bluuebunny/crossref_metadata_2025 huggingface dataset:
Importance of heptameric ring integrity for activity of Escherichia coli ClpP
Authors: Thompson Mark W. , Miller Jay , Maurizi Michael R. , Kempner Ellis
Published: March 2003

Radiation target analysis has been used to identify the minimal functional unit for expression of activity of ClpP, the proteolytic component of the ATP‐dependent ClpAP protease. Radiation target sizes determined for small peptide hydrolysis, for ClpA‐activated and nucleotide‐activated oligopeptide cleavage, and for ClpA‐activated ATP‐dependent protein degradation were 154, 118, and 160 kDa, respectively. Thus, the hydrolytic activity of ClpP, subunit Mr 21 500, is dependent on the native oligomeric structure. The quaternary structure of ClpP determined by electron microscopy and hydrodynamic studies consists of two face‐to‐face seven‐membered rings. The radiation target sizes are consistent with a requirement for conformational integrity of an entire ring for expression of hydrolytic activity. Radiation damage led to disruption of inter‐ring contacts, giving rise to isolated rings of ClpP. Thus, contacts between rings of ClpP are less stable and more easily disrupted than contacts between subunits within the rings. Our data suggest that cooperative interactions between subunits within the ClpP rings are important for maintaining the active conformation of the proteolytic active site.
UDP‐glucuronosyltransferase UGT1A7 induced in rat small intestinal mucosa by oral administration of 2‐naphthoflavone
Authors: Kobayashi Tsutomu , Yokota Hiroshi , Ohgiya Satoru , Iwano Hidetomo , Yuasa Akira
Published: March 2003

In the rat intestine, UDP‐glucuronosyltransferase (UGT) isoforms were highly induced by oral administration of 2‐naphthoflavone, as shown by intestinal UGT activity toward 1‐naphthol (1‐NA). The greatest increase in UGT activity occurred in the duodenum. Using UGT1A6 cDNA as a probe, we obtained three types of clones corresponding to UGT1A2, UGT1A6 and UGT1A7, in a ratio of 1 : 1 : 8, from a cDNA library constructed from the 2‐naphthoflavone‐treated rat intestine. The induction of each isoform was evaluated by means of Northern blotting with isoform‐specific probes. The mRNAs of UGT1A6 (glucuronizing various phenolic xenobiotics) and the mRNAs of UGT1A7 (glucuronizing the ultimate carcinogenic metabolite of benzo(a)pyrene) were expressed constitutively and were highly induced in the duodenum and proximal jejunum. S1 mapping showed that induction of the isoforms of the UGT1 family was more pronounced in the liver than in the small intestine and that UGT1A7 was the major UGT1 isoform in the small intestine of vehicle‐treated rats and in that of 2‐naphthoflavone‐treated rats. These results indicate that, in rats, UGT1A7 is expressed constitutively and is particularly inducible in the small intestine. In the light of these results, we believe that the UGT1A7 isoform would play an important role in glucuronidation in the small intestinal mucosa of rats.
Slow‐binding and competitive inhibition of 8‐amino‐7‐oxopelargonate synthase, a pyridoxal‐5′‐phosphate‐dependent enzyme involved in biotin biosynthesis, by substrate and intermediate analogs
Authors: Ploux Olivier , Breyne Olivier , Carillon Sophie , Marquet Andrée
Published: March 2003

8‐Amino‐7‐oxopelargonate synthase catalyzes the first committed step of biotin biosynthesis in micro‐organisms and plants. Because inhibitors of this pathway might lead to antibacterials or herbicides, we have undertaken an inhibition study on 8‐amino‐7‐oxopelargonate synthase using six different compounds. d‐Alanine, the enantiomer of the substrate of this pyridoxal‐5′‐phosphate‐dependent enzyme was found to be a competitive inhibitor with respect to l‐alanine with a Ki of 0.59 mm. The fact that this inhibition constant was four times lower than the Km for l‐alanine was interpreted as the consequence of the inversion‐retention stereochemistry of the catalyzed reaction. Schiff base formation between l or d‐alanine and pyridoxal‐5′‐phosphate, in the active site of the enzyme, was studied using ultraviolet/visible spectroscopy. It was found that l and d‐alanine form an external aldimine with equilibrium constants K = 4.1 mm and K = 37.8 mm, respectively. However, the equilibrium constant for d‐alanine aldimine formation dramatically decreased to 1.3 mm in the presence of saturating concentration of pimeloyl‐CoA, the second substrate. This result strongly suggests that the binding of pimeloyl‐CoA induces a conformational change in the active site, and we propose that this new topology is complementary to d‐alanine and to the putative reaction intermediate since they both have the same configuration. (±)‐8‐Amino‐7‐oxo‐8‐phosphonononaoic acid (1), the phosphonate derivative of the intermediate formed during the reaction, was our most potent inhibitor with a Ki of 7 µm. This compound behaved as a reversible slow‐binding inhibitor, competitive with respect to l‐alanine. Kinetic investigation showed that this slow process was best described by a one‐step mechanism (mechanism A) with the following rate constants: k1 = 0.27 × 103 m−1·s−1, k2 = 1.8 s−1 and half‐life for dissociation t1/2 = 6.3 min. The binding of compound 1 to the enzyme was also studied using ultraviolet/visible spectroscopy, and the data were consistent with the kinetic data (K = 4.2 µm). Among the other compounds tested, two potential transition state analogs, 4‐carboxybutyl(1‐amino‐1‐carboxyethyl)phosphonate (4) and 2‐amino‐3‐hydroxy‐2‐methylnonadioic acid (5) were found to be competitive inhibitors with respect to l‐alanine with Ki of 68 µm and 80 µm, respectively.
Protein interactions of Gts1p ofSaccharomyces cerevisiaethroughout a region similar to a cytoplasmic portion of some ATP‐binding cassette transporters
Authors: Kawabata Ken‐ichi , Mitsui Kazuhiro , Uno Takeshi , Tamura Kohji , Tsurugi Kunio
Published: March 2003

