bluuebunny/crossref_metadata_2025 huggingface dataset:
A two‐dimensional NMR study of Co(II)7 rabbit liver metallothionein
Authors: BERTINI Ivano , LUCHINAT Claudio , MESSORI Luigi , VASAK Milan
Published: March 2005

The 600‐MHz 1H‐NMR NOESY spectra on Co(II)7‐reconstituted metallothionein (Co7MT), exhibiting hyperfine signals in the range 350 ppm to – 50 ppm, with nuclear relaxation times of the order of a few milliséconds, have been measured and several interproton connectivities have been detected. To our knowledge, this is the largest spectral window ever reported for a two‐dimensional 1H‐NMR spectrum in the case of a paramagnetic metalloprotein. No scalar connectivities could be detected. The hyperfine‐shifted signals belong to the cysteine‐ligand protons of the Co4S11 cluster of Co7MT. Together with results from one‐dimensional NOE experiments, the two‐dimensional experiments allowed us to proceed with the pairwise assignment of the isotropically shifted signals of the CβH2 groups of the metal‐coordinated cysteines. With the aid of computer‐graphics inspection of the four‐metal‐cluster domain, based on the NMR solution structure of Cd7MT, it is possible to propose sequence‐specific assignments of a few hyperfine‐shifted 1H‐NMR signals. In particular, a tentative assignment is given for the six signals whose shifts exhibit an antiCurie temperature dependence. The assignment relies on the theoretical model that qualitatively rationalizes the isotropic‐shift pattern and its temperature dependence. Inferences on the solution structure of the Co4S11 cluster are drawn.
The cooperative binding of phenylalanine to phenylalanine 4‐monooxygenase studied by 1H‐NMR paramagnetic relaxation
Authors: MARTÍNEZ Aurora , OLAFSDOTTIR Sigridur , FLATMARK Torgeir
Published: March 2005

The effect of the paramagnetic high‐spin Fe(III) ion in phenylalanine 4‐monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1) on the water proton longitudinal relaxation rate has been used to study the environment of the iron center. The relaxation rate was measured as a function of the concentration of enzyme, substrate (phenylalanine), inhibitor (noradrenaline) and activator (lysolecithin), as well as of the temperature (18–40°C) and the external magnetic field strength (100–600 MHz). From the frequency dependence of the relaxation rate, an effective correlation time (τc) of 4.2(± 0.5) × 10−10 s was calculated for the enzyme‐substrate complex, which most likely represents the electron spin relaxation rate (τs) for Fe(III) (S= 5/2) in this complex. The relaxation rate was proportional to the concentration of enzyme (0.04–1 mM) both in the absence and presence of phenylalanine, but the paramagnetic molar relaxivity at 400 MHz and 22°C decreased from 2.2(± 0.05) × 103 s–1· M–1 in the enzyme as isolated to 1.2(± 0.06) × 103 s–1· M–1 in the presence of saturating concentrations of the substrate. The activation energy of the relaxation rate also decreased from 11.3 ± 0.8 kJ/mol to –1.5 ± 0.2 kJ/mol upon incubation of the enzyme with 5 mM phenylalanine. The results obtained can be interpreted in terms of a slowly exchanging water molecule coordinated to the catalytic paramagnetic Fe(III) in the native and resting enzyme, and that this water molecule seems to be displaced from coordination on the binding of substrate or inhibitor. Moreover, the effect of increasing concentrations of phenylalanine and noradrenaline on the water proton relaxation rate and on the hydrophobic surface properties of the enzyme indicate that substrate and inhibitor induce a similar cooperative conformational change upon binding at the active site. By contrast, the activator lysolecithin does not seem to affect the interaction of water with the catalytic Fe(III).
A Unique Autophosphorylation Site in the Platelet‐Derived Growth Factor α Receptor from a Heterodimeric Receptor Complex
Authors: Rupp Eva , Siegbahn Agneta , Rönnstrand Lars , Wernstedt Christer , Claesson‐Welsh Lena , Heldin Carl‐Henrik
Published: March 2005

The platelet‐derived growth factor (PDGF) α and β receptors undergo dimerization as a consequence of ligand binding. Depending on the PDGF isoform (PDGF‐AA, ‐AB or ‐BB), homodimers or heterodimers of receptors are formed. In this study, we have used transfected porcine aortic endothelial cells, coexpressing cDNAs for the α receptor and the β receptor at comparable levels, to investigate the properties of the αβ‐heterodimeric receptor complex. PDGF‐AB, which mainly induced αβ‐heterodimeric complexes, was the most efficient isoform for stimulating mitogenicity. Actin reorganization, in the form of circular membrane ruffling and chemotaxis, was induced by PDGF‐AB and PDGF‐BB, but not by PDGF‐AA, thus indicating that the β receptor in the homodimeric or heterodimeric configuration was required for induction of motility responses. The molecular basis for the apparent receptor dimer‐specific properties was examined by analyzing receptor autophosphorylation and phosphorylation of substrates. The α receptor was found to be phosphorylated at an additional tyrosine residue, Tyr754, in the heterodimeric complex as compared to the αα receptor homodimer. Phosphorylation of this tyrosine residue could permit the binding of a specific signal‐tranducing protein. A candidate is a 134000‐Mr protein, which was shown to associate preferentially with the α receptor in the heterodimeric receptor complex. It is possible that phosphorylated Tyr754 in the α receptor mediates activation of specific signal‐tranducing molecules like the 134000‐Mr substrate, and thereby initiates signal‐tranduction pathways from the αβ receptor heterodimer, which are distinct from those initiated via homodimeric receptor complexes.
Activation of the Silent Human Cytokeratin 17 Pseudogene‐Promoter Region by Cryptic Enhancer Elements of the Cytokeratin 17 Gene
Authors: Troyanovsky Sergey M. , Leube Rudolf E.
Published: March 2005

