bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficient purification and kinetic characterization of a bimodular derivative of the erythromycin polyketide synthase
Authors: Bycroft Matthew , Weissman Kira J. , Staunton James , Leadlay Peter F. 
Published: March 2003

Modular polyketide synthases (PKSs), such as the 6‐deoxyerythronolide B synthase (DEBS), are giant multienzymes that biosynthesize a number of clinically important natural products. The modular nature of PKSs suggests the possibility of a combinatorial approach to the synthesis of novel bioactive polyketides, but the efficacy of such a strategy depends critically on gaining fundamental insight into PKS structure and function, most directly through experiments with purified PKS proteins. Several recent investigations into important aspects of the activity of these enzymes have used only partially purified proteins (often 3–4% of total protein), reflecting how difficult it is to purify these multienzymes in amounts adequate for kinetic and structural analysis. We report here the steady‐state kinetic analysis of a typical bimodular PKS, 6‐deoxyerythronolide B synthase 1‐thioesterase (DEBS 1‐TE), purified from recombinant Saccharopolyspora erythraea JCB101 by a new, high‐yielding procedure consisting of three steps: ammonium sulfate precipitation, hydrophobic interaction chromatography and size‐exclusion chromatography. The method provides 13‐fold purification with a recovery of 11% of the applied PKS activity. The essentially homogeneous synthase exhibits an intrinsic methylmalonyl‐CoA hydrolase activity, which competes with polyketide chain extension.The most reliable value for the kcat for synthesis of (3S,5R)‐dihydroxy‐(2R,4R)‐dimethyl‐n‐heptanoic acid‐δ‐lactone is 0.84 min−1, and the apparent Km for (2RS)‐methylmalonyl‐CoA is 17 µm. This kcat is approximately 10‐fold lower than the value reported previously for a differently engineered version of the truncated PKS, DEBS 1+TE. The difference likely reflects the fact that the DEBS 1‐TE contains a hybrid acyl carrier protein (ACP) domain in its second module, which lowers its catalytic efficiency.
Structure of a glycerol teichoic acid‐like O‐specific polysaccharide of Proteus vulgaris O12
Authors: Perepelov Andrei V. , Torzewska Agnieszka , Shashkov Alexander S. , Senchenkova Sof’ya N. , Rozalski Antoni , Knirel Yuriy A. 
Published: March 2003

A phosphorylated O‐specific polysaccharide (O‐antigen) was obtained by mild acid degradation of Proteus vulgaris O12 lipopolysaccharide and studied by sugar and methylation analyses, 1H‐, 13C‐ and 31P‐NMR spectroscopy, including two‐dimensional COSY, TOCSY, NOESY, H‐detected 1H, 13C and 1H, 31P heteronuclear multiple‐quantum coherence experiments. It was found that the polysaccharide consists of pentasaccharide repeating units connected via a glycerol phosphate group, and has the following structure:where FucNAc is 2‐acetamido‐2,6‐dideoxygalactose and the degree of O‐acetylation at position 4 of GalNAc is ≈ 25%. Immunochemical studies with P. vulgaris O12 O‐antiserum suggested that the lipopolysaccharide studied shares common epitopes with the lipopolysaccharide core of P. vulgaris O8 and with the O‐antigens of P. penneri strains 8 and 63.
Structural analysis of the core region of lipopolysaccharides from Proteus mirabilis serotypes O6, O48 and O57
Authors: Vinogradov Evgeny , Perry Malcolm B. 
Published: March 2003

The structure of lipid A core region of the lipopolysaccharides (LPS) from Proteus mirabilis serotypes O6, O57 and O48 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of LPS:Incomplete substitutions are indicated by bold italic type. All sugars are present in pyranose form, α‐Hep is the residue of lglycero‐α‐dmanno‐Hep, α‐dd‐Hep is the residue of dglycero‐α‐dmanno‐Hep, l‐Ara4N is 4‐amino‐4‐deoxy‐l‐arabinose, Qui4NAlaAla is the residue of 4‐N‐(l‐alanyl‐l‐alanyl)‐4‐amino‐4,6‐dideoxyglucose. All sugars except l‐Ara4N have d‐configuration. β‐GalA* is partially present in the form of amide with 1,4‐diaminobutane (putrescine)‐HN(CH2)4NH2 or spermidine‐HN(CH2)3NH(CH2)4NH2.
Structure of the sialic acid‐containing O‐specific polysaccharide from Salmonella enterica serovar Toucra O48 lipopolysaccharide
Authors: Gamian Andrzej , Jones Christopher , Lipiński Tomasz , Korzeniowska‐Kowal Agnieszka , Ravenscroft Neil 
Published: March 2003

