bluuebunny/crossref_metadata_2025 huggingface dataset:
A Spectroscopic Study of the Structures of Latent, Active and Reactive‐Center‐Cleaved Type‐1 Plasminogen‐Activator Inhibitor
Authors: Schulze Andreas J. , Quarzago Daniela , Andreasen Peter A.
Published: July 2004

Type‐1 plasminogen‐activator inhibitor (PAI‐1) was studied by Fourier‐transform infrared spectroscopy, far‐ultraviolet CD spectroscopy, and fluorescence‐emission spectroscopy, with the aim to obtain structural information about its active form. The spectra of latent, active and reactive‐center‐cleaved forms of PAI‐1 produced by HT‐1080 cells were different. While the cleaved and the latent forms were similar with regard to their β‐structure content, comparison of the spectra of these forms with the spectra of active PAI‐1 suggested a much higher degree of unordered structure for the active form compared with the latent and reactive‐center‐cleaved forms than previously assumed. We discuss our results with reference to the known three‐dimensional X‐ray structures of latent PAI‐1, of reactive‐center‐cleaved serpins, including reactive‐center‐cleaved PAI‐1, and of intact serpins, and with reference to previous results on the differences in the affinity of mAbs for the different PAI‐1 forms. We interpret our results in favor of a global rearrangement of secondary structure during latency transition and reactive‐center cleavage in PAI‐1, not only involving the reactive‐center loop and parts of β‐sheets A and C, but also the ‘rear’ side of the molecule, such as helices H and G. Thus, we suggest flexibility in serpin structural elements that were previously regarded as rigid.
Characterization of the Binding Properties of Protein LG, an Immunoglobulin‐Binding Hybrid Protein
Authors: Kihlberg Britt‐Marie , Sjöholm Anders G. , Björck Lars , Sjöbring Ulf
Published: July 2004

Protein LG is a 50‐kDa hybrid molecule containing four Ig‐light‐chain‐binding domains from protein L of Peptostreptococcus magnus and two IgG‐Fc‐binding repeats from streptococcal protein G. Here we analyse the binding of protein LG to Ig from several mammalian species. Protein LG was shown to bind human IgG of all subclasses and other Ig classes that carry к chains. The binding to human IgG was only marginally influenced by changes in temperature (4–37°C) or salt concentration (0–1.6 M), and was stable over a wide pH range (pH 4–10). Protein LG bound to Ig from 11 of 12 mammalian species, including those of rabbit, mouse and rat. The affinity constants obtained for the interactions between protein LG and polyclonal IgG from rabbit (4.0×109 M−1), mouse (1.7×109 M−1) and rat (1.3×109 M−1) were similar to the value previously reported for the interaction between the hybrid protein and human polyclonal IgG (5.9×109M−1). The interaction between protein LG and a mouse IgG mAb was not influenced by the presence of the specific protein antigen, nor was the binding of this antibody to its ligand affected by protein LG. Inhibition experiments demonstrated that the Ig‐binding site of one of the fusion partners retained its ligand‐binding capacity when the other component was occupied. Protein LG selectively absorbed 85–90% of the total Ig present in human and rabbit sera and 75–80% of the Ig in sera from mouse and rat. Human serum depleted of C1q, factor D and properdin and preabsorbed by protein LG could be used as a source for other complement factors. These data demonstrate that protein LG is a very versatile Ig‐binding protein.
Studies on the Biosynthesis of Ovothiol A
Authors: Steenkamp Daniel J. , Weldrick Durand , Spies Hendrik S. C.
Published: July 2004

The recent discovery of N1‐methyl‐4‐mercaptohistidine (ovothiol A), a small aromatic thiol, in Crithi‐dia fasciculata made it possible to study its biosynthesis in an organism which can be cultured in large quantities and under defined growth conditions. Radiolabeling experiments using intact cells indicated that the methyl group in ovothiol A is derived from methionine, while 35S was incorporated from either cysteine or methionine. Three lines of evidence suggested that transsulfuration preceded the methylation step: (a) Crithidia fasciculata failed to convert radiolabeled Nπ‐methylhistidine to ovothiol A. (b) Ovo‐thiol A was poorly separated from a component which was labeled by [14C]histidine and by [35S]cysteine, but not by [methyl‐3H]methionine. (c) Dialysed crude extracts of C. fasciculata catalysed the conversion of histidine to a thiolated species in the presence of pyridoxal phosphate, iron and cysteine in the absence of S‐adenosylniethionine. The product of the in vitro reaction was isolated as the bimane derivative. Structural analysis using 1H and 1C‐NMR spectroscopy confirmed its identity as the bimane derivative of 4‐mercaptohistidine.
Probing the Domain Structure of Abrin‐a by Tryptic Digestion
Authors: Lin Song‐How , Chow Lu‐Ping , Chen Yung‐Liang , Liaw Yen‐Chywan , Chen Jeen‐Kuan , Lin Jung‐Yaw
Published: July 2004

Abrin‐a is a potent plant toxin that consists of A and B chains linked by a disulfide bond. The abrin‐a A chain (AaTA) has N‐glycosidase activity while the abrin‐a B chain (AaTB) has galactose‐binding activity. By partial tryptic digestion, the domain structure of abrin‐a was investigated. Seven tryptic fragments with molecular masses greater than 3500 Da were isolated and characterized. One fragment, designated T‐21 and consisting of 153 amino acid residues, contained the major part of the second domain of AaTB and, after cross‐linking of T‐21 with glutaraldehyde, the reaction product had the same level of hemagglutinating activity as native abrin. When the T‐21 fragment was conjugated with AaTA, the conjugate inhibited protein biosynthesis in HeLa cells. This suggests that the T‐21 fragment is able to bind specifically to cells; its conjugate facilitates membrane translocation of AaTA into cells and consequently inhibits protein biosynthesis. T‐21, with a molecular mass less than AaTB, is therefore a potentially useful substance for the preparation of immunotoxins.
Localization and Properties of Kinases in Clathrin‐Coated Vesicles from Zucchini Hypocotyls
Authors: Drucker Martin , Happel Nicole , Robinson David G.
Published: July 2004

