bluuebunny/crossref_metadata_2025 huggingface dataset:
The active site of purple acid phosphatase from sweet potatoes (Ipomoea batatas)
Authors: Durmus Atila , Eicken Christoph , Horst Sift Bernd , Kratel Andreas , Kappl Reinhard , Hüttermann Jürgen , Krebs Bernt
Published: March 2003

Purple acid phosphatase from sweet potatoes Ipomoea batatas (spPAP) has been purified to homogeneity and characterized using spectroscopic investigations. Matrix‐assisted laser desorption/ionization mass spectrometry analysis revealed a molecular mass of ≈ 112 kDa. The metal content was determined by X‐ray fluorescence using synchrotron radiation. In contrast to previous studies it is shown that spPAP contains a Fe(III)–Zn(II) center in the active site as previously determined for the purple acid phosphatase from red kidney bean (kbPAP). Moreover, an alignment of the amino acid sequences suggests that the residues involved in metal‐binding are identical in both plant PAPs. Tyrosine functions as one of the ligands for the chromophoric Fe(III). Low temperature EPR spectra of spPAP show a signal near g = 4.3, characteristic for high‐spin Fe(III) in a rhombic environment. The Tyr–Fe(III) charge transfer transition and the EPR signal are both very sensitive to changes in pH. The pH dependency strongly suggests the presence of an ionizable group with a pKa of 4.7, arising from an aquo ligand coordinated to Fe(III). EPR and UV/visible studies of spPAP in the presence of the inhibitors phosphate or arsenate suggest that both anions bind to Fe(III) in the binuclear center replacing the coordinated water or hydroxide ligand necessary for hydrolysis. The conserved histidine residues of spPAP corresponding to His202 and His296 in kbPAP probably interact in catalysis.
Efficient screening for catalytic antibodies using a short transition‐state analog and detailed characterization of selected antibodies
Authors: Takahashi Naoko , Kakinuma Hiroyuki , Hamada Katsumi , Shimazaki Kazuko , Takahashi Kyoko , Niihata Shigeo , Aoki Yoshiko , Matsushita Hajime , Nishi Yoshisuke
Published: March 2003

One of the major obstacles to acquiring catalytic antibodies is that it requires labor‐intensive procedures to select catalytic antibodies from huge repertories of antibodies. Here, we selected potential catalytic Abs by utilizing their affinity towards a short transition‐state analog which contained only the transition‐state structural element, and evaluated in detail its efficiency to enrich catalytic Abs. Hybridoma supernatants elicited against a phosphonate derivative, the TSA1, were screened by a three‐step screening process: step 1, ELISA for TSA1–BSA; step 2, ELISA for the short TSA4; and step 3, competitive‐inhibition by the short TSA2. Only 22.8% of positive mAbs from step 1 were found to be catalytic. The rate of catalytic Abs increased to 45.7% using screening steps 1 plus 2, and reached 83.3% using all three screening steps. This clearly suggests that our screening protocol is an efficient method to select potential catalytic Abs. Furthermore, we characterized the properties of both the catalytic Abs and the noncatalytic Abs in detail. The catalytic Abs tended to have lower Kd for TSA1 and the short TSA2 than noncatalytic Abs. It was also observed that catalytic Abs showed clear enantiospecificity toward substrate 6 containing d‐phenylalanine while noncatalytic Abs did not. The detailed analysis of kinetic and binding parameters for these antibodies gives us further insight into catalytic antibodies.
Characterization of a cDNA encoding a precursor polypeptide of a d‐amino acid‐containing peptide, achatin‐I and localized expression of the achatin‐I and fulicin genes
Authors: Satake Honoo , Yasuda‐Kamatani Yoshimi , Takuwa Kyoko , Nomoto Kyosuke , Minakata Hiroyuki , Nagahama Tomoaki , Nakabayashi Koichi , Matsushima Osamu
Published: March 2003

Achatin‐I and fulicin, isolated from the ganglia and atria of the giant land snail Achatina fulica, are a tetrapeptide and pentapeptide containing a d‐Phe and d‐Asn at position 2, respectively. We succeeded in cloning a cDNA encoding a precursor of achatin‐I from the Achatina ganglia, revealing that the d‐Phe present in achatin‐I is coded by a common l‐Phe codon, TTT or TTC. The deduced polypeptide was found to comprise seven repeats of the achatin sequence GFAD and one analog GFGD flanked on both sides by the typical endoproteolytic site KR. Northern blot analysis of transcripts and Southern blot analysis of reverse transcription (RT)–PCR products demonstrated that achatin‐I mRNA was localized in the subesophageal ganglia, whereas expression of fulicin mRNA was detected in the atrium as well as in the subesophageal ganglia. Furthermore, localization of the achatin gene transcript in the right and left pedal ganglia compartments was shown by in situ hybridization on sections of subesophageal ganglia, whereas the fulicin transcript was observed in the right and left parietal ganglia. These data suggested that achatin‐I plays an essential role in the regulation of the heart as a neurotransmitter or neurohormone through production in the pedal ganglia and transport to the atrium, whereas fulicin serves not only as a neurotransmitter or neurohormone but also as a novel atrial hormone.
Acn9 is a novel protein of gluconeogenesis that is located in the mitochondrial intermembrane space
Authors: Dennis Richard A. , McCammon Mark T.
Published: March 2003

