bluuebunny/crossref_metadata_2025_split huggingface dataset:
31P‐NMR spectroscopy of human and Paracoccus denitrificans electron transfer flavoproteins, and 13C‐ and 15N‐NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states
Authors: Griffin Kurt J. , Degala Gregory D. , Eisenreich Wolfgang , Müller Franz , Bacher Adelbert , Frerman Frank E. 
Published: March 2003

Human and Paracoccus denitrificans wild‐type electron transfer flavoproteins have been investigated by 31P‐NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein‐bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin−NADP+ reductase and, to a lesser degree, with those of NADPH−cytochrome P‐450 reductase.The wild‐type human electron transfer apoprotein was reconstituted with [2,4a‐13C2]FAD, [4,10a‐13C2]FAD, or [U‐15N4]FAD. The reconstituted proteins were studied by 13C‐ and 15N‐NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of flavoproteins published in the literature.In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X‐ray data, involves the C′(4)‐OH group of FAD. The sp2 hybridisation of N(10) is small compared to other flavoproteins.In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C′(4)‐OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein‐bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other flavoproteins.
Structure determination for octasaccharides derived from the carbohydrate−protein linkage region of chondroitin sulphate chains in the proteoglycan aggrecan from bovine articular cartilage
Authors: Huckerby Thomas N. , Lauder Robert M. , Nieduszynski Ian A. 
Published: March 2003

Five octasaccharides derived from the protein carbohydrate linkage region of chondroitin sulphate (CS) have been isolated from the large aggregating proteoglycan (aggrecan) extracted from the bovine articular cartilage of 6‐year‐old to 8‐year‐old animals. Following the purification of aggrecan the attached CS chains were digested with CS ABC endolyase and subsequently released from the protein core by β‐elimination. The individual oligosaccharides were purified by strong anion‐exchange chromatography and their structures determined by very high‐field one‐dimensional and two‐dimensional 1H‐NMR spectroscopy. They were found to be octasaccharides, comprised of tetrasaccharide repeat‐region extensions to the core tetrasaccharide linkage region structure. They have the following structures :

ΔUA(β1−3)GalNAc(β1−4)GlcA(β1−3)GalNAc(β1−4)GlcA(β1−3)Gal(β1−3)Gal(β1−4)Xyl‐ol,
ΔUA(β1−3)GalNAc(β1−4)GlcA(β1−3)GalNAc6S(β1−4)GlcA(β1−3)Gal(β1−3)Gal(β1−4)Xyl‐ol,
ΔUA(β1−3)GalNAc6S(β1−4)GlcA(β1−3)GalNAc(β1−4)GlcA(β1−3)Gal(β1−3)Gal(β1−4)Xyl‐ol,
ΔUA(β1−3)GalNAc6S(β1−4)GlcA(β1−3)GalNAc6S(β1−4)GlcA(β1−3)Gal(β1−3)Gal(β1−4)Xyl‐ol
and
ΔUA(β1−3)GalNAc4S(β1−4)GlcA(β1−3)GalNAc6S(β1−4)GlcA(β1−3)Gal(β1−3)Gal(β1−4)Xyl‐ol.They differ only in the nature of the sulphation of the GalNAc residues of the tetrasaccharide‐repeat‐region extension, which forms the first two disaccharides of the repeat region. No sulphation of any of the uronic acid residues has been identified and in one oligosaccharide neither of the GalNAc residues were sulphated. The majority of the linkage regions contained GalNAc residues which were fully 6‐sulphated. However, in a significant amount, only one of the residues was 6‐sulphated while the other was either unsulphated or 4‐sulphated. There was no evidence either for sulphation of the linkage region galactose residues or for phosphorylation of the xylose residue, through which the chain is attached to the core protein.
Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C
Authors: Benen Jacques A. E. , Kester Harry C. M. , Visser Jaap 
Published: March 2003

