bluuebunny/crossref_metadata_2025 huggingface dataset:
Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T‐protein
Authors: Chen Shuqing , Vincent Sarah , Wilson David B. , Ganem Bruce
Published: March 2003

The Escherichia coli bifunctional T‐protein transforms chorismic acid to p‐hydroxyphenylpyruvic acid in the l‐tyrosine biosynthetic pathway. The 373 amino acid T‐protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback‐inhibited by tyrosine. Fifteen genes coding for the T‐protein and various fragments thereof were constructed and successfully expressed in order to characterize the CM, PDH and regulatory domains. Residues 1–88 constituted a functional CM domain, which was also dimeric. Both the PDH and the feedback‐inhibition activities were localized in residues 94–373, but could not be separated into discrete domains. The activities of cloned CM and PDH domains were comparatively low, suggesting some cooperative interactions in the native state. Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain.
Nucleotide excision repair rates in rat tissues
Authors: Gospodinov Anastas , Ivanov Rumen , Anachkova Boyka , Russev George
Published: March 2003

We have determined and compared nucleotide excision repair capability of several rat tissues by a method based on restoration of the transformation activity of UV‐irradiated pBlueScript by incubation in repair‐competent protein extracts. After 3 h of incubation, plasmid DNA was isolated and used to transform competent Escherichia coli cells. Damaged plasmids showed low transformation efficiency prior to incubation in repair‐competent extracts. After incubation the transformation efficiency was restored to different extents permitting calculation of the repair capacity of the extracts. Our results showed that rapidly proliferating tissues such as liver, kidney and testis showed higher nucleotide excision repair capacity than slowly proliferating tissues such as heart, muscle, lung and spleen. When liver and splenocytes were stimulated to proliferation by partial hepatectomy and mitogen stimulation, their repair capability increased in parallel with the respective proliferative rates.
Characterization of two distinct P2Y receptors in human tracheal gland cells
Authors: Merten Marc D. , Saleh Ali , Kammouni Wafa , Marchand Stéphanie , Figarella Catherine
Published: March 2003

Human submucosal tracheal glands are now believed to play a major role in the physiopathology of cystic fibrosis, a genetic disease in which ATP is used as a therapeutic agent. However, actions of ATP on tracheal gland cells are poorly known. ATP‐binding characteristics, and ATP‐induced formation of cAMP were investigated in a cell line (MM39) of human tracheal gland cells. The binding of a radiolabelled non‐hydrolysable analogue of ATP Adenosine‐5′‐[35S]thiotriphosphate : [35S]ATP[γS] was rapid (within 30 min at 4 +C), stable and reversible. Scatchard analysis revealed two classes of [35S]ATP[γS]‐binding sites. Low‐affinity binding sites had a Kd1 of 20 ± 5 μM (Bmax = 150 nmol/106 cells) and the high‐affinity binding sites had a Kd2 of 2.5 ± 0.2 μM (Bmax = 52 nmol/106 cells). Competition experiments showed competition with ATP, ADP and 2‐methylthio‐ATP but no competition with UTP, AMP and adenosine. UTP stimulates protein secretion as well as it induced [Ca2+]i mobilization but did not affect the intracellular cAMP levels. ATP also caused induced [Ca2+]i mobilization and protein secretion but also caused an increase in cyclicAMP content of the cells, reaching a maximum after 1 min. ATP‐induced cAMP formation was concentration dependent and inhibited by the P2‐antagonist suramin. Reverse‐transcription‐PCR amplification revealed the presence of the transcripts of both the P2Y2 and the UTP‐specific P2Y4 receptors. In conclusion, P2Y2 receptors, UTP‐P2Y4 receptors and unidentified ATP‐specific receptors seem to be present in MM39 cells which appear to be coupled differently to intracellular second‐messenger systems.
Structure and tissue‐specific expression of genes encoding bovine copper amine oxidases
Authors: Høgdall Estrid V. S. , Houen Gunnar , Borre Martin , Bundgaard Jens R. , Larsson Lars‐Inge , Vuust Jens
Published: March 2003

A cDNA coding for an enzyme belonging to the family of copper amine oxidases was cloned from a bovine lung cDNA library using a PCR approach. The nucleotide sequence of this cDNA was found to be different from that of the previously published liver cDNA encoding bovine serum amine oxidase, another copper amine oxidase. Analyses using reverse transcription followed by PCR of RNA extracted from different bovine tissues confirmed that the copper amine oxidase gene expressed in bovine liver is closely related to, but different from, the copper amine oxidase gene expressed in bovine lung, kidney, spleen and heart. Northern blotting data showed that the level of copper amine oxidase expression in liver is considerably higher than in the other tissues tested. Southern blotting analyses of bovine chromosomal DNA suggested the existence of at least three copper amine oxidase genes. Two of these genes are apparently expressed in a tissue‐specific manner as outlined above. A fragment of a third copper amine oxidase gene is identified. The exon‐intron organization of the bovine copper amine oxidase genes analyzed is similar to that of the related human diamine oxidase gene, except that no intron in the position equivalent to that of the third intron in the human gene is found. In the third gene, a complete replacement of the third intron of the bovine copper amine oxidase gene (equivalent to the fourth intron of the human gene) has occurred.
Permeability properties of the porin of spinach leaf peroxisomes
Authors: Reumann Sigrun , Maier Elke , Heldt Hans W. , Benz Roland
Published: March 2003