We have previously described the three loci CK‐CA, CK‐CB and CK‐CC in the human genome that contain clustered type‐I cytokeratin genes and reported the complete nucleic acid sequences of the functional cytokeratin 17 gene located in CK‐CA and two closely related pseudogenes present in CK‐CB and CK‐CC [Troyanovsky, S. M., Leube, R. E. & Franke, W. W. (1992) Eur. J. Cell Biol. 59, 127–137]. By nucleic acid sequence analysis, we now show that extensive similarities between the functional gene and the pseudogenes exist in the 5′‐upstream region. However, despite the high degree of nucleic acid identity (94%), only the 5′‐upstream region of the functional gene was able to induce significant transcriptional activity in transfected cells of epithelial origin. Using chimeric upstream regions consisting of different fragments from the pseudogene and the functional gene, we made the surprising observation that cis elements in the proximal 5′‐upstream region of the pseudogene promoter can cooperate with distal enhancer elements of the functional gene to induce strong chloramphenicol‐O ‐acetyltransferase activity in transfected HeLa cells. A major site in the proximal upstream region was identified by deoxyribonuclease protection experiments to be necessary for this cooperative effect. The structure and properties of this element were further analysed by transfection of different chloramphenicol‐O ‐acetyltransferase gene constructs, and by nucleic acid sequence comparison to corresponding regions of the related cytokeratins 14 and 16. It is concluded that the upstream regions identified in this study contribute to the strong expression of the human cytokeratin 17 gene in a coordinated fashion.
Structure of the Human CALM1 Calmodulin Gene and Identification of two CALM1 ‐Related Pseudogenes CALM1P1 and CALM1P2
Authors: Rhyner Johannes A. , Ottiger Marcel , Wicki Roland , Greenwood Tammy M. , Strehler Emanuel E.
Published: March 2005

The human CALM1 calmodulin gene has been isolated and characterized. The gene contains six exons spread over about 10 kb of genomic DNA. The exon‐intron structure is identical to that of the human CALM3 and of the rat CALM1 and CALM3 genes. A cluster of transcription‐start sites was identified 200 bp upstream of the ATG translation‐start codon, and several putative regulatory elements were found in the 5′ flanking region as well as in intron 1. Sequence comparison with the rat CALM1 gene revealed significant similarities in the promoter regions of the two genes and an even more striking degree of identity (70%) in the available intron 1 sequences. A short CAG trinucleotide repeat region was identified in the 5′ untranslated region of the human CALM1 gene; this sequence is not conserved in the rat counterpart. Expression of the CALM1 gene was detected in all human tissues tested, although at varying levels. A 1.7‐kb mRNA was uniformly present at comparable levels, whereas a 4.2‐kb mRNA species was particularly abundant in brain and skeletal muscle. Clones for two different CALM1 ‐related pseudogenes CALM1P1 and CALM1P2 were also isolated and characterized. Both pseudogenes are intronless and non‐functional as judged from the presence of mutations abolishing the open reading frame. Genomic Southern analysis indicates that the human CALM1 gene/pseudogene subfamily comprises at least three but probably no more than four members. The entire family consists of three bona fide CALM genes, at least one expressed calmodulin‐like CALML gene as well as at least five pseudogenes.
Primary Structure of Lep D I, the Main Lepidoglyphus Destructor Allergen
Authors: Varela Javier , Ventas Pilar , Carreira José , Barbas Julio A. , Gimenez‐Gallego Guillermo , Polo Florentino
Published: March 2005

The most relevant allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been characterized. Lep d I is a monomer protein of 13273 Da. The primary structure of Lep d I was determined by N‐terminal Edman degradation and partially confirmed by cDNA sequencing. Sequence polymorphism was observed at six positions, with non‐conservative substitutions in three of them. No potential N‐glycosylation site was revealed by peptide sequencing, The 125‐residue sequence of Lep d I shows approximately 40% identity (including the six cysteines) with the overlapping regions of group II allergens from the genus Dermatophagoides, which, however, do not share common allergenic epitopes with Lep d I.
Thermodynamics of Ligand Binding to the Starch‐Binding Domain of Glucoamylase from Aspergillus Niger
Authors: Sigurskjold Bent W. , Svensson Birte , Williamson Gary , Driguez Hugues
Published: March 2005

The thermodynamics of ligand binding to the starch‐binding domain (SBD) of glucoamylase from Aspergillus niger has been studied using titration calorimetry. The ligand binding was studied both with the SBD fragment as well as glucoamylase G1 which contains both a catalytic domain and SBD. The ligands were β‐cyclodextrin and three thiopanose analogues [panose =α‐d‐Glcp‐(l→6)‐α‐d‐Glcp‐(1→4)‐d‐Glcp] each including an α‐(1→6) thioglycosidic linkage at the non‐reducing end. β‐Cyclodextrin binds more strongly than the thiopanose analogues and these have a slightly increasing binding constant with chain length. The reactions are enthalpy‐driven with unfavourable contributions from entropy and the variations in enthalpy and entropy compensate each other linearly. SBD was shown to have two binding sites that appear to bind identically and independently in the isolated binding domain, whereas they interact with each other in a negatively cooperative fashion when the catalytic domain of glucoamylase is present (glucoamylase G1). In glucoamylase G1 one site of SBD has an increased binding constant compared to the SBD fragment, whereas the other has the same association constant. The change in binding constant and induced cooperativity were not due to interactions with the catalytic binding site, since binding of β‐cyclodextrin was the same both when the catalytic site was occupied by the strong inhibitor acarbose and when the catalytic site was free.
Purification and Characterization of the Native and the Recombinant Leishmania Mexicana Glycosomal Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: Hannaert Véronique , Callens Mia , Opperdoes Fred R. , Michels Paul A. M.
Published: March 2005

