bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determination of the orientations of tryptophan analogues bound to the trp repressor and the relationship to activation
Authors: BORDEN Katherine L. B. , BECKMANN Pamela , LANE Andrew N. 
Published: March 2005

The antirepressor indole 3‐propanoate has been shown by X‐ray crystallography to bind in a different orientation compared with the natural corepressor for the trp repressor, l‐tryptophan (Lawson, C. L. & Sigler, P. B. (1988) Nature 333, 869–871). This suggests a simple difference between what constitutes a corepressor versus an antirepressor.We have used visible absorption and 1H‐NMR spectroscopy to characterise the nature of several ligand‐repressor complexes and DNA‐binding assays to assess the relative operator binding affinities. 5‐Fluorotryptophan binds with similar affinity and in the same orientation as l‐tryptophan, and is an equally effective corepressor. In contrast, the tight‐binding antirepressor indole 3‐acrylate binds in the same orientation as indole 3‐propanoate. Indole, also an antirepressor, also binds in the indole‐3‐propanoate orientation. 5‐Methyltryptamine, a corepressor, shows spectroscopic characteristics of both tryptophan and indoleacrylate, though NOEs indicate that the tryptophan orientation is preferred. These results indicate that the ammonium group in the side chain is essential both for activation and binding in the l‐tryptophan orientation. Antirepressors, lacking the ammonium group, bind in the more favourable indole‐3‐propanoate orientation.Differences in the NMR signatures of the different repressor‐ligand complexes indicate that the details of the conformations depend on the nature of the ligands and their orientation within the binding site. Despite any conformational rearrangement of the protein on binding, dissociation of ligands is facile: 5‐fluorotryptophan dissociates rapidly at 313 K. These findings complement and extend the X‐ray and thermodynamic analyses of ligand binding.
Heparinase II from Flavobacterium heparinum
Authors: MOFFAT Colin F. , McLEAN Maitland W. , LONG William F. , WILLIAMSON Frank B. 
Published: March 2005

Saccharides produced by the action of heparinase II on native pig mucosal heparin (heparin Is), de‐N‐sulphated heparin (heparin Ih), N‐acetylheparin (heparin Ia), de‐N/O‐sulphated heparin (heparin IVh), de‐O‐sulphated heparin (heparin IVs) and de‐O‐sulphated N‐acetylheparin (heparin IVa) were analysed by reversed‐phase HPLC using Spherisorb ODS2. Fractions obtained by gel filtration with Bio‐Gel P‐4 were similarly examined.Heparin Is gave ΔUA‐2S → GlcNS‐6S (Is) as the major unsaturated disaccharide and lesser amounts of ΔUA → GlcNS‐6S (IIs), ΔUA‐2S → GlcNS (IIIs), ΔUA → GlcNS (IVs), ΔUA‐2S → GlcNAc‐6S (Ia), ΔUA → GlcNAc‐6S (IIa), ΔUA‐2S → GlcNAc (IIIa) and ΔUA → GlcNAc (IVa).Heparins Ia, IVa and IVs gave as the predominant unsaturated disaccharide that corresponding to the major repeat structure of the polymer. These were respectively ΔUA‐2S → GlcNAc‐6S (Ia), ΔUA → GlcNAc (IVa) and ΔUA → GlcNS (IVs). Minor disaccharides from the heterogeneous structure in native pig heparin and from residual O‐sulphates after the de‐O‐sulphating process were detected.Heparin Ih was degraded more slowly than any of the N‐substituted heparins. The predominant unsaturated disaccharide was Ih, which was derived from the major repeating unit. In addition, disaccharides IIh, IIIh, Ia, IIa and IVa were detected.Heparin IVh showed little degradation, the unsaturated disaccharide IVh not being detected after 24 h. Disaccharide IVa was obtained from the heterogeneous sequence in heparin IVh.Several higher oligosaccharides were identified in the gel‐filtration fractions including saccharides from the linkage region (for heparin Is and IVa) and the anti‐thrombin binding site (for heparin Is only). A tetrasaccharide and hexasaccharide, with the structures ΔUA → GlcNAc → UA → GlcNAc and ΔUA → GlcNAc → UA → GlcNAc → UA → GlcNAc, were present in the HPLC profiles of heparins Ia and IVa.
Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria
Authors: RASMUSSON Allan G. , MØLLER Ian M. 
Published: March 2005

Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone‐insensitive internal oxidation of NADPH by inside‐out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD+‐dependent and NADP+‐dependent malate dehydrogenases were affected by the addition of Ca2+.The pH dependence and kinetics of Ca2+‐dependent NADPH oxidation by inside‐out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2.Half‐maximal stimulation of the NADPH oxidation was found at 3 μM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane‐bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+.Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone‐insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.
Characterization of a camptothecin‐resistant human DNA topoisomerase I in an in vitro system for Simian virus 40 DNA replication
Authors: ISHIMI Yukio , NISHIZAWA Miwako , ANDOH Toshiwo 
Published: March 2005

DNA topoisomerase I was required for bidirectional DNA replication in an in vitro system for Simian virus 40 (SV40) DNA replication with purified proteins in which the replication fork moved at the rate of 260 nucleotides/min on average. DNA topoisomerase I purified from camptothecin‐resistant human lymphoblastoid cells, which confers high resistance of cellular DNA replication to camptothecin [Andoh, T., Ishii, K., Suzuki, Y., Ikegami, Y., Kusunoki, Y., Takemoto, Y. & Okada, K. (1987) Proc. Natl Acad. Sci. USA 84, 5565–5569], was characterized using this system. The activity of stimulating bidirectional DNA replication was comparable between two topoisomerase I from parental and resistant cells, i.e. in its dose‐response relationship and in its time course for DNA synthesis.Camptothecin severely inhibited the leading as well as the lagging strand synthesis in the reaction containing the wild type topoisomerase I but not the mutant type topoisomerase I. The mutant type topoisomerase I was over 125‐fold as resistant to camptothecin as the wild type topoisomerase I. These results are in good agreement with those on the sensitivity of cellular DNA synthesis to camptothecin in the resistant cells. These findings suggest that topoisomerase I is involved in cellular DNA replication as a swivelase and the mutation conffering camptothecin‐resistance on the enzyme does not affect its functional efficiency in this system.
Lipoamide Dehydrogenase from Azotobacter vinelandii: site‐directed mutagenesis of the His450‐Glu455 diad
Authors: BENEN Jacques , van BERKEL Willem , ZAK Zdzislaw , VISSER Ton , VEEGER Cees , de KOK Arie 
Published: March 2005

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced by other residues. His450, the active‐site base, was changed into Ser, Tyr and Phe. Pro451, in cis conformation, was changed into Ala. Glu455 was replaced with Asp and Gln.Absorption, fluorescence and CD spectroscopy of the mutated enzymes in their oxidized state (Eox) showed only minor changes with respect to the wild‐type enzyme, wherease considerable changes were observed in the spectra of the two‐electron‐reduced (EH2) species of the enzymes upon reduction by the substrate dihydrolipoamide. Differences in extent of reduction of the flavin by NADH indicate that the redox potential of the flavin is altered by the mutations. Enzyme Pro455 Ala showed the greatest deviation from wild type. The enzyme is very easily over‐reduced to the four‐electron reduced state (EH4) by dihydrolipoamide. This is probably due to a change in the backbone conformation caused by the cis‐trans conversion.From studies on the pH dependence of the thiolate charge‐transfer absorption and the relative fluorescence of EH2 of the enzymes, it is concluded that mutation of His450 results in a relatively simple and easily interpreted distribution of electronic species at the EH2 level. For all three His450‐mutated enzymes an apparent pKa1 near 5.5 is calculated that is assigned to the interchange thiol. A second apparent pKa2 is calculated of 6.9, 7.5 and 7.1 for the His450 Phe, ‐Ser and ‐Tyr enzymes, respectively, and signifies the deprotonation of the tautomeric equilibrium between the interchange and charge‐transfer thiols. The difference in apparent pK2 values between the His45‐mutated enzymes is explained by changes in micropolarity.At the EH2 level the wild‐type enzyme consists of multiple electronic forms as reported for the Escherichia coli enzyme [Wilkinson, K. D. and Williams C. H. Jr (1979) J. Biol. Chem. 254, 852–862]. Based on the results obtained with the His450‐mutated enzymes, it is concluded that the lowest pKa is associated with the interchange thiol. A model for the equilibrium species of the wild‐type enzyme at the EH2 level is presented which takes three pKa values into account.The results of the pH dependence of the electronic species at the EH2 level of Glu455‐mutated enzymes essentially follow the model proposed for the wild‐type enzyme. However mutation of Glu455 shifts the tautomeric equilibrium of EH2 in favor of the charge‐transfer species. This effect is explained by impaired ion‐pair formation between the protonated His and the interchange thiolate and a lowering of the pka of His450.
An 100‐kDa arachidonate‐mobilizing phospholipase A2 in mouse spleen and the macrophage cell line J774
Authors: WIJKANDER Jonny , SUNDLER Roger 
Published: March 2005