Five major polypeptides of 70, 50, 47, 19 and 17 kDa and four minor polypeptides (100, 65, 45 and 39 kDa) become phosphorylated when clathrin‐coated vesicles (CCV) from zucchini hypocotyls are incubated in [γ32P]Mg‐ATP. After dissociation with 0.5 M Tris/HCl the CCV coat polypeptides were subjected to gel filtration in order to separate clathrin triskelions from β‐adaptin‐containing fractions. Only the latter bore kinase activities, with phosphorylated polypeptides of 39 kDa in addition to the 50, 19‐kDa and 17‐kDa polypeptides just mentioned. Heparin, an inhibitor of casein kinase II, permitted the phosphorylation of only the 19‐kDa and 17‐kDa polypeptides. Staurosporine, an inhibitor of protein kinase c‐like acitivities, prevented the phosporylation of the 70‐kDa polypeptide. When recombined with the triskelions the β‐adaptin fractions achieved the phosphorylation of the 45‐kDa and 70‐kDa polypeptides. Because of its heat stability and calcium‐binding properties we interpret the 45‐kDa polypeptide as being a clathrin light chain. Antibodies raised against the 70‐kDa group of heat‐shock proteins (Hsp70) recognize a 70‐kDa polypeptide in the β‐adaptin‐containing fractions. Because this polypeptide only phosphorylates in the presence of triskelions we consider it to be the uncoating ATPase, which is known to aggregate upon dissociation of the CCV coat. Our results therefore indicate that zucchini CCV contain a number of phosphorylable polypeptides equivalent to the β, μ and σ adaptins of bovine brain. Just as in bovine brain CCV a casein‐kinase‐II‐like activity is associated with the zucchini CCV 50/47‐kDa polypeptides, further pointing to their identity as plant μ2/μ1 adaptin equivalents.
Biosynthesis and Processing of Type XVI Collagen in Human Fibroblasts and Smooth Muscle Cells
Authors: Grässel Susanne , Timpl Rupert , Tan Elaine M. L. , Chu Mon‐Li
Published: July 2004

The α1(XVI) collagen chain, recently identified by cDNA cloning, exhibits structural similarity to a subgroup of collagens that associate with collagen fibrils. Recombinant α1(XVI) collagen chains produced in embryonic kidney cells are able to form stable homotrimers, which are rapidly converted into smaller polypeptides after secretion into the culture medium. In this study, we investigated the biosynthesis of native type XVI collagen by immunoprecipitation of metabolically labeled human cells. Dermal fibroblasts and arterial smooth muscle cells were precipitated with three antibodies raised against distinct regions in the N‐ and C‐terminal part of the human α1(XVI) collagen chain. A disulfide‐bonded polypeptide of 220 kDa was obtained from the culture medium, cells and extracellular matrix with all three antibodies. This polypeptide is sensitive to bacterial collagenase digestion and partially resistant to pepsin digestion, suggesting that it is the endogenous αl(XVI) collagen chain. Pulse/chase experiments showed that the newly synthesized αlXVI) chains are secreted into the medium and deposited in the extracellular matrix in a time‐dependent manner. Unlike the recombinant chain, the native type XVI collagen does not undergo extensive proteolytic processing upon secretion. Both cell types deposit a substantial amount of the newly synthesized αl(XVI) chain into the extracellular matrix, in which the 220‐kDa polypeptide is the only product immunoprecipitated. There is little evidence for the presence of another constituent chain. The data are consistent with a homotrimeric chain composition for type XVI collagen. No apparent difference exists in the rate of synthesis and secretion between fibroblasts and smooth muscle cells. Indirect immunofluorescence microscopy showed an extracellular distribution of type XVI collagen, which is located close to cells but not associated with fibrillar structures.
Cello‐Oligosaccharide Hydrolysis by Cellobiohydrolase II from Trichoderma Reesei
Authors: Harjunpää Vesa , Teleman Anita , Koivula Anu , Ruohonen Laura , Teeri Tuula T. , Teleman Olle , Drakenberg Torbjörn
Published: July 2004

The hydrolysis of soluble cello‐oligosaccharides, with a degree of polymerisation of 4–6, catalysed by cellobiohydrolase II from Trichoderma reesei was studied using 1H‐NMR spectroscopy and HPLC. The experimental progress curves were analysed by fitting numerically integrated kinetic equations, which provided cleavage patterns and kinetic constants for each oligosaccharide. This analysis procedure accounts for product inhibition and avoids the initial slope approximation. No glucose was detected at the beginning of the reaction indicating that only the internal glycosidic linkages are attacked. For cellotetraose only the second glycosidic linkage was cleaved. For cellopentaose and cellohexaose the second and the third glycosidic linkage from the non‐reducing end were cleaved with approximately equal probability. The degradation rates of these cello‐oligosaccharides, 1–12 s−1 at 27°C, are about 10–100 times faster than for the 4‐methylumbelliferyl substituted analogs or for cellotriose. No intermediate products larger than cellotriose were released. The degradation rate for cellotetraose were higher than its off‐rate, which accounts for the processive degradation of cellohexaose. A high cellohexaose/enzyme ratio caused slow reversible inactivation of the enzyme.
6‐Phosphogluconate Dehydrogenase from Trypanosoma Brucei
Authors: Hanau Stefania , Rippa Mario , Bertelli Massimo , Dallocchio Franco , Barrett Michael P.
Published: July 2004