Previous studies have indicated that the Acn9 protein is involved in gluconeogenesis. Yeast mutants defective in the ACN9 gene display phenotypes identical with mutants defective in metabolic enzymes required for carbon assimilation. These phenotypes include the inability to utilize acetate as a carbon and energy source, elevated levels of enzymes of the glyoxylate cycle, gluconeogenesis and acetyl‐CoA mobilization, and a deficiency in de novo synthesis of glucose from ethanol. The ACN9 gene was isolated by functional complementation of the acetate growth defect of an acn9 mutant. The open reading frame corresponds to YDR511w, and encodes a protein of unknown function. Homologs have been identified in human, mouse, and nematode databases. Two mutant alleles were sequenced. The mutations altered amino acid residues that are conserved among members of the new gene family. ACN9 gene expression was slightly repressed by glucose, and the level of the transcript was approximately 100‐fold lower than that of glyoxylate or tricarboxylic acid cycle enzymes. A functional epitope‐tagged form of Acn9 was expressed to study expression and the subcellular localization of the protein. The tagged protein was localized to the mitochondrial intermembrane space.
Organization and alternate splice products of the gene encoding nuclear inhibitor of protein phosphatase‐1 (NIPP‐1)
Authors: Van Eynde Aleyde , Pérez‐Callejón Encarna , Schoenmakers Eric , Jacquemin Marc , Stalmans Willy , Bollen Mathieu
Published: March 2003

Nuclear inhibitor of protein phosphatase‐1 (NIPP‐1) is one of two major regulatory subunits of protein phosphatase‐1 in mammalian nuclei. We report here the cloning and structural characterization of the human NIPP‐1 genes, designated PPP1R8P and PPP1R8 in human gene nomenclature. PPP1R8P (1.2 kb) is a processed pseudogene and was localized by in situ hybridization to chromosome 1p33–32. PPP1R8 is an authentic NIPP‐1 gene and was localized to chromosome 1p35. PPP1R8 (25.2 kb) is composed of seven exons and encodes four different transcripts, as determined from cDNA library screening, reverse transcriptase‐PCR (RT‐PCR) and/or EST (expressed sequence tag) database search analysis. NIPP‐1α mRNA represents the major transcript in human tissues and various cell lines, and encodes a polypeptide of 351 residues that only differs from the previously cloned calf thymus NIPP‐1 by a single residue. The other transcripts, termed NIPP‐1β, γ and δ, are generated by alternative 5′‐splice site usage, by exon skipping and/or by alternative polyadenylation. The NIPP‐1β/δ and NIPP‐1γ mRNAs are expected to encode fragments of NIPP‐1α that differ from the latter by the absence of the first 142 and 224 residues, respectively. NIPP‐1γ corresponds to ‘activator of RNA decay‐1’ (Ard‐1) which, unlike NIPP‐1α, displays in vitro and endoribonuclease activity and lacks an RVXF consensus motif for interaction with protein phosphatase‐1. While the NIPP‐1α/β/δ‐transcripts were found to be present in various human tissues, the NIPP‐1γ transcript could only be detected in human transformed B‐lymphocytes.
Cleavage of transcription factor SP1 by caspases during anti‐IgM‐induced B‐cell apoptosis
Authors: Rickers Anke , Peters Nils , Badock Volker , Beyaert Rudi , Vandenabeele Peter , Dörken Bernd , Bommert Kurt
Published: March 2003

Apoptosis is instrumental in the processes generating the diversity of the B‐cell repertoire. Autoreactive B‐cells are eliminated by anti‐IgM crosslinking after encountering self‐antigens, but precise mechanisms leading to B‐cell apoptosis are still not well understood. We report here the cleavage of the transcription factor SP1 in the human Burkitt lymphoma cell line BL60 during anti‐IgM‐induced apoptosis. Western blot analysis revealed two cleavage products of approximately 68 kDa and 45 kDa after induction of apoptosis. Cleavage could be completely inhibited by zDEVD‐fmk, an inhibitor specific for caspase 3‐like proteases. In‐vitro cleavage of recombinant SP1 by recombinant caspase 3 (CPP32) or caspase 7 (Mch 3) results in similar cleavage products as those observed in vivo. Recombinant caspase 6 (Mch 2) primarily generates a 68‐kDa cleavage product, as observed after calcium ionophore (CaI) induced B‐cell apoptosis. In contrast, caspase 1 (ICE) did not cleave SP1 in vitro. The time course of SP1 cleavage during anti‐IgM‐induced apoptosis is paralleled by an increase of caspase activity measured by DEVD‐p‐nitroanilide (DEVD‐pNA) cleavage. DNA band‐shift assays revealed a decrease in the intensity of the full length SP1/DNA complex and an increase in the intensity of a smaller complex due to the binding of one SP1 cleavage product. By Edman sequencing we could identify a caspase 3 cleavage site after Asp584 (D584AQPQAGR), generating a 22‐kDa C‐terminal SP1 protein fragment which still contains the DNA binding site. Our results show the cleavage of the human transcription factor SP1 in vivo and in vitro, underlining the central role of caspase 3‐like proteases during the process of anti‐IgM‐induced apoptosis.
Evidence of noncovalent dimerization of calmodulin
Authors: Lafitte Daniel , Heck Albert J. R. , Hill Tessa J. , Jumel Kornelia , Harding Stephen E. , Derrick Peter J.
Published: March 2003

Calcium‐binding proteins, such as S‐100, dimerize readily, and this phenomenon plays an important role in their regulation of target enzymes [Krebs, J., Quadroni, M. & Van Eldik, L.J. (1995) Nat. Struct. Biol.2, 711–714; Kilby, P.M., Van Eldik, L.J. & Roberts, G.C. (1996) Structure4, 1041–1052]. We have investigated by Fourier‐transform ion cyclotron resonance (FTICR) MS the conformational states of the calcium‐binding protein calmodulin, and present clear evidence for a calmodulin dimer formed as a result of noncovalent interactions between folded monomers. Ultra‐high‐resolution electrospray ionization (ESI) mass spectra for calmodulin, obtained with a 9.4 T FTICR mass spectrometer, are presented. With the use of denaturing solutions (1 : 1 acetonitrile/water + 1% formic acid), relatively high charge states (20 < z < 10) of monomeric calmodulin ions were detected, whereas when calmodulin was electrosprayed from buffer, monomers ions with only 5–10 charges were detected. CD measurements for calmodulin in buffered solution revealed that its α‐helical content was significantly higher than that for calmodulin in acetonitrile/water solutions, consistent with a proposition that changes in charge state distributions observed in the MS experiments reflect differing states of calmodulin folding.