Lipopolysaccharide was extracted from cells of Salmonella enterica serovar Toucra O48 and, after mild acid hydrolysis (1% AcOH, 1 h, 100 °C or 0.1 m NaOH‐AcOH, pH 4.5, 5 h, 100 °C), the O‐specific polysaccharide was isolated and characterized. The core and an oligosaccharide containing a fragment of the repeating unit linked to the core region were also obtained, depending on hydrolysis conditions. On the basis of sugar and methylation analyses and NMR spectroscopy of the hydrolysis products, the biological repeating unit of the O‐specific polysaccharide was shown to be the following trisaccharide:→4)‐α‐Neup5Ac(2→3)‐l‐α‐FucpNAc(1→3)‐d‐β‐GlcpNAc(1→The polysaccharide O‐chain was substituted with a single molar equivalent of O‐acetyl group, distributed between the Neu5Ac O‐9 and O‐7 positions, in an approximate ratio of 7 : 3.
The oxidation and reduction reactions of bovine serum amine oxidase
Authors: Bellelli Andrea , Morpurgo Laura , Mondovì Bruno , Agostinelli Enzo 
Published: March 2003

The presteady‐state and steady‐state kinetics of bovine serum amine oxidase (BSAO) were analyzed by stopped‐flow transient spectroscopy. A simplified model of the catalytic cycle was found to describe the experimental data and the rate constants of the individual steps were used to calculate Michaelis parameters that agree with the direct determinations. In spite of many studies on selected reactions from the catalytic cycle, this is amongst the first efforts to provide a comprehensive kinetic description of the reactions of BSAO, whose results can be compared with the steady‐state parameters.The reoxidation reaction by dioxygen is more complex than previously thought, in agreement with a recent report [Su, Q. & Klinman, J.P. (1998) Biochemistry37, 12513–12525], and occurs in at least two steps whose rate constants, previously undetermined, have been measured.The reaction of the oxidized enzyme with the amine substrate is poorly determined in this type of experiment, thus irreversible combination with aromatic hydrazine inhibitors was used as a model system, demonstrating that the mechanism and rate constants of their reaction is fully compatible with an accurate description of the catalytic cycle with the physiological substrate.These results constitute a simplified, yet complete and consistent, description of the catalytic cycle and offer an interesting comparison with those obtained on plant amine oxidases; two steps of the catalytic cycle are significantly slower in BSAO than in pea seedling or lentil seedling amine oxidases, namely the reoxidation and the trans‐iminative proton abstraction occurring in the enzyme–substrate complex. The former difference is rationalized as being due to the low to zero concentration of the semiquinolamine‐radical intermediate, while the latter is less easily interpreted.
The PKC targeting protein RACK1 interacts with the Epstein–Barr virus activator protein BZLF1
Authors: Baumann Matthias , Gires Olivier , Kolch Walter , Mischak Harald , Zeidler Reinhard , Pich Dagmar , Hammerschmidt Wolfgang 
Published: March 2003

Phorbol esters reactivate Epstein–Barr virus (EBV) from latently infected cells via transcriptional activation of the viral immediate‐early gene BZLF1. BZLF1 is a member of the extended AP‐1 family of transcription factors that binds to specific BZLF1‐binding motifs within early EBV promoters and to consensus AP‐1 sites. Regulation of BZLF1's activity is achieved at the transcriptional level as well as through post‐translational modifications. Recently, we reported that the transcriptional activity of BZLF1 is augmented by TPA [Baumann, M., Mischak, H., Dammeier, S., Kolch, W., Gires, O., Pich, D., Zeidler, R., Delecluse, H. J. & Hammerschmidt, W., (1998) J. Virol.72, 8105–8114]. The increase of BZLF1's activity depends on a single serine residue (S186) that is phosphorylated by protein kinase C (PKC) in vitro and in vivo after stimulation with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Here, we identified RACK1 as a binding partner of BZLF1 in a yeast interaction trap assay. RACK stands for receptor of activated C‐kinase and is involved in targeting activated PKCs and other signaling proteins. In vivo, RACK1 binds directly to the transactivation domain of BZLF1. Although a functional relationship between BZLF1 and PKC could be mediated by RACKs, RACK1 did not have a detectable effect on the phosphorylation status of BZLF1 in in vitro or in vivo phosphorylation assays. We suggest that RACK1 may act as a scaffolding protein on BZLF1 independently of activated PKCs.
The structure of the carbohydrate backbone of the lipopolysaccharides from Bordetella hinzii and Bordetella bronchiseptica
Authors: Vinogradov Evgeny 
Published: March 2003

