bluuebunny/crossref_metadata_2025 huggingface dataset:
cDNA Cloning and Primary Structure of Tryptase from Bovine Mast Cells, and Evidence for the Expression of Bovine Pancreatic Trypsin Inhibitor mRNA in the Same Cells
Authors: Pallaoro Michele , Gambacurta Alessandra , Fiorucci Laura , Mignogna Giuseppina , Barra Donatella , Ascoli Franca
Published: August 2004

A partial cDNA encoding bovine tryptase, an oligomeric serine proteinase previously isolated from bovine mast cells, was obtained by reverse transcription/polymerase chain reaction of mast cell mRNA, using combinations of primers designed on the basis of information obtained from partial sequencing of the purified protein. The complete amino acid sequence of bovine tryptase (245 residues) was deduced from a 711‐bp nucleotide sequence and from Edman degradation of the protein. Bovine tryptase primary structure has an identity of about 75% with tryptases from other species and includes all the essential residues of the active‐site regions; sequence data in the region of the putative substrate binding pocket suggest a rearrangement capable of maintaining the specificity of trypsin‐like proteinases. From the same mast cell mRNA, cDNA encoding bovine trypsin protease inhibitor (BPTI) was obtained and amplified with specific primers, confirming the synthesis of BPTI in these cells. Results are consistent with previous data on the presence of BPTI and bovine tryptase in the same granules of bovine mast cells and with their interaction in vitro.
Crystal Structures and Solution Studies of Oxime Adducts of Mitochondrial Aspartate Aminotransferase
Authors: Marković‐Housley Zora , Schirmer Tilman , Hohenester Erhard , Khomutov Alex R. , Khomutov Radii M. , Karpeisky Marat Y. , Sandmeier Erika , Christen Philipp , Jansonius Johan N.
Published: August 2004

The interaction of mitochondrial aspartate aminotransferase with hydroxylamine and five derivatives (in which the hydroxyl hydrogen is replaced by the side chain of naturally occurring amino acids) was investigated by X‐ray diffraction as well as by kinetic and spectral measurements with the enzyme in solution. The inhibitors react with pyridoxal 5′‐phosphate in the enzyme active site, both in solution and in the crystalline state, in a reversible single‐step reaction forming spectrally distinct oxime adducts. Dissociation constants determined in solution range from 10−8 M to 10−6 M depending on the nature of the side‐chain group.

The crystal structures of the adducts of mitochondrial aspartate aminotransferase with the monocarboxylic analogue of l‐aspartate in the open and closed enzyme conformation were determined at 0.23‐nm and 0.25‐nm resolution, respectively. This inhibitor binds to both the open and closed crystal forms of the enzyme without disturbing the crystalline order. Small differences in the conformation of the cofactor pyridoxal phosphate were detected between the structures of both oxime complexes and the 2‐methylaspartate adduct. The crystal structures indicate that the interaction between the ω‐carboxylate of the inhibitor and Arg292* of the neighbouring subunit is mainly responsible for the attainment of near‐coplanarity of the aldimine bond with the pyridine ring in the oxime adducts. Studies with a fluorescent probe aimed to detect shifts in the open/closed conformational equilibrium of the enzyme in oxime complexes showed that the hydroxylamine‐derived inhibitors, even those containing a carboxylate group, do not induce the ‘domain closure’ in solution. This is probably due to the absence of the α‐carboxylate group in the monocarboxylic hydroxylamine‐derived inhibitors, emphasizing that both carboxylates of the substrates l‐Asp and l‐Glu are essential for stabilizing the closed form of aspartate aminotransferase.
Two Distinct Long‐Chain‐Acyl‐CoA Synthetases in Guinea Pig Harderian Gland
Authors: Kono Mari , Hori Chiyo , Hashimoto Takashi , Hori Shinichiro , Seyama Yousuke
Published: August 2004

Two distinct long‐chain‐acyl–CoA synthetases which have different kinetic properties were identified in the guinea pig Harderian gland. One was localized in the microsomes and the other in the mitochondria. The relative Vmax values of the microsomal enzyme were 8.1, 1.7 and 1 and the apparent Km values were 66.7, 12.0 and 30.0 μM for palmitic, linoleic and arachidonic acids, respectively. The relative Vmax values of the mitochondrial enzyme were 2.7, 3.5 and 1 and the apparent Km values were 33.3, 29.9 and 30.0 μM for palmitic, linoleic and arachidonic acids, respectively. The relative Vmax values for the liver microsomal enzyme were 2.0, 2.5 and 1, while those of the liver mitochondrial enzyme were 4.1, 3.9 and 1 with palmitic, linoleic and arachidonic acids, respectively. There were no difference between the microsomal and the mitochondrial enzymes in the liver, regarding apparent Km values; these were 38.4, 29.9 and 22.0 μM for palmitic, linoleic and arachidonic acids, respectively. Thus, the substrate specificity and catalytic rate of the mitochondrial enzyme in Harderian gland for palmitic, linoleic and arachidonic acids were similar to the liver enzyme, but not to the microsomal enzyme in Harderian gland. On the other hand, the antiserum raised against the rat liver enzyme immune‐titrated and immuno‐blotted the enzymes from Harderian gland microsomes and liver, but not so the enzyme from Harderian gland mitochondria. Thus, the microsomal enzyme in Harderian gland had a common immunogenic epitope(s) with the liver enzyme, but the mitochondrial enzyme did not. The Harderian gland mitochondrial enzyme was a distinct protein from liver enzymes. The catalytic and immunogenic characteristics suggest that the enzyme proteins in the Harderian gland are unique, that is, different from that in the liver. The large Vmax value of the Harderian gland microsomal enzyme for palmitic acid suggests that it contributes to the synthesis of a large amount of the secretory lipid and the high Km value to maintenance of cellular lipid in this organ. The evidence that long‐chain‐acyl–CoA synthetase in the mitochondria is distinct from that in the microsomes was first found in guinea pig Harderian gland.
Modifications Occur at Different Structural Levels During the Heat Denaturation of β‐Lactoglobulin
Authors: Iametti Stefania , De Gregori Beatrice , Vecchio Giuseppe , Bonomi Francesco
Published: August 2004