The membrane of spinach leaf peroxisomes contains an anion‐selective channel. Reconstitution experiments were performed with lipid bilayer membranes to study its permeability properties. A variety of different monovalent inorganic and organic anions were found to be permeable through the porin channel. Its single‐channel conductance for these different ions suggested that the channel has a minimum diameter of about 0.6 nm. From selectivity measurement in KCl solution a ratio of the anion permeability to cation permeability of less than 0.04 was determined, indicating an almost ideal selectivity of the peroxisomal channel for chloride. The permeation of chloride through the peroxisomal channel could be blocked efficiently by the addition of increasing concentrations of organic anions to the aqueous phase. The results are consistent with a binding site for dicarboxylic anions inside the peroxisomal channel. A particular high stability constant for the binding was obtained for peroxisomal metabolites such as malate, oxaloacetate, succinate, and 2‐oxoglutarate, which have to cross the membrane of plant peroxisomes in vivo. Among these solutes maximal binding affinity was determined for C4 dicarboxylic anions. The results indicate that the peroxisomal channel does not form a general diffusion pore similarly to known eukaryotic porins, but has specific properties comparable to specific and inducible porins, which have been characterized in some gram‐negative bacteria.
A genetically engineered increase in fatty acid unsaturation in Synechococcus sp. PCC 7942 allows exchange of D1 protein forms and sustenance of photosystem II activity at low temperature
Authors: Sippola Katja , Kanervo Eira , Murata Norio , Aro Eva‐Mari
Published: March 2003

Photosystem II (PSII)‐reaction‐center protein D1 is encoded by three psbA genes in Synechococcus sp. PCC 7942. The psbAI gene encodes D1 form I (D1 : 1) and the psbAII and psbAIII genes encode the transiently expressed D1 form II (D1 : 2). We have studied the role of membrane‐lipid unsaturation in the expression of psbA genes at low temperature, using a Synechococcus transformant with an increased unsaturation level of membrane lipids. Transfer of the cells from 32 +C to 25 +C under growth light resulted in the exchange of D1 : 1, the prevailing form, for D1 : 2 in the wild‐type bacterium and the transformant, with no loss of PSII activity. Lowering the temperature further to 18 +C caused a drastic decrease in PSII activity in the wild‐type bacterium, whereas the transformant was much less affected. Similar decreases in psbAI transcripts and loss of D1 : 1 occurred in both strains at 18 +C, with concomitant accumulation of psbAII/III transcripts, the latter event being especially prominent in the wild‐type bacterium. However, the wild‐type bacterium was incapable of accumulating D1 : 2 to compensate for the loss of D1 : 1, which resulted in disassembly of PSII at low temperature. These results imply translational rather than transcriptional regulation of psbA gene expression in Synechococcus 7942 at low temperature, and demonstrate the crucial role of the degree of membrane‐lipid unsaturation in promotion of exchange of the D1 protein forms and thus the sustenance of PSII function at low temperature.
Identification and characterization of the natural electron donor ferredoxin and of FAD as a possible prosthetic group of benzoyl‐CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism
Authors: Boll Matthias , Fuchs Georg
Published: March 2003

Under anoxic conditions most aromatic compounds are metabolized via benzoyl‐CoA which becomes reduced by benzoyl‐CoA reductase (dearomatizing); this enzyme was recently described in the bacterium Thauera aromatica [Boll, M. & Fuchs, G. (1995) Eur. J. Biochem. 234, 921−933]. It catalyzes the reaction benzoyl‐CoA + 2 e− + 2 H+ + 2 MgATP + 2 H2O ← cyclohexa‐1,5‐diene‐1‐carboxyl‐CoA + 2 MgADP + 2 Pi. The iron‐sulfur protein has a native molecular mass of 160−170 kDa and consists of four different subunits. In addition a flavin may be present.

The nature of the potential prosthetic group and the natural electron donor were determined. Purified benzoyl‐CoA reductase preparations contained 0.25−0.3 mol FAD/mol enzyme. Cells grown anaerobically with aromatic substrates contained a ferredoxin which represented the main, if not the only ferredoxin present. It was purified from 200 g cells with a yield of 60 mg and its N‐terminal amino acid sequence was determined. The native molecular mass was 9659 ± 2 Da as determined by electrospray mass spectrometry. The protein contained 7.6 ± 0.6 mol iron and 7.6 ± 1 mol acid‐labile sulfur/mol. The ultraviolet−visible spectrum of the protein was typical for ferredoxins with maxima at 280 nm and 390 nm (in the oxidized state). The estimated molar absorption coefficients were 63 500 M−1 cm−1 at 280 nm and 40 500 M−1 cm−1 at 390 nm. The difference spectrum between the oxidized and the reduced form had a maximum at 415 nm with Δε415 = 8200 M−1 cm−1. 1 mol ferredoxin became reduced/mol dithionite added, suggesting the presence of two [4Fe‐4S] clusters. The average midpoint potential of the iron‐sulfur clusters was −450 mV.

The ferredoxin gene was cloned and sequenced. It was located in a gene cluster coding for enzymes involved in anaerobic aromatic metabolism. The amino acid sequence of the T. aromatica ferredoxin showed high similarities to several other ferredoxins containing 2[4Fe‐4S] clusters, e.g. from Clostridia and phototrophic bacteria. The reduced ferredoxin served as electron donor for benzoyl‐CoA reduction at a three times higher rate compared with the rate obtained with the artificial electron donor reduced methyl viologen. The turnover number with the natural electron donor of 5 s−1 can explain the bacterial growth rate with benzoate as substrate. Half‐maximal enzyme acitivity was obtained with 6 μM reduced ferredoxin, at an estimated cellular concentration of 70 μM ferredoxin. Both the low apparent Km value and the turnover number are consistent with the proposed role of ferredoxin in aromatic‐ring reduction.
Comparison of the kinetics of active efflux of 99mTc‐MIBI in cells with P‐glycoprotein‐mediated and multidrug‐resistance protein‐associated multidrug‐resistance phenotypes
Authors: Vergote Jackie , Moretti Jean‐Luc , De Vries Elisabeth G. E. , Garnier‐Suillerot Arlette
Published: March 2003

The overexpression of two membrane glycoproteins, P‐glycoprotein and multidrug‐resistance protein (MRP1) is a major cause of resistance to chemotherapeutic agents in the treatment of human cancers. Both proteins confer a similar multidrug‐resistant (MDR) phenotype.