TheGTS1gene product, Gts1p, has pleiotropic effects on the timing of budding, cell size, heat tolerance, sporulation and the lifespan of the yeastSaccharomyces cerevisiae. In this study, we found (using the yeast two‐hybrid system) that Gts1p forms homodimers throughout the 18‐amino acid region 296–313 which has considerable similarity to a region downstream of the Walker nucleotide‐binding motif A of some ATP‐binding cassette (ABC) transporters. The region contains two aspartic acid residues at 301 and 310 preceded by hydrophobic amino acid residues, and Gts1p with an Asp310 to Ala substitution showed considerably reduced homodimerization, as shown by the two‐hybrid assay. Overexpression of the point‐mutated Gts1p did not efficiently induce the Gts1p‐related phenotypes described above, suggesting that the homodimerization of Gts1p is required for it to functionin vivo. The C‐terminal cytoplasmic domain of the yeast ABC transporters Mdl1p (multidrug resistance‐like transporter) and Ycf1p (yeast cadmium factor or glutathione S‐conjugate pump) bound to Gts1p in the two‐hybrid system, and the heterodimerization activity of the Gts1p with the Asp301 to Ala substitution was more affected than the Gts1p with the Asp310 to Ala substitution. Overexpression ofGTS1considerably reduced, and disruption ofGTS1slightly decreased, cellular resistance to cycloheximide, cadmium, cisplatin and 1‐chloro‐2,4‐dinitrophenol, which (except for cycloheximide) are all substrates of Ycf1p. These results suggest that Gts1p interacts with some ABC transporters through the binding site overlapping that of homodimerization and modulates their activity.
mRNA stability and selenocysteine insertion sequence efficiency rank gastrointestinal glutathione peroxidase high in the hierarchy of  selenoproteins
Authors: Wingler Kirstin , Böcher Michael , Flohé Leopold , Kollmus Heike , Brigelius‐Flohé Regina
Published: March 2003

The recently described gastrointestinal glutathione peroxidase (GI‐GPx) is the fourth member of the family of the selenoenzymes glutathione peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical glutathione peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium‐dependent expression of GI‐GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo‐2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI‐GPx, phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter‐gene assay in human hepatoma cells and baby hamster kidney cells. GI‐GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium‐poor media, all GPx‐types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI‐GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI‐GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI‐GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI‐GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI‐GPx in the gastrointestinal tract.
A new model for microtubule‐associated protein (MAP)‐induced  microtubule assembly
Authors: Tokuraku Kiyotaka , Katsuki Miho , Nakagawa Hiroyuki , Kotani Susumu
Published: March 2003

The microtubule‐binding domains of microtubule‐associated protein (MAP) 2, tau, and MAP4 are divided into three distinctive regions: the Pro‐rich region, the AP sequence region and the tail region (Aizawa, H., Emori, Y., Murofushi, H., Kawasaki, H., Sakai., H., and Suzuki, K. (1990) J. Biol. Chem.265, 13849–13855). Electron microscopic observation showed that the taxol‐stabilized microtubules alone and those mixed with the A4T fragment (containing the AP sequence region and the tail region) had a long, wavy appearance, while those mixed with the PA4T fragment (containing the Pro‐rich region, the AP sequence region, and the tail region) or the PA4 fragment (containing the Pro‐rich region and the AP sequence region) were shorter and straighter. Stoichiometries of the binding between the fragments and the tubulin dimers were approximately between 1 and 2, suggesting that not all of the AP sequences in the AP sequence region bound to tubulin. Binding affinity of the PA4T fragment is only four times higher than that of the A4T fragment, while the microtubule nucleating activity of the PA4T fragment is far greater. Based on these results, we propose that the nucleation of microtubule assembly is promoted by the bridging activity of the Pro‐rich region in the MAPs.
Enzyme level of enterococcal F1Fo‐ATPase is regulated by pH at the step of assembly
Authors: Arikado Eri , Ishihara Hiroko , Ehara Teruyoshi , Shibata Chiyoko , Saito Hiromi , Kakegawa Tomohito , Igarashi Kazuei , Kobayashi Hiroshi
Published: March 2003

The amount of F1Fo‐ATPase in Enterococcus hirae (formerly Streptococcus faecalis) increases when the cytoplasmic pH is lowered below 7.6, and protons are extruded to maintain the cytoplasmic pH at around 7.6. In the present study, we found that the transcriptional activity of the F1Fo‐ATPase operon was not regulated by pH. The synthesis of F1 subunits was increased 1.65 ± 0.12‐fold by the acidification of medium from pH 8.0 to pH 5.3. Western‐blot analysis showed that there were F1 subunits in the cytoplasm, and the number of alpha plus beta subunits in the cytoplasm was 50% of the total number of the subunits in cells growing at pH 8.0. This decreased to 22% after shifting the medium pH to 5.3, with a concomitant 5.1‐fold increase in the level of membrane‐bound F1Fo‐ATPase. The cytoplasmic F1 subunits were shown to be degraded, and Fo subunits not assembled into the intact F1Fo complex were suggested to be digested. These data suggest that regulation of the enzyme level of F1Fo‐ATPase by the intracellular pH takes place mainly at the step of enzyme assembly from its subunits.
The cytochrome bc1 complex from Rhodovulum sulfidophilum is a dimer with six quinones per monomer and an additional 6‐kDa component
Authors: Montoya Guillermo , Kaat Kai te , Rodgers Simon , Nitschke Wolfgang , Sinning Irmgard
Published: March 2003