 Under buffered conditions, noncovalently bound calmodulin dimers were observed by ESI FTICR MS. Analytical ultracentrifugation experiments carried out in the same solution conditions as those used in the MS experiments were consistent with the proposed calmodulin dimer–monomer equilibrium. The ultra‐high mass resolution achieved with the 9.4 T FTICR mass spectrometer allowed unequivocal identification of the noncovalent, as opposed to covalent, character of the calmodulin dimer.
NMR studies of the C‐terminal secretion signal of the haem‐binding protein, HasA
Authors: Izadi‐Pruneyre Nadia , Wolff Nicolas , Redeker Virginie , Wandersman Cécile , Delepierre Muriel , Lecroisey Anne
Published: March 2003

HasA is a haem‐binding protein which is secreted under iron‐deficiency conditions by the gram‐negative bacterium Serratia marcescens. It is a monomer of 19 kDa (187 residues) able to bind free haem as well as to capture it from haemoglobin. HasA delivers haem to a specific outer‐membrane receptor HasR and allows the bacteria to grow in the absence of any other source of iron. It is secreted by a signal peptide‐independent pathway which involves a C‐terminal secretion signal and an ABC (ATP‐binding cassette) transporter. The C‐terminal region of the secretion signal containing the essential secretion motif is cleaved during or after the secretion process by proteases secreted by the bacteria.

 In this work, we study by 1H NMR the conformation of the C‐terminal extremity of HasA in the whole protein and that of the isolated secretion signal peptide in a zwitterionic micelle complex that mimicks the membrane environment. We identify a helical region followed by a random‐coil C‐terminus in the peptide–micelle complex and we show that in both the whole protein and the complex, the last 15 residues containing the motif essential for secretion are highly flexible and unstructured. This flexibility may be a prerequisite to the recognition of HasA by its ABC transporter. We determine the cleavage site of the C‐terminal extremity of the protein and analyse the effect of the cleavage on the haem acquisition process.
Platelet‐activating factor modulates brain sphingomyelin metabolism
Authors: Latorre Eduardo , Aragonés María D. , Fernández Inmaculada , Catalán R. Edgardo
Published: March 2003

In the present study the modulatory action of platelet‐activating factor (PAF) on sphingolipid metabolism in cerebral cortical slices was studied. PAF did not alter the basal levels of either sphingomyelin (SM) or ceramide. However, the SMase‐elicited reciprocal alterations in SM and ceramide levels were partially prevented by the PAF treatment. The PAF effect was dose‐dependent, with 10−8 m being the lowest effective concentration, and receptor‐mediated as it was abolished by WEB 2086, a PAF receptor antagonist. Neither N‐oleoylethanolamine (OE, ceramidase inhibitor) or d,l‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP, an inhibitor of glucosylceramide synthase and the formation of 1‐O‐acyl ceramides) prevented the action of PAF. Therefore, the effect of PAF was unlikely to be dependent upon transformation of ceramides into glycosphingolipids, 1‐O‐acyl ceramides or sphingosine. Experiments with different labeled compounds ([14C]serine, [14C]arachidonate and phosphatidyl [N‐methyl‐3H]choline) were also performed to test whether PAF could affect the resynthesis of SM. Data obtained agree with the idea that selective pools of both choline and ethanolamine phospholipids were used as precursors for the resynthesis of SM elicited by SMase treatment. PAF itself did not evoke any variation in the lipids analyzed but always prevented the SMase‐evoked alterations. Together the data suggest the interesting possibility that PAF increases the overall turnover of SM. In summary, the present data demonstrate that PAF is able to regulate the cellular ceramide levels in brain by accelerating the SM cycle.
Controlled cleavage of KLH1 and KLH2 by the V8 protease from Staphylococcus aureus
Authors: Gebauer Wolfgang , Harris J. Robin
Published: March 2003

The reassociation behaviour of protease V8‐cleaved peptides from KLH1 and KLH2, the two hemocyanin isoforms from the giant keyhole limpet Megathura crenulata, has been studied by transmission electron microscopy of negatively stained specimens and SDS/PAGE. Reassociation of the complete mixture of protease cleavage products and of combinations of peptide fragments purified by HPLC was performed in the presence of 100 mm CaCl2 and 100 mm MgCl2 at pH 7.4, over a period of 1 to 4 weeks. The V8 protease splits KLH1 into peptide fragments containing the functional units abc, def, defg, defgh, g and h. This mixture of peptide fragments reassociated to form helical tubular polymers, with a diameter of ≈ 25 nm. The single functional units g and h were not incorporated into the polymer. An essentially identical polymer was formed from the re‐mixed HPLC‐purified fragments abc, def and defg alone. As with uncleaved subunit, the tubular polymer of V8‐cleaved KLH1 forms bundles. The combination of peptides def and defg led to the formation of short arc‐like filamentous structures, which aggregated but showed little tendency to associate into larger polymers. The KLH1 peptide fragments abc and def alone, did not reassociate and in combination their potential to form polymers was very low. With KLH2, the V8 protease generated peptide fragments containing the functional units abc, defg, defgh and h, which in combination slowly reassociated to form a tubular polymer significantly different to that obtained from the KLH1 V8 fragments. The three‐functional unit fragment abc from KLH2 showed no tendency to polymerize and the combination of peptides defg + defgh generated only disordered aggregates, with some indication of malformed tubules. The combination of biochemical and electron microscopical methods enabled the characterization of these polymers with respect to peptide composition and higher order structure.
Site‐directed removal of N‐glycosylation sites in human gastric lipase
Authors: Wicker‐Planquart Catherine , Canaan Stéphane , Rivière Mireille , Dupuis Liliane
Published: March 2003

