bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutation of the distal arginine in Coprinus cinereus peroxidase
Authors: Neri Francesca , Indiani Chiara , Welinder Karen G. , Smulevich Giulietta
Published: March 2003

Heme peroxidases of prokaryotic, plant and fungal origin share the essential His and Arg catalytic residues of the distal cavity and a proximal His bound to heme iron. Spectroscopic techniques, in contrast to X‐ray crystallography, are well suited to detect the precise structure, spin and coordination states of the heme as influenced by its near environment. Resonance Raman and electronic absorption spectra obtained at various pH values for Fe3+ and Fe2+ forms of distal Arg51 mutants of the fungal Coprinus cinereus peroxidase are reported, together with the fluoride adducts at pH 5.0. This basic catalytic residue has been replaced by the aliphatic residue Leu, the polar residues Asn and Gln and the basic residue Lys (Arg51←Leu, Asn, Gln, and Lys, respectively). These mutations cause changes in the coordination and spin states of the heme iron, and in the ν(Fe‐Im) stretching frequency. The variations are explained in terms of pH‐dependent changes, charge location, size and hydrogen‐bonding acceptor/donor properties of the residue at position 51. The present work indicates that the hydrogen‐bond capability of the residue in position 51 influences the occupancy of water molecules in the distal cavity and the ability to form stable complexes between anionic ligands and the heme Fe atom.
Purification and characterization of a kinin‐releasing and fibrinogen‐clotting serine proteinase (KN‐BJ) from the venom of Bothrops jararaca, and molecular cloning and sequence analysis of its cDNA
Authors: Serrano Solange M. T. , Hagiwara Yuki , Murayama Nobuhiro , Higuchi Shigesada , Mentele Reinhard , Sampaio Claudio A. M. , Camargo Antonio C. M. , Fink Edwin
Published: March 2003

Two forms of a proteinase, KN‐BJ 1 and 2, were purified to homogeneity from the venom of Bothrops jararaca. In SDS/PAGE reduced KN‐BJ 1 and 2 migrated as single bands with molecular masses of 38 kDa and 39 kDa. The two enzymes have similar N‐terminal amino acid sequences and specific activities on synthetic chromogenic substrates, and both release bradykinin from bovine low‐molecular‐mass kininogen. KN‐BJ 1 and KN‐BJ 2 clot fibrinogen with specific activities of 245 NIH U/mg and 219 NIH U/mg, releasing only fibrinopeptide A. The amidolytic, kinin‐releasing and coagulant activities are inhibited by phenylmethylsulfonyl fluoride, demonstrating that KN‐BJ is a serine proteinase. Benzamidine derivatives, which are competitive inhibitors of trypsin‐like proteinases, also inhibited the amidolytic activity of KN‐BJ. A cDNA clone (HS104, 2.2 kb) has been isolated from a cDNA library of B. jararaca venom glands with an ORF of 771 bp. The deduced amino acid sequence contains segments that are identical to the sequences of the N‐terminus and three tryptic peptides of KN‐BJ 2. Therefore, the cDNA is believed to represent the gene of KN‐BJ 2. The deduced amino acid sequence indicates that KN‐BJ 2 is synthesized as a prezymogen of 257 amino acids with a putative signal peptide of 18 amino acids and an activating peptide of six amino acid residues. The sequence of 233 amino acids representing the mature enzyme exhibits high similarity to sequences of serine proteinases isolated from crotalid venoms.
Heat‐induced conformational changes of patatin, the major potato tuber protein
Authors: Pots André M. , de Jongh Harmen H. J. , Gruppen Harry , Hamer Rob J. , Voragen Alphons G. J.
Published: March 2003

This paper presents a first structural characterization of isolated patatin, the major potato tuber protein, at ambient and elevated temperatures. Isolated patatin at room temperature is a highly structured molecule at both secondary and tertiary levels. It is estimated from far‐ultraviolet circular dichroism data that about 33 % of the residues adopts an α‐helical and 46 % a β‐stranded structure. Patatin is thermally destabilized at temperatures exceeding 28 +C, as was indicated by near‐ultraviolet circular dichroism. It was shown that parts of the α‐helical contributions unfold in the 45−55 +C region, whereas the β‐stranded parts unfold more gradually at temperatures of 50−90 +C. This was confirmed with Fourier‐transform infrared spectroscopy. Differential scanning calorimetry indicated a cooperative transition between 50−60 +C, most likely reflecting the unfolding of α‐helical parts of the molecule. Furthermore, fluorescence spectroscopy confirmed a global unfolding of the protein between 45−55 +C. The observed unfolding of the protein coincides with the inactivation of the patatin enzyme activity and with the precipitation as occurs in the potato fruit juice upon heating. At high temperatures, patatin still contains some helical and stranded structures. Upon cooling the protein partly refolds, it was observed that mainly α‐helical structures were formed.
Induction of aromatase activity in human adipose tissue stromal cells by extracellular nucleotides
Authors: Schmidt Martin , Löffler Georg
Published: March 2003