Endopolygalacturonases I, II and C isolated from recombinant Aspergillus niger strains were characterized with respect to pH optimum, activity on polygalacturonic acid and mode of action and kinetics on oligogalacturonates of different chain length (n = 3–7). Apparent Vmax values using polygalacturonate as a substrate at the pH optimum, pH 4.1, were calculated as 13.8 µkat·mg–1, 36.5 µkat·mg–1 and 415 nkat·mg–1 for endopolygalacturonases I, II and C, respectively. Km values were < 0.15 mg·mL–1 for all three enzymes. Product progression analysis using polygalacturonate as a substrate revealed a random cleavage pattern for all three enzymes and suggested processive behavior for endopolygalacturonases I and C. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 5 or 6 for endopolygalacturonase I and endopolygalacturonase C, respectively. The bond‐cleavage frequencies obtained for the hydrolysis of the oligogalacturonates were used to assess subsite maps. The maps indicate that the minimum number of subsites is seven for all three enzymes. Using pectins of various degrees of esterification, it was shown that endopolygalacturonase II is the most sensitive to the presence of methyl esters. Like endopolygalacturonase II, endopolygalacturonases I, C and E, which was also included in this part of the study, preferred the non‐esterified pectate. Additional differences in substrate specificity were revealed by analysis of the reaction products of hydrolysis of a mixture of pectate lyase‐generated Δ4,5‐unsaturated oligogalacturonates of degree of polymerization 4–8. Whereas endopolygalacturonase I showed a strong preference for generating the Δ4,5‐unsaturated dimer, with endopolygalacturonase II the Δ4,5‐unsaturated trimer accumulated, indicating further differences in substrate specificity. For endopolygalacturonases C and E both the Δ4,5‐unsaturated dimer and trimer were observed, although in different ratios.
Structure of the O‐specific polysaccharide of a serologically separate strain Proteus penneri 2 from a new proposed serogroup O66
Authors: Arbatsky Nikolay P. , Shashkov Alexander S. , Toukach Filip V. , Moll Hermann , Zych Krystyna , Knirel Yuriy A. , Zähringer Ulrich , Sidorczyk Zygmunt 
Published: March 2003

O‐specific polysaccharide chain of Proteus penneri strain 2 lipopolysaccharide was studied by full and partial acid hydrolysis, Smith degradation, methylation analysis, and NMR spectroscopy, including two‐dimensional rotating‐frame NOE spectroscopy (ROESY) and 1H,13C heteronuclear multiple‐quantum coherence (HMQC) experiments. Together with d‐glucose and 2‐acetamido‐2‐deoxy‐d‐glucose, the polysaccharide was found to contain two rarely occurring sugars, 6‐deoxy‐l‐talose (L‐6dTal) and 2,3‐diacetamido‐2,3,6‐trideoxy‐l‐mannose (L‐RhaNAc3NAc), and the following structure of a non‐stoichiometrically O‐acetylated tetrasaccharide repeating unit was established:The O‐specific polysaccharide studied has a unique composition and structure and, accordingly, P. penneri 2 is serologically separate among Proteus strains. Therefore, we propose for P. penneri 2 a new Proteus O‐serogroup O66 where this strain is at present the single representative.
Mode of action of chitin deacetylase from Mucor rouxii on N‐acetylchitooligosaccharides
Authors: Tsigos Iason , Zydowicz Nathalie , Martinou Aggeliki , Domard Alain , Bouriotis Vassilis 
Published: March 2003