Human gastric lipase (HGL) is a highly glycosylated protein, as glycan chains account for about 15% of the molecular mass of the native HGL. Four potential N‐glycosylation consensus sites (Asn15, 80, 252 and 308) can be identified from the HGL amino acid sequence. We studied the functional role of the individual N‐linked oligosaccharide chains by removing one by one all the N‐glycosylation sites, via Ala residue replacement by site‐directed mutagenesis of Ser and Thr residues from the consensus sequences Asn‐X‐Ser/Thr. Mutagenized cDNA constructs were heterologously expressed in the baculovirus/insect cell system. Removal of oligosaccharides either at Asn15, 80 or 252 was found to have no significant influence on the enzymatic activity measured in vitro. However, the absence of glycosylation at Asn308, as well as a total deglycosylation, reduced the specific enzymatic activity of recombinant HGL (r‐HGL), measured on short‐ and long‐chain triglycerides, to about 50% of normal values. Furthermore, biosynthesis and secretion of r‐HGL markedly dropped when all four potential glycosylation sites were mutated. The kinetics of the interfacial adsorption of r‐HGL and the completely deglycosylated r‐HGL (four‐site mutant) were found to be identical when recording the changes with time of the surface pressure either at the air–water interface or in the presence of an egg phosphatidylcholine (PtdCho) monomolecular film spread at various initial surface pressures. This indicates that both recombinant HGLs are identical, as far as recognition of phospholipid film and adsorption on PtdCho are concerned. The N‐glycosylation of HGL may contribute to the enzyme stability in the stomach, as under acidic conditions the degradation by pepsin of the unglycosylated r‐HGL is increased.
Purification and cDNA cloning of a protein derived from Flammulina velutipes that increases the permeability of the intestinal Caco‐2 cell monolayer
Authors: Watanabe Hirohito , Narai Asako , Shimizu Makoto
Published: March 2003

A new protein that decreases transepithelial electrical resistance (TEER) in the human intestinal Caco‐2 cell monolayer was found in a water‐soluble fraction of the mushroom Flammulina velutipes. This protein, termed TEER‐decreasing protein (TDP), is not cytotoxic and does not induce cell detachment, but rapidly increases the tight junctional permeability for water‐soluble marker substances such as Lucifer Yellow CH (Mr 457) through the paracellular pathway. TDP was isolated and purified from the aqueous extract of F. velutipes by chromatographic means. Purified TDP was found to be a simple, nonglycosylated protein without intermolecular disulfide bonds, and the apparent molecular mass as estimated by SDS/PAGE and gel filtration is 30 kDa. It was revealed that the N‐terminal amino‐acid sequence of purified TDP is identical to the recently reported N‐terminal sequence of flammutoxin, a membrane‐perturbing hemolytic protein, for which the complete primary structure has not yet been reported [Tomita, T., Ishikawa, D., Noguchi, T., Katayama, E., and Hashimoto, Y. (1998) Biochem. J.333, 24794–24799]. The cDNA coding for TDP was cloned by 5′ and 3′ rapid amplification of cDNA ends. The ORF encodes a protein with 272 amino‐acid residues showing no homology to known proteins. Relevant studies using TDP cDNA will provide insight into the structure–function relationships of membrane pore‐forming toxins.
Phlorisovalerophenone synthase, a novel polyketide synthase from hop (Humulus lupulus L.) cones
Authors: Paniego Norma B. , Zuurbier Karin W. M. , Fung Suen‐Ying , van der Heijden Robert , Scheffer Johannes J. C. , Verpoorte Robert
Published: March 2003

Phlorisovalerophenone synthase (VPS), a novel aromatic polyketide synthase, was purified to homogeneity from 4.2 mg protein extract obtained from hop (Humulus lupulus L.) cone glandular hairs. The enzyme uses isovaleryl‐CoA or isobutyryl‐CoA and three molecules of malonyl‐CoA to form phlorisovalerophenone or phlorisobutyrophenone, intermediates in the biosynthesis of the hop bitter acids (α‐ and β‐acids). VPS is an homodimeric enzyme, with subunits of 45 kDa. The pI of the enzyme was 6.1. Km values of 4 µm for isovaleryl‐CoA, 10 µm for isobutyryl‐CoA and 33 µm for malonyl‐CoA, were found. The amino‐acid sequences of two peptides, obtained by digestion of VPS, showed that the enzyme is highly homologous to plant chalcone synthases. The functional and structural relationship between VPS and other aromatic polyketide synthases is discussed.
Cyanophycinase, a peptidase degrading the cyanobacterial reserve material multi‐L‐arginyl‐poly‐L‐aspartic acid (cyanophycin)
Authors: Richter Ralf , Hejazi Mahdi , Kraft Regine , Ziegler Karl , Lockau Wolfgang
Published: March 2003