The structure of the core‐lipid A region of the lipopolysaccharides from Bordetella hinzii and Bordetella bronchiseptica has been analyzed. Lipopolysaccharides were deacylated using strong alkaline hydrolysis, the products were separated by high performance anion‐exchange chromatography and analyzed by NMR and mass spectrometry. The following structure of the products can be deduced from the experimental results:where for the product from Bordetella hinzii N = H, R = H, β‐FucN4N‐ or partially N‐acetylated Sug‐(1–3)‐β‐FucN4N and for the product from Bordetella bronchiseptica N = α‐Hep, R = H, β‐FucN4N, β‐FucN4NMe or partially N‐acetylated Sug‐(1–3)‐β‐FucN4N or Sug‐(1–3)‐β‐FucN4NMe; Sug = 2,3‐diamino‐2,3,4‐trideoxy‐hex‐4‐enuronopyranosyl.
UV‐A‐related alterations of young and adult lens water‐insoluble α‐crystallin, plasma membranous and cytoskeletal proteins
Authors: Weinreb O. , Dovrat A. , Dunia I. , Benedetti E. L. , Bloemendal H. 
Published: March 2003

The damaging effects of UV‐A irradiation on lens water‐insoluble α‐crystallin, plasma membranous and cytoskeletal proteins derived from bovine lenses were studied. Young and adult bovine lenses were kept viable for 2 months in organ culture. After 24 h of incubation they were irradiated, and analyses of the proteins by one‐dimensional and two‐dimensional gel electrophoresis followed by Western blotting were carried out at several time intervals. RNA isolation, PCR and Northern blotting were also performed. We identified age‐related changes in water‐insoluble α‐crystallin, the major membrane protein MP26 and the cytoskeletal proteins vimentin, phakinin and actin between control and UV‐irradiated lenses. It appeared that adult lenses are more susceptible to UV light than young lenses, and protein modification occurred more frequently in adult lenses. UV‐A irradiation affects not only the cytoskeletal structure, as deduced by the abnormal arrangement of actin in the fiber cells, but also leads to degradation of actin mRNA. Furthermore, analysis of the expression of hsp25 and hsp70 revealed some alteration in the protein pattern of adult lenses. We suggest that degradation of the cytoskeletal proteins following irradiation is due to, at least in part, the decreased protective ability of heat shock proteins upon aging.
The protein kinase Cdk5
Authors: Maccioni Ricardo B. , Otth Carola , Concha Ilona I. , Muñoz Juan P. 
Published: March 2003

A set of different protein kinases have been involved in tau phosphorylations, including glycogen synthase kinase 3β (GSK3β), MARK kinase, MAP kinase, the cyclin‐dependent kinase 5 (Cdk5) system and others. The latter system include the catalytic component Cdk5 and the regulatory proteins p35, p25 and p39. Cdk5 and its neuron‐specific activator p35 are essential molecules for neuronal migration and for the laminar configuration of the cerebral cortex. Recent evidence that the Cdk5/p35 complex concentrates at the leading edge of axonal growth cones, together with the involvement of this system in the phosphorylation of neuronal microtubule‐asociated proteins (MAPs), provide further support to the role of this protein kinase in regulating axonal extension in developing brain neurons. Although the aminoacid sequence of p35 has little similarity with those of normal cyclins, studies have shown that its activation domain may adopt a conformation of the cyclin‐folded structure. The computed structure for Cdk5 is compatible with experimental data obtained from studies on the Cdk5/p35 complex, and has allowed predictions on the protein interacting domains. This enzyme exhibits a wide cell distribution, even though a regulated Cdk5 activity has been shown only in neuronal cells. Cdk5 has been characterized as a proline‐directed Ser/Thr protein kinase, that contributes to phosphorylation of human tau on Ser202, Thr205, Ser235 and Ser404. Cdk5 is active in postmitiotic neurons, and it has been implicated in cytoskeleton assembly and its organization during axonal growth. In addition to tau and other MAPs, Cdk5 phosphorylates the high molecular weight neurofilament proteins at their C‐terminal domain. Moreover, nestin, a protein that regulates cytoskeleton organization of neuronal and muscular cells during development of early embryos, and several other regulatory proteins appear to be substrates of Cdk5 and are phosphorylated by this kinase. Studies also suggest, that in addition to Cdk5 involvement in neuronal differentiation, its activity is induced during myogenesis, however, the mechanisms of how this activity is regulated during muscular differentiation has not yet been elucidated. Recent studies have shown that the β‐amyloid peptide (Aβ) induces a deregulation of Cdk5 in cultured brain cells, and raises the question on the possible roles of this tau‐phosphorylating protein kinase in the sequence of molecular events leading to neuronal death triggered by Aβ. In this context, there are evidence that Cdk5 is involved in tau hyperphosphorylation promoted by Aβ in its fibrillary form. Cdk5 inhibitors protect hippocampal neurons against both tau anomalous phosphorylations and neuronal death. The links between the studies on the Cdk5/p35 system in normal neurogenesis and its claimed participation in neurodegeneration, provide the framework to understand the regulatory relevance of this kinase system, and changes in its regulation that may be implicated in disturbances such as those occurring in Alzheimer disease.
Palmitate uptake by neonatal rat myocytes and hepatocytes
Authors: Elmadhoun Bassam M. , Wang Gu Qi , Kirshenbaum Lorrie A. , Burczynski Frank J. 
Published: March 2003

