bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary structure of a barley‐grain aspartic proteinase
Authors: RUNEBERG‐ROOS Pia , TÖRMÄKANGAS Kirsi , ÖSTMAN Anne
Published: March 2005

Two enzymatically active heterodimeric forms of an aspartic proteinase, a putative 32 kDa + 16 kDa precursor form and a putative 29 kDa + 11 kDa mature form, are present in resting barley grains (Sarkkinen, P., Kalkkinen, N., Tilgmann, C., Siuro, J., Kervinen, J. & Mikola, L., 1990, in the press). The cDNA corresponding to this enzyme has been cloned and sequenced. The full‐length 1863‐bp cDNA sequence codes for an open reading frame of 508 amino acids. The open reading frame consists of a 66‐amino acid preprosequence and a 442‐amino acid mature protein. Comparison of the N‐terminal amino acid sequences of the enzyme subunits with the sequence of the cDNA clone indicates that the heterodimeric enzyme is translated as a proenzyme which is processed into two subunits. The localisation of the experimentally determined N‐terminal amino acid sequences of all four subunits (32 kDa + 16 kDa and 29 kDa + 11 kDa) in the same transcript, as well as the detection of only one 2.0‐kb mRNA on Northern blots from resting seeds, clearly indicates that the larger (32 kDa + 16 kDa) enzyme is an intermediate precursor form of the smaller (29 kDa + 11 kDa) enzyme. The processing pattern of the barley enzyme, which is the first sequenced plant aspartic proteinase, differs from that of all other known aspartic proteinases. The barley enzyme is highly similar to mammalian and yeast aspartic proteinases, especially to human and porcine cathepsin D. This similarity is clearly dispersed over two regions, separated by a dissimilar, barley‐specific region of 104 amino acids.
Ornatins: potent glycoprotein IIb‐IIIa antagonists and platelet aggregation inhibitors from the leech Placobdella ornata
Authors: MAZUR Paul , HENZEL William J. , SEYMOUR Jana L. , LAZARUS Robert A.
Published: March 2005

The purification and characterization of six isoforms of ornatin, potent glycoprotein IIb‐IIIa (GP IIb‐IIIa) antagonists and platelet aggregation inhibitors are described. These isoforms were purified from whole leech homogenates of the leech Placobdella ornata, a North American leech commonly known as the turtle leech, by trichloroacetic acid precipitation, Sephadex G‐50 size exclusion chromatography, GP IIb‐IIIa affinity chromatography, and C18 reverse‐phase HPLC. Each of the five completely sequenced isoforms, which range from 41 to 52 residues in lenght, contains the Arg‐Gly‐Asp (RGD) sequence, a common recognition sequence in adhesion proteins, as well as 6 cysteine residues; the positions of both of these features are conserved in the primary sequences. The amino acid sequences of ornatin isoforms B, C, D, and E are highly conserved, whereas ornatin A2 and A3 are less similar and lack 9 residues at the N‐terminus. The ornatins are approximately 40% identical with decorsin, a GP IIb‐IIIa antagonist isolated from the leech Macrobdella decora [Seymour, J. L., Henzel, W. J., Nevins, B., Stults, J. T. & Lazarus, R. A. (1990) J. Biol. Chem. 265, 10143–10147]; furthermore, the RGD sequence and 5 out of 6 cysteine residues are maintained in the same relative positions in both decorsin and ornatin. The ornatin isoforms do not exhibit significant similarity to any members of the snake‐venomderived family of GP IIb‐IIIa antagonists [Dennis, M. S., Henzel, W. J., Pitti, R. M., Lipari, M. T., Napier, M. A., Deisher, T. A., Bunting, S. & Lazarus, R. A. (1990) Proc. Natl Acad. Sci. USA 87, 2471–2475] except in the RGD region of these proteins. The ornatin isoforms inhibit the binding of GP IIb‐IIIa to immobilized fibrinogen with IC50 values ranging over 2.9–5.3 nM; ornatin isoforms A2, C, and E inhibit ADP‐induced human platelet aggregation with IC50 values of about 130, 280, and 440 nM, respectively.
Refolding kinetics of pig muscle and yeast 3‐phosphoglycerate kinases and of their proteolytic fragments
Authors: SEMISOTNOV Gennady V. , VAS Maria , CHEMERIS Violetta V. , KASHPAROVA Natalija J. , KOTOVA Nina V. , RAZGULYAEV Oleg I. , SINEV Michael A.
Published: March 2005