A highly active, large‐scale preparation of cytochrome bc1 complex has been obtained from the photosynthetic purple bacterium Rhodovulum (Rhv.) sulfidophilum. It has been characterized using mass spectrometry, quinone and lipid analysis as well as inhibitor binding. About 35 mg of pure complex can be obtained from 1 g of membrane protein. EPR spectroscopy and optical titrations have been used to obtain the redox midpoint potentials of the cofactors. The Em‐value of 310 mV for the Rieske protein is the most positive midpoint potential for this protein in a bc1 complex so far. The bc1 complex from Rhv. sulfidophilum is very stable and consists of three subunits and a 6‐kDa polypeptide. The complex appears as a dimer in solution and contains six quinone molecules per monomer which are tightly bound. EPR spectroscopy shows that the Qo site is highly occupied. High detergent concentrations convert the complex into an inactive, monomeric form that has lost the Rieske protein as well as the quinones and the 6‐kDa component.
Mimicry of βII′‐turns of proteins in cyclic pentapeptides with one and without d‐amino acids
Authors: Weißhoff Hardy , Präsang Carsten , Henklein Peter , Frömmel Cornelius , Zschunke Adolf , Mügge Clemens
Published: March 2003

The solution structure of eight cyclic pentapeptides has been determined by two‐dimensional 1H‐NMR spectroscopy combined with spectra simulations and restrained molecular dynamic simulations. Six of the cyclic pentapeptides were derived from the C‐terminal cholecystokinin fragment CCK‐4 enlarged with Asp1 resulting in the sequence (Asp‐Trp‐Met‐Asp‐Phe), one
l‐amino acid after the other was substituted by its
d‐analog. In addition, two peptides, including an all‐
l‐amino‐acid‐containing cyclic pentapeptide, cyclo(Asp‐Phe‐Lys‐Ala‐Thr) and cyclo(Asp‐Phe‐Lys‐Ala‐
d‐Thr) were investigated. All
d‐amino‐acid‐containing peptides show βII′‐turn conformations with the
d‐amino acid in the i + 1 position, excepting the
d‐aspartic‐acid‐containing peptides. These two peptides are characterized by the lack of β‐turns at pH values less than 4, suggesting that
d‐aspartic acid in the full‐protonized state avoids the formation of β‐turns in these compounds. At pH values greater than 5, a conformational change into the βII′‐turn conformation was also observed for these peptides. Conformations without β‐turns are expected for cyclic all‐
l pentapeptides, but both cyclo(Asp‐Phe‐Lys‐Ala‐Thr) and the
d‐Thr analog cyclo(Asp‐Phe‐Lys‐Ala‐
d‐Thr) exhibit βII′‐turn conformations around Thr‐Asp and
d‐Thr‐Asp. Thus cyclic all‐
l pentapeptides and those with one
d‐amino acid are able to form similar structures preferably with a βII′‐turn. The β‐turn formation in cyclic pentapeptides containing a
d‐aspartic acid is dependent on the ionization state. The relevance of the work to the design of βΙΙ′‐turn mimetics is discussed.
Rapid purification of membrane extrinsic F1‐domain of chloroplast ATP synthase in monodisperse form suitable for 3D‐crystallization
Authors: Groth Georg , Schirwitz Katja
Published: March 2003

A new chromatographic procedure for purification of the membrane extrinsic F1‐domain of chloroplast ATP synthase is presented. The purification is achieved by a single anion exchange chromatography step. Determination of the enzyme‐bound nucleotides reveals only 1 mole of ADP per complex. The purified enzyme shows a latent Ca2+‐dependent ATPase activity of 1.0 µmol·mg−1·min−1 and a Mg2+‐dependent activity of 4.4 µmol·mg−1·min−1. Both activities are increased up to 8–10‐fold after dithiothreitol activation. Analysis of the purified F1‐complex by SDS/PAGE, silver staining and immunoblotting revealed that the preparation is uncontaminated by fragmented subunits or ribulose‐1,5‐bisphosphate carboxylase/oxygenase. Gel filtration experiments indicate that the preparation is homogenous and monodisperse. In order to determine the solubility minimum of the purified F1‐complex the isoelectric point of the preparation was calculated from pH mapping on ion exchange columns. In agreement with calculations based on the amino acid sequence, a slightly acidic pI of 5.7 was found. Using ammonium sulphate as a precipitant the purified CF1‐complex could be crystallized by MicroBatch.
Expression and regulation of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐ bisphosphatase isozymes in white adipose tissue
Authors: Bruni Paola , Vandoolaeghe Pascale , Rousseau Guy G. , Hue Louis , Rider Mark H.
Published: March 2003

The aim of this work was to identify the 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFK‐2/FBPase‐2) isozyme(s) present in white adipose tissue. Ion‐exchange chromatography of PFK‐2 from rat epididymal fat pads yielded an elution pattern compatible with the presence of both the L (liver) and M (muscle) isozymes. This was consistent with a study of the phosphorylation of the purified adipose tissue enzyme by cAMP‐dependent protein kinase, by specific labelling of the preparation with [2‐32P]fructose 2,6‐bisphosphate and by reaction with antibodies. Characterization of the PFK‐2/FBPase‐2 mRNAs showed that mature adipocytes express the mRNA that codes for the L isozyme and the two mRNAs that code for the M isozyme. Preadipocytes expressed mRNA that codes for the M isozyme. Incubation of rat epididymal fat pads with adrenaline stimulated glycolysis but decreased fructose 2,6‐bisphosphate concentrations without significant inactivation of PFK‐2. These results support previous findings showing that fructose 2,6‐bisphosphate is not involved in the adrenaline‐induced stimulation of glycolysis in white adipose tissue.
N‐Glycan structures of matrix metalloproteinase‐1 derived from human fibroblasts and from HT‐1080 fibrosarcoma cells
Authors: Saarinen Juhani , Welgus Howard G. , Flizar Catherine A. , Kalkkinen Nisse , Helin Jari
Published: March 2003