The role of extracellular binding proteins in the rate of [3H]palmitate uptake by neonatal cardiac myocytes and hepatocytes was investigated using a model‐independent approach. Binding proteins used in this study included α1‐acid glycoprotein [isoelectric point (pI) ≈ 2.7], conalbumin (pI ≈ 6.4), lysozyme (pI ≈ 11.0), albumin (pI ≈ 4.9), and albumin which had been modified to yield proteins with pI values of 3.5, 4.7, 7.5 and 8.6. All uptake studies were conducted at similar unbound ligand fractions. There was a linear relationship between the rate of neonatal hepatocyte [3H]palmitate clearance and protein pI (r2 = 0.98). In contrast, there was an overall poor relationship between neonatal cardiac myocyte [3H]palmitate‐clearance rate and protein pI (r2 = 0.48). However, the relationship improved when the data on [3H]palmitate‐clearance were analyzed using only the modified albumins. The study indicates that an ionic interaction between extracellular proteins and the hepatocyte surface enhances the overall uptake of [3H]palmitate. This interaction may be limited to albumin for neonatal cardiac myocytes.
The N‐terminal portion of the preToc75 transit peptide interacts with membrane lipids and inhibits binding and import of precursor proteins into isolated chloroplasts
Authors: Inoue Kentaro , Demel Rudy , de Kruijff Ben , Keegstra Kenneth 
Published: March 2003

Toc75 is an outer envelope membrane protein of chloroplasts. It is unusual among the outer membrane proteins in that its precursor form has a bipartite transit peptide. The N‐terminal portion of the Toc75 transit peptide is sufficient to target the protein to the stromal space of chloroplasts. We prepared a 45 amino‐acid peptide containing the stromal targeting domain of the Toc75 transit peptide in Escherichia coli, using the intein‐mediated system, and purified it by reverse‐phase HPLC. Its identity was confirmed by N‐terminal amino‐acid sequencing and matrix assisted laser desorption ionization mass spectrometry. In monolayer experiments, the peptide inserted into the chloroplastic membrane lipids sulfoquinovosyl diacylglycerol and phosphatidylglycerol and into a nonchloroplastic lipid phosphatidylethanolamine. However, it did not insert into other chloroplastic lipids, such as mono‐ and digalactosyl diacylglycerol, and phosphatidylcholine. Furthermore, the peptide significantly inhibited binding of radiolabeled precursors of Toc75 and the small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase to intact chloroplasts as effectively as did a bacterially produced precursor of the small subunit of 1,5‐bisphosphate carboxylase/oxygenase. The peptide also inhibited import of radiolabeled precursors into isolated chloroplasts, however, to a lesser extent than did nonlabeled precursor of the small subunit of 1,5‐bisphosphate carboxylase/oxygenase.
Spectroscopic characterization of metallothionein from the terrestrial snail, Helix pomatia
Authors: Dallinger Reinhard , Wang Yunjuan , Berger Burkhard , Mackay Elaine A. , Kägi Jeremias H. R. 
Published: March 2003