The time course of refolding of both pig muscle and yeast 3‐phosphoglycerate kinase (molecular masses about 47 kDa), as well as their proteolytic C‐terminal fragments (30 and 33 kDa, respectively) has been investigated. Very similar refolding kinetics (with half‐time between 80–120 s, at 20°C) were observed by fluorescence and ultraviolet absorbance spectroscopy, as well as by activity measurements, for the intact enzyme from both sources. This time course appears not to depend on the time the protein spends in the unfolded state, i.e. it is certainly not controlled by proline isomerization. Furthermore, after removal of a large N‐terminal part (molecular mass of about 18 kDa for pig muscle enzyme or 13 kDa for yeast enzyme) of the molecule by proteolysis, refolding of the remaining C‐terminal fragment of both proteins follows kinetics virtually indistinguishable from those of the intact protein molecule.
A thermodynamic model for the cooperative functional properties of the tetraheme cytochrome c3 from Desulfovibrio gigas
Authors: COLETTA Massimo , CATARINO Teresa , LeGALL Jean , XAVIER António V.
Published: March 2005

A thermodynamic model is presented to describe the redox behaviour of the tetraheme cytochrome c3 from Desulfovibrio gigas. This molecule displays different intrinsic redox potentials for the four hemes and during the redox titration Process, interactions among different hemes occur, thus altering the values of redox potentials according to which of the hemes are oxidized [Santos, H., Moura, J. J. G., Moura, I., LeGall, J. & Xavier, A. V. (1984) Eur. J. Biochem. 141, 283–296]. This complex cooperative behaviour [Xavier, A. V. (1986) J. Inorg. Biochem. 28, 239–243] has been analyzed here using an I2H4‐interaction network [Cornish‐Bowden, A. & Koshland, D. E. Jr (1970) J. Biol. Chem. 245, 6241–6250] coupled to a proton‐linked equilibrium between two tertiary structures. Such a formalism, which requires a reduced number of parameters, is able to fully account quantitatively for the pH dependence of the NMR redox‐titration curves. The ‘redox‐Bohr’ effect is discussed in terms of the available structure and thermodynamic data and a functional mechanism is proposed.
Kinetic study of the reduction mechanism for Desulfovibrio gigas cytochrome c3
Authors: CATARINO Teresa , COLETTA Massimo , LeGALL Jean , XAVIER António V.
Published: March 2005

The kinetic aspects of the reduction process in cytochrome c3 fromDesulfovibrio gigas have been investigated over a wide range of pH values ranging between pH 5.8 and pH 9.8. The data have been analyzed in the framework of an I2H4 interaction network coupled to a proton‐linked equilibrium between two tertiary structures (Cornish‐Bowden, A. & Koshland, D. E. Jr (1970) J. Biol. Chem. 245, 6241–6250). The kinetic rate constants for the reduction of the four hemes for the two tertiary conformations have been characterized in the framework of the thermodynamic network obtained from the equilibrium analysis (Coletta, M., Catarino, T., LeGall, J. J. & Xavier, A. V. (1991) Eur. J. Biochem. 202, 1101–1106). The intrinsic reduction rate constants determined by reaction with sodium dithionite for two hemes (namely heme 4 and heme 1) are significantly faster than those for the other two heme residues. In view of the equilibrium redox properties, heme 4 (with the fastest reduction rate) may then work as the kinetic electron‐capturing site for the electrons from sodium dithionite. The transfer to hemes 2 and 3 then occurs by virtue of their free‐energy levels at equilibrium. At our experimental conditions, there is also transfer of electrons to hemes 2 and 3 from heme 1, which is reduced at a slower rate than heme 4, thus contributing to the biphasic kinetics observed for the overall process. The kienetic parameters obtained are discussed in terms of the mechanism proposed for the coupling between the electron and proton transfer, as induced by the heme/heme cooperativity network.
Rate constants and equilibrium constants for binding of actin to the 1:1 gelsolin‐actin complex
Authors: SCHOEPPER Beate , WEGNER Albrecht
Published: March 2005