Heat‐induced modifications in the tertiary and quaternary structure of β‐lactoglobulin were followed at neutral pH for the protein at high temperature and for the protein that was heated and cooled. Fast changes in the environment of aromatic amino acids were apparent from near‐ultraviolet‐CD spectra of the heated protein and their intensity increased with increasing temperature. These modifications were irreversible only at temperatures higher than 65–70°C. Addition of iodoacetamide during the heating/cooling cycle greatly reduced the extent of irreversible modification of the tertiary structure of the protein. Reaction of the native‐lactoglobulin dimer with iodoacetamide or dithiobis(2‐nitrobenzoic acid) was only observed upon heating at temperatures higher than 40°C and resulted in progressive reaction of the unique sulfhydryl group in each of the two protein monomers. The sulfhydryl reagents induced release of a monomeric protein species that was no longer able to aggregate to the native dimeric form or to sequentially form polymers as found in the protein after heating at high temperature. Dimer dissociation was identified as the rate‐limiting step in the reaction of β‐lactoglobulin with sulfhydryl reagents. It occurred at temperatures much lower than those required for appreciable modification of the tertiary structure of the protein, and had an extremely high activation energy (Ea= 213 kJ/mol). These results are compared with other published data, and a general mechanism for the formation of early reactive species in heat‐treated β‐lactoglobulin at neutral pH is proposed which stresses the relevant role of a highly hydrophobic, molten‐globule‐like free monomer that has an exposed sulfhydryl group on its surface.
Glycosylation of Recombinant Ancrod from Agkistrodon rhodostoma after Expression in Mouse Epithelial Cells
Authors: Geyer Hildegard , Jacobi Ines , Linder Dietmar , Stirm Stephan , Bialojan Siegfried , Strube Karl‐Hermann , Geyer Rudolf
Published: August 2004

The thrombin‐like serine protease ancrod from the Malayan pit viper Agkistrodon rhodostoma was expressed in mouse epithelial cells (C127). Oligosaccharide constituents were liberated from tryptic glycopeptides by treatment with peptide‐N4‐(N‐acetyl–β‐glucosaminyl)asparagine amidase F. Neutral oligosaccharide alditols obtained after reduction and enzymic desialylation were separated by two‐dimensional HPLC and characterized by methylation analysis, liquid secondary‐ion mass spectrometry, matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and sequential degradation with exoglycosidases. In contrast to natural ancrod, the recombinant glycoprotein carries exclusively diantennary, triantennary and tetraantennary N‐glycans with Galβ4GlcNAcβ (type‐2) antennae which were, in part, further substituted by host‐cell‐specific structural elements such as Galα3 residues or N‐acetyllactosamine repeats. As a characteristic feature, a substantial proportion of the oligosaccharides bears a GalNAcβ4Glc‐NAc antenna. Studies at the level of individual N‐glycosylation sites demonstrated that glycans with N,N′‐diacetyllactosediamine units are not specifically attached but occur at all sites in varying amounts. Hence, the putative recognition signal (Pro70‐Lys‐Lys) for glycoprotein hormone N‐acetylgalactosaminyltransferase, present in this glycoprotein in close proximity to Asn79, does not convey site‐specific transfer of GalNAc residues in these cells.
Comparison of the Structural Features of Ubiquinone Reduction Sites Between Glucose Dehydrogenase in Escherichia coli and Bovine Heart Mitochondrial Complex I
Authors: Sakamoto Kimitoshi , Miyoshi Hideto , Matsushita Kazunobu , Nakagawa Masato , Ikeda Junko , Ohshima Michiyo , Adachi Osao , Akagi Toshio , Iwamura Hajime
Published: August 2004

To characterize the structural features of the ubiquinone reduction site of glucose dehydrogenase (GlcDH) in Escherichia coli, we performed structure/activity studies of a systematic set of synthetic ubiquinone analogues and specific inhibitors (synthetic capsaicins) of this site. Considering the proposed similarity of the quinone binding domain motif between GlcDH and one subunit of mitochondrial complex I [Friedrich, T., Strohdeicher, M., Hofhaus, G., Preis, D., Sahm, H. & Weiss, H. (1990) FEBS Lett. 265, 37–40], we compared the structure/activity profiles of the substrates and inhibitors for GlcDH with those for bovine heart mitochondrial complex I. With respect to GlcDH, replacement of one or both methoxy groups in the 2 and 3 positions of benzoquinone ring by ethoxy group(s) resulted in a drastic decrease in the electron accepting activity. The presence of a 5‐methyl group and the conformational property of the 6‐alkyl side chain did not significantly contribute to the activity. These results suggested that only half of the benzoquinone ring (the moiety corresponding to the 2 and 3 positions) is recognized by the quinone reduction site in a strict sense. In contrast, quinone analogues with structural modifications at all positions in the benzoquinone ring retained the activity with mitochondrial complex I. This finding indicated that the catalytic site of complex I is spacious enough to accommodate a variety of structurally different quinone derivatives. The correlation of the inhibitory potencies of a series of synthetic capsaicins between the two enzymes was very poor. These findings indicated that the binding environment of ubiquinone in GlcDH is very specific and differs from that in mitochondrial complex I.
Nucleocapsid Protein 10 Activates Dimerization of the RNA of Moloney Murine Leukaemia Virus in vitro
Authors: Bonnet‐Mathonière Bénédicte , Girard Pierre‐Marie , Muriaux Delphine , Paoletti Jacques
Published: August 2004