Here we describe properties of the P2‐purinoceptor, which is involved in the regulation of the key enzyme of estrogen biosynthesis, aromatase cytochrome P‐450, in stromal cells from human adipose tissue. Aromatase activity induced by cortisol and platelet‐derived growth factor BB (PDGF‐BB) is further increased by addition of ATP, ADP, AMP and, albeit with reduced potency, adenosine, GTP and adenosine(5′)tetraphospho(5′) adenosine. Stable P1‐purinoceptor agonists are inactive, whereas P2X‐purinoceptor agonists mimic the effects of purine(s) (nucleotides). Priorincubation of cells with a P2‐purinoceptor antagonist, but not P1‐purinoceptor antagonists, blocks augmentation of aromatase activity by all ligands. Nucleotides, but not adenosine, retain their ability to augment aromatase activity in the presence of adenosine deaminase, indicating that they do not act via their metabolite adenosine. These results lead to the conclusion, that at least one member of the P2‐subclass of purinoceptors exists in adipose tissue and is involved in modulation of aromatase activity in vitro. The pharmacological profile of the P2‐site differs from those reported for cloned P2‐purinoceptors, but is similar to that of the P2X‐subclass. Therefore, a combined response of different members of the P2X‐purinoceptor subclass or of members of different P2‐purinoceptor subclasses cannot be discounted. Purinoceptor activation triggers cytoplasmic calcium transients, which, in contrast to aromatase induction, are independent from the presence of cortisol and PDGF‐BB. Therefore the involved P2‐purinoceptor(s) seem(s) to be constitutively expressed in human adipose tissue stromal cells. P2‐purinoceptor(s) might provide a direct link between sympathetic nerve activity and estrogen biosynthesis in human adipose tissue. Furthermore it (or they) may contribute to the regulation of lipolysis.
α‐Glucan binding of potato‐tuber starch‐branching enzyme I as determined by tryptophan fluorescence quenching, affinity electrophoresis and steady‐state kinetics
Authors: Blennow Andreas , Viksø‐Nielsen Anders , Morell Matthew K.
Published: March 2003

The affinity of potato tuber starch‐branching enzyme‐I (PSBE‐I) for various linear malto‐oligosaccharides, cyclodextrins, (CDs) and macromolecular α‐glucans was investigated by α‐glucan induced fluorescence quenching of intrinsic PSBE‐I tryptophan residues and by affinity electrophoresis. α‐Glucan binding was characterised by distinct shifts towards shorter wavelengths of the PSBE‐I fluorescence emission spectrum and by concomitant reductions in fluorescence intensity. The magnitudes of both the maximum shift in emission spectrum and reduction in fluorescence intensity were dependent on the α‐glucan ligands used. Maximum Kd for a range of linear malto‐oligosaccharides analysed was 0.13 mM as found at a degree of polymerisation (DP) of 13. Large differences in dissociation constants were measured for CDs with DP 6 (α‐CD, 6.0 mM), DP 7 (β‐CD, 0.25 mM) and DP 8 (γ‐CD, 0.67 μM). The high‐molecular‐mass α‐glucans amylose and amylopectin, both substrates for PSBE‐I, showed apparent affinities of 0.018 and 0.066 mg/ml, respectively. Small linear and cyclic oligosaccharides competed with amylopectin in the affinity electrophoresis system and they were also competitive inhibitors for PSBE‐I activity. The affinities for oligosaccharides as measured by competition were, however, about 10‐fold lower than as measured by fluorescence quenching suggesting the existence of a separate oligosaccharide binding site on PSBE‐I. Affinity electrophoresis revealed multiform heterogeneity in the enzyme preparation with respect to α‐glucan interaction. starch; starch‐branching enzyme; substrate binding; fluorescence quenching; affinity electrophoresis.
Evaluation of the impact of S‐adenosylhomocysteine metabolic pools on cytosine methylation of the tobacco genome
Authors: Fojtová Miloslava , Kovařík Aleš , Votruba Ivan , Holý Antonín
Published: March 2003

We explored the possibility that the cytosine DNA methylation might be regulated by S‐adenosyl‐
L‐methionine (AdoMet) and S‐adenosyl‐
L‐homocysteine (AdoHcy) pools in plant cells. In order to change the AdoHcy/AdoMet ratio (methylation index), (S)‐9‐(2,3‐dihydroxypropyl)adenine was employed, a selective reversible inhibitor of cellular S‐adenosyl‐
L‐homocysteine hydrolase. Micromolar concentrations of the inhibitor increased dramatically (more than 1000‐fold) intracellular AdoHcy levels (and concominantly the AdoHcy/AdoMet ratio) in tobacco TBY‐2 cells. No toxic effect of the drug was observed and the cells displayed only marginal inhibition of growth at high AdoHcy levels. At near equal intracellular concentrations of AdoHcy and AdoMet, a significant reduction of cytosine methylation in transcribed (5SrDNA) and non‐transcribed (HRS60, NTRS) sequences was observed. Interestingly, the CpCpG and CpApG trinucleotide targets appeared to be most sensitive to changes in the methylation index. Methylation of cytosine residues at CpG sites was not affected even at AdoHcy/AdoMet ratio of > 10. These results support the possible regulation of DNA methylation via AdoHcy/AdoMet metabolic pathways in plant cells.
Detection of abscisic‐acid‐binding proteins in the microsomal protein fraction of Arabidopsis thaliana with abscisic‐acid−protein conjugates used as affinity probes
Authors: Pedron Jacques , Brault Mathias , Nake Christian , Miginiac Emile
Published: March 2003

A family of affinity probes has been generated to detect and purify abscisic‐acid (ABA)‐binding proteins, by coupling ABA onto carrier proteins (ovalbumin or BSA) through the C1 carboxyl group or the C4′ carbonyl group of ABA. ELISA detection showed that these ABA‐protein conjugates bound efficiently to the solubilized microsomal protein fraction of Arabidopsis thaliana, but not to the soluble protein fraction. Heat or proteolytic treatments inhibited the binding of the conjugates, indicating the protein nature of these binding sites. After membrane purification of the microsomes, the binding sites were found to be preferentially located in the plasma membrane fraction. The binding of the conjugates was independent of the nature of the carrier protein or the ABA‐carrier protein linker, but was competitively inhibited with an anti‐ABA mAb. Furthermore, the competitive inhibition of the binding of the conjugates with ABA, but not with the inactive ABA methyl ester analog, demonstrated the specificity of the binding and the saturability of the binding sites. The binding of the conjugates was strictly correlated to the ABA/carrier protein molar coupling ratio, confirming that the affinity of the conjugates to the ABA‐binding proteins was enhanced by the increase in the probability of binding events. The experimental approach permits a new insight into the nature of membrane‐associated ABA‐binding proteins.
Biosynthesis of C18 polyunsaturated fatty acids in microsomal membrane preparations from the filamentous fungus Mucor circinelloides
Authors: Jackson Frances M. , Fraser Thomas C. M. , Smith Mark A. , Lazarus Colin , Stobart A. Keith , Griffiths Gareth
Published: March 2003