Matrix metalloproteinase‐1 (MMP‐1) is a collagenolytic metalloproteinase capable of cleaving native triple‐helical forms of several collagen subtypes, as well as a number of non‐collagenous substrates. The role of MMP‐1 in various diseases affecting the connective tissue is well characterized. MMP‐1 is secreted as both glycosylated and unglycosylated species, and the two forms have been shown to be identical with respect to substrate specificity, specific activity and inhibitory profile. No function for the glycan moiety of the enzyme has been ascribed to date. In the present study, we report on the detailed characterization of MMP‐1‐derived oligosaccharides. Using strategies based on sequential exoglycosidase digestion combined with matrix‐assisted laser desorption ionization–time of flight MS and electrospray tandem MS, we have characterized the N‐glycan structures of MMP‐1, derived from human dermal fibroblasts and from the HT‐1080 fibrosarcoma cell line. MMP‐1 derived from fibroblasts was found to carry mainly α2,3‐sialylated complex‐type diantennary glycans. On the other hand, HT‐1080 cells produce MMP‐1 that has a heterogeneous glycosylation pattern, comprising diantennary glycans carrying Lewis X, LacdiNAc, sialylated LacdiNAc and GalNAcβ1,4(Fucα1,3)GlcNAc (LacdiNAc analogue of Lewis X) as terminal elements. We also show that, of the two potential glycosylation sites in the MMP‐1 sequence, only Asn120 is used.
Comparisons of flux control exerted by mitochondrial outer‐membrane carnitine palmitoyltransferase over ketogenesis in hepatocytes and mitochondria isolated from suckling or adult rats
Authors: New Karen J. , Krauss Stefan , Elliott Keith R. F. , Quant Patti A.
Published: March 2003

The primary aim of this paper was to calculate and report flux control coefficients for mitochondrial outer‐membrane carnitine palmitoyltransferase (CPT I) over hepatic ketogenesis because its role in controlling this pathway during the neonatal period is of academic importance and immediate clinical relevance. Using hepatocytes isolated from suckling rats as our model system, we measured CPT I activity and carbon flux from palmitate to ketone bodies and to CO2 in the absence and presence of a range of concentrations of etomoxir. (This is converted in situ to etomoxir‐CoA which is a specific inhibitor of the enzyme.) From these data we calculated the individual flux control coefficients for CPT I over ketogenesis, CO2 production and total carbon flux (0.51 ± 0.03; – 1.30 ± 0.26; 0.55 ± 0.07, respectively) and compared them with equivalent coefficients calculated by similar analyses [Drynan, L., Quant, P.A. & Zammit, V.A. (1996) Biochem. J.317, 791–795] in hepatocytes isolated from adult rats (0.85 ± 0.20; 0.23 ± 0.06; 1.06 ± 0.29). CPT I exerts significantly less control over ketogenesis in hepatocytes isolated from suckling rats than those from adult rats. In the suckling systems the flux control coefficients for CPT I over ketogenesis specifically and over total carbon flux (< 0.6) are not consistent with the enzyme being rate‐limiting. Broadly similar results were obtained and conclusions drawn by reanalysis of previous data {from experiments in mitochondria isolated from suckling or adult rats [Krauss, S., Lascelles, C.V., Zammit, V.A. & Quant, P.A. (1996) Biochem. J.319, 427–433]} using a different approach of control analysis, although it is not strictly valid to compare flux contol coefficients from different systems. Our overall conclusion is that flux control coefficients for CPT I over oxidative fluxes from palmitate (or palmitoyl‐CoA) differ markedly according to (a) the metabolic state, (b) the stage of development, (c) the specific pathway studied and (d) the model system.
Role of lysine and tryptophan residues in the biological activity of toxin VII (Ts γ) from the scorpion Tityus serrulatus
Authors: Hassani Oussama , Mansuelle Pascal , Cestèle Sandrine , Bourdeaux Madeleine , Rochat Hervé , Sampieri François
Published: March 2003

Toxin VII (TsVII), also known as Ts γ, is the most potent neurotoxin in the venom of the Brazilian scorpion Tityus serrulatus. It has been purified to homogeneity using a new fast and efficient method. Chemical modification of TsVII with the tryptophan‐specific reagent o‐nitrophenylsulfenyl chloride yielded three modified derivatives (residues Trp39, Trp50 and Trp54). Acetylation of TsVII mostly generated the monoacetylated Lys12 derivative. No side reactions were detected, as indicated by endoproteinase Lys‐C peptide mapping, Edman degradation and electrospray mass spectrometry. Circular dichroism and fluorimetric measurements showed that none of the chemical modifications altered the overall structure of the derivatives. The acetylation of Lys12 or the sulfenylation of Trp39 or Trp54 led to a loss of both toxicity in mice and apparent binding affinity for rat brain and cockroach synaptosomal preparations. Sulfenylation of Trp50, however, moderately affected the toxicity of TsVII in mice and had almost no effect on its binding properties. A 3‐dimensional model of TsVII was constructed by homology modeling. It suggests that the most reactive residues (Lys1 2 and Trp39 and Trp54) are all important in the functional disruption of neuronal sodium channels by TsVII, and are close to each other in the hydrophobic conserved region.
A novel human DNA‐binding protein with sequence similarity to a subfamily of redox proteins which is able to repress RNA‐polymerase‐III‐driven transcription of the Alu‐family retroposons in vitro
Authors: Kropotov Andrey , Sedova Vanda , Ivanov Vadim , Sazeeva Natalya , Tomilin Alexey , Krutilina Raisa , Oei Shiao Li , Griesenbeck Joachim , Buchlow Gerhard , Tomilin Nikolai
Published: March 2003