The kinetics of 6‐phosphogluconate dehydrogenase from Trypanosoma brucei was examined and compared to those of the same enzyme from lamb's liver. Variation of kinetic parameters as a function of pH suggests a chemical mechanism similar to other 6‐phosphogluconate dehydrogenases. The comparison extended to a detailed analysis of the effect on enzyme activity by several inhibitors including the trypanocidal drugs suramin, melarsoprol and analogues of these compounds. The T. brucei enzyme differs significantly from its mammalian counterpart with respect to several inhibitors, particularly the substrate analogue 6‐phospho‐2‐deoxygluconate and the coenzyme analogue adenosine 2′,5′‐bisphosphate which have respectively 170‐fold and 40‐fold higher affinity for the parasite enzyme.
Mutational Analysis of the Nor Gene Cluster which Encodes Nitric‐Oxide Reductase from Paracoccus denitrificans
Authors: De Boer Anthonius P. N. , Van Der Oost John , Reijnders Willem N. M. , Westerhoff Hans V. , Stouthamer Adriaan H. , Van Spanning Rob J. M.
Published: July 2004

The genes that encode the bc‐type nitric‐oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1‐type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu‐wenhoek 66, 111–127]. norC and norB encode the cytochrome‐c ‐containing subunit II and cytochrome‐b ‐containing subunit I of nitric‐oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP‐binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonus aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane α‐helices and has similarity to CoxIII, the third subunit of the aa3‐type cytochrome‐c oxidases. norF encodes a small protein with two putative transmembrane α‐helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as sub‐strate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild‐type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild‐type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad‐host‐range vector pEG400 were able to grow anaerobically, al‐though at a lower specific growth rate and with lower NOR activity compared with the wild‐type strain.
Analysis of the mRNA Cap‐Binding Ability of Human Eukaryotic Initiation Factor‐4E by Use of Recombinant Wild‐Type and Mutant Forms
Authors: Morino Shigenobu , Hazama Hisayoshi , Ozaki Masahide , Teraoka Yoshika , Shibata Satoshi , Doi Mitsunobu , Ueda Hitoshi , Ishida Toshimasa , Uesugi Seiichi
Published: July 2004

In order to identify the amino acid residues necessary for the selective recognition of the mRNA cap structure by human eukaryotic initiation factor‐4E (eIF‐4E), which plays a central role in the first step of mRNA translation, we prepared recombinant wild‐type and fourteen mutant forms and compared their cap‐binding abilities by affinity chromatography. By the direct expression of a synthetic gene encoding human eIF‐4E as the soluble form in Escherichia coli and the application on a 7‐methylguanosine‐5′‐triphosphate–Sepharose 4B cap affinity column, pure recombinant eIF‐4E was prepared; the optimum pH for the binding of the mRNA cap was 7.5. Among the amino acid residues conserved among various eIF‐4E species, each of 14 functional residues was replaced with a nonpolar amino acid (alanine or leucine). All mutant eIF‐4E genes, which were constructed by site‐directed niutagenesis, were expressed in the same way as the wild type, and their cap‐binding abilities were compared with that of the wild type. Consequently, all eight tryptophan residues, Glu103, and two histidine residues at positions 37 and 200 in human recombinant eIF‐4E were suggested to be important for the recognition of the mRNA cap structure through direct interaction and/or indirect contributions. Indirect contributions included the construction of the overall protein structure, especially the cap‐binding pocket.
Structural and Serological Characterisation of Two O‐Specific Polysaccharides of Acinetobacter
Authors: Vinogradov Evgeny V. , Pantophlet Ralph , Dijkshoorn Lenie , Brade Lore , Holst Otto , Brade Helmut
Published: July 2004

Extraction of dry bacteria of Acinetobacter strain 34 (DNA group 2) or Acinetobucter strain 108 (DNA group 13) by phenol/water yielded a polymer that was identified by means of serological studies and fatty acid analysis as S‐form lipopolysaccharide. Degradation of the lipopolysaccharides of strains 34 and 108 in 1% acetic acid and 5% acetic acid, respectively, and gel‐permeation chromatography gave the respective O‐antigenic polysaccharides, the structures of which were determined, by compositional analysis and NMR spectroscopy of the polysaccharide, as
image

for strain 34, and
image

for strain 108, where d‐FUCp 3NBUOH represents 3‐[(R)‐3‐hydroxybutyramido]‐3,6‐dideoxy‐d‐galactose and d‐Galp ANAc represents 2‐acetamido‐2‐deoxy‐D ‐galacturonic acid.

Both structures were specifically recognised in Western blots by polyclonal rabbit antisera and there was no cross‐reaction between these two structures.
Structural Characterisation of Human Recombinant Glycohormones Follitropin, Lutropin and Choriogonadotropin Expressed in Chinese Hamster Ovary Cells
Authors: Amoresano Angela , Siciliano Rosa , Orrù Stefania , Napoleoni Roberta , Altarocca Valter , De Luca Eva , Sirna Antonino , Pucci Piero
Published: July 2004

The α and β chains from human recombinant gonadotropins follitropin, lutropin and choriogonadotropin expressed in CHO cells have been structurally characterised both at the protein and at the carbohydrate level by using advanced mass spectrometric procedures. The three α chains share the same amino acid sequence while they display different glycosylation patterns. The oligosaccharide structures detected belong to the complex‐type glycans with different degree of sialylation. Partial proteolytic processing occurred at the N‐terminus of the follitropin β chain and at the C‐terminus of the lutropin β chain. The N‐linked glycans from the three β chains were found to contain fucosylated and sialylated diantennary and triantennary complex‐type structures. The follitropin β chain showed the presence of N‐acetyllactos‐amine repeats on the antennae.