Short RNA species that encompas the ψ domain of the retroviral genome spontaneously form dimers in vitro, and the retroviral nucleocapsid protein activates this dimerization in vitro. Addition of gag RNA sequences downstream of the 3′ end of the ψ domain decreases the level of spontaneous dimerization. Here, we report the effects of RNA length on dimerization in vitro, studied with RNA fragments from Moloney murine leukamia virus that contain the ψ domain and all or part of the gag sequence. Extension of the RNA leads to progressive inhibition of the in vitro dimerization process. Sequences located downstream of the 3′ end of the ψ domain seem to stabilize the monomeric structures. This stabilization participates in dimerization of the RNA sequences involved in the recognition of two RNA molecules. We studied the ability of nucleocapsid protein 10 to promote dimerization of such long RNA fragments, and found that the protein greatly enhances their dimerization in vitro. We propose that nucleocapsid protein 10 stimulates the overall dimerization process by reduction of the energy barrier that must be overcome to allow dimer formation. Our results show that dimerization of RNA form Moloney murine leukaemia virus in vitro is enhanced by nucleocapsid protein 10. This finding is in agreement with the involvement of the nucleocapsid protein in RNA dimerization in vivo.
Analysis of the Mechanism of Activation of cAMP‐Dependent Protein Kinase Through the Study of Mutants of the Yeast Regulatory Subunit
Authors: Zaremberg Vanina , Moreno Silvia
Published: August 2004

Spontaneous mutations in the gene which encodes the regulatory subunit of cAMP‐dependent protein kinase (PKA) of Saccharmyces cerevisiae (BCY1) have been isolated previously [Cannon, J. F., Gibbs, J. B. & Tatchell, K. (1986) Genetics 113, 247–264] by selection of ras2::LEU2 revertants that grew on non‐fermentable carbon sources. The revertants were placed into groups of increasing severity based on the number of PKA‐dependent traits affected [Cannon, J. F., Gitan, R. & Tatchell, K. (1990) J. Biol. Chem. 265, 11897–11904]. In this work the ras2 mutation has been crossed out in each bcy1 allele and the phenotypes of these mutants have been assessed. The order of severity of the mutants in both genetic backgrounds is maintained but the severity of each mutant in the normal background is higher than in the ras2∷LEU2 background. Total catalytic‐subunit and regulatory‐subunit activities were measured in crude extracts of the bcy1 ras2∷LEU2 mutants. With one exception (bcy 1‐6) the calculated regulatory subunit/catalytic subunit ratios of the bcy1 mutants relative to that of wild‐type cells were greater than one. The dependence of PKA activity on cAMP was measured in permeabilized cells. The strains show an activity ratio in the absence and presence of cAMP in the range 0.5–1 for Kemptide phosphorylation. Overexpression of the high‐affinity cAMP phosphodiesterase gene (PDE2) in the bcy1 ras2∷LEU2 strains did not alter their PKA‐dependent phenotypes. However, transformants were not observed from the parental ras2∷LEU2 strain and the bcyl‐6 ras2∷LEU2 strain. The results are discussed with respect to a hypothesis for the molecular mechanism of the differential reversal of ras2 phenotypes by the bcy1 alleles. Mutations in the regulatory subunit are predicted to affect the structure of the holoenzyme such that the catalytic subunit is capable of maintaining an active catalytic state, without the need to dissociate from the regulatory subunit.
A Characterization of Transcriptional Regulatory Elements in Chicken Ribosomal Protein L37a Gene
Authors: Toku Seikichi , Tanaka Tatsuo
Published: August 2004

Transcriptional control elements of chicken ribosomal protein L37a gene were characterized in terms of their activities to promote transcription and their protein binding activities. The region ‐120 to +168 was necessary for the maximal expression of the promoter‐less CAT gene in a transfected chicken cell line. Using the DNase I protection assay, we identified nine protein binding regions distributed in a wide range of ‐122 to +195. The sequences of most of the elements are conserved among many vertebrate ribosomal protein genes at similar positions of the promoters. These common control elements and their binding proteins may coordinate the expression of ribosomal protein genes.
Purification and Characterization of a p13Suc1‐Bound Serine/Threonine Kinase that is Expressed in Mature Rat Brain
Authors: Tamaru Teruya , Okacta Masato
Published: August 2004

We previously reported that the histone‐H1 kinase activity bound to p13suc1 increased dramatically during development of the rat brain. In the present work, an in situ kinase assay in an SDS/polyacrylamide gel that contained substrate proteins was employed to characterize the enzyme. Two major proteins of 45 kDa and 100 kDa were found to have p13suc1‐bound histone‐H1 kinase activity. The former (p45) exhibited strong activity towards histone H1 and had weak autophosphorylation activity, whereas the latter (p100) acted on myelin basic protein or histone H1 and underwent autophosphorylation. p45 was further purified from the nuclear‐enriched fraction of rat brain to near homogeneity through sequential column chromatographies. The purified enzyme retained its ability to bind specifically to p13suc1, which suggests that this binding does not require a cofactor. The immunochemical and enzymatic properties of p45 revealed that it differs from Cdk that are known to bind to p13suc1 with high affinity. However, in vitro p45 acted on the peptide motif that is conserved among substrates for cyclin‐dependent kinases (Cdk) and mitogen‐activated protein kinases, which implies that this protein might belong to the large family of proline‐directed kinases. The evidence obtained in this study suggest that p45 is a nuclear p13suc1‐bound kinase that has unique functions in the mature brain.
Sequence Requirements in the Reactive‐Center Loop of Plasminogen‐Activator Inhibitor‐1 for Recognition of Plasminogen Activators
Authors: Tucker H. Michael , Gerard Robert D.
Published: August 2004