The biosynthesis of C18 polyunsaturated fatty acids has been studied in the fungus Mucor circinelloides. Microsomal membrane preparations contained Δ9, Δ12 and Δ6 desaturase activities. The Δ9 desaturase exhibited characteristics similar to those of the animal and yeast Δ9 desaturases in being membrane bound and utilising stearoyl‐CoA as substrate. Cytochrome b5 (a soluble form lacking the 20‐amino‐acid hydrophobic C‐terminus) stimulated desaturation and was identified as a major cytochrome component of the membranes. A high ferricyanide reductase activity (indicative of NADH : cytochrome b5 reductase activity) coupled to inhibition by cyanide further supported the similarity with the mammalian and yeast enzymes. Time‐course studies with radiolabelled oleoyl‐CoA showed that the oleate [18 : 1(9)] was transferred to position sn‐2 of phosphatidylcholine (PtdCho) and was desaturated to linoleoyl‐PtdCho. Removal of the excess oleoyl‐CoA from the membranes prior to addition of reductant confirmed that oleoyl‐PtdCho is a substrate for the Δ12 desaturase. The entry of oleate at this position of the phospholipid was facilitated by the activity of lyso‐PtdCho : acyl‐CoA acyltransferase (LPCAT), which readily transferred oleate from oleoyl‐CoA to lyso‐PtdCho. Desaturation of oleate at the sn‐1 position of PtdCho was also demonstrated after the entry of oleate in to the phospholipid by the enzymes of the Kennedy pathway. Thus oleate at sn‐1 and sn‐2 positions served as substrate for the Δ12 desaturase and is consistent with observations in oil seed tissues. LPCAT activity was substantially higher than that observed with lysophosphatidylethanolamine : acyl‐CoA acyltransferase (LPEAT) indicating that oleate is less effectively channelled to phosphatidylethanolamine for linoleate synthesis. No desaturation on phosphatidylinositol could be demonstrated. Δ6 desaturase utilised linoleate at the sn‐2 position of exogenously supplied PtdCho presented to the membranes in the presence of reductant. Thus, the entry of substrates into PtdCho via LPCAT and the synthesis of linoleate [18 : 2(9,12)] and γ‐linolenate [18 : 3(6,9,12)] on this phospholipid is similar to that reported for oil seed membranes.
Isolation and characterization of the cDNA and the gene for eukaryotic translation initiation factor 4G from Drosophila melanogaster
Authors: Hernández Greco , del Mar Castellano María , Agudo Marta , Sierra José M.
Published: March 2003

Recent evidence supports the notion that the eukaryotic polypeptide chain initiation factor (eIF)4G plays a critical bridging role in coordinating other eIF involved in eukaryotic translation initiation. Here we report the isolation and characterization of a 5621‐bp cDNA encoding Drosophila eIF4G. The longest ORF predicts a polypeptide of 1666 amino acids with a molecular mass of 183 940 Da and shares 25 % amino acid identity with other eIF4G. The 5′ untranslated region is 386 nucleotides long and contains seven AUG codons out of frame. The in vitro transcription/translation of the cDNA yielded a major polypeptide, which was specifically immunoprecipitated with an antibody against Drosophila eIF4G. This polypeptide has the same electrophoretic mobility as eIF4G purified from Drosophila melanogaster embryos. A conserved eIF4E‐binding motif was found in Drosophila eIF4G. The gene maps at the 102E region of chromosome 4 and spans a genomic region of approximately 16 kb. It was found to contain 15 introns. A single RNA transcript of approximately 5.9 kb was detected by northern blotting of poly(A)‐rich RNA prepared from Drosophila adults. The sequence upstream of the transcription initiation site lacks the consensus TATA box, but contains several sequences possibly involved in the regulation of transcription.
Evidence that a peptide corresponding to the rat Muc2 C‐terminus undergoes disulphide‐mediated dimerization
Authors: Bell Sherilyn L. , Khatri Ismat A. , Xu Gongqiao , Forstner Janet F.
Published: March 2003

We have investigated the possibility that the intestinal mucin rat Muc2 forms dimers during biosynthesis via intermolecular disulphide bridging of its C‐terminal domains. Since the cysteine alignment of RMuc2 (and other secretory mucins) is similar to that of human von Willebrand factor, a similar C‐tail to C‐tail dimerization may occur in mucins. The C‐terminal domain of RMuc2 (534 amino acids) was expressed in COS‐1 cells, and the products monitored by SDS/PAGE and western blotting with three antibodies to different regions of the C‐terminal domain. In cells, the expressed domain was glycosylated and formed disulphide‐dependent dimers centred at approximately 150 kDa. The domain dimer, but not its precursor monomer, was secreted into the culture medium. The dimers in the media however, appeared to be 12−15‐kDa heavier (i.e. had a slower mobility) than in cell lysates. Initial N‐glycosylation, dimerization and secretion were inhibited by addition of tunicamycin to incubations, whereas benzyl‐α‐GalNAc did not interfere with these processes. However benzyl‐α‐GalNAc resulted in a decrease in the apparent size of secreted dimers, such that they now had the same mobility on gels as dimers normally seen in cell lysates (i.e. 150 kDa). A similar change in dimer size was observed after incubating untreated media samples with N‐acetylneuraminidase. This suggests that benzyl‐α‐GalNAc caused inhibition of sialylation of cell dimers just before they were secreted. In summary, the C‐terminal domain of RMuc2 can form disulphide‐dependent dimers, and N‐glycosylation is required for dimerization and subsequent secretion. A late sialylation event appears to precede the secretion of mucin domain dimers.
Thiol : fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum
Authors: Heim Steffen , Künkel Andreas , Thauer Rudolf K. , Hedderich Reiner
Published: March 2003