The rate constant and equilibrium constant of association of an actin monomer with 1:1 gelsolinactin complex isolated from chicken were measured by using fluorescently labeled actin. According to fluorescence stopped‐flow experiments, the rate constant of formation of the 1:2 gelsolin‐actin complex from 1:1 gelsolin‐actin complex and actin was found to be about 2 × 107 M‐1 s‐1 under conditions where gelsolin binds Ca2+. The rate of dissociation of one actin molecule from the 1:2 gelsolin‐actin complex was determined by exchange of actin for fluorescently labeled actin. The rate constant of dissociation was about 0.02 s‐1. Thus, the equilibrium constant for association of actin with 1:1 gelsolin‐actin complex can be calculated to be in the range of 109 M‐1. The rate of dissociation of actin from 1:2 gelsolin‐actin complex was independent of the Ca2+ concentration. Ca2+ affects only the rate of association of actin with 1:1 gelsolin‐actin complex.
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.
Fulvic acid disturbs processing of procollagen II in articular cartilage of embryonic chicken and may also cause Kashin‐Beck disease
Authors: YANG Chunlin , BODO Michael , NOTBOHM Holger , PENG An , MÜLLER Peter K.
Published: March 2005

Kashin‐Beck disease is an endemic osteoarthropathy in China which may lead to skeletal deformation and dwarfism. We have analysed articular cartilage from two patients and found an accumulation of the precursor molecule, pro‐pN‐collagen II (pN, peptide atached at the amino‐terminus) which was not present in extracts of control fetal cartilage. In addition, collagen II isolated from the same tissue by limited pepsin digestion had a decreased electrophoretic mobility, increased proline hydroxylation and decreased thermal stability. Previously, a genetic defect in pro‐pN‐collagen‐I processing has been described in calf and sheep (dermatosparaxis) and man (Ehlers‐Danlos, type VII) which caused an extreme fragility of the skin [Lenaers, A., Ansay, M., Nusgens, B. V. & Lapière, C. M. (1971) Eur. J. Biochem. 23, 533–541; Helle, O. & Nes, N. J. (1972) Acta Vet. Scand. 13, 443–445; Lichtenstein, J. R., Martin, G. R., Kohn, L. D., Byers, P. H. & McKusick, V. A. (1973) Science 182, 298–300]. Accordingly, one may assume that the impaired conversion of pro‐pN‐collagen II to collagen II and the structural alteration of collagen II, presumably caused by fulvic acid and other environmental factors, play an important role in the pathogenesis of Kashin‐Beck disease.
Characterization of the epoxide hydrolase from an epichlorohydrin‐degrading Pseudomonas sp.
Authors: JACOBS Mariken H. J. , VAN DEN WIJNGAARD Arjan J. , PENTENGA Marjan , JANSSEN Dick B.
Published: March 2005

An epoxide hydrolase was purified to homogeneity from the epichlorohydrin‐utilizing bacterium Pseudomonas sp. strain AD1. The enzyme was found to be a monomeric protein with a molecular mass of 35 kDa. With epichlorohydrin as the substrate, the enzyme followed Michaelis‐Menten kinetics with a Km value of 0.3 mM and a Vmax of 34 μmol · min‐1· mg protein‐1. The epoxide hydrolase catalyzed the hydrolysis of several epoxides, including epichlorohydrin, epibromohydrin, epoxyoctane and styrene epoxide. With all chiral compounds tested, both stereoisomers were converted. Amino acid sequencing of cyanogen bromide‐generated peptides did not yield sequences with similarities to other known proteins.
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.
Aldehyde dehydrogenase induction by glutamate in Escherichia coli
Authors: QUINTILLA Francesc Xavier , BALDOMA Laura , BADIA Josefa , AGUILAR Juan
Published: March 2005