A phospholipase A2 hydrolyzing arachidonic‐acid‐containing phospholipids has been purified 5600‐fold from mouse spleen and to near homogeneity from the macrophage cell line J774. A molecular mass of 100 kDa for the enzyme was estimated by SDS/PAGE, while it migrated as a 70‐kDa protein upon gel chromatography. The enzyme from both sources showed the same characteristics as that previously identified in murine peritoneal macrophages [Wijkander, J. & Sundler, R. (1989), FEBS Lett. 244, 51–56], i.e. it was totally dependent on Ca2+ with half‐maximal activity at approximately 0.7 μM and hydrolyzed arachidonoyl phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol equally well. Also, the platelet‐activating‐factor precursor, 1‐O‐alkyl‐2‐arachidonoylglycerophosphocholine, was hydrolyzed to a similar extent. A preference for arachidonoylphosphatidylcholine over oleoylphosphatidylcholine was seen both with sonicated vesicles and labeled macrophage membranes as substrate.Ca2+‐dependent interaction of the enzyme with sonicated vesicles composed of neutral phospholipids led to rapid initial hydrolysis, followed by loss of catalytic activity. Such inactivation did not occur with vesicles of pure anionic phospholipids, or with membranes prepared from macrophages.Phospholipase A2, purified from J774 cells, was rapidly phosphorylated by protein kinase C type‐II, leading to incorporation of approximately 0.5 mol phosphate/mol enzyme.
Carboxy‐terminal truncated rhuIFN‐γ with a substitution of Gln133 or Ser132 to leucine leads to higher biological activity than in the wild type
Authors: SLODOWSKI Otto , BÖHM Joachim , SCHÖNE Brigitte , OTTO Bernd 
Published: March 2005

The biological function of the 20 C‐terminal amino acids of human interferon‐γ (IFN‐γ) was examined by recombinant DNA methodology. Six truncated IFN‐γ analogues were produced by modification of the 3′ end of the coding sequence of the cloned gene, insertion into a vector with the trc promoter and expression of the recombinant IFN‐γ analogue genes in Escherichia coli strain JM 105. The IFN‐γ analogue proteins were shortened by 10 (C‐10L), 11 (C‐11, C‐11L), 14 (C‐14L), 19 (C‐19L) and 20 (C‐20) amino acid. Four of these constructs were modified to have a C‐terminal leucine.The expression rates of precipitating IFN‐γ variants in E. coli cells (wild type, C‐10L, C‐11, C‐11L) amount to 35–40% of the total protein, in contrast to 14–21% for the mainly soluble ones (C‐14L, C‐20). The variant C‐19L has an exceptional position in its solubility behaviour with a nearly 1:1 distribution between its soluble and insoluble form by an expression rate of 8%.The purification protocol of the insoluble variants contains a denaturing and a renaturation step. The characteristic step for purification soluble IFN‐γ is HPLC cation‐exchange chromatography.The antiviral activities of the variants lacking 14 or more amino acids are less than 2% of the wild‐type activity. The variants C‐10L, C‐11 and C‐11L show higher biological activities than wild‐type IFN‐γ. The most active variant, C‐10L, with leucine as the last C‐terminal amino acid, has a fourfold higher specific antiviral activity (A549 cells, encephalomyocarditis virus). Removal, but not replacement of the leucine, represented by the variant C‐11, reduces the biological activity compared with variant C‐10L. The activity of C‐11 is, nevertheless, higher than in the wild type.Comparing the secondary structures, as judged by CD analyses, no significant differences for C‐10L, C‐14L and C‐20, compared with wild type, are observed. Also, all molecules, including the wild‐type protein, exist as dimers under physiological conditions.There is a correlation between the grade of truncation and the pI values, which range from pI = 10.4 (wild type) to pI = 8.0 (C‐20). The variant C‐10L demonstrates a higher temperature stability (tm= 55°C) compared with wild type (tm= 53°C). Perhaps this higher stability will result in a longer half‐life in vivo.This investigation points out the importance of the basic amino acids cluster Lys128–Arg131. Partial or complete removal of this region leads to a drastic loss in biological activity, while truncations near to this region result in a significant increase in activity.
Mutation of the pseudo‐EF‐hand of calbindin D9k into a normal EF‐hand
Authors: JOHANSSON Charlotta , BRODIN Peter , GRUNDSTRÖM Thomas , FORSÉN Sture , DRAKENBERG Torbjön 
Published: March 2005