Most methanogenic Archaea contain an unusual cytoplasmic fumarate reductase which catalyzes the reduction of fumarate with coenzyme M (CoM‐S‐H) and coenzyme B (CoB‐S‐H) as electron donors forming succinate and CoM‐S‐S‐CoB as products. We report here on the purification and characterization of this thiol :fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum (strain Marburg). The purified enzyme, which was composed of two different subunits with apparent molecular masses of 58 kDa (TfrA) and 50 kDa (TfrB), was found to catalyze the following reactions : (a) the reduction of fumarate with CoM‐S‐H and CoB‐S‐H (150 U/mg); (b) the reduction of fumarate with reduced benzyl viologen (620 U/mg); (c) the oxidation of CoM‐S‐H and CoB‐S‐H to CoM‐S‐S‐CoB with methylene blue (95 U/mg); and (d) the reduction of CoM‐S‐S‐CoB with reduced benzyl viologen (250 U/mg). The flavoprotein contained 12 mol non‐heme iron and approximately the same amount of acid‐labile sulfur/mol heterodimer. The genes encoding TfrA and TfrB were cloned and sequenced. Sequence comparisons with fumarate reductases and succinate dehydrogenases from Bacteria and Eucarya and with heterodisulfide reductases from M. thermoautotrophicum and Methanosarcina barkeri revealed that TfrA harbors FAD‐binding motifs and the catalytic site for fumarate reduction and that TfrB harbors one [2Fe‐2S] cluster and two [4Fe‐4S] clusters and the catalytic site for CoM‐S‐H and CoB‐S‐H oxidation.
Phosphatidylinositol 4‐kinases
Authors: Gehrmann Thor , Heilmeyer Jr Ludwig M. G.
Published: March 2003

Polyphosphoinositides are involved in many signal transduction pathways in eukaryotic cells. The first committed step is catalysed by phosphatidylinositol 4‐kinase leading to the formation of phosphatidylinositol 4‐phosphate. In the last four years, ten cDNA molecules have been cloned which code isoforms of phosphatidylinositol 4‐kinase; some of which are highly related. Characteristically, they contain a C‐terminal catalytic domain which is similar to that of (poly)phosphoinositide 3‐kinases and to that of more distantly related lipid/protein kinases. Alignment has characterised cDNAs from Chaenorabditis, Dictyostelium and Schizostaphyloccus pombe as those of phosphatidylinositol 4‐kinases also. All these lipid kinases are related to the superfamily of protein kinases. Several amino acids are highly conserved in catalytic domains of lipid and protein kinases. Employing the catalytic subunit of the cAMP‐dependent protein kinase as template, these residues can be assigned functionally. On the basis of the alignment, a phylogenetic tree of the superfamily of phosphatidylinositol kinases has been constructed. Three families, the phosphatidylinositol 4‐kinases, phosphoinositide 3‐kinases, and the phosphatidylinositol related lipid/protein kinases, can be recognised. Each family comprises two subfamilies. The involvement of the phosphatidylinositol 4‐kinases in signal transduction processes is summarised and a new hypothesis for the function of their isoforms in polyphosphoinositide signalling is presented. The involvement of phosphatidylinositol 4‐kinases in formation of lipid−protein interactions with cytoskeleton proteins and the metabolism of polyphosphoinositide in the nucleus is discussed.
Mutant analysis reveals an alternative pathway for N‐linked glycosylation in Drosophila melanogaster
Authors: Roberts David B. , Mulvany Wendy J. , Dwek Raymond A. , Rudd Pauline M.
Published: March 2003

The mas‐1 gene of Drosophila melanogaster encodes Golgi mannosidase I (MAS‐1), and flies homozygous for small deletions of the gene are viable. They show but few abnormalities and those have a low penetrance [Kerscher, S., Albert, S., Wucherpfennig, D., Heisenberg, M. & Schneuwly, S. (1995) Dev. Biol. 168, 613−626]. Here we sequence the N‐linked oligosaccharides associated with a reporter protein, and with membrane proteins prepared from wild type and MAS‐1 null organisms. The results show that the null organisms synthesise the same range of oligosaccharides as wild type, albeit with different ratios. There is an accumulation of the Man8GlcNAc2 which is one of the substrates for the MAS‐1 enzyme. This supports the suggestion of Kerscher et al. that the lack of severe phenotypic disturbances in the null organisms is due to the presence of an alternative pathway(s), but it also shows that this alternative pathway(s) does not entirely compensate for the normal pathway.
Inhibition of growth and aggregation of calcium oxalate crystals in vitro
Authors: Grover Phulwinder K. , Moritz Robert L. , Simpson Richard J. , Ryall Rosemary L.
Published: March 2003

The aim of this study was to compare directly, in the absence of interfering contaminants, the inhibitory effects of Tamm‐Horsfall glycoprotein (THG), human serum albumin (HSA), α1‐microglobulin and prothrombin fragment 1 (PTF1) on calcium oxalate crystallization. These proteins have been detected in urinary calculi, and with the exception of THG in calcium oxalate crystals generated from undiluted human urine. THG was isolated from the urine of healthy men, while PTF1 was purified from Prothrombinex‐HT, a human blood concentrate; HSA and α1‐microglobulin were obtained from commercial sources. The effects of these proteins were determined, separately, at the same final concentration (32 nM) on calcium oxalate crystallization in a seeded, inorganic reaction system, using Coulter Counter and [14C]oxalate analysis. Analysis of [14C]oxalate data showed that THG, HSA and α1‐microglobulin had no measurable effect on deposition of calcium oxalate. However, PTF1 significantly inhibited mineral deposition by 19.6 %. The average size of the particles precipitated was reduced from the control value of 8.6 μm to 7.3, 5.9, 5.6 and 4.0 μm in the presence of α1‐microglobulin, HSA, THG and PTF1 respectively. These findings were confirmed by scanning electron microscopy, which also revealed that the smaller particles deposited in the presence of the proteins resulted from reduced crystal aggregation rather than a decrease in the size of the individual crystals. It was concluded that, on a molar basis, PTF1 is a more potent inhibitor of calcium oxalate crystal aggregation than THG, HSA and α1‐microglobulin. Moreover, unlike those proteins it significantly inhibits the deposition of calcium oxalate. These findings have implications for the putative role of urinary proteins in the formation of calcium oxalate stones.
Protein translocation into the endoplasmic reticulum (ER)
Authors: Kalies Kai‐Uwe , Hartmann Enno
Published: March 2003