Plasminogen activator inhibitor‐1 (PAI‐1) is a member of the serpin superfamily of proteins and is the fast acting inhibitor of both urinary plasminogen activator and tissue‐type plasminogen activator. We have assessed the functional significance of reactive center residues on the carboxy‐terminal side of the cleavage site of recombinant human PAI‐1. Using site‐directed mutagenesis, the P1′–P5′ residues (P1′ is the first residue on the carboxy‐terminal side of the protease cleavage site) of the wild‐type PAI‐1 reactive center sequence were replaced with the corresponding sequences of plasminogen activator inhibitor‐2, antithrombin, α2‐antiplasmin and protease nexin I. Rate constants of inhibition of the serine proteases urinary plasminogen activator, tissue‐type plasminogen activator, plasmin and thrombin by the variants were determined. The results suggest a crucial role for both reactive center length and sequence in the inhibition of plasminogen activators by PAI‐1. Analysis of substitutions at positions P4′ and P5′ both confirms and extends our previous work demonstrating a favorable electrostatic interaction between these residues and tissue‐type plasminogen activator. None of the variants show dramatic increases in the rate constants of inhibition of other serine proteases, suggesting that these residues alone are not sufficient to confer protease specificity on PAI‐1. Apparently, the determinants of the rapid inhibitory specificity of PAI‐1 are localized to the P1′–P5′ region of the reactive center and these residues act synergistically to produce the exquisite specificity of PAI‐1 for plasminogen activators.
Conformational Requirements of a Recombinant Ferredoxin‐NADP+ Reductase Precursor for Efficient Binding to and Import into Isolated Chloroplasts
Authors: Ceccarelli Eduardo A. , Krapp Adriana R. , Serra Esteban C. , Carrillo Néstor
Published: August 2004

The cytosolic precursor of the chloroplast flavoprotein ferredoxin‐NADP+ reductase was expressed in Escherichia coli rendering a soluble protein that contained bound FAD and could be imported by isolated chloroplasts. The mechanism of plastid translocation was studied under defined conditions using this recombinant precursor holoprotein and intact pea chloroplasts. The first step in the import pathway, namely, binding of the reductase precursor to isolated chloroplasts, was saturable at about 2000 molecules/ plastid, and showed a high‐affinity interaction with a dissociation constant Kd of approximately 5 nM. Binding was not affected by the addition of soluble leaf extracts or by prior denaturation of the precursor with urea. Analysis of the initial import rates at different precursor concentrations indicated the existence of a single translocation system for this protein. Inclusion of leaf extracts in the assay resulted in a threefold increase of the maximal import rates to 14000 molecules · min‐1· chloroplast‐1, with a concomitant decrease in the apparent Km for the recombinant precursor, from 1 μM to 100–150 nM. Comparison of Km and Kd values under various conditions indicated that the binding step of the translocation process is largely irreversible, favouring import and processing. In the absence of extract, a denatured precursor obtained by incubation with urea was a better substrate for plastid import than the holoprotein. Treatment of the precursor with either extract or urea resulted in similar increases in import efficiency (V/Km), suggesting that stimulation by leaf extracts is probably related to unfolding of the precursor prior to translocation.
Thermal Stability of Individual Domains in Platelet Glycoprotein IIbIIIa
Authors: Makogonenko Eugeny M. , Yakubenko Valentin P. , Ingham Kenneth C. , Medved Leonid V.
Published: August 2004

Thermal denaturation of platelet glycoprotein IIbIIIa (integrin αIIbβ3) was investigated by spectrofluorimetry and differential scanning calorimetry (DSC). Two forms of the protein were compared: active IIbIIIa, i.e., that fraction that binds to RGD‐Sepharose, and inactive IIbIIIa, the non‐binding fraction. At pH 8.5 in the presence of octyl glucoside and Ca2+ both forms exhibited a broad complex endotherm consisting of a well expressed low‐temperature heat‐absorption peak in the range of 40–65°C followed by a broad peak stretching over 65–110°C. Each endotherm could be deconvoluted into at least eight transitions reflecting the melting of at least this many independently folded domains. The first two transitions in the region of the low‐temperature peak had similar positions in both forms while at least some of the other transitions occurred at higher temperature in the active protein suggesting that some of the domains are more stable in the latter. When both fractions of IIbIIIa were heated in the fluorometer a sigmoidal transition was observed in the region of the first endothermic peak where the two thermolabile domains melt. This transition was destabilized by 15°C in the presence of EDTA, suggesting that these domains are formed by the 243–468 region of the IIb subunit which contains four Ca2+‐binding motifs. It was further stabilized by 3°C upon addition of the GRGDSPK peptide in the presence of Ca2+ while in EDTA the peptide had no effect. This is consistent with the involvement of Ca2+‐binding region in the formation of the ligand‐binding site. A 66‐kDa chymotryptic fragment, containing the 17‐kDa NH2‐terminal portion of the IIIa subunit disulfide‐linked to its 50‐kDa COOH‐terminal portion including the cysteine‐rich core, exhibited a fluorescence‐detected Ca2+‐independent transition in the region where the higher temperature DSC‐detected transitions occur suggesting that some of the latter may be connected with the melting of the corresponding portions of IIbIIIa.
Hydrogen Exchange in the Carbon Monoxide Complex of Soybean Leghemoglobin
Authors: Morikis Dimitrios , Wright Peter E.
Published: August 2004