The branched polypeptide multi‐l‐arginyl‐poly‐l‐aspartic acid, also called cyanophycin, is a water‐insoluble reserve material of cyanobacteria. The polymer is degraded by a specific hydrolytic enzyme called cyanophycinase. By heterologous expression in Escherichia coli, a gene encoding cyanophycinase has been identified in the sequenced genome of Synechocystis sp. PCC 6803. The gene, designated cphB, codes for a protein of 29.4 kDa. The high level of expression of active cyanophycinase in E. coli from the Synechocystis gene allowed for its purification to electrophoretic homogeneity. The enzyme, which appears to be specific for cyanophycin, hydrolysed the polymer to a dipeptide consisting of aspartic acid and arginine. Based on inhibitor sensitivity and primary sequence, cyanophycinase appears to be a serine‐type exopeptidase related to dipeptidase E [Conlin, C.A., Haakensson, K., Liljas, A. & Miller, C.G. (1994) J. Bacteriol.176, 166–172].
Cytochrome c‐dependent methacrylate reductase from Geobacter sulfurreducens AM‐1
Authors: Mikoulinskaia Oxana , Akimenko Vasily , Galouchko Alexander , Thauer Rudolf K. , Hedderich Reiner
Published: March 2003

Geobacter sulfurreducens AM‐1 can use methacrylate as a terminal electron acceptor for anaerobic respiration. In this paper, we report on the purification and properties of the periplasmic methacrylate reductase, and show that the enzyme is dependent on the presence of a periplasmic cytochrome c (apparent Km = 0.12 µm). The methacrylate reductase was found to be composed of only one polypeptide with an apparent molecular mass of 50 kDa and to contain, bound tightly but not covalently, 1 mol of FAD per mol. The N‐terminal amino acid sequence showed sequence similarity to a periplasmic fumarate reductase from Shewanella putrefaciens. However, methacrylate reductase did not catalyze the reduction of fumarate. The periplasmic cytochrome c, which was also purified, had an apparent molecular mass of 30 kDa and contained ≈ 4 mol of heme·mol−1. Cells of G. sulfurreducens AM‐1 grown on acetate and methacrylate as an energy source were found to contain all the enzymes required for the oxidation of acetate to CO2 via the citric acid cycle.
Chromatin assembly during DNA replication in somatic cells
Authors: Krude Torsten
Published: March 2003

Newly replicated DNA is assembled into chromatin through two principle pathways. Firstly, parental nucleosomes segregate to replicated DNA, and are transferred directly to one of the two daughter strands during replication fork passage. Secondly, chromatin assembly factors mediate de‐novo assembly of nucleosomes on replicating DNA using newly synthesized and acetylated histone proteins. In somatic cells, chromatin assembly factor 1 (CAF‐1) appears to be a key player in assembling new nucleosomes during DNA replication. It provides a molecular connection between newly synthesized histones and components of the DNA replication machinery during the S phase of the cell division cycle.
Modulation of murine phenobarbital‐inducible CYP2A5, CYP2B10 and CYP1A enzymes by inhibitors of protein kinases and phosphatases
Authors: Posti Katja , Leinonen Susanna , Tetri Sami , Kottari Sami , Viitala Pirkko , Pelkonen Olavi , Raunio Hannu
Published: March 2003

Phenobarbital causes a multitude of effects in hepatocytes, including increased cell proliferation, inhibition of apoptosis and upregulation of xenobiotic and endobiotic metabolizing enzymes. In this study, the involvement of several protein kinase and phosphatase pathways on constitutive and phenobarbital‐induced activities of CYP2A5, CYP2B10 and CYP1A1/2 in primary mouse hepatocytes was determined using well‐defined chemical modulators of intracellular protein phosphorylation and desphosphorylation events. A 48‐h treatment of the hepatocytes with 2‐aminopurine, a nonspecific serine/threonine kinase inhibitor, elicited dose‐dependent increases in both basal and phenobarbital‐induced CYP2A5 catalytic activity (assayed as coumarin 7‐hydroxylation), the maximal induction being 60‐fold greater than the control value upon cotreatment with 1.5 mm phenobarbital and 10 mm 2‐aminopurine. In contrast, phenobarbital induction of CYP2B10 (pentoxyresorufin O‐deethylase) and CYP1A1/2 (ethoxyresorufin O‐deethylase) activities were blocked by 2‐aminopurine. Increases in CYP2A5 activity were also observed after exposure of the hepatocytes to other protein kinase inhibitors affecting the cell cycle, i.e. roscovitine, K‐252a and rapamycin. Inhibitors of protein kinases A and C, as well as tyrosine kinases, did not appreciably affect CYP2A5 activity levels. The serine/threonine phosphatase inhibitors tautomycin, calyculin A and okadaic acid all reduced both basal and phenobarbital‐induced CYP2A5, CYP2B10 and CYP1A1/2 activities. These results further strengthen the concept that hepatic CYP2A5 is regulated in a unique way compared with CYP2B10 and CYP1A.
Purified apolipoprotein B gene regulatory factor‐3 is DNA topoisomerase I
Authors: Chuang Samuel S. , Banerjee Deben , Das Hriday K.
Published: March 2003

Hepatic cell‐specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis‐acting elements located between positions −128 and +122 [Chuang, S. S., & Das, H. K. (1996) Biochem. Biophys. Res. Commun.220, 553–562]. A negative cis‐acting element (+20 to +40) is located in the first nontranslated exon of the human apoB gene, and apoB regulatory factor‐3 (BRF‐3) interacts with this. In this paper, we report the purification and characterization of BRF‐3 from rat liver nuclear extracts. BRF‐3 has been purified to apparent homogeneity by DEAE–cellulose, heparin–agarose, and DNA‐specific affinity chromatography. Purified BRF‐3 produced two polypeptide bands with apparent molecular masses of 70 kDa and 67 kDa in SDS/PAGE as detected by silver staining. Both 70‐kDa and 67‐kDa proteins have been found to hybridize specifically with labeled double‐stranded oligonucleotide containing BRF‐3 binding site in a South‐Western blot. Double‐stranded oligonucleotide containing mutations in the BRF‐3 binding site was found to abolish DNA binding by these two proteins. Amino acid sequences of tryptic peptides derived from affinity purified 70‐kDa and 67‐kDa rat BRF‐3 proteins were found to have 100% sequence homologies with DNA topoisomerase I. These data suggest that the 70‐kDa and 67‐kDa forms of BRF‐3 are derived by proteolytic cleavage of topoisomerase I, and therefore, topoisomerase I may play an important role in transcriptional regulation of apoB.
Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum
Authors: Schröder Ilka , Thauer Rudolf K.
Published: March 2003