In this study we identified a novel protein which may contribute to the transcriptional inactivity of Alu retroposons in vivo. A human cDNA clone encoding this protein (ACR1) was isolated from a human expression library using South‐western screening with an Alu subfragment, implicated in the regulation of Alu in vitro transcription and interacting with a HeLa nuclear protein down‐regulated in adenovirus‐infected cells. Bacterially expressed ACR1 is demonstrated to inhibit RNA polymerase III (Pol III)‐dependent Alu transcription in vitro but showed no repression of transcription of a tRNA gene or of a reporter gene under control of a Pol II promoter. ACR1 mRNA is also found to be down‐regulated in adenovirus‐infected HeLa cells, consistent with a possible repressor function of the protein in vivo. ACR1 is mainly (but not exclusively) located in cytoplasm and appears to be a member of a weakly characterized redox protein family having a central, highly conserved sequence motif, PGAFTPXCXXXXLP. One member of the family identified earlier as peroxisomal membrane protein (PMP)20 is known to interact in a sequence‐specific manner with a yeast homolog of mammalian cyclosporin‐A‐binding protein cyclophilin, and mammalian cyclophilin A (an abundant ubiquitously expressed protein) is known to interact with human transcriptional repressor YY1, which is a major sequence‐specific Alu‐binding protein in human cells. It appears, therefore, that transcriptional silencing of Alu in vivo is a result of complex interactions of many proteins which bind to its Pol III promoter.
Drosophila melanogaster transferrin
Authors: Yoshiga Toyoshi , Georgieva Teodora , Dunkov Boris C. , Harizanova Nedjalka , Ralchev Kiril , Law John H.
Published: March 2003

Drosophila melanogaster transferrin cDNA was cloned from an ovarian cDNA library by using a PCR fragment amplified by two primers designed from other dipteran transferrin sequences. The clone (2035 bp) encodes a protein of 641 amino acids containing a signal peptide of 29 amino acids. Like other insect transferrins, Drosophila transferrin appears to have a functional iron‐binding site only in the N‐terminal lobe. The C‐terminal lobe lacks iron‐binding residues found in other transferrins, and has large deletions which make it much smaller than functional C‐terminal lobes in other transferrins. In‐situ hybridization using a digoxigenin labeled transferrin cDNA probe revealed that the gene is located at position 17B1‐2 on the X chromosome. Northern blot analysis showed that transferrin mRNA was present in the larval, pupal and adult stages, but was not detectable in the embryo. Iron supplementation of the diet resulted in lower levels of transferrin mRNA. When adult flies were inoculated with bacteria (Escherichia coli), transferrin mRNA synthesis was markedly increased relative to controls.
Solution structure of a conformationally constrained Arg‐Gly‐Asp‐like motif inserted into the α/β scaffold of leiurotoxin I
Authors: Kellenberger Esther , Mer Georges , Kellenberger Christine , Marguerie Gérard , Lefèvre Jean‐François
Published: March 2003

A monoclonal antibody, AC7, directed against the RGD‐binding site of the GPIIIa subunit of the platelet fibrinogen receptor, interacts with activated platelet. The H3 region (H3, RQMIRGYFDV sequence) of the complementarity‐determining region 3 heavy chain of AC7 inhibits platelet aggregation and fibrinogen binding to platelet. H3 contains the arginine, glycine and aspartate residues, but in an unusual order. The solution structure of the decapeptide has been studied by proton NMR. The NMR data suggested a helical equilibrium. To test whether the helical structure of H3 was biologically relevant, a conformationally constrained peptide with the RGD‐like motif was designed. The sequence of a scorpion toxin (leiurotoxin I) has been modified in order to constrain the H3 sequence in a rigid helical conformation. The structure of leiurotoxin I consists of a β‐sheet and an α‐helix, linked by three disulfide bridges. The structural feature of the chimeric peptide (H3‐leiurotoxin) has been determined by standard two‐dimensional NMR techniques. H3‐Leiurotoxin structure closely resembles that of leiurotoxin I.
Half‐of‐the‐sites reactivity of outer‐membrane phospholipase A against an active‐site‐directed inhibitor
Authors: Ubarretxena‐Belandia Iban , Cox Ruud C. , Dijkman Ruud , Egmond Maarten R. , M. Verheij Hubertus , Dekker Niek
Published: March 2003

The reaction of a novel active‐site‐directed phospholipase A1 inhibitor with the outer‐membrane phospholipase A (OMPLA) was investigated. The inhibitor 1‐p‐nitrophenyl‐octylphosphonate‐2‐tridecylcarbamoyl‐3‐ethanesulfonyl‐amino‐3‐deoxy‐sn‐glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active‐site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active‐site serines, as judged from the amount of p‐nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half‐of‐the‐sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl‐N,N‐dimethyl‐1‐ammonio‐3‐propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half‐of‐the‐sites reactivity is discussed in view of the dimeric nature of this enzyme.
Binding and catalytic properties of the Cdc2 and Crp proteins of Dictyostelium
Authors: Sharma Shiv K. , Michaelis Christine , Lee Ki‐Young , Wang Jerry H. , Weeks Gerald
Published: March 2003