Hydrogen/deuterium exchange rates for individual amide protons have been measured for the carbon monoxide complex of soybean leghemoglobin. Fast two‐dimensional NOESY experiments were performed, with 5.2‐min data‐collection time for each spectrum, which made possible the measurement of NOE cross‐peaks of relatively rapidly exchanging amide protons at early time points. Exchange rates were measured for 61 backbone amides, and protection factors were calculated to provide information on the packing and local stability of the protein. The data are consistent with the presence of transient cooperative local unfolding of helical segments. The B‐, E‐, G‐ and H‐helices have extensive regions of slow‐, medium‐ and fast‐exchanging amide protons. For each of these helices, there is a progressive decrease in protection on moving from the helix center to the termini. This is consistent with a stable helix center, with dynamic fraying at the ends. Amide exchange from the A‐helix and C‐helix is rapid except in small local regions. The F‐helix, which is located on the proximal side of the heme pocket and is well formed in solution as demonstrated by characteristic medium range NOE connectivities [Morikis, D., Lepre, C. A. & Wright, P. E. (1994) Eur. J. Biochem. 219, 611–626], exhibits fast exchange for all amide protons. The implied flexibility and low stability of the F‐helix may be functionally important in facilitating movement of the helix upon ligand binding. Fast exchange has also been observed for all amide protons in the CE‐loop and in turns, as expected for flexible or solvent exposed regions. A strong tertiary contact has been established between the A‐, G‐ and H‐helices by the presence of a slowly exchanging indole NεH of Trpl29.
Translation is Enhanced After Silent Nucleotide Substitutions in A+T‐Rich Sequences of the Coding Region of CD46 cDNA
Authors: Milland Julie , Christiansen Dale , Thorley Bruce R. , Mckenzie Ian F. C. , Loveland Bruce E.
Published: August 2004

Specific sequences in the coding region of CD46 (membrane cofactor protein) transcripts have been shown to have a marked effect on translation. Two A+T‐rich regions of CD46 cDNA were altered by mutation without changing the CD46 amino acid sequence (silent nucleotide substitution). In one region, the A+T content was reduced from 78% to 55% and in the other a putative polyadenylation addition sequence was disrupted. In each example, mutated sequences transfected into COS‐7 cells produced significantly more soluble or cell surface protein (up to a 20–fold increase) than wild‐type sequences. The amount of cellular plasmid DNA and CD46 mRNA was not increased, suggesting that the effect was not due to increased transfection efficiency, or transcript synthesis or stability. Biosynthetically labelled transfected cells showed an increase in translation rate but cell‐free in vitro translation studies demonstrated that wild‐type and mutated transcripts were translated with similar efficiency. The data show that translation of CD46 is affected by specific mRNA coding sequences, 400–540 bases from the initiation codon, and suggest that these sequences require the structural integrity of the cell to exert their effect.
Protein Purification, and Cloning and Characterization of the cDNA and Gene for Xylose Isomerase of Barley
Authors: Kristo Paula , Saarelainen Ritva , Fagerström Richard , Aho Sirpa , Korhola Matti
Published: August 2004

The first eukaryotic xylose isomerase protein was purified from barley Hordeum vulgare. The enzyme requires Mn2+ for its activity and is fairly thermostable, with the optimum temperature being 60°C. It showed maximum activity over a broad pH range (7.0–9.0). The molecular mass of the monomer was about 50000 Da based on the SDS/PAGE, and the calculated value from the cDNA‐deduced polypeptide sequence was 53620 Da. A relative mass estimation of 100000 Da was obtained from the Superose 12 chromatography, suggesting that the barley enzyme is a dimer. The cloned corresponding cDNA sequence of 1710 nucleotides encoded a polypeptide of 480 amino acids. The genomic sequence of 4473 nucleotides, revealed that the isomerase gene contained 20 introns, all starting with GT and ending with AG. One large intron was located in the 5′untranslated region. The barley isomerase has an insertion of about 40 residues at its amino terminus when compared to the prokaryotic cluster (family) II isomerases; cluster (family) I and cluster (family) II isomerases vary from the former in an insertion of around 50 residues at their amino termini. Comparison of the barley protein with the prokaryotic isomerases shows that the conserved catalytic and metal binding regions are also well conserved in barley.
Structure/Function Analysis of Human Factor XII Using Recombinant Deletion Mutants
Authors: Citarella Franca , Ravon Dorothea M. , Pascucci Barbara , Felici Angelina , Fantoni Antonio , Hack C. Erik
Published: August 2004

The binding site of human factor XII (FXII) for negatively charged surfaces has been proposed to be localized in the N‐terminal region of factor XII. We have generated two recombinant factor XII proteins that lack this region: one protein consisting of the second growth‐factor‐like domain, the kringle domain, the proline‐rich region and the catalytic domain of FXII (rFXII‐U‐like), and another consisting of only 16 amino acids of the proline‐rich region of the heavy‐chain region and the catalytic domain (rFXII‐lpc). Each recombinant truncated protein, as well as recombinant full‐length FXII (rFXII), were produced in HepG2 cells and purified by immunoaffinity chromatography. The capability of these recombinant proteins to bind to negatively charged surfaces and to initiate contact activation was studied. Radiolabeled rFXII‐U‐like and, to a lesser extent, rFXII‐lpc bound to glass in a concentration‐dependent manner, yet with lower efficiency than rFXII. The binding of the recombinant proteins was inhibited by a 100‐fold molar excess of non‐labeled native factor XII. On native polyacrylamide gel electrophoresis, both truncated proteins appeared to bind also to dextran sulfate, a soluble negatively charged compound. Glass‐bound rFXII‐U‐like was able to activate prekallikrein in FXII‐deficient plasma (assessed by measuring the generation of kallikrein–C1‐inhibitor complexes), but less efficiently than rFXII. rFXII‐U‐like and rFXII‐lpc exhibited coagulant activity, but this activity was significantly lower than that of rFXII. These data confirm that the N‐terminal part of the heavy‐chain region of factor XII contains a binding site for negatively charged activating surfaces, and indicate that other sequences, possibly located on the second epidermal‐growth‐factor‐like domain and/or the kringle domain, contribute to the binding of factor XII to these surfaces.
Escherichia coli Isocitrate Dehydrogenase Kinase/Phosphatase
Authors: Rittinger Katrin , Negre Didier , Divita Gilles , Scarabel Marie , Bonod‐Bidaud Christelle , Goody Roger S. , Cozzone Alain J. , Cortay Jean‐Claude
Published: August 2004