In Escherichia coli, an aldehyde dehydrogenase that catalyzes the oxidation of l‐lactaldehyde to l‐lactate is induced not only by l‐fucose, l‐rhamnose or d‐arabinose, but also by growth in the presence of glutamate or amino acids yielding glutamate, with the exception of proline. Induction by these amino acids requires glutamate accumulation. 4‐Aminobutyric acid also induces this aldehyde dehydrogenase through its transamination to glutamate. Growth on 2‐oxoglutarate, the tricarboxylic acid cycle intermediate with which glutamate is in equilibrium, also induces this aldehyde dehydrogenase. Conditions in which the conversion of 2‐oxoglutarate into glutamate is highly restricted displayed unchanged rates of induction by 2‐oxoglutarate, indicating that glutamate induces the aldehyde dehydrogenase through 2‐oxoglutarate formation. Evidence is presented showing that l‐fucose‐ and 2‐oxoglutarate‐inducing systems share the same regulatory protein. Induction by growth on either of these two compounds is repressed both by glucose and by glycerol. Addition of cAMP to these cultures partially recovers the glucose‐repressed aldehyde dehydrogenase activity, while this nucleotide has no effect on the glycerolmediated repression. These results indicate that ald is under carbon regulation mediated by at least two different mechanisms.
Interference with myosin subfragment‐1 binding by site‐directed mutagenesis of actin
Authors: ASPENSTRÖM Pontus , KARLSSON Roger
Published: March 2005

Three N‐terminal double mutants of β‐actin expressed in the yeast Saccharomyces cerevisiae have been characterized with respect to DNase‐I interaction, N‐terminal post‐translational modification, polymerizability and myosin subfragment‐1 binding. The results strongly support earlier suggestions that the acidic residues at the N‐terminus of actin are part of the myosin‐binding site, while they seem to be of no importance for the other aspects of actin biochemistry tested. The suitability of this expression system for production of recombinant actin in general is discussed.
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.
Evidence for the endogenous GTP‐dependent ADP‐ribosylation of the α‐subunit of the stimulatory guanyl‐nucleotide‐binding protein concomitant with an increase in basal adenylyl cyclase activity in chicken spleen cell membrane
Authors: OBARA Seiji , YAMADA Kazuo , YOSHIMURA Yasuro , SHIMOYAMA Makoto
Published: March 2005

We investigated the endogenous GTP‐dependent ADP‐ribosylation of the α‐subunit of the stimulatory guanylnucleotide‐binding protein (Gsα) concomitant with an increase of basal adenylyl cyclase activity in chicken spleen cell membranes. When these membranes were incubated with [adenylate‐32P]NAD, there was significant incorporation of [32P]ADP‐ribose into a 45‐kDa acceptor protein in the membranes. This reaction was inhibited when 20 mM arginine was present during the incubation. When the membranes were incubated with unlabelled NAD, subsequent ADP ribosylation by cholera toxin was diminished significantly. Thus, chicken spleen cell membranes have the potential to endogenously ADP‐ribosylate the arginine residue of Gsα. The endogenous ADP‐ribosylation of Gsα was enhanced by the addition of 0.1 mM GTP or 0.1 mM guanosine 5′‐[γ‐thio]triphosphate (GTP[S]), but not 0.1 mM GDP, 0.1 mM ATP or 0.1 mM ADP. The endogenous GTP‐dependent ADP‐ribosylation of Gsα stimulated basal adenylyl cyclase activity. Furthermore, NAD‐induced stimulation of basal adenylyl cyclase activity was suppressed, when the membranes were incubated with NAD in the presence of novobiocin, an inhibitor of arginine‐specific ADP‐ribosyltransferase. These data represent the first demonstration that a eukaryotic cell membrane contains an ADP‐ribosyltransferase which can catalyze the endogenous GTP‐dependent ADP‐ribosylation of the arginine residue of Gsα and that this modification enhances basal adenylyl cyclase activity in the membrane. In light of this evidence, the possible control of basal adenylyl cyclase activity via endogenous GTP‐dependent ADP‐ribosylation in eukaryotic cells warrants further attention.
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.
Effects of ethanol and clofibrate on expression of cytochrome P‐450 enzymes and epoxide hydrolase in cultures and cocultures of rat hepatocytes
Authors: PERROT Nathalie , CHESNÉ Christophe , DE WAZIERS Isabelle , CONNER Joseph , BEAUNE Philippe Henri , GUILLOUZO André
Published: March 2005