99mTc‐MIBI, a myocardial imaging agent, which is also useful for the detection of a variety of tumours, has been shown to be a substrate for P‐glycoprotein and MRP1. It thus may provide additional information about the P‐glycoprotein and MRP1 status of tumour cells.

In order to obtain information on the substrate specificity of these proteins, we have studied the transport kinetics of Tc‐MIBI in two cell lines, K562/ADR and GLC4/ADR, which overexpress P‐glycoprotein and MRP1, respectively. The mean active efflux coefficient ka, which is proportional to the ratio of maximal efflux rate VM to the apparent Michaelis‐Menten constant Km, used to characterise the efficiency of the active efflux, was very similar being 1.9 ± 0.6×10−11 s−1 cells ml and 1.3 ± 0.5×10−11 s−1 cells ml for drug‐resistant K562 and GLC4, respectively. These values are 50−100‐times lower than for daunorubicin and other anthracycline derivatives, strongly suggesting that the efficiency of both transporters to pump Tc‐MIBI is by far less than that to efflux anthracyclines.

Our data show that (a) P‐glycoprotein and MRP transporter efficiencies to wash out Tc‐MIBI are similar, in spite of a different suspected mechanism of its transport and (b) that both transporters are less efficient to pump Tc‐MIBI than to pump anthracyclines (the ka parameter is about 100‐times lower for TC‐MIBI than for anthracycline).
Characterization of a gene that is inversely correlated with estrogen receptor expression (ICERE‐1) in breast carcinomas
Authors: Thompson Devon A. , Weigel Ronald J.
Published: March 2003

Differential display was used to compare patterns of gene expression in two estrogen receptor (ER)‐positive breast carcinoma cell lines (MCF7 and T‐47D) and two ER‐negative breast carcinoma cell lines (MDA‐MB‐231 and HBL‐100). A 377‐bp fragment was identified that was overexpressed in the ER‐negative cell lines. Sequence analysis of this clone and comparison with the GenBank/EMBL databases indicated that it did not match any genes published previously. The expression pattern of this gene was inversely correlated with the expression of ER and has been termed ICERE‐1 (inversely correlated with estrogen receptor expression). A longer clone of ICERE‐1 was isolated from a MDA‐MB‐231 cDNA library and sequence analysis indicated that this 2168‐bp cDNA contained an ORF encoding a protein of 234 amino acids that bears little similarity with any previously described protein sequence. Northern blot analysis of a panel of breast cancer cell lines demonstrated that an ICERE‐1 mRNA of approximately 2.2 kb was abundantly expressed in the ER‐negative breast carcinoma cell lines, MDA‐MB‐231 and HBL‐100, and the ER‐negative cell lines, HEC‐1‐B, HeLa, and 293. Expression of ICERE‐1 was absent or minimal in the ER‐positive breast carcinoma cell lines MCF7, T‐47D, MDA‐MB‐361, ZR‐75−1, BT‐474 and BT‐20. Reverse transcription/PCR was used to examine ICERE‐1 expression in 29 primary breast carcinomas, 15 of which had been designated as ER positive and 14 as ER negative by immunohistochemistry. The expression level of ICERE‐1 was significantly lower (P < 0.001) in the ER‐positive tumors compared with the ER‐negative tumors. The pattern of expression of ICERE‐1 indicates that this gene may be involved in tumor biology specific to hormonally unresponsive breast cancers.
Transient up‐regulation of a prolyl endopeptidase activity in the microsomal fraction of rat liver during postnatal development
Authors: Matsubara Youco , Ono Tomio , Tsubuki Satoshi , Irie Shinkichi , Kawashima Seiichi
Published: March 2003

To identify proteases which are involved in cell proliferation and differentiation of liver cells, we assayed various protease activities in rat liver during its postnatal development. We found that a protease activity specific to proline residues in the microsomal fraction of rat liver was transiently increased around postnatal day 8 and decreased thereafter, indicating that the enzyme activity is highly correlated to the proliferation and differentiation of liver cells. This protease was purified to an apparent homogeneity from the microsomal fraction of the postnatal‐day‐8 rat liver. The purified enzyme gave a single band with an apparent molecular mass of 65 000 on SDS/PAGE. It cleaved several peptide 4‐methylcoumaryl‐7‐amide substrates and bioactive peptides at the C‐terminus of proline residues. The enzyme did not hydrolyze any protein substrate examined suggesting that it is a peptidase rather than a proteinase. Although the best substrate among those tested was succinyl‐Gly‐Pro‐Leu‐Gly‐Pro‐4‐methylcoumaryl‐7‐amide (‐NH‐Mec), the purified enzyme did not hydrolyze succinyl‐Gly‐Pro‐NH‐Mec, indicating that the enzyme has different substrate specificity from any hitherto known prolyl endopeptidases. These results suggest that the purified enzyme is a unique prolyl endopeptidase which may be involved in proliferation and differentiation of liver cells.
Atomic mutations in annexin V
Authors: Budisa Nediljko , Huber Robert , Golbik Ralph , Minks Caroline , Weyher Elisabeth , Moroder Luis
Published: March 2003

We have recently developed methods for specific and high‐level replacement of methionine with 2‐aminohexanoic acid, selenomethionine and telluromethionine as isosteric and atomic analogues for structural investigations of human recombinant annexin V. The variants formed isomorphic crystals and retained the parent three‐dimensional fold and bioactivities. Folding parameters were determined from thermal and chemical unfolding to partially denatured states. Stabilities estimated from guanidinium chloride unfolding equilibria are not changed significantly for the atomic mutants (S←Se←Te) while the denaturation midpoint is shifted toward lower values with an increase of the m values at the increase of hydrophobicity. In contrast, stabilities in urea are considerably affected by the atomic substitutions, decreasing together with the m and [D]1/2 values. The methylene and selenium variants are identical within the limits of error of all measurements performed here. The physical parameters of the amino acid analogues and the values derived from the slopes of the unfolding data are highly correlated. This approach demonstrates how systematic variations in atomic number at the site of replacement (atomic mutations) can provide a method to probe specific folding properties of proteins.
Bdellastasin, a serine protease inhibitor of the antistasin family from the medical leech (Hirudo medicinalis)
Authors: Moser Matthias , Auerswald Ennes , Mentele Reinhard , Eckerskorn Christoph , Fritz Hans , Fink Edwin
Published: March 2003