The aceK gene of Escherichia coli, which encodes the isocitrate dehydrogenase kinase/phosphatase (IDH K/P), was cloned in the pQE30 expression vector to overproduce a protein tagged with six histidine residues at its N‐terminus. By using a one‐step chromatographic procedure, the IDH K/P was purified to near homogeneity. The IDH K/P, which contains nine Trp residues, exhibited a characteristic intrinsic tryptophan fluorescence with a low maximal emission at 326 nm. The low value of the Stern‐Volmer quenching constant in the presence of acrylamide (Ksv= 2.1 M−1) indicated that the tryptophan residues were deeply buried in the protein. Furthermore, the intrinsic tryptophan fluorescence was very sensitive to the binding of nucleotide. The quenching of protein fluorescence induced by the binding of nucleotide together with an increased intrinsic fluorescence of fluorescent nucleotide analogues, methylanthraniloyl derivatives ADP, ATP, GDP and GTP and adenosine‐5′‐triphosphoro‐1‐(5‐sulfonic‐acid) naphthylamidate, were used to investigate the interaction with IDH K/P. The IDH K/P dimer was shown to contain two identical nucleotide binding sites, one on each subunit, with a Kd in the range of 1.7–2.5 μM for unmodified ADP or ATP and of 2.5–3.7 μM for fluorescently labelled nucleotides. In contrast, the affinity for GDP or GTP was 10‐fold lower than for adenine nucleotides. The nucleotide binding site was located within residues 315–340 by using limited proteolysis of IDH K/P by endoproteinase Lys‐C. Only one main site of cleavage was obtained: the peptide bond K346‐E347 which was strongly protected in the presence of ATP.
Two Distinct Quinoprotein Amine Oxidases are Induced by n‐Butylamine in the Mycelia of Aspergillus niger AKU 3302
Authors: Frébort Ivo , Tamaki Hisanori , Ishida Hiroki , Peč Pavel , Luhová Lenka , Tsuno Hiroko , Halata Martin , Asano Yasuhisa , Kato Yasuo , Matsushita Kazunobu , Toyama Hirohide , Kumagai Hidehiko , Adachi Osao
Published: August 2004

Two distinct quinoprotein amine oxidases were found in Aspergillus niger mycelia grown on n‐buty‐lamine medium and purified using chromatographic techniques. The respective enzymes were termed AO‐I, which had already been isolated, and AO‐II, a new enzyme found in this study. HPLC indicated that their molecular masses are 150 kDa and 80kDa, respectively. On SDS/PAGE, the enzymes gave a similar but distinct mobility, which corresponds to 75 kDa for the subunit of dimeric AO‐I and 80 kDa for monomeric AO‐II. The absorption spectra of both enzymes were different from each other; the absorption maxima in the visible region were at 490 nm for AO‐I and 420 nm for AO‐II. The enzymes showed positive quinone staining, comparable substrate specificity, and sensitivity to inhibitors typical for copper/ topa quinone‐containing amine oxidases, but they had different copper contents and also differed in their N‐terminal sequences. Their peptide maps showed almost identical patterns, with the exception of two additional bands for AO‐II. Among the peptides obtained from digestion of AO‐II, peptides with sequences corresponding to the N‐terminal part of AO‐I were detected. Polyclonal antibodies raised against AO‐I and AO‐II recognized both enzymes, but with different specificities. Using precipitation with AO‐I, the antibody prepared against AO‐II was purified and was shown to be specific only for AO‐II. The cDNA of AO‐I was cloned and sequenced. A highly conserved tetrapeptide sequence, Asn‐Tyr‐Glu‐Tyr, was identified in which the first tyrosine residue (Tyr404) that could be converted to topa quinone was present in the 670‐residue deduced amino acid sequence. Northern blot analysis indicated that AO‐I was highly expressed in A. niger grown on n‐butylamine as a single nitrogen source. Genomic Southern blot analysis confirmed that both enzymes are likely to be encoded by the same gene.
Microheterogeneity of the Hydrophobic and Hydrophilic Part of the Glycosylphosphatidylinositol Anchor of Alkaline Phosphatase from Calf Intestine
Authors: Armesto Julio , Hannappel Ewald , Leopold Klaus , Fischer Werner , Bublitz Renate , Langer Lydia , Cumme Gerhard A. , Horn Anton
Published: August 2004

Digestion of calf intestine alkaline phosphatase with pronase and subsequent dephosphorylation of the released peptidyl‐(Etn‐P)2‐glycosyl‐PtdIns with HF generated 8 glycosyl‐Ins species the largest of which (G1 and G2) have the following proposed structures:

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G3 and G5 are lower homologues of G1 and G2, respectively, being one αl‐2 linked mannopyranosyl residue shorter. G4 is analogous to G2 lacking the N ‐acetylgalactosaminyl residue and G6 is the next lower homologue of G4. Most of G4 and G6 occur substituted with a palmitoyl (G4, G6) or a myristoyl residue (G6) probably attached to the inositol moiety. Thus, the basic ManxGlc‐Ins species are either substituted with an N ‐acetylgalactosaminyl residue or a fatty acid ester. The structures were deduced from compositional analysis, molecular‐mass determination by matrix‐assisted laser desorption MS, sequential hydrolysis with appropriate exoglycosidases and treatment with CrO3. Purification of the glycosylinositol species was achieved by a novel reverse‐phase HPLC technique using fluorescent fluoren‐9‐yl‐methoxy‐carbonyl (Fmoc) derivatives. These stable derivatives were susceptible to hydrolysis with exoglycosidases which allowed sequential cleavages to be carried out and kinetics to be followed at the picomole level.