The Cd‐sequestering metallothionein (MT) isoform isolated from the midgut gland of Roman snails exposed to Cd supplements in the feed was characterized by compositional and spectroscopic analysis. The preparations contained nearly 5 mol of Cd, small amounts of Cu and about 1 mol of Zn per chain mass of 6620 Da, in numerical agreement with the apoprotein's measured capacity of firmly binding a maximum of 6 equivalents of Cd per molecule. As with other Cd‐containing MTs the occurrence of a prominent Cd‐mercaptide‐specific shoulder at 250 nm in its absorption spectrum showed that Cd is complexed in tetrahedral symmetry by the cysteine residues of the protein, and the multiphasic ellipticity profile in the CD spectrum revealed that these complexes are joined to form one or more oligonuclear Cd‐mercapto clusters. Both spectral features vanished with the removal of the metal but were reconstituted to maximum amplitudes by readdition of Cd to the metal‐free apoprotein, provided precautions were taken to prevent air oxidation of the latter. Quantitative analysis of snail MT reconstituted with Cd established that the 18 cysteine side chains bind the metal in a 3‐to‐1 ratio; spectroscopic studies on fractionally restored forms demonstrated that the six Cd ions were bound to the apoprotein molecule in succession in two sets of three Cd ions each. Thus, one can infer from the observed stoichiometry and the coordinating preferences of Cd that this gastropod MT, like the Cd‐bearing MTs of marine crustaceans, harboured the metal in two separate cyclically constructed Cd3Cys9 clusters. The snail clusters differeed, however, from other MTs in their response to acidification. Their protolytic dissociation proceeded through two separate protonation steps with the manifestation of spectroscopically distinguishable intermediate forms. Thus, this snail isoform displays in its metal composition and its chemical and spectroscopic features both similarities and differences to other animal kingdom MTs. Its properties suggest that it serves an important role in the protection of the terrestrial gastropod from Cd.
Identification of protein‐coding genes in the genome of Vibrio cholerae with more than 98% accuracy using occurrence frequencies of single nucleotides
Authors: Wang Ju , Zhang Chun‐Ting 
Published: March 2003

The published sequence of the Vibrio cholerae genome indicates that, in addition to the genes that encode proteins of known and unknown function, there are 1577 ORFs identified as conserved hypothetical or hypothetical gene candidates. Because the annotation is not 100% accurate, it is not known which of the 1577 ORFs are true protein‐coding genes. In this paper, an algorithm based on the Z curve method, with sensitivity, specificity and accuracy greater than 98%, is used to solve this problem. Twenty‐fold cross‐validation tests show that the accuracy of the algorithm is 98.8%. A detailed discussion of the mechanism of the algorithm is also presented. It was found that 172 of the 1577 ORFs are unlikely to be protein‐coding genes. The number of protein‐coding genes in the V. cholerae genome was re‐estimated and found to be ≈ 3716. This result should be of use in microarray analysis of gene expression in the genome, because the cost of preparing chips may be somewhat decreased. A computer program was written to calculate a coding score called VCZ for gene identification in the genome. Coding/noncoding is simply determined by VCZ > 0/VCZ < 0. The program is freely available on request for academic use.
Structure of the O‐specific polysaccharide of Proteus mirabilis D52 and typing of this strain to Proteus serogroup O33
Authors: Zych Krystyna , Toukach Filip V. , Arbatsky Nikolay P. , Kołodziejska Katarzyna , Senchenkova Sof'ya N. , Shashkov Alexander S. , Knirel Yuriy A. , Sidorczyk Zygmunt 
Published: March 2003

The acidic O‐specific polysaccharide chain (O‐antigen) of the lipopolysaccharide (LPS) of Proteus mirabilis strain D52 was studied using chemical analyses along with 1H‐NMR and 13C‐NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H‐detected 1H,13C and 1H,31P HMQC experiments. The polysaccharide was found to contain d‐ribitol 5‐phosphate (d‐Rib‐ol‐5‐P) and ethanolamine phosphate (Etn‐P) and has the following structureThis structure is identical with that of the O‐polysaccharide of P. mirabilis O33 strain 59/57, and, hence, P. mirabilis D52 belongs to the same Proteus serogroup O33. Serological studies with O‐antiserum against P. mirabilis D52 confirmed this but showed that the LPS species of P. mirabilis 59/57 and D52 are not identical, having different epitopes in the core region. A serological cross‐reactivity of P. mirabilis D52 O‐antiserum was observed with LPS of two other Proteus strains, P. mirabilis O16 and P. penneri 103, which have structurally different O‐polysaccharides. The role of charged groups, Rib‐ol‐5‐P and Etn‐P in the immunospecificity is discussed.