The gene coding for the glycosomal glyceraldehyde‐3‐phosphate dehydrogenase from Leishmania mexicana has been cloned into vector pET3A and expressed as a soluble and active protein in Escherichia coli BL21(DE3) in which the endogenous gene has been inactivated by mutation. The recombinant enzyme was purified to near homogeneity by ammonium sulphate precipitation, followed by hydrophobic and cation‐exchange chromatography. From a 1‐L culture of E. coli cells, 25 mg purified protein was obtained with a specific activity of 125 units/mg. The recombinant protein restores the natural E. coli phenotype when expressed at low level. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. The recombinant and the native proteins show identical mobilities on SDS/PAGE, and have the same isoelectric point and similar pH‐activity profiles. The kinetics of both enzymes are very similar, the most important aspect being their lower apparent affinity for the cofactor NAD when compared to all other homologous enzymes studied, with the exception of glycosomal glyceraldehyde‐3‐phosphate dehydrogenase from Trypanosoma brucei.
Expression of GDP‐l‐Fuc: Gal(β1–4)GlcNAc‐R (Fuc to GlcNAc) α‐1,3‐fucosyltransferase and its Relationship to Glycoprotein Structure in a Human Erythroleukemia Cell Line, HEL
Authors: Giuntoli Robert L. , Stoykova Lidia I. , Gillies Donald R. B. , Glick Mary Catherine
Published: March 2005

Terminal glycosylation may be a mechanism to control the function of specific biologically active glycoproteins. The biosynthesis of terminal sialyl and fucosyl residues on certain glycoproteins has been linked to the expression of the respective glycosyltransferase. In contrast, a human erythroleukemia cell line, HEL, contained a highly active GDP‐l‐Fuc: Gal(β1‐4)GlcNAc‐R (Fuc to GlcNAc) α‐1,3‐fucosyltransferase (α‐1,3‐fucosyltransferase) but no detectable α‐1,3‐linked fucosyl residues on the glycoproteins. The α‐1,3‐fucosyltransferase gave apparent Km values for Fuc(α1‐2)Gal(β1‐4)GlcNAc β‐O ‐benzyl, Gal(β1‐4)GlcNAc and GDP‐fucose of 0.04, 0.68 and 0.12 mM, respectively. The lack of detectable fucosyl residues in α‐1,3‐linkage to GlcNAc on the [3H]fucose‐labeled glycoproteins was shown with the use of almond α‐1,3/4‐fucosidase and internal controls to verify that the enzyme was active. Using Western‐blot analysis, HEL cell glycoproteins reacted with blood group H type‐2 antibody, confirming the presence of Fuc(α1‐2)Gal(β1‐4)GlcNAc as reported by others and the presence of the preferred substrate for the enzyme. It is proposed that controls for terminal glycosylation in addition to glycosyltransferase expression are operative in HEL cells and that they are part of a multi‐regulated process controlling terminal modifications of glycoproteins.
Modulation of the Intracellular Ca2+‐dependent Proteolytic System is Critically Correlated with the Kinetics of Differentiation of Murine Erythroleukemia Cells
Authors: Sparatore Bianca , Passalacqua Mario , Pessino Anna , Melloni Edon , Patrone Mauro , Pontremoli Sandro
Published: March 2005

Calpain has been identified as the intracellular proteinase that catalyzes the selective down‐regulation of protein kinase C (PKC) isoforms, occurring in the early stages of commitment to terminal erythroid differentiation of murine erythroleukemia (MEL) cells induced by hexamethylenebisacetamide. This conclusion has been reached through direct experiments performed with two MEL cell clones, one characterized by a high and the other by a low rate of differentiation. In both cell types, introduction of an anti‐calpain antibody resulted in a significant delay in the onset of down‐regulation of PKC isoforms, and in an increase in the latent period that precedes differentiation. Both cell lines also displayed reduced rates of PKC decay and accumulation of mature erythroid cells. Furthermore, in the fast‐responding clone, calpastatin, the natural calpain‐inhibitor protein, was found to be almost completely absent, resulting in activation and expression of proteolytic activity of calpain even at micromolar concentrations of Ca2+, a condition not sufficient to trigger calpain activation in the slowly responding clone which contains high levels of calpastatin. The fast‐responding MEL cell clone, enriched with calpastatin, displayed a lower rate of cell differentiation, with a kinetics almost identical to that observed following introduction of the anti‐calpain antibody.

It is proposed that Ca2+‐dependent proteolysis plays a crucial role for the progress of MEL cell differentiation through the specific degradation of PKC isozymes.
Processing of the Pre‐β‐amyloid Protein by Cathepsin d is Enhanced by a Familial Alzheimer's Disease Mutation
Authors: Dreyer Robert N. , Bausch Kathryn M. , Fracasso Paul , Hammond Lisa J. , Wunderlich David , Wirak Dana O. , Davis Gary , Brini Carla M. , Buckholz Thomas M. , König Gerhard , Kamarck Michael E. , Tamburini Paul P.
Published: March 2005

A major pre‐β‐amyloid protein695 (APP695) processing activity from Alzheimer's disease brain extracts was identified and found to be indistinguishable from the activity of cathepsin D.APP695 processing activity cleaved APP695 into a series of fragments that reacted on immunoblots to a monoclonal antibody (C286.8a) against β‐amyloid‐(1–7)‐peptide and cleaved N ‐dansyl‐APP‐(591–601)‐amide at the Glu‐Val and Met‐Asp bonds. Fragments of 5.5 kDa and 10–12 kDa were formed from the cleavage of APP695 by cathepsin D at the Glu593‐Va1594 bond, and had the same N‐terminus as a minor form of β‐amyloid released by cells. The Lys595→Asn and Met596→Leu substitutions found in a pedigree of familial Alzheimer's disease, increased the cathepsin d‐catalyzed rate of accumulation of 5.5 kDa and 10–12 kDa C286.8a‐reactive fragments 5–10fold. This substitution also increased the rate of N‐dansyl‐APP‐(591–601)‐amide cleavage at the Xaa‐Asp bond by up to 41‐fold. These observations suggest a role of cathepsin D in β‐amyloid formation under certain circumstances.
ATPase Strongly Bound to Higher Eukaryotic Ribosomes
Authors: Rodnina Marina V. , Serebryanik Alexander I. , Ovcharenko Galina V. , El'skaya Anna V.
Published: March 2005