The overall structure of the recombinant glycohormones corresponds to their natural counterparts with the exception that sulphated terminal glycosylation is missing.
Abundant Calcium Homeostasis Machinery in Rat Dental Enamel Cells
Authors: Hubbard Michael J.
Published: July 2004

Enamel cells handle large amounts of calcium, particularly during the developmental phase (termed maturation) when dental enamel is hypermineralized. The extent of intracellular calcium burden, and the nature of calcium homeostasis machinery used to accommodate it, are largely unknown. Here, the calcium‐binding capacity of enamel cell cytosol was found to increase during development, in parallel with the putative transcellular flux of calcium. At maturation, the abundance of calcium‐binding proteins in enamel cells exceeded that in brain and other established calcium‐oriented tissues, which implies a large calcium burden. A search for likely cytosolic calcium transporters revealed only one high‐affinity calcium‐binding protein (12 kDa, distinguished from α‐parvalbumin) that was up‐regulated during maturation, but its low abundance (0.02% of soluble protein) precluded a major calcium transport or cytoprotec‐tive role. Two low‐affinity calcium‐binding proteins up‐regulated during maturation (by 1.8‐fold and 2.1‐fold, respectively) were identified as calreticulin and endoplasmin, both residents of the endoplasmic reticulum. Together, calreticulin and endoplasmin constituted an exceptionally high proportion (5%) of soluble protein during maturation, which gives an inferred calcium capacity 67‐fold higher than that of the principal cytosolic calcium‐binding protein, 28‐kDa calbindin. Evidence that endoplasmin expression varied inversely with serum calcium concentration, and that the inositol trisphosphate receptor also was highly expressed during maturation, supported the novel hypothesis that non‐mitochondrial calcium stores play a major role in transcellular calcium transport. In conclusion: (a) enamel cells contain a general high abundance of calcium homeostasis proteins, consistent with a heavy intracellular calcium burden; (b) the expression pattern (phenotype) of calcium‐binding proteins varies with enamel cell function; (c) enamel cells appear to contain unusually large non‐mitochondrial calcium stores; (d) contrary to the prevailing view that calcium passes mainly through the cytosol of calcium‐transporting cells, the findings imply a route through the endoplasmic reticulum. This study gives novel information about how a highly calcium‐oriented tissue avoids calcium toxicity, and provides a new focus for investigations into the mechanisms of transcellular calcium transport.
Transient Non‐Native Interactions in Early Folding Intermediates Do Not Influence the Folding Kinetics of Escherichia Coli Tryptophan Synthase β2 Subunits
Authors: Planchenault Thierry , Navon Amiel , Schulze Andreas J. , Goldberg Michel E.
Published: July 2004

Local structures formed in early intermediates are thought to play a key role in orientating the protein folding pathway. Here, we test whether non‐native interactions formed by eight N‐terminal residues in early folding intermediates of tryptophan synthase β chains [Navon, A., Schulze, A. J., Guillou, Y., Zylinskii, C. A., Baleux, F., Expert‐Bezanqon, N., Friguet, B., Djavadi‐Ohaniance, L. & Goldberg, M. E. (1995) J. Biol. Chem. 270, 4255‐4261] influence the folding kinetics. The kinetics of in vitro renaturation of wild‐type β chains and truncated β chains lacking residues 2–9 were compared. Each step analyzed (molten globule formation, tryptophan shielding, closing up of distant residues, and complete renaturation) occurred with similar kinetics in the normal and truncated proteins. Thus, non‐native interactions formed early during the folding of β chains seem to influence neither the rate and efficiency of the complete renaturation, nor the kinetics of the early folding steps, which suggests that such non‐native early intermediates are poorly stable and highly dynamic. This observation is consistent with the current view that productive folding should avoid the formation of stable intermediates.
Contribution of Specific Disulphide Bonds to Two Epitopes of Thyrotropin β‐Subunit Associated with Receptor Recognition
Authors: Fairlie W. Douglas , Stanton Peter G. , Hearn Milton T. W.
Published: July 2004

In previous studies, we have shown that two epitopes of bovine thyrotropin β‐subunit that are recognised by the monoclonal antibodies designated mAb 279 and mAb 299 are also associated with the receptor‐binding site of bovine thyrotropin. The present investigation has examined the role of the six disulphide bonds of bovine thyrotropin β‐subunit in the conformational stabilisation of these two epitopes, and hence assessed the relative contribution that these disulphide bonds make to the stabilisation of the receptor‐binding region of the β‐subunit. The experimental procedure involved the production of several bovine thyrotropin β‐subunit‐related derivatives in which an increasing number of the disulphide bonds were selectively reduced with dithiothreitol and alkylated with iodoacetic acid. Antibody‐binding properties of these derivatives were then evaluated in thyrotropin β‐subunit‐specific immunoassays based on the use of the well characterised mAb 279 and mAb 299, to determine the effect of disulphide bond reduction and alkylation on each epitopic specificity. In separate experiments, the residual disulphide bonds that remained intact following these selective partial reductive alkylation procedures were then fully reduced and alkylated with the fluorescent reagent 5‐N‐[(iodoacetamidoethyl)amino)naphthalene 1‐sulphonic acid. The relative contribution of individual disulphide bonds in the stabilisation of each epitope could then be assessed after application of reverse‐phase HPLC peptide mapping methods. Epitope recognition by mAb 279 was not dependent on the preservation of the so‐called determinant loop Cys88–Cys95 disulphide bond nor directly involved binding interactions via the Cys2–Cys52, Cys27–Cys83, and Cys31–Cys85 disulphide bonds. However, the experimental results indicated that the mAb 279 epitope was stabilised by the Cys19–Cys105 and Cys16–Cys67 disulphide bonds, which is consistent with other data on the role of the C‐terminal region of the thyrotropin β‐subunit in this epitope. In contrast, the presence of an intact Cys88–Cys95 disulphide bond was required for the stabilisation of the mAb 299 epitope, although the location of this disulphide bond is distal to the hairpin loop structure that constitutes the mAb 299 epitope. These results on the relative contribution of these disulphide bonds are also discussed in terms of their relationship to the stabilisation of thepredicted region of bovine thyrotropin β‐subunit involved in receptor binding.
Location of Cysteine and Cystine Residues in S ‐Ribonucleases Associated with Gametophytic Self‐Incompatibility
Authors: Ishimizu Takeshi , Norioka Shigemi , Kanai Michiko , Clarke Adrienne E. , Sakiyama Fumio
Published: July 2004