Methanobacterium thermoautotrophicum is a methane‐forming archaeon that grows on H2 and CO2 as sole carbon and energy source. Cell extracts of the methanogen were found to contain methylcobalamin: homocysteine methyltransferase activity which was purified 3000‐fold to a specific activity of ≈ 500 U·mg−1 protein. SDS/PAGE revealed the presence of a polypeptide with an apparent molecular mass of 34 kDa. Via its N‐terminal amino acid sequence, the 34‐kDa polypeptide was identified as the metE gene product. The metE gene was heterologously expressed in Escherichia coli. The overproduced protein was recovered in the inclusion body fraction and was found to be inactive. The protein could be partially solubilized by unfolding in 8 m urea and then refolding. The solubilized protein had a specific activity of 450 U·mg−1. It exhibited first‐order kinetics with respect to methylcobalamin concentration and Michaelis–Menten kinetics with respect to l‐homocysteine concentration (apparent Km 0.1 mm). The enzyme was specific for l‐homocysteine as methyl acceptor. Methylcobalamin could be substituted with methylcobinamide as methyl donor.
Functional and structural diversities of C‐reactive proteins present in horseshoe crab hemolymph plasma
Authors: Iwaki Daisuke , Osaki Tsukasa , Mizunoe Yoshimitsu , Wai Sun N. , Iwanaga Sadaaki , Kawabata Shun‐ichiro
Published: March 2003

Limulin, a sialic‐acid‐binding and phosphorylethanolamine‐binding hemagglutinin in the hemolymph plasma of the American horseshoe crab (Limulus polyphemus), is a hemolytic C‐reactive protein [Armstrong, P.B., Swarnakar, S., Srimal, S., Misquith, S., Hahn, E.A., Aimes, R.T. & Quigley, J.P. (1996) J. Biol. Chem. 271, 14717–14721]. We have now identified three types of C‐reactive protein in the plasma of the Japanese horseshoe crab (Tachypleus tridentatus), based on different affinities against fetuin–agarose and phosphorylethanolamine–agarose determined by quantitative precipitin assays using fetuin and an artificial phosphorylethanolamine–protein conjugate. Partial amino acid sequences of the isolated C‐reactive proteins identified homologous proteins which were named Tachypleus tridentatus CRP‐1 (tCRP‐1), tCRP‐2 and tCRP‐3, each of which possibly constitute isoprotein mixtures. tCRP‐2 and tCRP‐3, but not tCRP‐1, agglutinated mammalian erythrocytes. tCRP‐1, the most abundant C‐reative protein in the plasma, exhibited the highest affinity to the phosphorylethanolamine–protein conjugate but lacked both sialic‐acid‐binding and hemolytic activities. tCRP‐2 bound to both fetuin–agarose and phosphorylethanolamine‐agarose, and exhibited Ca2+‐dependent hemolytic and sialic‐acid‐binding activities, suggestive of limulin‐like properties. Furthermore, tCRP‐2 exhibited a higher affinity to colominic acid, a bacterial polysialic acid. By contrast, tCRP‐3 shows stronger hemolytic, sialic‐acid‐binding and hemagglutinating activities than tCRP‐2. tCRP‐3 has no affinity to phosphorylethanolamine–agarose, phosphorylethanolamine–protein conjugate and colominic acid. This suggests tCRP‐3 is a novel hemolytic C‐reactive protein lacking a common characteristic of phosphorylethanolamine–agarose binding affinity. Twenty‐two clones of tCRPs with different deduced amino acid sequences were obtained by PCR using oligonucleotide primers based on the N‐terminal and C‐terminal sequences of tCRPs and with templates including genomic DNA and cDNA of hemocytes or hepatopancreas derived from one individual. The translation products of the tCRP clones possess high molecular diversity which falls into three related groups, consistent with classification based on their biological activities. Only tCRP‐3 contained a unique hydrophobic nonapeptide sequence that appears in the transmembrane domain of a major histocompatibility complex class I heavy chain of rainbow trout, suggesting the importance of the hydrophobic patch to the hemolytic activity of tCRP‐3. The structural and functional diversities of tCRPs provide a good model for studying the properties of innate immunity in invertebrates, which survive without the benefit of acquired immunity.
Pigment‐free NADPH:protochlorophyllide oxidoreductase from Avena sativa L
Authors: Klement Harald , Helfrich Michael , Oster Ulrike , Schoch Siegrid , Rüdiger Wolfhart
Published: March 2003

The enzyme NADPH:protochlorophyllide oxidoreductase (POR) is the key enzyme for light‐dependent chlorophyll biosynthesis. It accumulates in dark‐grown plants as the ternary enzyme–substrate complex POR‐protochlorophyllide a‐NADPH. Here, we describe a simple procedure for purification of pigment‐free POR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n‐octyl‐β‐d‐glucoside and one chromatographic step with DEAE‐cellulose. We show, using pigment and protein analysis, that etioplasts contain a one‐to‐one complex of POR and protochlorophyllide a. The preparation of 13 analogues of protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by POR. The kinetics of the light‐dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for protochlorophyllide a and for the analogues.
Inhibitor peptide SNP‐1 binds to a soluble form of BST‐1/CD157 at a 2 : 2 stoichiometry
Authors: Sato Atsushi , Yamamoto Sumie , Kajimura Naoko , Oda Masayuki , Usukura Jiro , Jingami Hisato
Published: March 2003