Protein translocation into the endoplasmic reticulum (ER) occurs cotranslationally with the ribosome tightly bound at the membrane, or post‐translationally. Transport of polypeptides is performed by an elaborate structure in the ER membrane consisting of numerous proteins. Both pathways have been reconstituted in vitro using proteoliposomes that contain purified components. Primary structures of these key components have been identified and recent electron microscopic data provide a first impression of the tertiary structure of the translocation apparatus. However, the precise function of most subunits of the translocation apparatus is still a mystery and little is known about structural changes in the translocation site during a transport cycle.
Primary structure and high expression of human agrin in basement membranes of adult lung and kidney
Authors: Groffen Alexander J. A. , Buskens Carin A. F. , van Kuppevelt Toin H. , Veerkamp Jacques H. , Monnens Leo A. H. , van den Heuvel Lambert P. W. J.
Published: March 2003

Agrin is a heparan sulfate proteoglycan involved in the development of the neuromuscular junction during embryogenesis. In addition to this well‐characterized function, agrin may have additional functions in other tissues and during other stages in development. In this study we present the cDNA sequence of human agrin, and demonstrate a high agrin content in adult basement membranes. The N‐terminal domain of human agrin is highly similar to that of chick agrin, suggesting a similar function in laminin binding. The presence of three SGXG sequences supports serine‐linked glycosylation of the core protein, two sites being particularly favorable for heparan sulfate attachment. Comparison of levels of agrin mRNA in fetal and adult human tissues showed a remarkable upregulation in adult kidney and lung. In both tissues truncated agrin transcripts were detected, lacking the region that encodes the laminin‐binding domain. The high transcription levels in lung and kidney corresponded with the accumulation of agrin in the alveolar and glomerular basement membranes, suggesting a filtration‐associated function. These data provide new directions for investigating the role of agrin in its different physiological environments, including the basement membranes of the neuromuscular junction, kidney and lung.
Evolution of tetrameric lectin Ricinus communis agglutinin from two variant groups of ricin toxin dimers
Authors: Hegde Ramesh , Podder Sunil K.
Published: March 2003

Seeds of Ricinus communis contain two types of lectins; the toxin ricin (≈60 kDa) and R. communis agglutinin (≈120 kDa). The toxin is a heterodimer composed of a toxic A subunit and a lectin B subunit, while R. communis agglutinin is a tetramer, constituted of two ricin‐like dimers held together by non‐covalent forces. The lactamyl Sepharose affinity‐purified ricin consists of two major groups of variants designated ricin II and III [Hegde, R. & Podder, S. K. (1992) Eur. J. Biochem. 204, 155−164]. The purified A subunits of all the variants of ricins and R. communis agglutinin show heterogeneity in the molecular mass as shown for ricin before [Fulton, J. R., Blakey, C. D., Knowles, P. P., Uhr, J. W., Thorpe, P. E. & Vitetta, E. S. (1986) J. Biol. Chem. 261, 5314−5319]. Since the isoelectric points of the R. communis agglutinin variants fall between the isoelectric points of ricin II and III, we investigated the possibility that this lectin is made up of ricin II and III. The isoelectric points of the purified B subunits of R. communis agglutinin matched well with those of ricin II and III on urea‐polyacrylamide isoelectric focussing gel. Further, two‐dimensional electrophoretic analysis of the ricin constituants of R. communis agglutinin in the presence of 9 M urea, showed two protein bands, differing by nearly pH 2 in their isoelectric points, the more alkaline one corresponding to that of ricin III analyzed under the same conditions, while the other, although a higher molecular mass variant, corresponding well with ricin II in its isoelectric point. Based on these results and those obtained from adenine binding to A chains of both ricin and R. communis agglutinin, we provide a plausible evolutionary relationship between R. communis agglutinin and two groups of ricin variants; ricin II and III. The model predicts that one half of R. communis agglutinin is derived from ricin I and II, and the other half from ricin III. The results presented, contrary to the existing notion, unequivocally show that the two halves of R. communis agglutinin are not identical protein units, but differ both in surface charge and molecular mass.
X‐ray crystal structure of a dipeptide‐chymotrypsin complex in an inhibitory interaction
Authors: Kashima Akiko , Inoue Yoshihisa , Sugio Shigetoshi , Maeda Iori , Nose Takeru , Shimohigashi Yasuyuki
Published: March 2003