We have reported earlier the isolation and amino acid composition of bdellin A from medical leech, and characterised it as an inhibitor of trypsin, plasmin and acrosin [Fritz, H., Gebhardt, M., Meister, R. & Fink, E. (1971) in Proceedings of the international research conference on proteinase inhibitors (Fritz, H. & Tschesche, H., eds) pp. 271−280, Walter de Gruyter, Berlin]. In the present study, one of several chromatographic forms of this inhibitor was isolated from a semi‐pure preparation. Elucidation of its amino acid sequence revealed that bdellin A is a member of the antistasin family. Therefore, it was renamed bdellastasin to avoid confusion with bdellin B, which is another trypsin‐plasmin inhibitor from the medical leech, but of the Kazal type. Furthermore, a synthetic gene of bdellastasin was constructed, and the protein expressed in Saccharomyces cerevisiae with yields of 29 mg/l. The recombinant bdellastasin was purified by hydrophobic interaction and anion‐exchange chromatography. Comparison by mass spectroscopy, far‐ultraviolet circular dichroism studies, sequence determination, and inhibition characteristics demonstrated the identity of recombinant and native bdellastasin. The Ki values of bdellastasin for inhibition of bovine trypsin and human plasmin are in the nanomolar range; no inhibition was detected for factor Xa, thrombin, tissue kallikrein, plasma kallikrein and chymotrypsin. Circular dichroism analyses indicated that bdellastasin is devoid of secondary‐structural elements.
Molecular cloning and characterization of mouse caspase‐8
Authors: Sakamaki Kazuhiro , Tsukumo Shin‐ichi , Yonehara Shin
Published: March 2003

Fas (APO‐1/CD95) is a transmembrane receptor protein which induces apoptosis upon activation. In apoptosis triggered by Fas, a subset of cysteine proteases designated caspases is activated, playing a central role as effector molecules. Among these caspases, human caspase‐8 (FLICE/MACH/Mch5) has been isolated and shown to be indispensable for Fas‐mediated apoptotic signaling. In this study, we isolated the mouse homologue to human caspase‐8 from a BaF3 cell cDNA library. This molecule conserved the death effector domain (DED) and protease domain as detected in human caspase‐8, and was capable of inducing apoptosis in KB and Rat‐1 cells when overexpressed. Expression of caspase‐8 was detected in the various tissues of adult mouse and in embryos at 9.5 days and 17.5 days of development by Northern‐blot analysis. Further, we isolated a chromosomal gene for caspase‐8 from a mouse genomic library and analyzed the genomic structure of the isolated gene. This gene consisted of eight exons and seven introns spanning about 26 kb in the coding region.
Bactericidal activity of Lys49 and Asp49 myotoxic phospholipases A2 from Bothrops asper snake venom
Authors: Páramo Leandro , Lomonte Bruno , Pizarro‐Cerdá Javier , Bengoechea José‐Antonio , Gorvel Jean‐Pierre , Moreno Edgardo
Published: March 2003

Mammalian group‐II phospholipases A2 (PLA2) of inflammatory fluids display bactericidal properties, which are dependent on their enzymatic activity. This study shows that myotoxins II (Lys49) and III (Asp49), two group‐II PLA2 isoforms from the venom of Bothrops asper, are lethal to a broad spectrum of bacteria. Since the catalytically inactive Lys49 myotoxin II isoform has similar bactericidal effects to its catalytically active Asp49 counterpart, a bactericidal mechanism that is independent of an intrinsic PLA2 activity is demonstrated. Moreover, a synthetic 13‐residue peptide of myotoxin II, comprising residues 115−129 (common numbering system) near the C‐terminal loop, reproduced the bactericidal effect of the intact protein. Following exposure to the peptide or the protein, accelerated uptake of the hydrophobic probe N‐phenyl‐N‐naphthylamine was observed in susceptible but not in resistant bacteria, indicating that the lethal effect was initiated on the bacterial membrane. The outer membrane, isolated lipopolysaccharide (LPS), and lipid A of susceptible bacteria showed higher binding to the myotoxin II‐(115−129)‐peptide than the corresponding moieties of resistant strains. Bacterial LPS chimeras indicated that LPS is a relevant target for myotoxin II‐(115−129)‐peptide. When heterologous LPS of the resistant strain was present in the context of susceptible bacteria, the chimera became resistant, and vice versa. Myotoxin II represents a group‐II PLA2 with a direct bactericidal effect that is independent of an intrinsic enzymatic activity, but adscribed to the presence of a short cluster of basic/hydrophobic amino acids near its C‐terminal loop.
Rearrangement reactions in the biosynthesis of molybdopterin
Authors: Rieder Christoph , Eisenreich Wolfgang , O'Brien John , Richter Gerald , Götze Eva , Boyle Peter , Blanchard Sylvain , Bacher Adelbert , Simon Helmut
Published: March 2003