Recently we have identified a 15‐mer peptide, SNP‐1, by a random phage library that can bind to bone marrow stromal cell antigen‐1 (BST‐1)/CD157 [Sato, A., Yamamoto, S., Ishihara, K., Hirano, T. & Jingami, H. (1999) Biochem. J. 337, 491–496]. SNP‐1 inhibits BST‐1 ADP‐ribosyl cyclase activity uncompetitively with a Ki value of 180 ± 40 nm. In this study we analysed biophysically the SNP‐1 binding to a soluble form of BST‐1 (sBST‐1). Equilibrium binding data of wild‐type SNP‐1 from surface plasmon resonance studies gave a Kd value of 500 ± 35 nm. Titration calorimetry analysis showed that the binding reaction is exothermic at 20 °C. The values of Kd = 211 nm, enthalpy change, ΔH = –18.68 kcal·mol−1, and saturated molar ratio of bound SNP‐1 per sBST‐1, N = 0.8 mol·mol−1 were obtained. On the basis of the molecular masses of SNP‐1 and sBST‐1 calculated by analytical ultracentrifugation, the stoichiometry of the binding was determined to be 2 : 2. Electron microscopy also revealed the dimer form of sBST‐1. To delineate the core residue of SNP‐1 responsible for binding, each amino acid residue has been replaced by alanine. A region from amino acid residues 7–12 appeared to be critical for the SNP‐1 binding to sBST‐1. The substitution of the first residue, His, to Ala led to a reduction in binding, suggesting that the N‐terminal residue is also crucial.
Stability, refolding and Ca2+ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei
Authors: Schwerdtfeger Ruth M , Chiaraluce Roberta , Consalvi Valerio , Scandurra Roberto , Antranikian Garabed
Published: March 2003

The unfolding and refolding of the extremely heat‐stable pullulanase from Pyrococcus woesei has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation and the transition midpoint for guanidinium chloride‐induced unfolding was determined to be 4.86 ± 0.29 m for intrinsic fluorescence and 4.90 ± 0.31 m for far‐UV CD changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 m guanidinium chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 m in the first dilution step. Particular attention has been paid to the role of Ca2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca2+ ions with a Kd of 0.080 ± 0.010 µm and a Hill coefficient H of 1.00 ± 0.10. This cation enhances significantly the stability of the pullulanase against guanidinium chloride‐induced unfolding and the ΔGH2OD increased from 6.83 ± 0.43 to 8.42 ± 0.55 kcal·mol−1. The refolding of the pullulanase, on the other hand, was not affected by Ca2+.
Selective Sp1 and Sp3 binding is crucial for activity of the integrin αV promoter in cultured endothelial cells
Authors: Czyz Malgorzata , Cierniewski Czeslaw S.
Published: March 2003

Human integrin αvβ receptors are expressed in a number of cells and their expression is regulated at the level of transcription and by post‐transcriptional mechanisms. A substantial body of research exists on the structure, function, molecular biology and physiological significance of αv integrin receptors. However, the importance of particular cis‐acting DNA elements or trans‐acting nuclear factors in the regulation of the αv gene promoter is still not adequately understood. Previous functional analysis of the αv gene 5′ flanking region in transfected cultured cells identified cis elements critical for αv transcription within a 222‐bp region. To define further the location of this enhancing element, we performed DNase I footprinting of the human αv gene promoter between −522 and the translation initiation site. For this purpose, nuclear extracts of αvβ3‐positive cells, human umbilical vein endothelial cells, were used. Nuclear proteins of endothelial cells strongly protected essentially one region corresponding to the sequence between −194 and −172 of the αv promoter region. Electrophoretic mobility shift assays with different oligonucleotides, and competition analysis identified a CTCCTCCTC sequence that is directly involved in the transcriptional activity of the αv promoter. Purified Sp1 alone produced an identical footprint, and DNA binding assays using anti‐Sp1 and anti‐Sp3 antibodies showed that transcription factors Sp1 and Sp3 were the major nuclear proteins bound to this region.
Cyclic organization of the carbohydrate metabolism in Sinorhizobium meliloti
Authors: Portais Jean‐Charles , Tavernier Patricia , Gosselin Isabelle , Barbotin Jean‐Noël
Published: March 2003

The pathways of polysaccharide biosynthesis were investigated in cells of Sinorhizobium meliloti (strain Su47) using a stable isotope approach. The isotopic labeling of the periplasmic β‐1,2‐glucans synthesized from glucose labeled at various positions evidenced the involvement of catabolic pathways, namely the pentose‐phosphate and Entner–Doudoroff pathways, into the early steps of polysaccharide synthesis. The exopolysaccharides produced at the same time had a labeling pattern similar to that of the β‐glucans, indicating similar early steps for both polysaccharides. The results emphasized a cyclic organization of the carbohydrate metabolism in S. meliloti, in which the carbons of the initial hexose were allowed to re‐enter the catabolic pathways many times. The metabolic incidences of such metabolic topology are discussed.
Translational control by the La antigen
Authors: James Marion C. , Jeffrey Ian W. , Pruijn Ger J. M. , Thijssen José P. H. , Clemens Michael J.
Published: March 2003