The two Ca2+‐binding sites in calbindin D9k, a protein belonging to the calmodulin superfamily of intracellular proteins, have sligtly different structure. The C‐terminal site (amino acids 54–65) is a normal EF‐hand as in the other proteins of the calmodulin superfamily, while the N‐terminal site (amino acids 14–27) contains two additional amino acids, one of which is a proline.We have constructed and studied five mutants of calbindin D9k modified in the N‐terminal site. In normal EF‐hand structures the first amino acid to coordinate calcium is invariantly an Asp. For this reason Ala 15, is exchanged by an Asp in all mutants and the mutants also contain various other changes in this site. The mutants have been characterized by 43Ca, 113Cd and 1H NMR and by the determination of the calcium binding constants using absorption chelators. In two of the mutants (one where Ala14 is deleted, Ala15 is replaced by Asp and Pro20 is replaced by Gly, the other where, in addition, Asn21 is deleted), we find that the structure has changed considerably compared to the wild‐type calbindin. The NMR results indicate that the calcium coordination has changed to mainly side‐chain carboxyls, from being octahedrally coordinated by mainly back‐bone carbonyls, and/or that the coordination number has decreased. The N‐terminal site has thus been turned into a normal EF‐hand, in which the calcium ion is coordinated by side‐chain carboxyls. Furthermore, the calcium binding constants of these two mutant proteins are almost as high as in the wild‐type calbindin D9k. That is, the extensive alterations in the N‐terminal site have not disrupted the calcium binding ability of the proteins.
Secondary structure of the membrane‐bound form of the pore‐forming domain of colicin A
Authors: GOORMAGHTIGH Erik , VIGNERON Laurence , KNIBIEHLER M. , LAZDUNSKI Claude , RUYSSCHAERT Jean‐Marie 
Published: March 2005

The structure of the pore‐forming domain of the bacterial toxin colicin A was studied by attenuated total‐reflection polarized Fourier‐transform infrared spectroscopy. This channel‐forming fragment interacts with dimyristoylglycerophosphoglycerol (Myr2GroPGro) vesicles and forms disk‐like complexes. Analysis of the shape of the amide I' band indicates that its secondary structure is not affected by the pH 5.0–7.2. However, 5–10% of the peptide amino acids adopt an α‐helical structure upon complex formation with Myr2GroPGro, while the random‐coil and β‐sheet structure contents decrease.Interestingly, the increase in α‐helical content is essentially due to an increase in the high‐frequency component of the α‐helical domain of amid I'.The fact that only this component was 90° polarized (i.e. the helix is parallel to the acyl chain) suggests that only this particular type of helix is associated with the Myr2GroPGro bilayer.
Purification and properties of F420‐and NADP+‐dependent alcohol dehydrogenases of Methanogenium liminatans and Methanobacterium palustre, specific for secondary alcohols
Authors: BLEICHER Karl , WINTER Josef 
Published: March 2005