Dictyostelium expresses at least two proteins of the cyclin‐dependent kinase (Cdk) family, Cdc2 and Crp. Cdc2 levels remain relatively constant during differentiation, whereas the levels of Crp increase dramatically as differentiation progresses. Crp is highly related to the mammalian Cdk5, and p25 (a truncated form of p35, the activating subunit of Cdk5 from mammalian brain) stimulates the histone H1 kinase activity of GST‐Crp by several fold. In contrast, p25 does not stimulate the histone H1 kinase activity of GST‐Cdc2 or the Cdc2 activity present in cell extracts from vegetative Dictyostelium cells. GST‐Cdc2, in vitro translated Cdc2 and Cdc2 from all stages of differentiation bind to p13suc1. In contrast, GST‐Crp, in vitro translated Crp and the Crp protein present in cell extracts do not bind to p13suc1. We have confirmed a previous report by Arakane and Maeda [J. Plant Res. (1997) 110, 81–85] that there is a peak of p13suc1 bound histone H1 kinase activity during late development, but we found that there was no corresponding peak of p13suc1 bound Cdc2 protein that corresponds to this activity. Taken together, these data suggest that neither Cdc2 nor Crp is responsible for the late developmental peak of histone H1 kinase activity that binds to p13suc1.
31P Nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase
Authors: Evrard Adeline , Zeghouf Mahel , Fontecave Marc , Roby Claude , Covès Jacques
Published: March 2003

SiR‐FP60, the monomeric form of the Escherichia coli sulfite reductase flavoprotein component (SiR‐FP), has been analysed by 31P‐NMR spectroscopy. This protein was reported previously as a reliable simplified model for native SiR‐FP [Zeghouf, M., Fontecave, M., Macherel, D., & Covès, J. (1998) Biochemistry37, 6117–6123]. SiR‐FP60 was examined in its native form, as a complex with NADP+ and after monoelectronic reduction either with NADPH or dithionite. In these latter cases, the stabilized FMN semiquinone radical offers a natural and internal paramagnetic probe. The paramagnetic effect of added manganese was also studied. In each case, the NMR parameters were extracted from digitalized data by a deconvolution procedure and compared with those obtained previously with cytochrome P450 reductase. Evolution of the NMR parameters and of calculated relaxation rate constants upon biochemical modifications of SiR‐FP60 led us to propose that the reactive center is more compact than the one of cytochrome P450 reductase, with the redox components, FMN, FAD and NADPH, in a tighter spatial arrangement, close to the protein surface. This underlies some subtle differences between the two proteins for which a very similar overall structure is likely considering their common genetic origin and common operating cycle.
Ectodomain cleavage and shedding of the type III transforming growth factor‐β receptor in lung membranes
Authors: Philip Anie , Hannah Rita , O’Connor‐McCourt Maureen
Published: March 2003

Previous studies from our laboratory[Philip, A. & O’Connor‐McCourt, M. D. (1991) J. Biol. Chem.266, 22290‐‐22296] have shown that the lung exhibited the highest uptake of circulating [125I]‐transforming growth factor‐β1 (TGF‐β1) on a per gram basis. This observation, together with the lack of information on TGF‐β receptor expression in the lung, prompted us to attempt to characterize TGF‐β receptors in this tissue. In the present report we show that the type III TGF‐β receptor is the most abundant TGF‐β binding protein in rat lung membranes and that it exhibits a 10‐fold higher affinity for TGF‐β2 than for TGF‐β1. We observed that the majority of the type III receptor population in lung membranes is cleaved at a site in the central portion of the ectodomain, the resulting two fragments (95 kDa and 58 kDa) being held together by disulfide bonds. Furthermore, we demonstrate that a soluble form of the ectodomain of the type III receptor is shed from rat lung membranes in an efficient manner, with protease cleavage occurring at a site close to the transmembrane domain. This shedding is controllable by temperature, thus providing a system to study the mechanism of ectodomain release. Using this system, we show that the shedding is inhibited by prior ligand binding and by membrane solubilization. The identification of a membrane preparation which exhibits controllable and quantitative release of the type III receptor ectodomain provides a unique cell‐free system for further studies of the mechanism of shedding of the type III TGF‐β receptor ectodomain.
Structure‐function studies of new C‐20 epimer pairs of vitamin D3 analogs
Authors: Väisänen Sami , Ryhänen Sanna , Saarela Janne T. A. , Mäenpää Pekka H.
Published: March 2003

A growing number of calcitriol (1α,25‐dihydroxyvitamin D3) analogs have become available in recent years. Many of these analogs exhibit lower calcemic effects than calcitriol and inhibit cell proliferation and enhance cell differentation more efficiently than calcitriol. We have compared structure‐function relationships of a series of new C‐20 epimer (20‐epi) vitamin D3 analogs with their natural C‐20 counterparts. In human MG‐63 osteosarcoma cells, quantification of cellular osteocalcin mRNA levels by Northern blot analysis and osteocalcin biosynthesis by radioimmunoassay indicated that most studied analogs at a concentration of 10 nm induced osteocalcin gene expression more efficiently than the parent compound, calcitriol. Interestingly, when the biological responses were compared with the binding affinities of the analogs to in‐vitro translated human vitamin D receptor and with their ability to protect the receptor against partial proteolytic digestion, significant correlations were not observed. Further, molecular modelling of the compounds by energy minimization did not reveal marked differences in the three‐dimensional structures of the analogs. These results suggest that higher than normal ligand binding affinity or ‘natural’ conformation of the ligand‐receptor complex are not necessarily required for the ‘superagonist’ transactivation activity. The mechanism of action of the efficient analogs may involve stabilization and/or differential binding of transcriptional coactivators or transcription intermediary factors to the hVDR during transactivation.
An ethanol‐inducible MDR ethanol dehydrogenase/acetaldehyde reductase in Escherichia coli
Authors: Shafqat Jawed , Höög Jan‐Olov , Hjelmqvist Lars , Oppermann Udo C. T. , Ibáñez Carlos , Jörnvall Hans
Published: March 2003