80 S ribosomes from a number of higher eukaryotic organisms are able to hydrolyse ATP and GTP without the addition of soluble protein factors. ATPase seems to be an intrinsic activity of the ribosome, as indicated by the findings that ATPase activity is not diminished upon dissociation of ribosomes and reassociation of subunits, by washing with 0.66 M (KCl + NH4Cl) or 0.6 M LiCl treatment and ethanol precipitation; 1.5 M LiCl treatment removes only 40% ATPase activity. 80 S ribosomes are able to bind a variety of NTPs, NDPs and NTP analogues, with a preference for ATP. Effective inhibitors of the ribosomal ATPase are ammonium metavanadate and alcaloid emetine. The ATPase activity is present on both ribosomal subunits, which may reflect the existence of two catalytical sites for ATP on the 80 S ribosome. Ribosomal ATPase is stimulated by the occupancy of the A site, in particular with charged tRNA. The ATPase inhibitor adenylylimidodiphosphate almost completely prevents elongation‐factor(EF)‐1‐dependent binding of Phe‐tRNAPhe to the A site. The hydrolysis of ATP, therefore, is likely to be involved in the mechanism of tRNA binding to the A site of the 80 S ribosome. As far as wide substrate specificity and possible participation in tRNA interaction with the ribosome are concerned, the ribosomal ATPase seems to be similar to EF‐3 found in fungi. A synergism in ATPase activities of yeast EF‐3 and rabbit liver ribosomes at high ATP concentration and certain ribosome/EF‐3 ratios have been observed. Rabbit liver ribosomes seem to stimulate the ATPase activity of yeast EF‐3 similar to the mechanism in yeast ribosomes, though less efficiently.
Gene XV of Bacteriophage PRD1 Encodes a Lytic Enzyme with Muramidase Activity
Authors: Caldentey Javier , Hänninen Anna‐Liisa , Bamford Dennis H.
Published: March 2005

Bacteriophage PRD1 is a lipid‐containing virus that infects a variety of Gram‐negative bacteria, including Escherichia coli. The phage lyses its host by virtue of a virally‐encoded lytic enzyme, the synthesis of which has been assigned to gene XV on the basis of complementation analysis and experiments with mutant phages. We report here the cloning of gene XV into an expression plasmid and the purification of its product, protein P15, to near homogeneity. The purified protein P15, identified by N‐terminal sequence analysis, showed a strong lytic activity against chloroform‐treated Gram‐negative cells. No activity against Gram‐positive bacterial species could be detected. The pH optimum of the enzyme was between 7.0–8.0. Protein P15 was readily inactivated at temperatures above 4°C as well as by increasing the ionic strength of the buffers. The analysis of cell wall digests indicated that P15 is a glycosidase that cleaves the β(1–4) linkage between N‐acetylmuramic acid and N‐acetylglucosamine, thus displaying muramidase activity.
Diversity of Vertebrate Class I Alcohol Dehydrogenase
Authors: Estonius Mats , Hjelmqvist Lars , Jörnvall Hans
Published: March 2005

Class I alcohol dehydrogenase has been characterized from ostrich liver in order to evaluate enzyme variability between two independent lines, mammalian forms of class I alcohol dehydrogenase as a group, and a sufficient number of the enzyme from the most recent animal class (Aves, birds) as another. Between the two enzyme groups, patterns are consistent and mutually similar. This indicates conserved metabolic and catalytic properties of class I alcohol dehydrogenase, suggesting its metabolic role to be distinct, in spite of its protein variability. The new structure has a microheterogeneity (position 112, Arg/Cys) in a variable Zn‐binding loop. In addition, it also establishes further native variants at active‐site positions, including one thus far unique residue at the inner part of the substrate‐binding pocket (Ala141), and a replacement at position 271 (giving His271), which is also the site of a human alcohol dehydrogenase γ1/γ2 isozyme variability. The data correlate with functional differences in catalytic properties, the ostrich enzyme having a comparatively high Km for ethanol (5.9 mM at pH 10), and emphasize the importance of single positions in substrate and coenzyme binding, paralleling isozyme variability with protein variability within the class I enzymes.
Structural Comparison of phospholipase‐A2‐binding Regions in phospholipase‐A2 Receptors from Various Mammals
Authors: Higashino Ken‐ichi , Ishizaki Jun , Kishino Junji , Ohara Osamu , Arita Hitoshi
Published: March 2005

We determined the nucleotide sequence of a mouse cDNA encoding the receptor for pancreatic group I phospholipase A2 (PLA2‐I). Interspecies structural comparison of the mouse receptor with bovine PLA2‐I receptor, whose structure had been clarified, revealed that the fourth carbohydrate‐recognition domain (CRD)‐like domain (CRD‐like 4) was the most conserved among the domains in the PLA2‐I receptor, suggesting the functional importance of CRD‐like 4. A transient expression experiment with a truncated form of the receptor consisting of three CRD‐like domains, from the third to the fifth, demonstrated that the PLA2‐I‐binding site of the receptor is constituted from these three CRD‐like domains, supporting the functional indispensability of CRD‐like 4 in the receptor. Since the PLA2‐I‐binding region was thus assigned to be CRD‐like domains 3–5, we further analyzed the structures of the PLA2‐I‐binding regions in the PLA2‐I receptors from the rat, rabbit and human. Furthermore, the obtained PLA2‐I receptor cDNA fragments from these animals made it possible to examine the tissue expression patterns of this receptor in various mammals. The results, together with the results of the genomic structural analysis of this gene, indicated that a PLA2 receptor recently characterized by Lambeau et al. [Lambeau, G., Ancian, P., Barhanin, J. & Lazdunski, M. (1994) J. Biol. Chem. 269, 1575–1578] is a rabbit counterpart of the PLA2‐I receptor although these two PLA2 receptors have distinctive PLA2‐binding specificities.
Purification and Characterization of the Thyrotropin‐releasing‐hormone‐degrading Ectoenzyme
Authors: Bauer Karl
Published: March 2005

The membrane‐bound enzyme which catalyzes the degradation of thyrotropin‐releasing hormone (TRH; Glp‐His‐Pro‐NH2) could be released from membranes of rat and pig brain by treatment with trypsin under very mild incubation conditions. The solubilized enzyme was purified 200000‐fold, with an overall yield of 20%, by conventional chromatographic methods.