The dipeptide D‐leucyl‐
L‐phenylalanyl p‐fluorobenzylamide (
D‐Leu‐Phe‐NH‐BzlF) inhibits chymotrypsin strongly in a competitive manner with the Ki value of 0.61 μM [Shimohigashi, Y., Maeda, I., Nose, T., Ikesue, K., Sakamoto, H., Ogawa, T., Ide, Y., Kawahara, M., Nezu, T., Terada, Y., Kawano, K. & Ohno, M. (1996) J. Chem. Soc. Perkin Trans. 1, 2479−2485]. The structure/activity studies have suggested a unique inhibitory conformation, in which the C‐terminal benzyl group fits the chymotrypsin S1 site and the hydrophobic core constructed by the side chains of
D‐Leu‐Phe fits the S2 or S.es.cf1.′.rb.ei1.rb site. To verify this assumption, the molecular structure of the complex between the dipeptide and γ‐chymotrypsin has been determined crystallographically. γ‐Chymotrypsin itself was first crystallized and refined at 1.6‐Å resolution. The refined structure was virtually identical to the conformation reported and the electron density at the active site was interpreted as a pentapeptide Thr‐Pro‐Gly‐Val‐Tyr derived from autolysis of the enzyme (residues 224−228). The chymotrypsin‐dipeptide complex was obtained by soaking the crystals of γ‐chymotrypsin in a solution saturated with the dipeptide inhibitor. The crystal structure of the complex has been refined at 1.8‐Å resolution to a crystallographic R‐factor of 18.1 %. The structure of γ‐chymotrypsin in the complex agreed fairly well with that of γ‐chymotrypsin per se with a rmsd of 0.13 Å for all the Cα carbons. Two inhibitor molecules were assigned in an asymmetric unit, i.e. one in the active site and the other at the interface of two symmetry‐related enzyme molecules. In both sites dipeptides adopted very similar folded conformations, in which side chains of
D‐Leu‐Phe are spatially proximal. In the active site where the binding of dipeptide was judged to be a direct cause of inhibition, C‐terminal p‐fluorobenzylamide group of the dipeptide, NH‐BzlF, was found in the S1 hydrophobic pocket. At the bottom of this pocket, the p‐fluorine atom hydrogen bonded with a water molecule, probably to enhance the inhibitory activity. The stereospecific interaction of R and S isomers of the dipeptide with C‐terminal NH‐C*H(CH3)‐C6H5 was well explained by the space available for methyl replacement in the complex. The hydrophobic core constructed by side chains of
D‐Leu‐Phe was found at the broad S2 site. Interestingly, a novel interaction was found between the inhibitor Phe residue and chymotrypsin His57, the phenyl of Phe and the imidazole of His being in a π‐π stacking interaction at a distance 3.75 Å.
Structural characterization of Cu(I) and Zn(II) sites in neuronal‐growth‐inhibitory factor by extended X‐ray absorption fine structure (EXAFS)
Authors: Bogumil Ralf , Faller Peter , Binz Pierre‐Alain , Vašák Milan , Charnock John M. , Garner C. David
Published: March 2003

Neuronal‐growth‐inhibitory factor (GIF) is a metalloprotein specific to the central nervous system which has been linked to Alzheimer's disease. The high metal content, approximately seven metal atoms/protein molecule, and 70 % sequence identity to mammalian metallothioneins (MT), including a preserved array of 20 cysteinyl residues, place GIF in the family of MT. In contrast to MT, native GIF isolated from human or bovine brain contains an unusual metal composition, viz. four Cu(I) and three Zn(II) per polypeptide chain. Cu and/or Zn K‐edge X‐ray absorption spectra have been recorded for native Cu, Zn‐GIF, Zn‐substituted GIF, and these metals bound to the 32‐residue N‐terminal domain, Cu4‐, Cu6‐ or Zn3‐GIF‐(1−32) at 77 K. The results are consistent with the metals being bound to the protein by cysteinyl residues in every case. The Cu‐S distance is approximately 2.25 Å and the EXAFS is considered to be consistent with primarily trigonal coordination of the Cu(I); Cu    Cu backscattering is observed at approximately 2.67 Å, indicative of the formation of Cux(Scys)y clusters. Thus, the Cu(I) environment is similar to that observed in MT. This is also the case for Zn(II), with 4 S at approximately 2.34 Å. However, in contrast to Zn‐MT for Zn‐substituted GIF and Zn3‐GIF‐(1−32), Zn    Zn backscattering is observed at approximately 3.28 Å. The significance of these results are discussed with respect to the specific biological activity of GIF.
Structure determination of the capsular polysaccharide from Vibrio vulnificus strain 6353
Authors: Reddy G. Prabhakar , Hayat Unaiza , Xu Qiuwei , Reddy K. Vasavi , Wang Yuhui , Chiu Kou‐Wei , Morris Jr J. Glenn , Bush C. Allen
Published: March 2003

Vibrio vulnificus is a pathogenic gram‐negative bacterium, endemic to brackish waters, which is often isolated from sediments, from the water column or from shellfish. It is associated with wound infections and septicemia in humans and the virulence of V. vulnificus has been strongly associated with encapsulation. The capsular polysaccharide purified from a virulent strain of V. vulnificus 6353 did not show cross reactivity with antibodies to the capsular polysaccharide of a related pathogenic strain of V. vulnificus (MO6‐24) the structure of which was recently reported. NMR spectroscopic analysis of the purified polysaccharide from strain 6353 showed that the polymer is composed of four sugar residues per repeating subunit including 2,6‐dideoxy‐2‐N‐acetylamino‐α‐
D‐glucose (QuiNAc), 2‐deoxy‐2‐N‐acetylamino‐α‐
D‐galactose (α‐
D‐GalNAc), 2‐deoxy‐2‐N‐acetylamino‐α‐
D‐galcturonic acid (α‐
D‐GalNAcA) and 2‐N‐acetylamino‐α‐
D‐glucuronamide (α‐
D‐GlcNAcANH2). The 1H‐ and 13C‐NMR spectra were completely assigned by homonuclear and heteronuclear NMR spectroscopy. Sugar types and anomeric configurations were determined from proton homonuclear coupling constants and glycosidic linkages were determined from 1H−13C heteronuclear multiple bond correlation spectra. Sugar identities were confirmed by high performance anion‐exchange chromatography and absolute configurations were determined by gas chromatography in combination with molecular modeling and NMR spectroscopy. The structure of the polysaccharide repeating unit is :[←4)‐α‐.cf15.D.cf15..cf1.‐Gal.cf2.p.cf2..cf1.NAc‐(1←3)‐α‐.cf15.D.cf15..cf1.‐Gal.cf2.p.cf2..cf1.NAcA‐(1←3)‐α‐.cf15.D.cf15..cf1.‐Qui.cf2.p.cf2..cf1.NAc‐(1←].cf8.n.cf8..cf1.α‐
D‐GlcpNAcANH2 (1←4)‐↗