S‐Ribonucleases (S‐RNases) that cosegregate with S‐alleles in the styles of solanaceous and rosaceous plants are associated with gametophytic self‐incompatibility (GSI). The amino acid sequences of many S‐RNases have been derived from cDNA sequences, but the state of half‐cystines has not been clarified. We report the locations of the two free cysteine residues and four disulfide bridges of tobacco S6‐RNase and of the four disulfide bridges of Japanese pear S4‐RNase. The protein was first S‐pyridylethylated at a low pH to selectively modify the free cysteines without thiol‐disulfide exchange. The S‐pyridylethylated protein (PE‐protein) was digested with Achromabucter protease I (API) at pH 6.5 then analyzed by liquid chromatography/electrospray‐ionization mass spectrometry (LC/ESI‐MS). This analysis showed that tobacco S6‐RNase has two free cysteine residues, Cys77 and Cys95, and four disulfide bonds at Cysl6‐ Cys21, Cys45‐Cys94, Cysl53‐Cysl82 and Cys16S‐Cys176. Similarly, four disulfide bonds were identified for pear S4‐RNase, which bears no free cysteine, at Cys15‐Cys22, Cys48‐Cys92, Cys156‐Cys195 and Cys172‐ Cys183. The eight cysteines forming these four disulfide bonds are conserved in all the known S‐RNases, indicative that these cross‐links are important in stabilizing the tertiary structures of the self‐incompatibility‐associated glycoproteins in the solanaceous and rosaceous families. The LC/ESI‐MS analysis also provided some structural informations regarding sugar chains attached to the S ‐RNases.
Characterization of Glycoprotein PAS‐6/7 from Membranes of Bovine Milk Fat Globules
Authors: Hvarregaard Jane , Andersen Mikkel H. , Berglund Lars , Rasmussen Jan T. , Petersen Torben E.
Published: July 2004

Glycoprotein components PAS‐6 and PAS‐7 were purified from bovine milk‐fat‐globule membranes and the amino acid sequence of their common polypeptide core, PAS‐6/7, was determined by peptide and cDNA sequencing. The cDNA encoded a signal peptide of 18 amino acid residues and a mature PAS‐6/7 protein of 409 amino acid residues. A cDNA splice variant was identified by reverse transcription/PCR. Results obtained by amino acid analyses, amino‐acid‐sequence analyses, carbohydrate‐composition determinations, and MS analyses of glycopeptides revealed that both proteins were glycosylated with a carbohydrate structure that contained galactose, N‐acetylgalactosamine and fucose, and which was O‐linked to Ser9 in PAS‐6 and to Thr16 in PAS‐7. In addition, PAS‐6 and PAS‐7 were N‐glycosylated at Am41 with a hybrid‐type‐carbohydrate structure. A high‐mannose glycan was N‐linked to Asn209 of PAS‐6. The sequence of PAS‐617 contained two epidermal growth factor (EGF)‐like domains in the N‐terminal region, the second of which contained an RGD cell‐adhesion sequence in an extended loop. The EGF‐like domains were followed by a C‐terminal tandem repeat, which showed 60–63% similarity to the C1‐C2 domain of blood‐clotting factors V and VIII. The disulfide bonds within the C1‐C2 domain were identified.
Different Oligomeric States are Involved in the Allosteric Behavior of Uracil Phosphoribosyltransferase from Escherichia Coli
Authors: Jensen Kaj Frank , Mygind Bente
Published: July 2004

Uracil phosphoribosyltransferase, catalyzing the formation of UMP and pyrophosphate from uracil and 5‐phosphoribosyl‐α‐1‐diphosphate (PP RibP), was purified from an overproducing strain of Escherichia coli. GTP was shown to activate the enzyme by reducing Km for PP RibP by about fivefold without affecting Vmax. When started by addition of enzyme, the reactions accelerated over an extended period of time, while enzyme solutions incubated first with GTP and PP RibP displayed constant velocities. This indicated that PP RibP and GTP influenced the structure of the enzyme. Gel‐filtration and sedimentation analyses showed that the apparent oligomeric state of uracil phosphoribosyltransferase is defined by a dynamic equilibrium between a slowly sedimenting form (dimeric or trimeric) that has only a little activity, and a more highly aggregated form (pentameric or hexameric), which is more active. It appears that the smaller form predominates in the absence of substrates, while the larger form predominates in the presence of GTP and PP RibP. Guanosine‐3′,5′‐bis(diphosphate) was found to activate the enzyme much like GTP.
Two Potential Indole‐3‐Acetaldehyde Dehydrogenases in the Phytopathogenic Fungus Ustilago maydis
Authors: Basse Christoph W. , Lottspeich Friedrich , Steglich Wolfgang , Kahmann Regine
Published: July 2004