The enzyme preparation appeared to be electrophoretically homogenous since SDS/PAGE analysis revealed a single band with a molecular mass of 116000 Da. By gel‐filtration chromatography, a molecular mass of 230000 Da was estimated, suggesting that the enzyme consists of two identical subunits.

The enzyme could be identified as a glycoprotein by lectin‐binding analysis and by the reduction of the molecular mass to 97000 Da upon treatment of the denatured enzyme with endoglycosidase‐F/N‐glycosidase F. In its native form, however, the enzyme was only partially deglycosylated and retained full enzymic activity.

In addition to TRH, the enzyme also hydrolyzed l‐5‐oxoprolyl‐β‐naphthylamide, and thus a convenient fluorimetric assay could be established to determine high enzyme activities. The hydrolysis of both substrates was found to obey Michaelis‐Menten kinetics, but considerable differences in the respective Km and Vmax values were noticed.
Induction and Turnover of Fructose 1,6‐Bisphosphatase in HL‐60 Leukemia Cells by Calcitriol
Authors: Mizunuma Hideo , Tashima Yohtalou
Published: March 2005

Fructose 1,6‐bisphosphatase mRNA and enzyme activity in HL‐60 cells were rapidly and markedly induced by calcitriol (formerly known as 1α,25‐dihydroxyvitamin D3). The activity reached 70–80 times the basal level after 96 h. The enzyme activity in the cells incubated for 96 h with calcitriol decreased immediately after its withdrawal but after a 24‐h incubation the activity in the cells continued to increase slightly and then decreased slowly. Calcitriol increased the enzyme activity dose‐dependently with maximal stimulation at 10 nM and half‐maximal at 2.1 nM. The rate of synthesis of fructose 1,6‐bisphosphatase almost paralleled the increase in mRNA level during treatment with calcitriol. When calcitriol was removed from media after incubation for either 24 h or 96 h, fructose‐1,6‐bisphosphatase mRNA and fructose‐1,6‐bisphosphatase synthesis decreased rapidly to the basal level. The enzyme was only slightly degraded in the cells incubated with calcitriol for 24 h followed by the subsequent culture without calcitriol but it was degraded with a half‐life estimated to be approximately 64 h in the same cells followed by culturing with calcitriol. In the cells incubated for 96 h, the same degradation rate (i.e. half‐life ≈64 h) was observed irrespective of the following culture with or without calcitriol. Calcitriol did not affect the degradation rate of total soluble proteins.
Structural and Catalytic Properties of the Four Phenylalanine Ammonia‐Lyase Isoenzymes from Parsley (Petroselinum Crispum Nym.)
Authors: Appert Christoph , Logemann Elke , Hahlbrock Klaus , Schmid Jürg , Amrhein Nikolaus
Published: March 2005

Near‐full‐length cDNAs for the four phenylalanine ammonia‐lyase (PAL) isoenzymes in parsley (Petroselinum crispum Nym.) were cloned and the complete amino acid sequences deduced. Fusion proteins with glutathione S ‐transferase were expressed in Escherichia coli, purified and cleaved. All of the resulting phenylalanine ammonia‐lyase proteins, as well as the fusion proteins, were catalytically active. The turnover number of one selected isoenzyme, PAL‐1, was estimated to be around 22 s−1 for each active site. In contrast to a certain degree of differential expression in various parts of parsley plants, the four phenylalanine ammonia‐lyase isoenzymes exhibited very similar apparent Km, values for L‐phenylalanine (15–24.5 μM) as well as identical temperature (58° C) and pH (8.5) optima. All of them were competitively inhibited by (E)‐cinnamate with similar efficiency (Ki, values: 9.1–21.5 μM), lacked cooperative behaviour, and accepted L‐tyrosine as a substrate with low affinity (Km, values: 2.6–7.8 μM). These results suggest that the occurrence of multiple gene copies has a function other than encoding isoenzymes with different enzyme kinetic properties.
Gene Cloning and Characterization of PepC, a Cysteine Aminopeptidase from Streptococcus thermophilus, with sequence Similarity to the Eucaryotic Bleomycin Hydrolase
Authors: Chapot‐Chartier Marie‐Pierre , Rul Françoise , Nardi Michèle , Gripon Jean‐Claude
Published: March 2005