The genes moaABC of Escherichia coli were ligated into the expression vector pNCO113. The resulting plasmid was transformed into a moeA mutant of E. coli. From cultures of the recombinant strain, a pteridine designated compound Z could be isolated at 5 mg/liter. Compound Z is a product of precursor Z, a biosynthetic precursor of molybdopterin. Cultures of the recombinant E. coli strain were supplied with [U‐13C6]glucose, [U‐13C5]ribulose 5‐phosphate, or [7‐15N,8‐13C]guanine. The culture medium also contained a large excess of unlabeled glucose. Compound Z as well as nucleosides obtained by hydrolysis of RNA were isolated from the bacterial cultures, and their heavy isotope distribution was investigated by one‐dimensional and two‐dimensional NMR spectroscopy. The labelling patterns of compound Z show that the carbon atoms of a pentose or pentulose are diverted to the ring atoms C6 and C7 and to the side chain atoms C2′, C3′ and C4′ of compound Z. Carbon atom C1′ of compound Z is derived from carbon atom C8 of a guanine derivative. The remodeling of the carbon skeleton of the pentose and purine moieties proceed via intramolecular rearrangement reactions.
Mapping of the binding of platelet‐derived growth factor to distinct domains of the basement membrane proteins BM‐40 and perlecan and distinction from the BM‐40 collagen‐binding epitope
Authors: Göhring Walter , Sasaki Takako , Heldin Carl‐Henrik , Timpl Rupert
Published: March 2003

A surface plasmon resonance assay was used to analyze the binding of platelet‐derived growth factor (PDGF)‐AA and PDGF‐BB to various proteins of the extracellular matrix. This identified several collagen types; laminin‐1, nidogen, perlecan and BM‐40 as potential ligands for PDGF with Kd values in the range 2−3200 nM. Perlecan and BM‐40 were used to examine the domain specificity and other parameters of the interactions. Recombinant human and mouse BM‐40 were shown to bind both PDGFs in a similar manner with a Kd of about 5−10 nM. Studies with deletion mutants of human BM‐40 demonstrated binding to its C‐terminal extracellular calcium‐binding (EC) module, yet the interaction did not require calcium. This distinguishes this from the binding of the EC module to various collagen types, which is strictly calcium dependent. Furthermore, deletion of helix αC or two point mutations in helix αA of the EC module either enhanced or abolished binding to collagen IV. Since these mutations had no effects on binding to PDGF, it demonstrated the presence of two different binding epitopes. Binding of PDGF‐BB to the perlecan core protein could be mapped to its domain III‐2 (Kd = 8 nM) with lower affinities shown for domains I, IV‐1 and V (Kd = 34−64 nM). Other perlecan domains (II, III‐1, III‐3, IV‐2) were inactive. PDGF‐AA was also shown to bind domain III‐2 but not III‐1. Neither nidogen, BM‐40 or perlecan domain III‐2 interfered with the binding of PDGF to its α and β receptors, however, suggesting that these interactions may be mainly used for storage of PDGF in the extracellular matrix.
Identification and proteolytic processing of procardosin A
Authors: Ramalho‐Santos Miguel , Veríssimo Paula , Cortes Luísa , Samyn Bart , Van Beeumen Jozef , Pires Euclides , Faro Carlos
Published: March 2003

Plant aspartic proteinases contain a plant‐specific insert (PSI) of about 100 amino acids of unknown function with no similarity with the other aspartic proteinases but with significant similarity with saposins, animal sphingolipid activator proteins. PSI has remained elusive at the protein level, suggesting that it may be removed during processing. To understand the molecular relevance of PSI, the proteolytic processing of cardosin A, the major aspartic proteinase from the flowers of cardoon (Cynara cardunculus L.) was studied. Procardosin A, a 64‐kDa cardosin A precursor containing PSI and the prosegment was identified by immunoblotting using monospecific antibodies against PSI and the prosegment. Procardosin A undergoes proteolytic processing as the flower matures. PSI was found to be removed before the prosegment, indicating that during processing the enzyme acquires a structure typical of mammalian or microbial aspartic proteinase proforms. In vitro studies showed that processing of PSI occurs at pH 3.0 and is inhibited by pepstatin A and at pH 7.0. Sequence analysis allowed the identification of the cleavage sites, revealing that PSI is removed entirely, probably by an aspartic proteinase. Cleavage of the PSI scissile bonds requires, however, a conformation specific to the precursor since isolated cardosins and pistil extracts were unable to hydrolyse synthetic peptides corresponding to the cleavage sites. In view of these results, a model for the proteolytic processing of cardosin A is proposed and the molecular and physiological relevance of PSI in plant aspartic proteinase is discussed.
Unique progastrin processing in equine G‐cells suggests marginal tyrosyl sulfotransferase activity
Authors: Johnsen Anders H. , Sandin Andreas , Rourke Ian J. , Bundgaard Jens R. , Nilsson Göran , Rehfeld Jens F.
Published: March 2003

Previous studies have indicated that equine G‐cell processing of progastrin differs from that of other species. Since the difference may be due to structural features, we have identified equine gastrin‐17 and ‐34 (<ELGLQGSPHLVADLSKKQGPWLEKEEAAYGWMDF‐NH2), and cloned a corresponding 451‐bp cDNA that encodes a 107‐amino‐acid preprogastrin. Comparison with other mammalian gastrins shows a high degree of conservation, but instead of four or five acidic residues preceding the bioactive carboxyamidated C‐terminal heptapeptide, equine gastrin contains the remarkable substitution of Lys for Glu in this presumed invariant region. In contrast with known mammalian gastrins, which are all significantly Tyr‐sulphated, the equine antral gastrins are virtually non‐sulphated. Transfection of the equine preprogastrin cDNA into an endocrine cell line resulted in highly sulphated gastrins, indicating that the absence of in situ sulphation is not due to the structure of gastrin, but occurs rather because the equine antral G‐cells are unique with respect to tyrosyl sulfotransferase activity. Furthermore, the marginal sulphation may explain the high proportion of gastrin‐34 versus gastrin‐17 in the equine antrum, since tyrosyl sulphation has been shown to promote the endoproteolytic processing of prohormones.
Genes coding for the benzoyl‐CoA pathway of anaerobic aromatic metabolism in the bacterium Thauera aromatica
Authors: Breese Klaus , Boll Matthias , Alt‐Mörbe Juliane , Schägger Hermann , Fuchs Georg
Published: March 2003