The phytopathogenic basidiomycete Ustilago maydis produces indole‐3‐acetic acid (IndCH2COOH) and indole‐3‐pyruvic acid (Ind‐Prv) from tryptophan. Indole‐3‐acetaldehyde (IndCH2CH2O) is the common intermediate in the conversion of Ind‐Prv and tryptamine to IndCH2COOH. We purified an enzyme (Iadl) from U. maydis that catalyzes the NAD+‐dependent conversion of IndCH2CH2O to IndCH2COOH and isolated corresponding cDNA and genomic clones. The identity of the cDNA clone was confirmed by expression in Escherichia coli and demonstration of enzymatic activity. In U. maydis, iadl‐null mu‐tants were generated by gene replacement. The ability to convert IndCH2CH2O to IndCH2COOH was at least 100‐fold reduced in U. maydis iadlnull mutants grown in medium with glucose as carbon source. However, the iadl ‐null mutants were not diminished in their capacity to produce IndCH2COOH from tryptophan, indicating that IndCH2COOH formation from tryptophan apparently proceeds in the absence of IndCH2CH2O dehydrogenase activity under these conditions. Iadl expression was strongly induced during growth on ethanol while under these conditions Iadl ‐null mutants were unable to grow. This reveals that Iadl is primarily engaged in the conversion of ethanol to acetate. In iadl ‐null mutants we detected an additional NAD+‐dependent IndCH2CH2O dehydrogenase activity that was induced during growth on L‐arabinose but repressed in the presence of D‐glucose. In arabinose‐containing medium the conversion of tryptophan to IndCH2COOH was approximately 5‐fold reduced in wild‐type strains but 10–15‐fold reduced in iadl ‐null mutant strains compared to IndCH2COOH formation in glucose‐containing medium. In addition, the formation of Ind‐Prv from tryptophan was abolished in wild‐type and iadl‐null mutant strains. During growth on arabinose, the conversion of tryptamine to IndCH2COOH was strongly favored suggesting that the glucose‐repressible IndCH2CH2O dehydrogenase is required to convert IndCH2CH2O derived from tryptamine to IndCH2COOH.
Molecular Cloning and Characterization of a Putative Glutathione Reductase Gene, the PfGR2 Gene, from Plasmodium falciparum
Authors: Färber Petra M. , Becker Katja , Müller Sylke , Schirmer R. Heiner , Franklin Richard M.
Published: July 2004

Recently, glutathione reductase (GR) has emerged as a promising target for antiparasitic drugs. The central role of GR in cellular antioxidant defence, the particular susceptibility of intracellular parasites like Plasmodium falciparum to oxidative stress, and successful inhibitor studies substantiate this approach. However, more information is required on the structural and functional characteristics of GR from malarial parasites and differences from the enzyme of host erythrocytes.

We have identified a putative P. falciparum GR gene coding for a polypeptide (PfGR2) of 500 ami no acids that exhibits 40–45% sequence identity with GR enzymes from other species. 18 out of 19 residues contributing to glutathione binding are identical in the putative PfGR2 and human GR.

According to Southern blot analysis, the PfGR2 gene is present as a single‐copy gene. It is expressed during the intraerythrocytic life cycle. Stage‐specific Northern blot analysis demonstrates that the PfGR2 gene is only weakly transcribed in ring, early trophozoite, and segmenter stages; major transcription occurs in the late trophozoite/early schizont stage. This is consistent with the high glutathione reductase activity found in early schizonts. Other data also suggest that PfGR2 corresponds to the enzyme isolated from parasitized erythrocytes. These criteria include the subunit molecular mass (56.2 kDa), the N‐terminal sequence (VYDLIVIGGGSGGMA), the presence of specific sequence motifs at ligand‐binding sites, and, as demonstrated by Western blotting, the occurrence of a unique chain segment in the core of the central domain.

In view of these data, the function(s) of PfGR2 as well as PfGR1, the product of another GR‐like gene of P falciparum (Müller et al., 1995) should be carefully assessed.
Mutational Studies of the Amino Acid Residues in the Combining Site of Erythrina corallodendron Lectin
Authors: Adar Rivka , Sharon Nathan
Published: July 2004

High‐resolution X‐ray crystallography of the complex of the Gal/GalNAc‐specific Erythrina corallodendron lectin with lactose identified the amino acid side chains that form contacts with the galactose moiety of the disaccharide. The contribution of these amino acids to the binding of different monosaccha‐rides and oligosaccharides by the lectin was examined by site‐directed mutagenesis.

Replacement of Phe131, on which the galactose is stacked, by tyrosine, gave a mutant with the same hemagglutinating activity and carbohydrate specificity as the parent lectin, but replacement by alanine or valine resulted in loss of activity. Mutations of Ala88, Asp89, and Asn133 produced mutants that were also inactive whereas those of the other combining site residues, Tyr106, Ala218, and Gln219, were biologically active. None of the active mutants interacted with mannose or glucose. Thus, contiary to an earlier assumption, Ala218 is not responsible for the inability of E. corallodendron lectin to bind these sugars. Our findings also demonstrate that Gln219 is not involved in galactose binding in solution, even though this is implicated by the crystal data. Instead, our data suggest that Gln219 assists in the ligation of N ‐acetyllactosamine to the lectin, by interacting with the acetamide group of the disaccharide.

Comparison with other legume lectins specific for mannose/glucose, galactose, N‐acetylgalactosam‐ine, l‐fucose or N ‐acetylglucosamine, shows that only three of the combining site residues of E. corallodendron lectin occupy invariant positions both in their primary and tertiary structures. These residues are an aspartic acid and an asparagine corresponding to positions 89 and 133, respectively, in E. corallodendron lectin, and an aromatic residue, either phenylalanine (as Phe131 in this lectin), tyrosine or tryptophan. We therefore postulate that these three residues are essential for ligand binding by all such lectins, irrespective of their specificity.
α‐Oxidation of 3‐Methyl‐Substituted Fatty Acids in Rat Liver
Authors: Croes Kathleen , Casteels Minne , De Hoffmann Edmond , Mannaerts Guy P. , Van Veldhoven Paul P.
Published: July 2004