The mode of action of chitin deacetylase from the fungus Mucor rouxii on N‐acetylchitooligosaccharides with a degree of polymerization 1–7 has been elucidated. Identification of the sequence of chitin oligomers following enzymatic deacetylation was verified by the alternative use of two specific exo‐glycosidases in conjunction with HPLC. The results were further verified by 1H‐NMR spectroscopy. It was observed that the length of the oligomer is important for enzyme action. The enzyme cannot effectively deacetylate chitin oligomers with a degree of polymerization lower than three. Tetra‐N‐acetylchitotetraose and penta‐N‐acetylchitopentaose are fully deacetylated by the enzyme, while in the case of tri‐N‐acetylchitotriose, hexa‐N‐acetylchitohexaose and hepta‐N‐acetylchitoheptaose the reducing‐end residue always remains intact. Furthermore, the enzyme initially removes an acetyl group from the nonreducing‐end residue of all chitin oligomers with a degree of polymerization higher than 2, and further catalyses the hydrolysis of the following acetamido groups in a processive fashion. The results are in agreement with the mode of action that the same enzyme exhibits on partially deacetylated water soluble chitosan polymers.
Structural and membrane‐binding properties of saposin D
Authors: Tatti Massimo , Salvioli Rosa , Ciaffoni Fiorella , Pucci Piero , Andolfo Annapaola , Amoresano Angela , Vaccaro Anna Maria 
Published: March 2003

Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor, called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has been reported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yet been clearly established. In the present study we examined structural and membrane‐binding properties of saposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionic phospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization of the lipid surface and, conversely, the association with the membrane markedly affected the fluorescence properties of saposin D. The presence of phosphatidylserine‐containing vesicles greatly enhanced the intrinsic tyrosine fluorescence of saposin D, which contains tyrosines but not tryptophan residues. The structural properties of saposin D were investigated in detail using advanced MS analysis. It was found that the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked in the following order: Cys5–Cys78, Cys8–Cys72 and Cys36–Cys47. The disulfide pattern of saposin D is identical with that previously established for two other saposins, B and C, which also exhibit a strong affinity for lipids. The common disulfide structure probably has an important role in the interaction of these proteins with membranes. The analysis of the sugar moiety of saposin D revealed that the single N‐glycosylation site present in the molecule is mainly modified by high‐mannose‐type structures varying from two to six hexose residues. Deglycosylation had no effect on the interaction of saposin D with phospholipid membranes, indicating that the glycosylation site is not related to the lipid‐binding site. The association of saposin D with membranes was highly dependent on the composition of the bilayer. Neither ceramide nor sphingomyelin, sphingolipids whose hydrolysis is favoured by saposin D, promoted its binding, while the presence of an acidic phospholipid such as phosphatidylserine or phosphatidic acid greatly favoured the interaction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles where saposins are localized, anionic phospholipids may be determinants of the saposin D topology and, conversely, saposin D may affect the lipid organization of anionic phospholipid‐containing membranes.
Characterization of the ternary complex between Rab7, REP‐1 and Rab geranylgeranyl transferase
Authors: Alexandrov Kirill , Simon Iris , Yurchenko Vyacheslav , Iakovenko Andrei , Rostkova Elena , Scheidig Axel J. , Goody Roger S. 
Published: March 2003

Geranylgeranylation is a post‐translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to the Rab geranylgeranyl transferase (RabGGTase) which covalentely modifies the Rab protein with two geranylgeranyl moieties. After prenylation, the Rab protein remains in complex with REP and is delivered to the target membrane by the latter. In this work, we show that RabGGTase can form a stable complex with Rab7–REP in the absence of its lipid substrate geranylgeranyl pyrophosphate. In order to characterize this interaction, we developed three fluorescence assays reporting on the interaction of RabGGTase with the Rab7–REP complex. For this interaction we determined a Kd value of about 120 nm. Association of RabGGTase with the Rab7–REP complex occurs with a rate constant of ≈ 108 m−1·s−1. We demonstrate that the state of the nucleotide bound to Rab7 does not influence the affinity of RabGGTase for the Rab7–REP‐1 complex. Finally, we address the issue of substrate specificity of RabGGTase. Titration experiments demonstrate that, in contrast with farnesyl transferase, RabGGTase does not recognize a defined C‐terminal sequence motif. Experiments using Rab7 mutants in which the last 16 amino acids were either mutated or truncated revealed that the distal part of the C‐terminus makes only a limited contribution to the binding affinity between RabGGTase and the Rab7–REP‐1 complex. This demonstrates the functional dissimilarity between RabGGTase and geranylgeranyl transferase I and farnesyl transferase, which interact specifically with the C‐terminus of their substrates. Based on these experiments, we propose that RabGGTase recognizes the overall structure arising from the association of Rab and REP and then ‘scans’ the flexible C‐terminus to position the proximal cysteines into the active site.
The responses of rat hepatocytes to glucagon and adrenaline
Authors: Ainscow Edward K. , Brand Martin D. 
Published: March 2003