An ethanol‐active medium‐chain dehydrogenase/reductase (MDR) alcohol dehydrogenase was isolated and characterized from Escherichia coli. It is distinct from the fermentative alcohol dehydrogenase and the class III MDR alcohol dehydrogenase, both already known in E. coli. Instead, it is reminiscent of the MDR liver enzyme forms found in vertebrates and has a Km for ethanol of 0.7 mm, similar to that of the class I enzyme in humans, however, it has a very high kcat, 4050 min−1. It is also inhibited by pyrazole (Ki = 0.2 µm) and 4‐methylpyrazole (Ki= 44 µm), but in a ratio that is the inverse of the inhibition of the human enzyme. The enzyme is even more efficient in the reverse direction of acetaldehyde reduction (Km = 30 µm and kcat = 9800 min−1), suggesting a physiological function like that seen for the fermentative non‐MDR alcohol dehydrogenase. Growth parameters in complex media with and without ethanol show no difference. The structure corresponds to one of 12 new alcohol dehydrogenase homologs present as ORFs in the E. coli genome. Together with the previously known E. coli MDR forms (class III alcohol dehydrogenase, threonine dehydrogenase, ζ‐crystallin, galactitol‐1‐phosphate dehydrogenase, sensor protein rspB) there is now known to be a minimum of 17 MDR enzymes coded for by the E. coli genome. The presence of this bacterial MDR ethanol dehydrogenase, with a structure compatible with an origin separate from that of yeast, plant and animal ethanol‐active MDR forms, supports the view of repeated duplicatory origins of alcohol dehydrogenases and of functional convergence to ethanol/acetaldehyde activity. Furthermore, this enzyme is ethanol inducible in at least one E. coli strain, K12 TG1, with apparently maximal induction at an enthanol concentration of ≈17 mm. Although present in several strains under different conditions, inducibility may constitute an explanation for the fairly late characterization of this E. coli gene product.
Structure and expression of the highly repetitive histone H1‐related sperm chromatin proteins from winter flounder
Authors: Watson Catherine E. , Gauthier Sherry Y. , Davies Peter L.
Published: March 2003

In the late stages of spermatogenesis, winter flounder produce a family of high molecular mass (80–200 kDa) basic nuclear proteins (HMrBNPs) that combine with the normal complement of histones to produce condensed sperm chromatin with an increased nucleosomal repeat length. The HMrBNPs have a biased amino‐acid composition in which Arg, Ser, Lys and Pro are abundant because of their presence in many simple peptide repeats. The organization of these repeats was deduced by cDNA cloning. The predominant repeating units are related 26‐ and 30‐amino‐acid sequences that in turn are linked by 6‐amino‐acid spacers to form 58‐ and 62‐amino‐acid repeats. Subsets of these repeats are also present, such as a dispersed 20‐amino‐acid repeat and a tandem array of nine heptapeptides at the C‐terminus. The HMrBNPs appear to have evolved from an extreme H1 variant that has an N‐terminal tail of HMrBNP‐like sequence linked to an H1 globular region. Based on sequences of the most abundant HMrBNP cDNAs, and the lack of hybridization between HMrBNP mRNAs and a DNA probe for the H1 globular region, the latter domain appears to have been lost during expansion and amplification of the HMrBNP‐like repeats. Transcripts of the HMrBNP and H1 variant genes are present in testis RNAs only during the mid‐spermatid stage of spermatogenesis, at the same time that HMrBNPs in their highly phosphorylated form first appear in the nucleus. Judging by the lack of a lag between HMrBNP mRNA synthesis and translation, the mRNAs for these highly basic proteins are not stored for any length of time. Instead, the deposition of HMrBNPs onto DNA, which coincides with the major reorganization and silencing of the chromatin, may be controlled by dephosphorylation.
Protein kinase C antagonizes pertussis‐toxin‐sensitive coupling of the calcitonin receptor to adenylyl cyclase
Authors: Shyu Jia‐Fwu , Zhang Zhiyuan , Hernandez‐Lagunas Laura , Camerino Claudia , Chen Yan , Inoue Daisuke , Baron Roland , Horne William C.
Published: March 2003

The calcitonin receptor is known to couple to Gs and Gq, activating adenylyl cyclase and phospholipase C, respectively. The observation of pertussis‐toxin‐sensitive responses to calcitonin suggests that the receptor is capable of coupling to Gi/o as well. However, the calcitonin‐dependent activation of adenylyl cyclase in HEK‐293 cells that stably express the cloned rabbit calcitonin receptor, as in many other cells that express calcitonin receptors, shows little pertussis toxin sensitivity. Calcitonin treatment of these cells stimulates protein kinase C, which is reported to antagonize the receptor‐dependent activation of Gi. The possibility that protein kinase C could be antagonizing Gαi–adenylyl cyclase coupling was tested by examining the effects of protein kinase C inhibitors (chelerythrine chloride and sphingosine) or of chronic treatment with phorbol ester to deplete protein kinase C. All three treatments led to a reduction of calcitonin‐induced adenylyl cyclase activity that was reversed by pertussis toxin. Inhibiting or depleting protein kinase C had no effect on the activation of adenylyl cyclase by cholera toxin, indicating that Gs and adenylyl cyclase were not affected by these treatments. Calcitonin treatment of HEK‐293 cells, that stably express a myc‐tagged rabbit calcitonin receptor, induced the formation of complexes of the receptor and Gαi subunits, confirming that the calcitonin receptor interacts with Gi. Thus, the calcitonin receptor can couple to Gi, but the inhibition of adenylyl cyclase by Gαi is negatively regulated by protein kinase C.
Activities of human alcohol dehydrogenases in the metabolic pathways of ethanol and serotonin
Authors: Svensson Stefan , Some Margareta , Lundsjö Anders , Helander Anders , Cronholm Tomas , Höög Jan‐Olov
Published: March 2003