Many aromatic compounds are anaerobically oxidized to CO2 via benzoyl‐CoA as the common aromatic intermediate. In Thauera aromatica, the central benzoyl‐CoA pathway comprises the ATP‐driven two‐electron reduction of the benzene ring; this reaction uses a ferredoxin as electron donor and is catalyzed by benzoyl‐CoA reductase. The first intermediate, cyclohex‐1,5‐diene‐1‐carboxyl‐CoA, is subsequently hydrated by dienoyl‐CoA hydratase to 6‐hydroxycyclohex‐1‐ene‐1‐carboxyl‐CoA. Formation of the main product produced by cell extracts, 3‐hydroxypimelyl‐CoA, requires at least two further steps; the oxidation of a hydroxyl group and the hydrolytic carbon ring cleavage of a CoA‐activated β‐oxoacid. In addition, enoyl‐CoA hydratase may come into play. A cluster of eight adjacent genes, which are transcribed in the same direction and may form an operon, was found in this bacterium. The cluster codes for proven and postulated enzymes of the benzoyl‐CoA pathway. The genes for the enzymes code for ferredoxin, four subunits of benzoyl‐CoA reductase, dienoyl‐CoA hydratase, 6‐hydroxycyclohex‐1‐ene‐1‐carboxyl‐CoA dehydrogenase (NAD+), and the ring hydrolyzing enzyme. The deduced amino acid sequences of these proteins were 35−86 % similar to the corresponding sequences found in Rhodopseudomonas palustris. Benzoyl‐CoA reductase subunits exhibit distinct similarities with 2‐hydroxyglutaryl‐CoA dehydratase and its ATP‐hydrolysing activase protein of Acidaminococcus fermentans as well as with open reading frames of unknown function in other bacteria. Conversion of benzoyl‐CoA to 3‐hydroxypimelyl‐CoA can be explained by a minimal model of the benzoyl‐CoA pathway assuming the four enzymes whose genes were characterized and an additional enoyl‐CoA hydratase. In R. palustris the dienoyl‐CoA hydratase gene is lacking suggesting the operation of a modified benzoyl‐CoA pathway with cyclohex‐1‐ene‐1‐carboxyl‐CoA as intermediate.
PACSIN, a brain protein that is upregulated upon differentiation into neuronal cells
Authors: Plomann Markus , Lange Rita , Vopper Gaby , Cremer Harold , Heinlein Uwe A. O. , Scheff Stephen , Baldwin Stanley A. , Leitges Michael , Cramer Matthias , Paulsson Mats , Barthels Dagmar
Published: March 2003

To identify genes that are differentially expressed during self‐repair processes in mouse brain, we screened a subtracted cDNA library enriched for brain‐specific clones. One of these clones, H74, detected a 4.4‐kb mRNA predominantly expressed in brain and dorsal root ganglia neurons. Expression increased continuously during the lifespan and the state of differentiation, but decreased after entorhinal‐cortex lesion. A full‐length cDNA clone was isolated from a cerebellum cDNA library and characterized. Sequence analysis and database search revealed high sequence similarity to FAP52, a protein expressed in focal‐adhesion contacts, and uncharacterized Echinococcus and Caenorhabditis elegans gene products. Furthermore, peptide sequences derived from human cDNA fragments showed up to 65 % sequence identity at the amino acid level. The presence of a C‐terminal src homology 3 (SH3) domain and its phosphorylation by casein kinase 2 (CK2) and protein kinase C (PKC) imply a role in signaling. Here we demonstrate that the gene encodes a phosphoprotein, referred to as PACSIN, with a restricted spatial and temporal expression pattern.
Structural analysis of the CD5 antigen
Authors: McAlister Mark S. B. , Brown Marion H. , Willis Antony C. , Rudd Pauline M. , Harvey David J. , Aplin Robin , Shotton David M. , Dwek Raymond A. , Barclay A. Neil , Driscoll Paul C.
Published: March 2003

CD5 is a type‐I transmembrane glycoprotein found on thymocytes, T‐cells and a subset of B‐cells. The extracellular region consists of three domains belonging to the scavenger receptor cysteine‐rich (SRCR) superfamily, for which no three‐dimensional structure has been obtained. Recombinant soluble CD5 domain 1 (CD5d1), the N‐terminal SRCR domain, has been expressed in both chinese hamster ovary (CHO) cells and Pichia pastoris. CD5d1 was shown to be correctly folded by binding to the CD5 monoclonal antibody Leu1. Circular dichroism and NMR analyses indicate that CD5d1 has a high β‐sheet content. CD5d1 from both CHO cells and P. pastoris have very similar properties. The disulphide bonding pattern was determined and is consistent with that found for the group‐A SRCR domain of type‐1 macrophage scavenger receptor and MARCO, the macrophage receptor with collagenous structure. Observations have been made of the role of glycosylation of CD5. P. pastoris expression provides large quantities of correctly folded recombinant CD5d1 for multidimensional NMR and for X‐ray crystallographic studies. The whole extracellular region of CD5, expressed as a chimaera with rat CD4 domains 3 and 4 (cCD5d1−3‐CD4d3+4), was studied by electron microscopy and carbohydrate analysis to gain an overview of the structure of the extracellular portion of intact CD5. Carbohydrate analysis identified N‐linked glycans on CD5 domains 1 and 2, and sialylated O‐linked glycans on the linker peptide between domains 1 and 2. Electron microscopy and carbohydrate analysis together suggest that the extracellular region of CD5 forms a rod‐like structure with domain 1 distal from the cell surface and separated from domains 2 and 3 by an O‐glycosylated peptide linker region.
Okadaic acid‐induced lens epithelial cell apoptosis requires inhibition of phosphatase‐1 and is associated with induction of gene expression including p53 and bax
Authors: Li David Wan‐Cheng , Fass Uwe , Huizar Isham , Spector Abraham
Published: March 2003