The internal control of hepatocyte metabolism has been previously analysed using metabolic control analysis. The aim of this paper is to extend this analysis to include the responses of the cells to hormonal stimulus. Hepatocyte metabolism was divided into nine reaction blocks: glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, proton leak, mitochondrial phosphorylation and ATP consumption, linked by five intermediates: mitochondrial membrane potential, cytoplasmic NADH/NAD and total cellular ATP, glucose 6‐phosphate and pyruvate. The kinetic responses of the reaction blocks to the intermediates were determined previously in the absence of added hormones. In this study, the changes in flux and intermediate levels that occurred upon addition of either glucagon or adrenaline were measured. From comparison of the fractional changes in fluxes and intermediate levels with the known kinetics of the system, it was possible to determine the primary sites of action of the hormones. The results show that the majority of processes in the cell are responsive to the hormones. The notable exception to this is the failure of adrenaline to have a direct effect on glycolysis. The activity change of each metabolic block observed in the presence of either hormone was quantified and compared to the indirect effects on each block caused by changes in metabolite levels. The second stage of the analysis was to use the calculated activity changes and the known control pattern of the system to give a semiquantitative analysis of the regulatory pathways employed by the hormones to achieve the changes in fluxes and metabolite levels. This was instructive in analysing, for example, how glucagon caused a decrease in flux through glycolysis and an increase in oxidative phosphorylation without large changes in metabolite levels (homeostasis). Conversely, it could be seen that the failure of adrenaline to maintain a constant glucose 6‐phosphate concentration was due to the stimulation of glycogen breakdown and inhibition of glucose release.
Structural studies utilizing 13C‐enrichment of the O‐antigen polysaccharide from the enterotoxigenic Escherichia coli O159 cross‐reacting with Shigella dysenteriae type 4
Authors: Linnerborg Malin , Weintraub Andrej , Widmalm Göran 
Published: March 2003

The structure of the O‐antigen polysaccharide from Escherichia coli O159 has been determined using primarily NMR spectroscopy of the 13C‐enriched polysaccharide. The sequence of the sugar residues could be determined by heteronuclear multiple bond connectivity NMR experiments. The polysaccharide is composed of a pentasaccharide repeating unit with the following structure:Matrix assisted laser desorption ionization mass spectrometry was performed on intact lipopolysaccharide and from the resulting molecular mass the O‐antigen part was estimated to contain approximately 23 repeating units. Cross‐reactivity of this O‐antigen to that of Shigella dysenteriae type 4 was confirmed using enzyme‐linked immunoabsorbant assay.
Two proteins, a goldfish 20S proteasome subunit and the protein interacting with 26S proteasome, change in the meiotic cell cycle
Authors: Tokumoto Mika , Horiguchi Ryo , Nagahama Yoshitaka , Ishikawa Katsutoshi , Tokumoto Toshinobu 
Published: March 2003