While there are some common features shared among the structures of the capsular polysaccharides of pathogenic strains of V. vulnificus, there are distinct differences in the detailed structures.
Drug resistance of herpes simplex virus type 1
Authors: Kussmann‐Gerber Susanna , Kuonen Oliver , Folkers Gerd , Pilger Beatrice D. , Scapozza Leonardo
Published: March 2003

Several drug‐resistant strains of herpes simplex virus type 1 (HSV1) isolated in vivo or from tissue culture, have exhibited a mutated thymidine kinase (TK). Moreover, various site‐directed‐mutagenesis experiments conducted on HSV1 TK allowed the assignment of specific amino acid residues to specific functional properties. From this, a range of hypotheses was generated related to substrate binding of TK at the molecular level. A site‐directed‐mutagenesis study on Q125 was performed to clarify the contribution of this residue to the binding of thymidine or aciclovir beyond the hydrogen‐bonding pattern observed in the crystal structure. While Q125L is only able to phosphorylate thymidine, Q125N accepts thymidine and aciclovir as substrates. Q125E shows no phosphorylation activity. Several mutations identified previously as relevant in drug resistance were studied in an attempt to further understand their role in these processes. Four amino acid positions are described (T63, A168, R176 and C336) that confer drug resistance when mutated; however, the molecular mechanisms are considerably different in each case. Analysis of the crystal structures and the molecular modeling presented in this paper suggest that T63 is essential for the binding of Mg2+ and thus the catalytic activity of the enzyme, while A168 limits steric accessibility and if mutated to a bulkier residue will exclude binding of larger substrate analogues. R176 appears to be essential for electrostatic balance within the active site, and C336, which is located at the surface of TK and directed toward the ATP‐binding site, disrupts the three‐dimensional structure of the whole active site by shifting the LID‐domain. The present work contributes to a detailed understanding of nucleoside binding to TK, thereby facilitating the rational design of substrates for HSV1 TK and of drug‐specific TK for gene therapy.
Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase
Authors: Wilquet Valérie , Gaspar Joe A. , van de Lande Monica , Van de Casteele Mark , Legrain Christianne , Meiering Elizabeth M. , Glansdorff Nicolas
Published: March 2003

We have overexpressed the gene for dihydrofolate reductase (DHFR) from Thermotoga maritima in Escherichia coli and characterized the biochemical properties of the recombinant protein. This enzyme is involved in the de novo synthesis of deoxythymidine 5′‐phosphate and is critical for cell growth. High levels of T. maritima DHFR in the new expression system conferred resistance to high levels of DHFR inhibitors which inhibit the growth of non‐recombinant cells. The enzyme was purified to homogeneity in the following two steps : heat treatment followed by affinity chromatography or cation‐exchange chromatography. Most of the biochemical properties of T. maritima DHFR resemble those of other bacterial or eukaryotic DHFRs, however, some are unique to T. maritima DHFR. The pH optima for activity, Km for substrates, and polypeptide chain length of T. maritima DHFR are similar to those of other DHFRs. In addition, the secondary structure of T. maritima DHFR, as measured by circular dichroism, is similar to that of other DHFRs. Interestingly, T. maritima DHFR exhibits some characteristics of eukaryotic DHFRs, such as a basic pI, an excess of positively charged residues in the polypeptide chain and activation of the enzyme by inorganic salts and urea. Unlike most other DHFRs which are monomeric or part of a bifunctional DHFR‐thymidylate synthase (TS) enzyme, T. maritima DHFR seems to generally form a dimer in solution and is also much more thermostable than other DHFRs. It may be that dimer formation is a key factor in determining the stability of T. maritima DHFR.
Carboxymethylated phosphatidylethanolamine in mitochondrial membranes of mammals
Authors: Pamplona Reinald , Requena Jesús R. , Portero‐Otín Manuel , Prat Joan , Thorpe Suzanne R. , Bellmunt Maria Josep
Published: March 2003

The non‐enzymatic modification of aminophospholipids with lipoperoxidation‐derived aldehydes and glycoxidation‐derived products have been reported previously. However, it remains unknown whether intracellular membranes are damaged by these glycoxidation‐derived products. To investigate this issue, we tested whether aminophospholipids from mitochondrial membranes are damaged by glycoxidative stress the mitochondrion being identified as the major site of reactive‐species production in the cell. We have used a selected‐ion‐monitoring/gas‐chromatography/mass‐spectrometry assay for carboxymethyl‐ethanolamine (CM‐Etn) detection, and provide evidence for the presence of CM‐Etn in mitochondrial phospholipids. Further, as a physiological approach to evaluate the influence of mitochondrial oxidative stress in CM‐Etn formation, we also present the comparative levels of CM‐Etn in mitochondrial membranes of ten mammalian species ranging in maximum life‐span from 3.5 years to 100, since the rate of mitochondrial reactive‐oxygen‐species production is inversely correlated to the maximum life‐span. Our results show that CM‐Etn levels correlate in a logarithmic fashion with the maximum‐life‐span {[CM‐Etn] = 0.51 + 0.50 x′, where x′ = log(maximum‐life‐span); r = 0.81, P < 0.004}. The data demonstrate the intracellular ocurrence of glycoxidative processes affecting membrane lipids. Moreover, these data show that longer‐lived mammals contain higher levels of CM‐Etn in mitochondrial membrane aminophospholipids. This trend could result from differences in rates of CM‐Etn accumulation and/or phospholipid turnover.
The transient association of ERp57 with N‐glycosylated proteins is regulated by glucose trimming
Authors: van der Wal Fimme J. , Oliver Jason D. , High Stephen
Published: March 2003