Streptococcus thermophilus CNRZ 302 contains at least three general aminopeptidases able to hydrolyze Phe‐β‐naphthylamide substrate. The gene encoding one of these aminopeptidases was cloned from a total DNA library of S. thermophilus CNRZ 302 constructed in Escherichia coli TG1 using pBluescript plasmid. The wild‐type TG1 strain, although not deficient in aminopeptidase activity, is unable to hydrolyze the substrate Phe‐β‐naphthylamide, and thus the library could be screened with an enzymic plate assay using this substrate. One clone was selected which was shown to express an aminopeptidase, identified as a PepC‐like enzyme on the basis of cross‐reactivity with polyclonal antibodies directed against the lactococcal PepC cysteine aminopeptidase. The gene was further subcloned and sequenced. A complete open reading frame coding for a 445‐residue (50414 Da) polypeptide was identified. 70% identity was found between the deduced amino acid sequence and the sequence of PepC from Lactococcus lactis subspecies cremoris, confirming the identity of the cloned gene. High sequence similarity (38% identity) was also found with an eucaryotic enzyme, bleomycin hydrolase. In addition, the predicted amino acid sequence of the streptococcal PepC showed a region of strong similarity to the active site of cysteine proteinases with conservation of the residues involved in the catalytic site. The product of the cloned pepC gene was overproduced in E. coli and was purified from a cellular extract. Purification to homogeneity was achieved by two‐step ion‐exchange chromatography. Biochemical characterization of the pure recombinant enzyme confirms that the cloned peptidase is a thiol aminopeptidase possessing a broad specificity. The enzyme has a molecular mass of 300 kDa suggesting an hexameric structure. On the basis of sequence similarities as well as common biochemical and enzymic properties, the bacterial PepC‐type enzymes and the eucaryotic bleomycin hydrolase constitute a new family of thiol aminopeptidases among the cysteine peptidases.
Crystallographic Studies of Savinase, a Subtilisin‐like Proteinase, at pH 10.5
Authors: Lange Gudrun , Betzel Christian , Branner Sven , Wilson Keith S.
Published: March 2005

The effect of high pH (pH 10.5) on Savinase, a subtilisin‐like serine proteinase, has been investigated using X‐ray crystallography. The structures of two Savinase mutants were determined at two different pH values, namely pH 6.0, where the enzyme is inactive (this is the pH at which most of the structural work has been carried out on other serine proteinases), and pH 10.5, where Savinase is active. Comparison of these high resolution (0.16 nm) structures showed four related sets of changes between the two pH values. First, the difference in protonation state of the active‐site histidine leads to a change in conformation of the active‐site triad. Secondly, there are resulting changes in the water structure around this histidine residue with much increased mobility of the water in the active‐site at pH 10.5. Thirdly, the two substrate‐binding loops on either side of the binding site are less well linked by ordered water molecules at pH 10.5 and show substantially increased flexibility. Finally, the first substrate‐binding loop, residues at positions 99–104, moves slightly so as to widen the substrate channel.
Proteasome‐associated RNAs are non‐specific
Authors: Pamnani Vashu , Haas Bernd , PÜHler Gabriela , SÄNger Heinz Ludwig , Baumeister Wolfgang
Published: March 2005

The RNA isolated from RNase‐treated proteasome preparations from human erythrocytes, HeLa cells, the archaeon Thermoplasma acidophilum and also from recombinant proteasomes of T. acidophilum expressed in Escherichia coli was characterized. The RNA associated with structurally similar protein particles, namely with the two molecular chaperones, groEL from E. coli and with the thermosome from T. acidophilum, served as controls. Electrophoretic analysis on polyacrylamide gels of the radioactively endlabelled RNA revealed a very similar size distribution pattern, irrespectively of the protein particles from which they had been isolated. The predominant RNA species were in the size ranges 80 nucleotides and 120 nucleotides, respectively. Partial sequencing of their terminal regions by mobility‐shift analysis revealed that, of the proteasomes from human erythrocytes, the approximately 80‐ucleotide‐long RNA consists of a heterogenous population of mostly tRNA species because they carried the tRNA‐specific 3′‐terminal sequence motif 5′‐CCA‐3′. The RNA in the size range 120 nucleotides isolated from the proteasomes of human erythrocytes and of T. acidophilum was also heterogeneous and displayed, in the terminal regions, a remarkable sequence similarity to the corresponding regions of the 5S rRNA from the same and different organisms. The total content of RNA of all the protein particles was quantified and found to be consistently sub‐stoichiometric. All these findings strongly suggest that RNA associated with the proteasomes and with the molecular chaperones originate from the abundant cellular pool of the tRNAs and 5S rRNAs which bind non‐specifically to these large protein particles.
Bombesin, endothelin and platelet‐derived growth factor induce rapid translocation of the myristoylated alanine‐rich C‐kinase substrate in Swiss 3T3 cells
Authors: Herget Thomas , Rozengurt Enrique
Published: March 2005

We analyzed the effect of growth factors on the localization of the 80‐kDa acidic myristoylated alanine‐rich C‐kinase substrate (80‐kDa MARCKS), the major protein kinase C (PKC) substrate, in Swiss 3T3 fibroblasts. Virtually all 80‐kDa MARCKS of quiescent cultures of these cells was membrane bound. However, within 40min after addition of bombesin (10 nM) to these cells, the content of 80‐kDa MARCKS in the cytoplasmic fraction increased 25‐fold. Phosphorylated 80‐kDa MARCKS was detectable in the cytoplasmic fraction as early as 30 s after addition of bombesin and the translocation was sustained for 6 h i.e. until 80‐kDa MARCKS became down‐regulated. The ability of bombesin to stimulate translocation of 80‐kDa MARCKS was dose‐dependent (concentration required to produce 50% of the effect was 0.6 nM bombesin) and was abolished by the specific antagonist [Leu14,13Ψ14CH2NH]bombesin. Furthermore, platelet‐derived growth factor (PDGF) stimulated a dose‐dependent (concentration required to produce 50% of the effect was 3 ng/ml) translocation which was comparable to that induced by bombesin in terms of kinetics and magnitude. Translocation was independent of continuous protein synthesis, but dependent on active PKC. Depletion or inhibition of PKC activity abolished the 80‐kDa MARCKS translocation induced by either bombesin or PDGF. Furthermore, the neuropeptides β‐endothelin, bradykinin, and vasopressin, which are known to stimulate PKC activity, also promoted translocation. In contrast, epidermal growth factor, insulin and forskolin, which do not activate PKC, failed to cause such an effect. Translocation of 80‐kDa MARCKS was also observed in Rat1 cells treated with phorbol ester, PDGF and β‐endothelin. We conclude that the translocation of 80‐kDa MARCKS from the membrane to the cytosol is an early response to a variety of growth‐promoting factors that stimulate PKC through different signal‐transduction pathways.
Molecular Characterization of Escherichia coli Malate Synthase G
Authors: Molina Isabel , Pellicer María‐Teresa , Badia Josefa , Aguilar Juan , Baldoma Laura
Published: March 2005