The F420‐dependent alcohol dehydrogenase (ADH) of Methanogenium liminatans and the NADP+‐dependent ADH of Methanobacterium palustre were purified to homogeneity. The native F420‐dependent ADH of Mg. liminatans had a molecular mass of 150 kDa and consisted of four (presumably identical) subunits with a mass of 39 kDa. The temperature optimum was 42°C, the optimum pH 6.0 and NaCl or KCl were inhibitory. The NADP+‐dependent ADH of Mb. palustre had a molecular mass of 175 kDa and consisted also of four (presumably identical) subunits with a mass of 44 kDa. The temperature optimum was 60°C, the optimum pH 8.0 and optimal activity was observed in the presence of 500 mM NaCl or KCl.The ADHs of both organisms catalysed the oxidation of various secondary and cyclic alcohols to the corresponding ketones and the reverse reaction. No primary alcohols were apparently oxidized. The NADP+ ‐dependent ADH of Mb. palustre contained 4–8 mol atoms zinc/mol enzyme and was inhibited by low concentrations of iodoacetate and 4‐hydroxymercuribenzoate, whereas the F420 ‐dependent ADH of Mg. liminatans presumably contained no zinc ions and was inhibited by 1,10‐phenanthroline or high concentrations (e.g. 100 μM) of 4‐hydroxymercuribenzoate.Polyclonal antibodies against the NADP+ ‐dependent ADH of Mb. palustre precipitated only the homologour ADH. A precipitation of the NADP+ ‐dependent ADH of Methanocorpusculum parvum required a 10‐fold higher antibody concentration, showing at least a distant relationship of both ADHs. Antibodies against the NADP+ ‐dependent ADH of Mcp. parvum, however, formed precipitates with the homologous ADH of Mcp. parvum and with the NADP+ ‐dependent ADH of Mb. palustre. They also formed precipitates with the ADH of Thermoanaerobium brockii, which is not related to methane bacteria. Antibodies against the F420‐dependent ADH of Mg. liminatans reacted only with the homologous enzyme and did not form precipitates with NADP+ ‐dependent ADHs. No immunological relation of the NADP+ ‐ or F420‐dependent ADHs of methanogens with ADH of yeast or horse liver was found.In accordance with the immunological data, the N‐terminal amino acid sequences of the NADP+ ‐dependent ADHs of Mb. palustre and Mcp. parvum had a high degree of similarity, whereas the N‐terminal amino acid sequence of the ADH of Mg. liminatans revealed no similarity with the two NADP+ ‐dependent enzymes.
2,4‐Diamino‐7‐hydroxy‐pteridines, a new class of catabolites of methotrexate
Authors: DHONDT Jean‐Louis , HAYTE Jean‐Marie , MILLOT Frédéric , KLEIN Roger , BLAIS Jean‐Claude , PFLEIDERER Wolfgang 
Published: March 2005

Methotrexate remains a commonly used drug in the chemotherapy of various malignancies. The known catabolites are 7‐hydroxy‐methotrexate, formed in the liver, and diamino‐methyl‐pteroic acid formed in the gut.We report for the first time evidence that 2,4‐diamino‐7‐hydroxy‐pteridine derivatives are present in the biological fluids of patients on high‐dose methotrexate protocols. So far, two major derivatives have been identified as 2,4‐diamino‐6‐hydroxymethyl‐7‐hydroxy‐pteridine and 2,4‐diamino‐6‐methyl‐7‐hydroxy‐pteridine. In regard to the actual knowledge of the catabolism of pteridines, these compounds are presumably formed by intestinal bacteria during enterohepatic circulation of the drug.Their slow clearance from the body raises the question of possible interference of these compounds on pteridine‐dependent enzymes, which might explain in part some of the toxic effects of methotrexate.
New site‐directed reversible inhibitors of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase
Authors: SIXT Bernhard , EGGERER Hermann 
Published: March 2005

CoA‐thioether analogues of 3‐hydroxy‐3‐methylglutaryl‐CoA containing an additional methyl group at positions 2, 6 (methyl at C3) or 4 of the acyl residue were prepared. To probe for hydrophobic interaction, their inhibitory properties were determined with 3‐hydroxy‐3‐methylglutaryl‐CoA reductase purified from baker's yeast. The CoA‐thioethers were purely competitive inhibitors whose affinity to the reductase was near to that of the physiological substrate.
CoA‐sulfoxides derived from the CoA‐thioethers displayed affinities to the reductase superior to that of the physiological substrate (Km= 7 μM). Depending on the degree of recognition of diastereomers by the enzyme, the inhibitor constants of the two best inhibitors vary from Ki= 200 nM and Ki= 80 nM (diastereomeric mixtures) to 25 nM and 20 nM, respectively (if only one diastereomer would interact with the enzyme).