The thiol‐dependent reductase ERp57 has been shown to interact specifically with in vitro synthesised glycoproteins imported into canine pancreatic microsomes. On this basis, it was proposed that ERp57 forms part of a glycoprotein‐specific folding ’machinery', present in the lumen of the endoplasmic reticulum (ER). In this study, we have investigated the interaction of ERp57 with newly synthesised proteins using semi‐permeabilised mammalian cells (SP cells), in which the ER remains essentially intact and, hence, resembles that of a living cell. We demonstrate that ERp57 interacts preferentially with the glycosylated versions of soluble and membrane proteins, and that this interaction occurs in combination with calnexin and calreticulin. For the first time, we have performed a detailed analysis of the kinetics of ERp57 binding to newly synthesised glycoproteins. We find that ERp57 associates transiently with glycoproteins − a characteristic of molecular chaperones. Using mutant SP cells deficient in glucosidase I, we confirm that the binding of ERp57 to glycoproteins depends upon glucose trimming. We also demonstrate, for the first time, that the release of ERp57 from glycoprotein substrates is dependent upon glucose trimming. These data are combined to present a unified model for the role of ERp57/ER lectin complexes during glycoprotein folding in vivo.
Solution structure of the cellulose‐binding domain of endoglucanase I from Trichoderma reesei and its interaction with cello‐oligosaccharides
Authors: Mattinen Maija‐Liisa , Linder Markus , Drakenberg Torbjörn , Annila Arto
Published: March 2003

The solution structure of a synthetic 38‐residue cellulose‐binding domain (CBD) of endoglucanase I from Trichoderma reesei (CBDEGI) was determined by two‐dimensional 1H‐NMR spectroscopy. 100 structures were generated from a total of 599 NOE derived distance restraints and 28 φ and 14 χ dihedral angle restraints. For the final set of 19 selected structures, the rms deviation about the mean structure was 0.83 ± 0.26 Å for all atoms and 0.50 ± 0.22 Å for the backbone atoms. The structure of CBDEGI was very similar to that of CBD of cellobiohydrolase I from T. reesei (CBDCBHI). The backbone trace of CBDEGI followed closely the irregular triple‐stranded antiparallel β‐sheet structure of CBDCBHI. Moreover, apart from the different side chains of Trp7 (CBDEGI) and Tyr5 (CBDCBHI), the cellulose‐binding face of CBDEGI was similar to that of CBDCBHI within the precision of the structures. Finally, the interaction between CBDEGI and soluble sugars was investigated using cellopentaose and cellohexaose as substrates. Experiments showed that the interactions between CBDEGI and cellobiose units of sugars are specific, supporting the previously presented model for the CBD binding to crystalline cellulose.
The crystal structure of anionic salmon trypsin in complex with bovine pancreatic trypsin inhibitor
Authors: Helland Ronny , Leiros Ingar , Berglund Gunnar I. , Willassen Nils P. , Smalås Arne O.
Published: March 2003

The complex formed between anionic salmon trypsin (ST) and bovine pancreatic trypsin inhibitor (BPTI) has been crystallised, and the X‐ray structure has been solved using the molecular replacement method. The crystals are hexagonal and belong to space group P6122 with lattice parameters of a = b = 83.12 Å and c = 222.15 Å. Data have been collected to 2.1 Å and the structure has been refined to a crystallographic R‐factor of 20.6 %. Catalysis by salmon trypsin is distinguished by a Km value 20‐fold lower than that for mammalian trypsins, and a kcat twice as high. The present ST‐BPTI complex serves as a model for the Michaelis‐Menten complex, and has been compared with corresponding bovine and rat trypsin (RT) complexes. The binding of BPTI to salmon trypsin is characterised by stronger primary interactions in the active site, and a somewhat looser secondary binding.
Mutual conversion of fatty‐acid substrate specificity by a single amino‐acid exchange at position 527 in P‐450Cm2 and P‐450Alk3A
Authors: Zimmer Thomas , Scheller Ulrich , Takagi Masamichi , Schunck Wolf‐Hagen
Published: March 2003

The two eukaryotic fatty‐acid hydroxylases P‐450Cm2 and P‐450Alk3A, which represent CYP52A4 variants naturally occurring in the yeast Candida maltosa, were characterized with respect to their substrate specificity. Whereas P‐450Cm2 was found to catalyse lauric acid ω‐hydroxylation with greater efficiency, P‐450Alk3A had higher palmitic acid turnover numbers compared to P‐450Cm2, resulting in ratios of lauric acid to palmitic acid turnover rates of nearly 11 and 3 for P‐450Cm2 and P‐450Alk3A, respectively. As shown by means of chimeric enzymes and site‐directed mutagenesis, the key residue determining these differences in substrate specificity was found to be a single amino acid at position 527. Interestingly, the mutual exchange of valine (P‐450Cm2) and leucine (P‐450Alk3A) led to a direct transposition of specificity, suggesting that amino acids at this site may determine the efficiency of fatty‐acid hydroxylation relatively independently of other active‐site residues. This was further supported by the finding that P‐450Cm2 and P‐450Alk3A with methionine at position 527 displayed almost identical hydroxylation activities. Moreover, methionine to leucine substitutions at the corresponding alignment position in P‐450Cm1 (CYP52A3), P‐450Alk2A (CYP52A5) and P‐450Alk5A (CYP52A9) altered the fatty‐acid specificity of these enzymes. In comparison to the structure of the bacterial P‐450BM3 (CYP102), we propose that the amino acid at position 527 may serve to close the substrate‐binding pocket near to the haem in the fatty‐acid‐ω‐hydroxylating P‐450 of the CYP52 family.