Two genes encoding the enzymes malate synthase G and glycolate oxidase, have been linked to locus glc (64.5 min), responsible for glycolate utilization in Escherichia coli. The gene encoding malate synthase G, for which we propose the notation glcB, has been cloned, sequenced and found to correspond to a 2262‐nucleotide open‐reading frame, which can encode a 723‐amino‐acid polypeptide, clearly different from the isoenzyme malate synthase A, which has 533 amino acids. Northern‐blot experiments indicate that glcB was expressed as an apparently monocistronic transcript, inducible by glycolate. Malate synthase G was purified to near homogeneity. The molecular mass determined by gel filtration yielded a value of 82 kDa for the purified enzyme and the same value as for the crude extract enzyme, indicating a monomeric structure. Despite the lower sequence similarity between malate synthase G and the other reported malate synthases, three out of nine consensus boxes defined in most of these enzymes are conserved in addition to a cysteine residue that has been reported to be important for the catalytic mechanisms.
The capacity of reducing‐equivalent shuttles limits glycolysis during ethanol oxidation
Authors: Berry Michael N. , Gregory Roland B. , Grivell Anthony R. , Phillips John W. , SchÖN Arne
Published: March 2005

The inhibition of glycolysis during ethanol oxidation has been examined in isolated hepatocytes from fasted rats. Glycolytic flux was measured by determining the rate of release of tritium from [6‐3H]glucose. During ethanol oxidation, the rate of glycolysis was inhibited 80% in freshly prepared hepatocytes, in which shuttle intermediates are depleted, but was depressed only about 20% in the presence of asparagine, a condition under which activity of the malate/aspartate shuttle was restored to normal levels. The inhibition of glycolysis was also partially released by addition of pyruvate and when alcohol dehydrogenase activity was depressed by 4‐methylpyrazole. Titrations with this inhibitor revealed inverse linear relationships between the rates of glycolysis and ethanol oxidation. For any given rate of ethanol oxidation, glycolytic flux was lowest and the [lactate]/[pyruvate] ratio highest in the presence of aminooxyacetate, an inhibitor of the malate/aspartate shuttle, whereas flux was highest and the ratio lowest in the presence of asparagine. During these titrations with 4‐methylpyrazole the inhibition of ethanol oxidation and concomitant restoration of glycolysis were accompanied by a decline in the [lactate]/[pyruvate] ratio, a substantial fall in the rate of reducing‐equivalent transfer from cytoplasm to mitochondria and an increase in lactate accumulation. These findings imply that the reducing equivalents generated during ethanol oxidation compete with those arising in glycolysis for transfer to the mitochondria. This competition leads to an inhibition of aerobic glycolysis, and at the same time contributes to a rise in cytoplasmic NADH and fall in NAD+ that results in depression of anaerobic glycolysis. Allosteric inhibition of 6‐phosphofructo‐1‐kinase due to a decrease in the concentration of fructose 2,6‐bisphosphate did not appear to play a primary role in the inhibition of glycolysis by ethanol. Ethanol oxidation had no effect on glucose phosphorylation as measured with [2‐3H] glucose, but induced a substantial increase in cycling between glucose and glucose 6‐phosphate.
Structure/Function Implications for the Aminopeptidase Specificity of Aleurain
Authors: Rothe Markus , Zichner Angelika , Auerswald Ennes A. , Dodt Johannes
Published: March 2005

The cysteine protease aleurain, a member of the papain superfamily, was characterized by its specificity constants, kcat/Km, for the hydrolysis of different substrates of the type H‐P1‐NH‐Mec (NH‐Mec, 4‐methylcoumaryl‐7‐amide). The determined constants for the different substrates decrease in the order citrulline > Arg = Phe ≫ Ala. A 75‐fold loss of specificity was observed when the substrate Bz‐Arg‐NH‐Mec (Bz, benzoyl), with a blocked N‐terminus, was used instead of H‐Arg‐NH‐Mec. The pH dependence of kcat/Km for H‐Arg‐NH‐Mec was bell‐shaped with pKa1, and pKa2values of 5.81 and 7.27, respectively, at 25°C. The residue corresponding to a pKa1, value of 5.81 could be identified by its ionisation enthalpy, Hion, of 15 kJ/mol as a carboxylate group of the enzyme interacting electrostatically with the residue with pKa2 7.27, attributed to the α‐amino group of the substrate by its Hion, value of 48 kJ/mol. Aleurain can be titrated at the active site with l‐trans‐epoxy‐succinylleucylamido(4‐guanidino)butane, and the reaction was characterized by its association rate constant of 19000 M−1· s−1. Native chicken cystatin inhibited aleurain competitively with Ki 133 nM. Recombinant chicken cystatin variants Ala‐Glu‐Phe‐[Met1, Ile29, Leu89] chicken egg‐white cystatin, (variant 1) and the N‐terminally truncated form des‐(S1‐P11)‐[Ala12, Glu12, Phe14, Met15, Ile29, Leu89]‐chicken egg‐white cystatin, (variant 2), inhibited aleurain competitively with Ki, values of 125 nM and 5 μM, respectively. Implications for the aminopeptidase activity of aleurain are discussed using cathepsin H for comparison.
Human Erythrocyte Membrane Protein 4.2 is Palmitoylated
Authors: Das Amit K. , Bhattacharya Raja , Kundu Manikuntala , Chakrabarti Parul , Basu Joyoti
Published: March 2005