α‐Oxidation of 3‐methyl‐substituted fatty acids in rat liver was studied in intact and permeabilized rat hepatocytes, and in homogenates and subcellular fractions. The experiments revealed that the primary end product of α‐oxidation is formic acid, which is then converted to CO2, Rates of α‐oxidation identical to those observed in intact hepatocytes were obtained in the permeabilized hepatocytes and liver homogenates when ATP, Mg2+ and CoA, and Fe2+, 2‐oxoglutarate and ascorbate were added, suggesting that α‐oxidation involves a fatty acid activation reaction and a dioxygenase reaction. Subcellular fractionation by differential and density gradient centrifugation demonstrated that α‐oxidation is confined to peroxisomes, which produce formic acid that is converted to CO2, mainly in the cytosol. α‐Oxidation in broken cell systems went hand in hand with the formation of a 2‐hydroxy‐3‐methylacyl‐CoA ester. Formation of the metabolite was strictly dependent on the presence of the above‐mentioned cofactors, was confined to peroxisomes and was inhibited by fenoprofen and propyl gallate, inhibitors of α‐oxidation in intact cells, indicating that the 2‐hydroxyacyl‐CoA ester is a bona fide intermediate of α‐oxidation. Selective omission of cofactors from the reaction mixture and analysis of the incubation mixtures for 3‐methyl fatty acids, 3‐methyl fatty acyl‐CoAs and their respective 2‐hydroxy derivatives revealed that the activation reaction precedes the dioxygenase (hydroxylase) reaction.

Our experiments demonstrate that α‐oxidation is a peroxisomal process that consists of at least three reactions: fatty acid activation, hydroxylation and the reaction(s) involved in the release of formic acid.
Exploring the Substrate Specificities of α‐2,6–and α‐2,3–Sialyltransferases using Synthetic Acceptor Analogues
Authors: Van Dorst Johannes A. L. M. , Tikkanen Jaana M. , Krezdorn Christian H. , Streiff Markus B. , Berger Eric G. , Van Kuik J. Albert , Kamerling Johannis P. , Vliegenthart Johannes F. G.
Published: July 2004

The acceptor specificities of rat liver Gal(β1‐4)GlcNAc α‐2,6‐sialyltransferase, recombinant fulllength human liver Gal(β1‐4)GlcNAc α‐2,6‐sialyltransferase, and a soluble form of recombinant rat liver Gal(β1‐3/4)GlcNAc α‐2,3‐sialyltransferase were studied with a panel of analogues of the trisaccharide Gal(β1‐4)GlcNAc(βl‐2)Man(α1‐O)(CH2)7CH3. These analogues contain structural variants of D‐galactose, modified at either C3, C4 or C5 by deoxygenation, fluorination, O‐methylation, epimerization, or by the introduction of an amino group. In addition, the enantiomer of D‐galactose is included. The α‐2,6‐sialyltransferases tolerated most of the modifications at the galactose residue to some extent, whereas the α‐2,3‐sialyltransferase displayed a narrower specificity. Molecular dynamics simulations were performed in order to correlate enzymatic activity to three‐dimensional structure. Ineffective acceptors for rat liver α‐2,6‐sialyltransferase were shown to be inhibitory towards the enzyme; likewise, the α‐2,3‐sialyltransferase was found to be inhibited by all non‐substrates. Modified sialyloligosaccharides were obtained on a milligram scale by incubation of effective acceptors with one of each of the three enzymes, and characterized by 500‐MHz 1H‐NMR spectroscopy.
Structures of the Antigenic O‐Polysaccharides of Lipopolysaccharides Produced by Actinobacillus actinomycetemcomitans Serotypes a, c, d and e
Authors: Perry Malcolm B. , Maclean Leann M. , Brisson Jean‐Robert , Wilson Mark E.
Published: July 2004

Actinobacillus actinomycetemcomitans is a gram‐negative capnophilic coccobacillus that has been implicated in the etiology of certain forms of early‐onset periodontitis as well as non‐oral infections, mostly bacterial endocarditis. Five distinct serotypes of A. actinomycetemcomitans have been described. Although the O‐polysaccharide (O‐PS) of lipopolysaccharide (LPS) has been shown to define the serologic specificity for this species. only the structure of the O‐PS of serotype b has been characterized. The focus of the current study was to define the structures of the O‐PS of A. actinomycetemcomitans serotypes a, c, d and e. Structure determination was accomplished through the use of methylation, periodate oxidation, and one‐dimensional and two‐dimensional NMR methods.

The O‐PS of A. actinomycetemcomitans (OMZ‐542) serotype d had [α]D+15° and was composed of D‐glucose, D‐mannose and L‐rhamnose (L‐Rha) in a molar ratio of 1:2:1. Methylation, periodate oxidation, and two‐dimensional 1H‐NMR and 13C‐NMR studies showed that the antigenic O‐PS was a high‐molecular‐mass polymer composed of repeating tetrasaccharide units with the structure:
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The O‐PS of the LPS produced by A. actinomycetemcomitans (ATCC 29523) serotype a had [α]D+ 150° and was found to contain 6‐deoxy‐D‐talose (6dTalp) and O ‐acetyl (2:1) and was a high‐molecular‐mass polymer composed of O ‐acetyl–substituted repeating disaccharide units with the structure:
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The O‐PS of the LPS of A. actinomycetemcomitans (SUNY 67) serotype c had [α]D ‐170° and was composed of 6‐deoxy‐L‐talose and O ‐acetyl (2:1). Structural analysis showed that the O‐PS was a high‐molecular‐mass polymer of repeating disaccharide units with the structure:
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The O‐PS of the LPS of A. actinomycetemcomitans (OMZ 534) serotype e had [4aL]D+57° and was composed of 2‐acetamido‐2‐deoxy‐D‐glucose and L‐rhamnose (1:1) and by chemical and NMR analysis was found to be a polymer of repeating disaccharide units with the structure:
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Cytochrome c′ of Methylococcus Capsulatus Bath
Authors: Zahn James A. , Arciero David M. , Hooper Alan B. , Dispirito Alan A.
Published: July 2004