To investigate the regulatory mechanism for the proteasome in the meiotic cell cycle, we purified the 26S proteasome from immature (in G2‐phase) and mature (in M‐phase) oocytes, and compared its subunits by immunoblotting. At least two protein bands, at 30 kDa (detected by GC3β antibody) and 62 kDa (detected by 1‐4D5 antibody), differed between 26S proteasomes. A monoclonal antibody, GC3β cross‐reacted with two bands in the 26S proteasome from immature oocytes, however, the upper band was absent in the 26S proteasome from mature oocytes. The 62‐kDa protein band detected by 1‐4D5 antibody was not detected in the immature oocyte 26S proteasome; however, a band was detected in mature oocyte 26S proteasome. The cDNAs encoding these proteins were isolated by an immunoscreening method using the monoclonal antibodies. The 30‐kDa protein was an α4 subunit, which is one of the α‐subunit group of the 20S proteasome, and the 62‐kDa protein was a homologue of CCTε, one of the components of eukaryotic molecular chaperones. Phosphatase treatment of the 26S proteasome revealed that a part of the α4 subunit of goldfish 20S proteasome, α4_ca, is phosphorylated in G2‐phase and dephosphorylated in M‐phase. A binding assay using a recombinant goldfish CCTε revealed that unmodified CCTε interacts with the 26S proteasome.Fertilization triggers a transition from meiotic metaphase to mitotic interphase. During fertilization, a GC3β cross‐reacting upper band reappeared. The 62‐kDa band dissociated from the 26S proteasome. As a result, the 26S proteasome changed to an immature type from a mature type during fertilization. These results suggest that the 26S proteasome is changed reversibly during the meiotic cell cycle by modification of its subunits and interactions between regulators.
Characterization of an α‐macroglobulin‐like glycoprotein isolated from the plasma of the soft tick Ornithodoros moubata
Authors: Kopáček Petr , Weise Christoph , Saravanan Thangamani , Vítová Kateřina , Grubhoffer Libor 
Published: March 2003

We report the identification of the first representative of the α‐2‐macroglobulin family identified in terrestrial invertebrates.An abundant acidic glycoprotein was isolated from the plasma of the soft tick Ornithodoros moubata. Its molecular mass is about 420 kDa in the native state, whereas in SDS/PAGE it migrates as one band of 190 kDa under nonreducing conditions and a band of 92 kDa when reduced. Chemical deglycosylation reveals that it is composed of two different subunits, designated A and B.The N‐terminal amino‐acid sequence of subunit A is similar to the N‐terminus of invertebrate α‐2‐macroglobulin. Sequence analysis of several internal peptides confirms that the tick protein belongs to the α‐2‐macroglobulin family, and the protein is therefore referred to as tick α‐macroglobulin (TAM). Functional analyses strengthen this assignment. TAM inhibits trypsin and thermolysin cleavage of the high‐molecular‐weight substrate azocoll in a manner similar to that of bovine α‐2‐macroglobulin. This effect is abolished by pre‐treatment of TAM with methylamine. In the presence of TAM, trypsin is protected against active‐site inhibition by soybean trypsin inhibitor.We cloned and sequenced a PCR product containing sequences from both subunits and spanning the N‐terminus of subunit B and the putative ‘bait region’ (a segment of α‐2‐macroglobulin which serves as target for various proteases). This indicates that the two subunits are generated from a precursor polypeptide by post‐translational processing.
Alkaline phosphatase from the Antarctic strain TAB5
Authors: Rina Maria , Pozidis Charalambos , Mavromatis Konstantinos , Tzanodaskalaki Maria , Kokkinidis Michael , Bouriotis Vassilis 
Published: March 2003

The gene encoding alkaline phosphatase (AP) from the psychrophilic strain TAB5 was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1125 base pairs which encodes a polypeptide consisting of signal peptide of 22 amino acids and a mature protein of 353 amino acids was identified. The deduced protein sequence of AP exhibits a 38% identity to the AP III and AP IV sequences of Bacillus subtilis and conserves the typical sequence motifs of the core structure and active sites of APs from various sources. Based on the crystal structure of the mutated Escerichia coli AP D153H, a homology‐based 3D model of the TAB5 AP was constructed on the basis of which various features of the enzyme amino‐acid sequence can be interpreted in terms of potential psychrophilic adaptations.The AP gene was expressed in E. coli BL21(DE3) cells, the recombinant protein was isolated to homogeneity from the membrane fraction of the cells and its properties were examined. The purified TAB5 AP shows typical features of a cold enzyme: high catalytic activity at low temperature and a remarkable thermosensitivity.The use of this heat‐labile enzyme, for dephosphorylation of nucleic acids, simplifies dephosphorylation protocols.