It is well established that phosphorylation and dephosphorylation are key cellular events which regulate important metabolic activities such as gene expression, cell cycle progression, and apoptosis. The polyether fatty acid, okadaic acid has been shown previously to activate apoptosis in a variety of cell lines. Although this marine sponge toxin is known to inhibit protein phosphatase (PP)‐2A and PP‐1, it is not certain in most cases whether inhibition of PP‐1 or PP‐2A is necessary to activate apoptosis. Furthermore, it is not clear how inhibition of these phosphatases leads to apoptosis. Here we present evidence that inhibition of PP‐2A by okadaic acid does not activate apoptosis in the lens system. However, when PP‐1 is inhibited by okadaic acid, rabbit lens epithelial cells undergo rapid apoptosis. Associated with this process is the several‐fold up‐regulation of the tumor suppressor gene p53 and the pro‐apoptotic gene bax at both mRNA and protein levels. Analyses of the temporal pattern of expression of the two genes reveal that the up‐regulation is maximized in a few hours after treatment with okadaic acid, when the majority of the treated cells become committed to apoptosis. A brief treatment of the cells with a protein synthesis inhibitor can abolish okadaic acid‐induced up‐regulation of both P53 and Bax proteins. Concomitant with this inhibition, okadaic acid‐induced apoptosis is also temporarily blocked. These results suggest that okadaic acid‐induced expression of p53, bax, and other genes are necessary for the activation of the apoptotic programs in lens systems.
Two hexameric cyanoprotein subunits from an insect, Riptortus clavatus
Authors: Miura Ken , Shinoda Tetsuro , Yura Mamoru , Nomura Sen , Kamiya Katsumi , Yuda Masao , Chinzei Yasuo
Published: March 2003

Hemolymph of a hemipteran insect, Riptortus clavatus, contains four distinct hexameric proteins (cyanoproteins) composed of two distinct subunits, CPα and CPβ, which show profiles of abundance depending on developmental stage, diapause status controlled by juvenile hormone, and sex. We have isolated cDNA clones encoding the two subunits and determined the complete sequences. The nucleotide sequences predict polypeptides of 693 and 691 residues for CPα and CPβ, respectively, including identical 16‐residue signal peptides. The deduced amino acid sequences of both CPα and CPβ have significant similarity to other hemocyanin‐related proteins, indicating that the cyanoproteins represent hexamerins. Phylogenetic analyses show that the cyanoprotein subunits can be grouped together with other hexamerins from exopterygote insects. Developmental Northern‐blot analyses suggest that the expression of the cyanoproteins is regulated at the level of transcript abundance. In addition, the expression of the CPα subunit in female adults has been shown to be enhanced by juvenile hormone (JH) while the expression of the CPβ subunit is suppressed by the same hormone. To our knowledge, the CPα subunit of R. clavatus is the first case of a JH‐enhanceable hexamerin whose sequence has been determined.
Preferential inhibition of inducible nitric oxide synthase in intact cells by the 4‐amino analogue of tetrahydrobiopterin
Authors: Schmidt Kurt , Werner‐Felmayer Gabriele , Mayer Bernd , Werner Ernst R.
Published: March 2003

In the present study we demonstrate that the 4‐amino analogue of tetrahydrobiopterin, 2,4‐diamino‐5,6,7,8‐tetrahydro‐6‐(l‐erythro‐1,2‐dihydroxypropyl)pteridine (4‐amino‐H4biopterin) binds with high affinity to recombinant endothelial NO synthase and concomitantly inhibits enzyme activity [IC50 = 14.8 ± 7.5 µm in the presence of added 5,6,7,8‐tetrahydro‐l‐erythrobiopterin (H4biopterin) 10 µm] as efficiently as previously shown for inducible NO synthase [Mayer, B., Wu, C.Q., Gorren, A.C.F., Pfeiffer, S., Schmidt, K., Clark, P., Stuehr, D.J. & Werner, E.R. (1997) Biochemistry36, 8422–8427]. In cultured porcine endothelial cells, however, 4‐amino‐H4biopterin was less effective in inhibiting NO formation (IC50 = 420 ± 36 µm) as compared with inhibition of the inducible isoform in murine fibroblasts (IC50 = 15 ± 4.9 µm) and in human DLD‐1 adenocarcinoma cells (IC50 = 55 ± 10.3 µm). In all cells investigated, the inhibitory effect of 4‐amino‐H4biopterin was markedly enhanced by depletion of intracellular H4biopterin and could be overcome by increasing intracellular H4biopterin concentrations. Endothelial cells contained lower amounts of H4biopterin [5.2 ± 0.3 pmol·(mg protein)−1] than fibroblasts [19.4 ± 2.7 pmol·(mg protein)−1] and DLD‐1 cells [8.3 ± 1.1 pmol·(mg protein)−1], so that the selectivity of 4‐amino‐H4biopterin towards inducible NO synthase was not explained by differences in the H4biopterin levels. Because 4‐amino‐H4biopterin did not suppress expression of NO synthase in cytokine‐treated cells, we suggest that high‐affinity binding of the inhibitor during protein expression may be responsible for the preferential inhibition of the inducible isozyme in intact cells.
Physicochemical and immunological studies of the N‐terminal domain  of the Torpedo acetylcholine receptor α‐subunit expressed in  Escherichia coli
Authors: Alexeev Timophey , Krivoshein Arcadius , Shevalier Alexander , Kudelina Irina , Telyakova Olga , Vincent Angela , Utkin Yuri , Hucho Ferdinand , Tsetlin Victor
Published: March 2003