Protein 4.2 is a major protein of the human erythrocyte membrane. It has previously been shown to be N‐myristoylated. After labeling of intact human erythrocytes with [3H]palmitic acid, radioactivity was found to be associated with protein 4.2 by immunoprecipitation of peripheral membrane proteins extracted at pH 11 from ghosts with anti‐(4.2) sera, followed by SDS/PAGE and fluorography. The fatty acid linked to protein 4.2 was identified as palmitic acid after hydrolysis of protein and thin‐layer chromatography of the fatty acid extracted in the organic phase. Protein 4.2 could be depalmitoylated with hydroxylamine, suggesting a thioester linkage. Depalmitoylated protein 4.2 showed significantly decreased binding to protein‐4.2‐depleted membranes, compared to native protein 4.2.
The DNA‐binding domain of the yeast Saccharomyces cerevisiae CYP1(HAP1) transcription factor possesses two zinc ions which are complexed in a zinc cluster
Authors: Timmerman Johanna E. , Guiard Bernard , Shechter Evelyne , Delsuc Marc‐André , Lallemand Jean‐Yves , Gervais Michel
Published: March 2005

Various fragments of the N‐terminal, DNA‐binding domain of the yeast Saccharomyces cerevisiae transcriptional activator CYP1(HAP1) have been cloned and expressed in Escherichia coli. The corresponding polypeptides have been analysed biochemically and we have undertaken a more extensive physical study of a fragment consisting of amino acids 49–126 [CYP1(49–126)]. We show that this CYP1(49–126) peptide requires zinc or cadmium in the growth medium in order to maintain a stable structure. A method to purify CYP1(49–126) is presented. We demonstrate that the purified CYP1(49–126) fragment contains two zinc ions/fragment or two cadmium ions/fragment, which are necessary for DNA binding. 113Cd one‐dimensional NMR data suggest that CYP1(HAP1) has a tetrahedral coordination, and that it forms a zinc‐cluster complex like GAL4.
Penicillin‐binding Protein 7/8 of Escherichia coli is a dd‐endopeptidase
Authors: Romeis Tina , Höltje Joachim‐Volker
Published: March 2005

Penicillin‐binding protein 7 (PBP7) and its proteolytic degradation product PBP8 are shown to be soluble proteins, which can be set free from whole cells of Escherichia coli by an osmotic shock. The proteins are loosely associated with the membranes and are totally released into the supernatant in the presence of 1 M NaCl. Partial purification of PBP8 was accomplished by hydroxyapatite, heparin‐Sepharose and Monos chromatography. Murein meso‐diaminopimelate‐d‐alanine dd‐endopeptidase activity was demonstrated for both PBP7 and PBP8, which specifically hydrolyse the dd‐diaminopimelatealanine bonds in high‐molecular‐mass murein sacculi but fail to cleave these bonds in isolated dimeric muropeptides. The enzyme is inhibited by the ‘penem’β‐lactam antibiotic CGP31608 at a concentration of 0.25 μg/ml by 50%. Thus besides PBP4 and the mepA gene product, a third endopeptidase exists in E. coli.
The Complete Amino Acid Sequence and Disulphide Bond Arrangement of Oat Alcohol‐soluble Avenin‐3
Authors: Egorov Tsezi A. , Musolyamov Aleksander K. , Andersen Jens S. , Roepstorff Peter
Published: March 2005

We have elucidated the complete amino acid sequence of one of the avenin components, avenin‐3, isolated from oat (Avena sativa L.), variety Narymsky 943. The sequence of the protein was determined by sequencing of CNBr and trypsin‐generated peptides in combination with mass spectrometry. The protein is a single polypeptide chain, consisting of 201 amino acid residues with Mr 23252.8. The N‐terminal amino acid residue of the protein is blocked with 5‐oxoproline (pyroglutamic acid). All eight cysteine residues in avenin‐3 are involved in disulphide bonds. The positions of these bonds were established by identification of a CNBr cleavage product of the intact avenin containing all the disulfide bonds (S‐S core). Subsequent subdigestion of this S‐S core allowed isolation of disulphide bonded peptides detected by differential reverse‐phase HPLC before and after reduction. As a result, all four disulphides

,

,

and

were identified. Comparison of avenins with other prolamins demonstrates a high degree of similarity, which is especially pronounced around the cysteine residues. Avenins differ slightly from other prolamins in having unique N‐terminal sequences and some differences in the repeated sequence motifs.
Covalent modification of the interleukin‐5 receptor by isothiazolones leads to inhibition of the binding of interleukin‐5
Authors: Devos René , Guisez Yves , Plaetinck Geert , Cornelis Sigrid , Tavernier Jan , Van Der Heyden José , Foley Louise H. , Scheffler Julie E.
Published: March 2005

Using a fusion protein of the human interleukin‐5‐receptor α chain (hILSRα) and the human IgG Cγ3 chain (hIL5Rα‐hγ3), we have developed a solid‐phase assay for high‐flux screening of a collection of synthetic compounds. We report on the identification of isothiazolone derivatives as potent inhibitors of binding of interleukin‐5 (IL5) to the hIL5Ra, as measured in a solid‐phase assay (soluble hIL5Rα or hIL5Rα‐hγ3) or on COS‐1 cells expressing the hIL5Rα on the cell membrane. The binding of ML4 and human granulocyte macrophage colony‐stimulating factor (hGM‐CSF) to their respective receptors is not inhibited by the isothiazolones in similar assay systems. Scatchard analysis revealed that these compounds caused a decrease in affinity of the IL5Rα for IL5. The inhibition of binding IL5 to its receptor by the isothiazolone derivatives is abrogated by free‐sulfhydryl‐containing compounds such as dithiothreitol, indicating that the isothiazolones react with the sulfhydryl group of free cysteine residues in the hIL5Rα. Mutation of Cys66 led to a receptor which still binds hIL5, but which was insensitive to the inhibition by isothiazolones. Mutation of Cys249 and Cys296 to serine resulted in complete loss of IL‐5‐binding activity. The use of radio‐labeled isothiazolone confirmed that Cys66, present in the first domain of the receptor, is the target for covalent modification leading to a decrease in affinity.