dsRNA, only deletion of dsRBD1 severely reduced the basal eIF2α kinase activity. Removal of as few as 25 residues at the C‐terminal junction of dsRBD2 dramatically increased eIF2α kinase activity and characterization of larger deletions that included dsRBD1 demonstrated that removal of these negative‐acting sequences could bypass the dsRBD1 requirement for in vitro phosphorylation of eIF2α. Heparin, a known in vitro activator of PKR, enhanced eIF2α phosphorylation by PKR mutants lacking their entire N‐terminal sequences, including the dsRBDs. The results indicate that induction of PKR activity is mediated by multiple mechanisms, one of which involves release of inhibition by negative‐acting sequences in PKR.
Thermodynamic analysis of binding of p‐substituted benzamidines to trypsin
Authors: Talhout Reinskje , Engberts Jan B. F. N.
Published: March 2003

Understanding the structural basis of inhibitor–enzyme interactions, important for the design of new drugs, requires a complete thermodynamic characterization of the binding process as well as a description of the structure of the complex. In this paper, the binding of p‐substituted benzamidinium derivatives to the structurally well‐characterized serine proteinase bovine pancreatic trypsin has been studied using isothermal titration calorimetry. These experiments have permitted a complete characterization of the temperature dependence of the inhibitor‐binding thermodynamics. At 25 °C, both the enthalpy and entropy of binding are favourable for all studied derivatives, but this is only true for a relatively narrow temperature range. As binding is characterized by a negative change in heat capacity, the process is characterized by enthalpy–entropy compensation, resulting in a change of the net thermodynamic driving force for association from entropic to enthalpic with increasing temperature. These phenomena are not unusual when hydrophobic forces play an important role. The trend in the relative binding potencies can, to a significant extent, be attributed to the electron‐donating/withdrawing character of the substituent at the para position, as shown by the Hammett

 plot for the different inhibitors; the more polar the p‐substituted benzamidine, the less potent it will be as a trypsin inhibitor. This behaviour might result from a bulk solvation effect, meaning that the more polar, lower potency inhibitors will be more stabilized in water than the less polar, higher potency inhibitors.
A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI‐1
Authors: Takai Masaki , Kamimura Kazuo , Sugio Tsuyoshi
Published: March 2003

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI‐1 in an electrophoretically homogenous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (α), 28 kDa (β), 24 kDa (γ), 20 kDa (δ), and 17 kDa (ε). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n‐dodecyl‐β‐d‐maltopyranoside (DM), a native iron oxidase purified from strain TI‐1 seems to be a homodimeric enzyme (αβγδε)2. Optimum pH and temperature for iron oxidation were pH 3.0 and 45 °C, respectively. The Km of iron oxidase for Fe2+ was 1.06 mm and Vmax for O2 uptake was 13.8 µmol·mg−1·min−1. The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI‐1 is a new iron oxidase in which electrons of Fe2+ were transferred to haem a and then to the molecular oxygen.
Site‐directed mutagenesis of human membrane‐associated ganglioside sialidase
Authors: Wang Yan , Yamaguchi Kazunori , Shimada Yukiko , Zhao Xuejian , Miyagi Taeko
Published: March 2003

Unlike microbial sialidases, mammalian sialidases possess strict substrate specificity, for example the human membrane‐associated sialidase, which hydrolyzes only gangliosides. To cast light on the molecular basis of this narrow substrate preference, predicted active site amino‐acid residues of the human membrane sialidase were altered by site‐directed mutagenesis. When compared with the active site amino‐acid residues proposed for Salmonella typhimurium sialidase, only five out of 13 residues were found to be different to the human enzyme, these being located upstream of the putative transmembrane region. Alteration of seven residues, including these five, was followed by transient expression of the mutant enzymes in COS‐1 cells and characterization of their kinetic properties using various substrates. Substitution of glutamic acid (at position 51) by aspartic acid and of arginine (at position 114) by glutamine or alanine resulted in retention of good catalytic efficiency toward ganglioside substrates, whereas other substitutions caused a marked reduction. The mutant enzyme E51D exhibited an increase in hydrolytic activity towards GM2 as well as sialyllactose (which are poor substrates for the wild‐type) with change to a lower Km and a higher Vmax. R114Q demonstrated a substrate specificity shift in the same direction as E51D, whereas R114A enhanced the preference for gangliosides GD3 and GD1a that are effectively hydrolyzed by the wild‐type. The inhibition experiments using 2‐deoxy‐2,3‐didehydro‐N‐acetylneuraminic acid were consistent with the results in the alteration of substrate specificity. The findings suggest that putative active‐site residues of the human membrane sialidase contribute to its substrate specificity.
Mechanism of phosphoryl transfer by nucleoside diphosphate kinase
Authors: Schneider Benoit , Babolat Manuel , Xu Ying Wu , Janin Joël , Véron Michel , Deville‐Bonne Dominique
Published: March 2003

Nucleoside diphosphate (NDP) kinase phosphorylates nucleoside diphosphates with little specificity for the base and the sugar. Although nucleotide analogues used in antiviral therapies are also metabolized to their triphosphate form by NDP kinase, their lack of the 3′‐hydroxyl of the ribose, which allows them to be DNA chain terminators, severely impairs the catalytic efficiency of NDP kinase. We have analyzed the kinetics parameters of several mutant NDP kinases modified on residues (Lys16, Tyr56, Asn119) interacting with the γ‐phosphate and/or the 3′‐OH of the Mg2+‐ATP substrate. We compared the relative contributions of the active‐site residues and the substrate 3′‐OH for point mutations on Lys16, Tyr56 and Asn119. Analysis of additional data from pH profiles identify the ionization state of these residues in the enzyme active form. X‐ray structure of K16A mutant NDP kinase shows no detectable rearrangement of the residues of the active site.