We observed recently that native alkaline phosphatase separates on octyl‐Sepharose into four distinct fractions of increasing hydrophobicity (F1–F4). Here we show that all four fractions contain G1–G6. The acylated species G4 and G6 were restricted to F2 and F4 which had been shown earlier to contain, on average, 2.5 and 3 fatty acid residues/subunit, respectively. In all four fractions the diradylglycerol moiety was predominantly diacylglycerol, alkylacylglycerol being less than 10% which is in contrast to most glycosyl‐PtdIns–anchored proteins of mammalian origin.
Structures of the Glycolipid Antigens of Members of the third Biovariant Complex of Mycobacterium Fortuitum
Authors: Lanéelle Marie‐Antoinette , Silve Gaby , López Marín Luz M. , Daffé Mamadou
Published: August 2004

Among the fast‐growing mycobacteria, members of the Mycobacterium fortuitum complex are the most‐commonly cited opportunistic human pathogens, notably in post‐surgical infections. Previous studies showed that this complex was composed of four well‐identified species and a group of isolates that did not correspond to recognized species, which has been referred to as the third biovariant complex. The occurrence and chemical structure of the glycolipid antigens of six strains that belong to this latter group were examined in the present study. Based on the TLC profiles, resistance to alkali and seroreactivities of their glycolipids, the examined strains were classified into three groups: one group was devoid of species‐specific glycolipid and the two other groups contained alkali‐stable or alkali‐labile glycoconjugates. The structures of the major glycolipid antigens of the latter two groups were elucidated by fastatom‐bombardment MS, one‐dimensional and two‐dimensional NMR spectroscopy and conventional chemical analyses. The alkali‐stable glycolipids were structurally identical to the C‐mycoside‐type glycopeptidolipids characterized in the taxonomically related species Mycobacterium peregrinum. The major alkali‐labile glycolipid was identified as β‐Glcp ‐(1→6)‐α‐Glcp 2Acyl‐(l→l)‐α‐Glcp 3,4,6Acyl3. The acyl substituents consisted of one acetyl group and three fatty acyl residues composed mainly of tetradecanoyl residues, but significant amounts of 2‐methylhexadecanoyl and 2‐methyloctadecanoyl substituents were also present. The heterogeneity of the glycolipid content of members of the third biovariant complex of M. fortuitum demonstrated in the present study confirms the heterogeneity of the complex. In addition, the occurrence of a species‐specific glycolipid in some strains supports the hypothesis that some strains of this complex of M. fortuitum may belong to a new mycobacterial species.
Stereoselectivity of Pigment Exchange with 132‐Hydroxylated Tetrapyrroles in Reaction Centers of Rhodobacter Sphaeroides R26
Authors: Storch Kai‐Florian , Cmiel Edrmund , Schäfer Wolfram , Scheer Hugo
Published: August 2004

Bacteriochlorophyll a and bacteriopheophytin a carry a stereochemically labile asymmetric carbon at position C132. The steric requirements of photosynthetic reaction centers from Rhodobacter sphaeroides R26 have been probed by exchange experiments with the respective epimeric 132‐hydroxylated pigments, in which epimerisation is blocked. (132S)‐132‐Hydroxy‐bacteriochlorophyll a is accepted at both monomeric binding sites, BA, B, (132S)‐l32‐hydroxy‐bacteriopheophytin a exclusively at the inactive site HB. The orientation of the 132‐COOCH3 substituents in these pigments is the same as in the native (132R)‐bacteriochlorophylls and (132R)‐bacteriopheophytins. In no cases are the unnaturally configured 132‐hydroxylated (132R)‐epimers accepted, even if a large excess (>95%) is offered. It is concluded that the three binding sites always require the 132‐COOCH3 group on the opposite side of the macrocycle (down) than the 17‐propionic ester side chain (up).
Low‐spin Heme A in the Heme A Biosynthetic Protein CtaA from Bacillus Subtilis
Authors: Svensson Birgitta , Anderson Kristoffer K. , Hederstedt Lars
Published: August 2004

Synthesis of heme A from heme B (protoheme IX) most likely occurs in two steps with heme O as an intermediate. Bacillus subtilis CtaB, an integral membrane protein, functions in farnesylation of heme B to form heme O. CtaA, also a membrane protein, is required for heme A synthesis from heme O and appears to be a monooxygenase and/or a dehydrogenase. Wild‐type ctaA and ctaB expressed together from plasmids in B. subtilis resulted in CtaA containing equimolar amounts of low‐spin heme B and heme A; this form of CtaA was named cyt ba ‐CTA. A mutant ctaB gene was identified and characterised. It encodes a truncated CtaB polypeptide. Wild‐type ctaA and the mutant ctaB gene on plasmids resulted in CtaA containing mainly low‐spin heme B; this variant was named cyt b ‐CTA. The heme B component in cyt ba ‐CTA and cyt b ‐CTA showed identical properties; a mid‐point redox potential of +85 mV, an EPR gmax signal at 3.7, and a split α‐band light absorption peak. The heme A component in cyt ba ‐CTA showed a mid‐point potential of +242 mV, an EPR gmax signal at 3.5, and the α‐band light absorption peak at 585 nm. It is suggested that the CtaA protein contains two heme binding sites, one for heme B and one for substrate heme. The heme B would play a role in electron transfer, i.e. function as a cytochrome, in the monooxygenase and/or dehydrogenase reaction catalysed by CtaA whereas heme O/heme A would be substrate/product.
Human Bone Morphogenetic Protein 2 Contains a Heparin‐Binding Site which Modifies Its Biological Activity
Authors: Ruppert Rainer , Hoffmann Elke , Sebald Walter
Published: August 2004