The La antigen is a protein which can bind both single‐stranded and double‐stranded forms of RNA and has regulatory effects on gene expression at the levels of transcription and translation. It was previously shown to inhibit the activation of the dsRNA‐dependent protein kinase PKR by sequestering and/or unwinding double‐stranded RNA. Here, we demonstrate that, as predicted by these properties, the La antigen can rescue protein synthesis in the reticulocyte lysate system from inhibition by low concentrations of dsRNA. This effect is reversed by higher concentrations of dsRNA. Using a series of deletion mutants we have investigated the structural features of the La antigen that are required for these effects. The ability to bind dsRNA is influenced by regions within both the previously characterized N‐terminal RNP motif and the C‐terminal half of the protein. La mutants with either N‐terminal or C‐terminal deletions retain the ability to inhibit the protein kinase activity of PKR and to rescue protein synthesis from inhibition by dsRNA. It is notable that sequences in the C‐terminal half of the La antigen, including a phosphorylation site at Ser366, which are needed for other regulatory effects of the protein on gene expression are dispensable for the effects of La on PKR. We suggest that La regulates PKR activity solely as a result of its ability to act as an RNA‐binding protein that can compete with PKR for limiting amounts of dsRNA.
A Drosophila gene encoding multiple splice variants of Kazal‐type serine protease inhibitor‐like proteins with potential destinations of mitochondria, cytosol and the secretory pathway
Authors: Niimi Tomoaki , Yokoyama Hiroaki , Goto Akira , Beck Konrad , Kitagawa Yasuo
Published: March 2003

A Drosophila gene (KAZ1), mapped to cytological position 61A1‐2 on chromosome 3, has been cloned and found to encode multiple splice variants of Kazal‐type serine protease inhibitor‐like proteins. KAZ1 consists of five exons and four alternatively retained introns to produce six transcripts of type AB, C1, C2, C3, D and E. The AB transcript contains two ORFs, of which the upstream one produces a polypeptide α, which has a mitochondrial sorting signal. Localization to mitochondria was confirmed by expression in COS1 cells. The downstream ORF is shared partially with type C1, C2, C3, D and E transcripts and produces polypeptides β, γ, δ and ε when expressed in Drosophila cells. Type C1, C2 and C3 transcripts differ only in the 5′‐noncoding sequence and thus all produce type γ. Polypeptides γ and ε have a signal sequence at their N‐termini and are secreted into the medium while β and δ lack this sequence and remain in the cytoplasm. Isoforms β and ε share a common C‐terminal sequence distinct from that shared by polypeptides γ and δ. The N‐terminal sequences of isoforms β to ε contain a PEST region which could induce rapid intracellular degradation of isoforms β and δ. Sequence analysis of the Kazal‐type domain suggests a similar folding pattern as observed for rhodniin and SPARC/BM‐40. Northern analysis and in situ hybridization showed that the type C3 transcript is predominant and the expression is highest in midgut at larval stage.
The self‐association of protein SV‐IV and its possible functional implications
Authors: Stiuso Paola , Metafora Salvatore , Facchiano Angelo M. , Colonna Giovanni , Ragone Raffaele
Published: March 2003

The protein SV‐IV, a major protein secreted from the rat seminal vesicle epithelium, is a basic protein with immunomodulatory, anti‐inflammatory, and procoagulant activity. Predictions suggested that this protein is very flexible, with its three tyrosyl residues presumably located in water‐exposed segments of the primary structure. The solution behaviour of the protein was investigated by two types of spectroscopic techniques. Modifications of the spectral characteristics of tyrosyl residues induced by changes of protein concentration were demonstrated by absorption and fluorescence experiments. In addition, secondary structure rearrangements associated with a possible self‐association equilibrium were highlighted by far‐UV CD spectra. The equilibrium, confirmed by chromatographic techniques, appears to control some biological properties of the protein.
Interactions of Alzheimer amyloid‐β peptides with glycosaminoglycans
Authors: McLaurin JoAnne , Franklin Trudy , Zhang Xiaoqiong , Deng Jianping , Fraser Paul E.
Published: March 2003

Proteoglycans and their constituent glycosaminoglycans are associated with all amyloid deposits and may be involved in the amyloidogenic pathway. In Alzheimer’s disease, plaques are composed of the amyloid‐β peptide and are associated with at least four different proteoglycans. Using CD spectroscopy, fluorescence spectroscopy and electron microscopy, we examined glycosaminoglycan interaction with the amyloid‐β peptides 1–40 (Aβ40) and 1–42 (Aβ42) to determine the effects on peptide conformation and fibril formation. Monomeric amyloid‐β peptides in trifluoroethanol, when diluted in aqueous buffer, undergo a slow random to amyloidogenic β sheet transition. In the presence of heparin, heparan sulfate, keratan sulfate or chondroitin sulfates, this transition was accelerated with Aβ42 rapidly adopting a β‐sheet conformation. This was accompanied by the appearance of well‐defined amyloid fibrils indicating an enhanced nucleation of Aβ42. Incubation of preformed Aβ42 fibrils with glycosaminoglycans resulted in extensive lateral aggregation and precipitation of the fibrils. The glycosaminoglycans differed in their relative activities with the chondroitin sulfates producing the most pronounced effects. The less amyloidogenic Aβ40 isoform did not show an immediate structural transition that was dependent upon the shielding effect by the phosphate counter ion. Removal or substitution of phosphate resulted in similar glycosaminoglycan‐induced conformational and aggregation changes. These findings clearly demonstrate that glycosaminoglycans act at the earliest stage of fibril formation, namely amyloid‐β nucleation, and are not simply involved in the lateral aggregation of preformed fibrils or nonspecific adhesion to plaques. The identification of a structure–activity relationship between amyloid‐β and the different glycosaminoglycans, as well as the condition dependence for glycosaminoglycan binding, are important for the successful development and evaluation of glycosaminoglycan‐specific therapeutic interventions.