Alcohols and aldehydes in the metabolic pathways of ethanol and serotonin are substrates for alcohol dehydrogenases (ADH) of class I and II. In addition to the reversible alcohol oxidation/aldehyde reduction, these enzymes catalyse aldehyde oxidation. Class‐I γγ ADH catalyses the dismutation of both acetaldehyde and 5‐hydroxyindole‐3‐acetaldehyde (5‐HIAL) into their corresponding alcohols and carboxylic acids. The turnover of acetaldehyde dismutation is high (kcat = 180 min−1) but saturation is reached first at high concentrations (Km = 30 mm) while dismutation of 5‐HIAL is saturated at lower concentrations and is thereby more efficient (Km = 150 µm; kcat = 40 min−1). In a system where NAD+ is regenerated, the oxidation of 5‐hydroxytryptophol to 5‐hydroxyindole‐3‐acetic acid proceeds with concentration levels of the intermediary 5‐HIAL expected for a two‐step oxidation. Butanal and 5‐HIAL oxidation is also observed for class‐I ADH in the presence of NADH. The class‐II enzyme is less efficient in aldehyde oxidation, and the ethanol‐oxidation activity of this enzyme is competitively inhibited by acetate (Ki = 12 mm) and 5‐hydroxyindole‐3‐acetic acid (Ki = 2 mm).

 Reduction of 5‐HIAL is efficiently catalysed by class‐I γγ ADH (kcat = 400 min−1; Km = 33 µm) in the presence of NADH. This indicates that the increased 5‐hydroxytryptophol/5‐hydroxyindole‐3‐acetic acid ratio observed after ethanol intake may be due to the increased NADH/NAD+ ratio on the class‐I ADH.
Multiple cloning sites from mammalian expression vectors interfere with gene promoter studies in vitro
Authors: Béliveau A. , Leclerc S. , Rouleau M. , Guérin S. L.
Published: March 2003

When performing transcriptional analyses, reporter gene‐expression vectors are used to insert promoter fragments through the selected use of a multiple cloning site (MCS) located upstream of the reporter gene. The MCS from pBluescript™ has frequently been transferred into reporter plasmids (usually bearing the chloramphenical acetyltransferase reporter gene) and used to subclone various promoter fragments from diverse genes. Analyses in electrophoretic mobility shift assay using this MCS as labeled probe revealed that it specifically binds multiple nuclear proteins from a whole array of widely used cell types. Moreover, the presence of the MCS sequence dramatically altered promoter activity in a totally unpredictable fashion that depends on the distance between the MCS and the basal promoter start site of the gene, leading to severe misinterpretation of the transfection data. Finally, we provide evidence that the BamHI/SmaI/PstI restriction site combination is likely one of the major binding site for nuclear proteins on the pBluescript™ MCS, therefore suggesting that this particular combination of restriction sites should be avoided in the MCS from plasmids that are to be used in promoter studies.
Identification and characterization of a tri‐partite hydrophobin from Claviceps fusiformis
Authors: de Vries Onno M. H. , Moore Sabine , Arntz Claudia , Wessels Joseph G. H. , Tudzynski Paul
Published: March 2003

A new type of hydrophobin is encoded by an abundant mRNA of Claviceps fusiformis. The predicted amino‐acid sequence of the protein, dubbed CFTH1, shows a putative signal sequence for secretion, followed by three class II hydrophobin domains each preceded by glycine/asparagine rich regions. SDS/PAGE analysis of 60% ethanol extractions of C. fusiformis mycelia from shaken cultures showed CFTH1 at the 50–55‐kDa position. N‐terminal sequencing of both untreated mature CFTH1 and of a fragment obtained by trypsin digestion revealed that CFTH1 is not processed between the hydrophobin domains. Mass spectroscopy showed a mass of about 36 500 Da, which is about 1500 Da higher than the mass predicted from the constituent amino acids, indicating post‐translational modification but not glycosylation. Purified CFTH1 self‐assembled at hydrophilic/hydrophobic interfaces and, after assembly at a water/air interface, it was found to be highly surface active. Antibodies raised against CFTH1 localized the protein in a mucilageous coat surrounding submerged vegetative hyphae in liquid shaken culture and, as a discrete layer of about 10 nm thickness at the surface of aerial hyphae of standing cultures, suggesting a role in the formation of aerial hyphae.
Structures and co‐regulated expression of the genes encoding mouse cytosolic chaperonin CCT subunits
Authors: Kubota Hiroshi , Yokota Shin‐ichi , Yanagi Hideki , Yura Takashi
Published: March 2003

The chaperonin‐containing TCP‐1 (CCT) is a hetero‐oligomeric molecular chaperone that mediates protein folding in the cytosol of eukaryotes. Eight (or nine in testis) subunit species are assembled in the CCT hexadecamer complex. We have cloned seven CCT subunit genes, Cctb, Cctd, Ccte, Cctz‐1, Cctz‐2 (testis specific), Ccth and Cctq, from mouse genomic DNA libraries, in addition to the Ccta and Cctg genes reported previously, and the entire nucleotide sequences of these DNA clones were determined. These genes are ≈15–20 kb in length except for Cctz‐2 which is longer than 35 kb, and all the Cct genes consist of 11–16 exons. Primer extension analyses of testis RNA indicate one to several potential transcription start sites 50–150 bp upstream from the translation start codon of each Cct gene. There are several possible Sp1‐binding sequences, but no obvious TATA box was observed around the potential start sites. From 5′‐flanking regions to the first introns, the Cct genes are rich in CpG dinucleotides. In reporter gene assays using these regions, five of eight Cct genes showed strong transcriptional activity comparable with the combination of SV40 promoter and enhancer in HeLa cells. We also show, by Western and Northern blot analyses, that CCT expression levels vary widely among different tissues but the expression patterns are very similar among the eight subunit species. It is likely that expression levels of the eight different subunits are tightly co‐regulated to maintain a constant ratio of these subunits which constitute the CCT hexadecamer complex with a fixed subunit arrangement.