Bone morphogenetic protein 2 (BMP‐2) plays a decisive role during bone regeneration and repair as well as during various stages of embryonal development. A cDNA encoding mature human BMP‐2 could be efficiently expressed in Escherichia coli, and after renaturation a dimeric BMP‐2 protein of Mr 26000 was prepared with a purity greater 98%. The recombinant BMP‐2 was functionally active as demonstrated by the induction of alkaline phosphatase activity in the C3H10T1/2 fibroblast cell line (EC50 of 70 nM) and proteoglycan synthesis in embryonic chicken limb bud cells (EC50 of 15–20 nM). A peptide 1–17 representing the N‐terminal basic part of BMP‐2 as well as heparin increased the specific activity of the protein about fivefold in the limb bud assay. These observations suggested that the N‐terminal reduce the specific activity of BMP‐2, probably by interacting with heparinic sites in the extracellular matrix. This conclusion was supported by a variant EHBMP‐2, where the N‐terminal residues 1–12 of BMP‐2 had been substituted by a dummy sequence of equal length and which showed an EC50 value of around 1 nM which was affected neither by heparin nor by peptide 1–17. A physical interaction between BMP‐2 and heparin could be seen in biosensor experiments, where BMP‐2 bound to immobilized heparin with a dissociation constant, Kd, of approximately 20 nM, whereas the heparin‐binding of variant EHBMP‐2 was negligible. These results identify the basic N‐terminal domains of dimeric BMP‐2 as heparin‐binding sites that are not obligatory for receptor activation but modulate its biological activity.
Synthetic Alleles at Position 121 Define a Functional Domain of Human Interleukin‐1β
Authors: Ambrosetti Davide‐Carlo , Palla Emanuela , Mirtella Adriana , Galeotti Cesira , Solito Egle , Navarra Pierluigi , Parente Luca , Melli Marialuisa
Published: August 2004

The non‐conservative substitution of the tyrosine residue at position 121 of human interleukin‐1β (IL‐1β generates protein mutants showing strong reduction of the capacity to induce (a) prostaglandin E2 (PGE2) release from fibroblasts and smooth muscle cells, (b) murine T‐cells proliferation and (c) activation of interleukin‐6 (TL‐6) gene expression. It is generally accepted that these functions are mediated by the type‐I interleukin‐1 receptor (IL‐1RI). However, the mutant proteins maintain the binding affinity to the types‐I and II IL‐1 receptors, which is the same as the control IL‐1β, suggesting that this amino acid substitution does not alter the structure of the molecule, except locally.

Thus we have identified a new functional site of IL‐1β different from the known receptor binding region, responsible for fundamental IL‐1β functions. Moreover, we show that the same mutants maintain at least two hypothalamic functions, that is, the in vitro short‐term PGE2 release from rat hypothalamus and the induction of fever in rabbits. This result suggests that there is yet another site of the molecule responsible for the hypothalamic functions, implying that multiple active sites on the IL‐1β molecule, possibly binding to more than one receptor chain, trigger different signals.
Successive Amino‐Terminal Proteolysis of the Large Subunit of Ribulose 1,5‐bisphosphate Carboxylase/Oxygenase by Vacuolar Enzymes from French Bean Leaves
Authors: Yoshida Tomoe , Minamikawa Takao
Published: August 2004

Mainly using the protein immunoblot technique, we observed the decrease in amounts of the large subunit (LSU) and the small subunit (SSU) of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) in detached primary leaves of French bean plants during senescence under the light or in darkness, but detected no significant degradation products of these subunits. Treatment of the detached leaves with 0.6% (mass/vol.) dimethyl sulfoxide, 0.05% (mass/vol.) Tween 80 considerably promoted the senescence, as estimated by the reduction in content of the soluble protein, and also in the amounts of LSU and SSU, but no degradation product of either subunit was found. When extracts prepared from the primary leaves were incubated at pH 5.4 or pH 7.4, the amount of LSU of 53 kDa decreased and concurrently 50‐kDa and 42‐kDa polypeptides were formed. Since the results suggested that Rubisco may be degraded by vacuolar enzymes, we incubated Rubisco with vacuolar lysates prepared from the senescing primary leaves and found that the LSU, but not SSU, was degraded to a 41‐kDa polypeptide through three intermediates of 50 kDa, 48 kDa and 42 kDa. Determination of amino‐terminal amino acid sequences of these fragments indicated that each of the proteolysis steps occurred by removal of a small amino‐terminal peptide. Experiments with various inhibitors of proteases as well as with a purified Vigna mungo vacuolar protease, termed SH‐EP [Mitsuhashi, W. & Minamikawa, T. (1989) Plant Physiol. 89, 274–279] suggested the involvement of two types of proteases in these steps: a cysteine protease that is the same type of enzyme as SH‐EP catalyzes the steps from the LSU to the 48‐kDa polypeptide through the 50‐kDa polypeptide, and a serine protease catalyzes the steps from the 48–kDa polypeptide to the 41–kDa polypeptide through the 42–kDa polypeptide.
Potassium Ions and the Molecular‐Chaperone Activity of DnaK
Authors: Feifel Bastian , Sandmeier Erika , Schönfeld Hans‐Joachim , Christen Philipp
Published: August 2004

Potassium ions stabilize the DnaK · ADP complex that forms on incubation of nucleotide‐free DnaK with ADP or ATP. Generation of the crystallographically defined Mg2+ cluster [Wilbanks, S. M. & McKay, D. B. (1995) J. Biol. Chem. 270, 2251–2257], in which two K+ and the nucleotide are bound together with Mg2+ in the ATPase site, appears to be essential for the ATP‐induced acceleration of binding of peptide ligands, the ATP‐induced release of peptide ligands and for the peptide‐induced increase in ATPase activity. Thus, K+ is instrumental in signal transmission between the ATPase site and the peptide‐binding site.