The nicotinic acetylcholine receptor (AChR) from the electric organ of Torpedo species is an oligomeric protein composed of α2βγδ subunits. Although much is known about its tertiary and quaternary structure, the conformation of the large extracellular domains of each of the subunits has not been analysed in detail. In order to obtain information about the spatial structure of the extracellular domain, we have expressed the N‐terminal fragment 1–209 of the Torpedo californica AChR α‐subunit in Escherichia coli. Two vectors coding for a (His)6 tag, either preceding or following the 1–209 sequence, were used and the recombinant proteins obtained (designated α1‐209pET and α1‐209pQE, respectively) were purified by affinity chromatography on a Ni2+‐agarose column. The chemical structure of both proteins was verified by Edman degradation and mass spectrometry. The proteins were soluble in aqueous buffers but to make possible a comparison with the whole AChR or its isolated subunits, the recombinant proteins were analyzed both in aqueous solution and with the addition of detergents. The two proteins bound [125I]α‐bungarotoxin with equal potency (KD≈ 130 nm, Bmax≈ 10 nmol·mg–1). Both were shown to interact with several monoclonal antibodies raised against purified Torpedo AChR. The circular dichroism (CD) spectra of the two proteins in aqueous solution revealed predominantly β‐structure (50–56%), the fraction of α‐helices amounting to 32–35%. Nonionic (β‐octylglucoside) and zwitterionic (CHAPS) detergents did not appreciably change the CD spectra, while the addition of SDS or trifluoroethanol decreased the percentage of β‐structure or increased the α‐helicity, respectively. The predominance of β‐structure is in accord with recent data on the N‐terminal domain of the mouse muscle AChR α‐subunit expressed in the mammalian cells [West et al. (1997) J. Biol. Chem.272, 25 468]. Thus, expression in E. coli provides milligram amounts of the protein that retains several structural characteristics of the N‐terminal domain of the Torpedo AChR α‐subunit and appears to share with the latter a similar secondary structure. The expression of recombinant polypeptides representing functional domains of the AChR provides an essential first step towards a more detailed structural analysis.
Replacement of terminal cysteine with histidine in the metallothionein α and β domains maintains its binding capacity
Authors: Romero‐Isart Núria , Cols Neus , Termansen Marie K. , Gelpí Josep Lluís , González‐Duarte Roser , Atrian Sílvia , Capdevila Mercè , Gonzàlez‐Duarte Pilar
Published: March 2003

To generate novel forms of metal‐binding proteins, six mutant mouse metallothionein (MT) 1 fragments, in which a terminal cysteine residue was replaced by histidine, were expressed in Escherichia coli. The spectroscopic and analytical results showed that the αMT (C33H, C36H, C41H, C57H) and βMT (C5H, C13H) mutant forms bound 4 and 3 Zn(II) atoms per molecule of protein to the nearest integer, even though in C41H and C5H, species of lower stoichiometry were also detected. In Cd(II) titrations, all the Zn(II) ions bound to the mutant proteins were displaced from the binding sites, giving rise to Cd‐mutated MT forms with 4 and 3 Cd(II), respectively. However, although Cys‐to‐His substitutions maintained the binding capacity of the MT fragments, they caused structural changes with respect to the wild‐type proteins. While C13H, C36H and C57H seem to contain Zn(II)‐aggregates that are closely related to those of the wild‐type proteins, only C41H and C57H gave rise to Cd(II)‐aggregates similar to those of Cd4‐αMT, where the His residue plays the role of the substituted Cys. Despite the structural implications of the Cys‐to‐His replacement, the dissociation constants showed no major decrease in the Cd‐binding affinity in any of the mutants assayed compared with the wild‐type.
Probing the function of the conserved tryptophan in the flexible loop of the Yersinia protein‐tyrosine phosphatase
Authors: Keng Yen‐Fang , Wu Li , Zhang Zhong‐Yin
Published: March 2003

The involvement of the strictly conserved Trp354 residue in the catalysis of the Yersinia protein tyrosine phosphatase (PTPase) has been investigated by site‐directed mutagenesis and kinetic studies. Crystallographic structural data have revealed that Trp354 interacts with the active site Arg409 and is located at one of the hinge positions of the flexible surface loop (WpD loop) which also harbors the general acid/base (Asp356) essential for catalysis [Schubert, H. L., Fauman, E. B., Stuckey, J. A., Dixon, J. E. & Saper, M. A. (1995) Protein Sci. 4, 1904–1913]. Two mutants were constructed and expressed that contained the Trp354→Phe and Trp354→Ala substitutions. The Km of the W354F and W354A mutants were not significantly different from that of the wild‐type. However, a major decrease in the affinity for oxyanions was observed for the mutants, which is consistent with Trp354 playing a role in aligning Arg409 for oxyanion binding. In addition, replacement of Trp354 with Phe or Ala caused a decrease in kcat of 200‐fold and 480‐fold, respectively, and impaired the ability of the mutant enzymes to stabilize the negative charge in the leaving group at the transition state. In fact, the W354F and W354A mutants exhibited catalytic efficiency and leaving group dependency similar to those observed for the general acid‐deficient PTPase D356N. These results indicate that Trp354 is an important residue that keeps the WpD loop in a catalytically competent conformation and positions the general acid/base Asp356 in the correct orientation for proton transfer.
Production in Escherichia coli and site‐directed mutagenesis of a 9‐kDa nonspecific lipid transfer protein from wheat
Authors: Lullien‐Pellerin Valérie , Devaux Carine , Ihorai Tania , Marion Didier , Pahin Valérie , Joudrier Philippe , Gautier Marie‐Françoise
Published: March 2003

The sequence encoding a wheat (Triticum durum) nonspecific lipid transfer protein of 9 kDa (nsLTP1) was inserted into an Escherichia coli expression vector, pET3b. The recombinant protein that was expressed accumulated in insoluble cytoplasmic inclusion bodies and was purified and refolded from them. In comparison with the corresponding protein isolated from wheat kernel, the refolded recombinant protein exhibits a methionine extension at its N‐terminus but has the same structure and activity as demonstrated by CD, lipid binding and lipid transfer assays. Using the same expression system, four mutants with H5Q, Y16A, Q45R and Y79A replacements were produced and characterized. No significant changes in structure or activity were found for three of the mutants. By contrast, lipid binding experiments with the Y79A mutant did not show any increase of tyrosine fluorescence as observed with the wild‐type nsLTP1. Comparison of the two tyrosine mutants suggested that Tyr79 is the residue involved in this phenomenon and thus is located close to the lipid binding site as expected from three‐dimensional structure data.