Cytochrome c′ was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The native and subunit molecular masses of the cytochrome were 34.9 kDa and 16.2 kDa, respectively, with an isoelectric pH of 7.0. The amino acid composition and N‐terminal amino acid sequence were consistent with identification of the protein as a cytochrome c′. The electron paramagnetic resonance spectrum of the monoheme cytochrome indicated the presence of a high spin, S= 5/2, heme center that is diagnostic of cytochromes c′. The optical absorption spectra of ferric or ferrous cytochrome c′ were also characteristic of cytochromes c′. The ferrocytochrome bound carbon monoxide and nitric oxide, but not isocyanide, cyanide, or azide. Changes in physical properties due to binding of CO or NO to some other c′‐type cytochromes have been interpreted as an indication of dimer dissociation. In the case of cytochrome c′ from M. capsulatus Bath, analytical ultracentrifugation of the ferricytochrome, the ferrocytochrome, and the ferrocytochrome–CO complex indicate that the changes induced by binding of CO are conformational and are not consistent with dimer dissociation.

EPR spectra show that cytochrome c′ was reduced in the presence of hydroxylamine only when in a complex with cytochrome P‐460. The value of the midpoint potential, Em 7.0 was –205 mV for cytochrome c′ from M. capsulatus Bath, which is well below the range of values reported for other cytochromes c′. The values of midpoint potentials for cytochrome P‐460 (Em 7.0=–300 mV to –380 mV) and cytochrome c555 (Em 7.0=+175 mV to +195 mV) are less than and greater than, respectively, the value for cytochrome c′ and suggest the possibility that the latter may function as an electron shuttle between cytochrome P‐460 and cytochrome c555.
Purification and Partial Characterisation of a Reversible Artificial Mediator Accepting NADH Oxidoreductase from Clostridium thermoaceticum
Authors: Bayer Manfred , Walter Katrin , Simon Helmut
Published: July 2004

An NAD(H)‐dependent artificial mediator accepting pyridine nucleotide oxidoreductase present in Clostridium thermoaceticum has been purified 50‐fold by three chromatographic steps to apparent electro‐phoretical homogeneity with a yield of 25%. By PAGE and gel filtration the molecular mass of the native enzyme was estimated to be 200 kDa and 210 kDa, respectively. By SDS/gel electrophoresis, a single band was found at 17000 Da, suggesting a homododecamer. Reducing carbamoyl methylviologen or hexa‐cyanoferrate(III) with NADH, the enzyme was most active at pH 10 and the specific activities were 100 μmol min‐1 mg‐1 protein and 800 μmol min‐1 mg‐1 protein, respectively. The Km values for hexacya‐noferrate(III), carbamoylmethylviologen and NADH at pH 8.5 were determined to be 0.40, 0.55 and 1.1 mM, respectively. Other electron acceptors for the dehydrogenation of NADH were 2,6‐dichlorophe‐nolindophenol, anthraquinone‐2,6‐disulphonate, ubiquinone 0 and FAD. In the reduction of NAD+ with reduced methyl viologen (MV+), the specific activity was about 225 μmol min‐1 mg‐1 protein at the pH maximum of 5.0. The Km values for reduced methylviologen, NADH and NAD+ were 1.0, 1.1 and 0.25 mM, respectively.

The enzyme had 10.6 atoms iron and 12.7 atoms sulphur per dodecamer. A significant content of flavin or molybdopterin cofactor could not be detected.

The first 45 amino acids of the oxidoreductase show a surprisingly high degree of identity or similarity with the ribosomal L12 protein of various eubacteria, the acyl carrier proteins of microorganisms, but also with bovine heart mitochondria and a 3‐phosphoglycerate dehydrogenase as well as a gyceraldehyde‐3‐phosphate dehydrogenase from bacteria and pea chloroplasts, respectively.
The Enoyl‐[Acyl‐Carrier‐Protein] Reductase (FabI) of Escherichia coli, which Catalyzes a Key Regulatory Step in Fatty Acid Biosynthesis, Accepts NADH and NADPH as Cofactors and is Inhibited by Palmitoyl‐CoA
Authors: Bergler Helmut , Fuchsbichler Sandra , Högenauer Gregor , Turnowsky Friederike
Published: July 2004

Reduction of enoyl–acyl‐carrier‐protein (ACP) substrates by enoyl‐ACP reductase is a key regulatory step in fatty acid elongation of Escherichia coli. Two enoyl‐ACP reductase activities have been described in E. coli, one specific for NADH, the other for NADPH as cofactor. Because of their distinct enzymatic properties, these activities were ascribed to two different proteins. The NADH‐dependent enoyl‐ACP reductase of E. coli has previously been identified as the FabI protein, which is the target of a group of antibacterial compounds, the diazaborines. We now demonstrate that both enoyl‐ACP reductase activities reside in FabI. In crude cell extracts of FabI‐overproducing strains, both NADH‐dependent and NADPH‐dependent enoyl‐ACP reductase activities are increased. Mutations in the fabI gene that lead either to temperature‐sensitive growth or diazaborine resistance result in the reduction of both activities. When FabI is purified in pH 6.5 buffers, the protein exhibits NADH‐dependent and NADPH‐dependent reductase activities. Both enzymatic activities are inhibited by diazaborine. The NADPH‐dependent enoyl‐ACP reductase activity, however, turned out to be approximately eight times more resistant to diazaborine. The difference in sensitivity indicates that binding of either NADPH or NADH to FabI results in distinct changes in the configuration of the protein or, alternatively, it is different due to the different charge of the cofactors. These effects might be responsible for the differences in the enzymatic properties. Both reductase activities of the FabI protein are inhibited by physiologically relevant concentrations of palmitoyl‐CoA, which might be important in regulating endogenous fatty acid biosynthesis in E. coli in the presence of exogenous fatty acids.