Cultured and cocultured rat hepatocytes were used to study the effects of ethanol and clofibrate on cytochrome P‐450 (P‐450) enzymes and epoxide hydrolase. We showed that in the presence of ethanol, clofibrate or both compounds, rat hepatocytes were able to express, after 3 days of pure culture, quantitatively and qualitatively reasonable levels of most cytochrome P‐450 enzymes and epoxide hydrolase, compared to freshly isolated hepatocytes. However, ethanol induced the P‐450IA subfamily, and clofibrate the P‐450‐IVA subfamily. In cocultures, after 6 days, most P‐450 enzymes were still expressed while P‐450IIC11 was completely lost. Ethanol and clofibrate had the same effect as in pure culture. These results show, by modifying culture medium conditions and cell‐cell interactions, that it is possible to maintain reasonable xenobiotic‐metabolizing‐enzyme expression; however, these conditions have to be improved in order to preserve better P‐450 expression. The mechanism of these effects and the inducibility of these systems remain to be elucidated by a study at molecular level.
Affinity purification of a rat‐brain junctional protein, connexin 43
Authors: DUPONT Emmanuel , EL AOUMARI Abdelhakim , FROMAGET Catherine , BRIAND Jean‐Paul , GROS Daniel
Published: March 2005

Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap‐junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti‐peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity‐chromatography purification procedure of the 41‐kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314–322) of rat heart CX43 were covalently bound to a protein‐A–Sepharose‐CL‐4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41‐kDa protein associated with low amounts of high‐molecular‐mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5–17, 55–56 and 314–322 of rat heart CX43. The NH2‐terminal partial sequence for the 41‐kDa protein was determined by microsequencing and shown to be similar to α1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41‐kDa protein is a CX43 gene product.
The primary structure of fatty‐acid‐binding protein from nurse shark liver
Authors: MEDZIHRADSZKY Katalin F. , GIBSON Bradford W. , KAUR Surinder , YU Zhonghua , MEDZIHRADSZKY Denes , BURLINGAME Alma L. , BASS Nathan M.
Published: March 2005

The primary structure of a fatty‐acid‐binding protein (FABP) isolated from the liver of the nurse shark (Ginglymostoma cirratum) was determined by high‐performance tandem mass spectrometry (employing multichannel array detection) and Edman degradation. Shark liver FABP consists of 132 amino acids with an acetylated N‐terminal valine. The chemical molecular mass of the intact protein determined by electrospray ionization mass spectrometry (Mr= 15124 ± 2.5) was in good agreement with that calculated from the amino acid sequence (Mr= 15121.3). The amino acid sequence of shark liver FABP displays significantly greater similarity to the FABP expressed in mammalian heart, peripheral nerve myelin and adipose tissue (61 – 53% sequence similarity) than to the FABP expressed in mammalian liver (22% similarity). Phylogenetic trees derived from the comparison of the shark liver FABP amino acid sequence with the members of the mammalian fatty‐acid/retinoid‐binding protein gene family indicate the initial divergence of an ancestral gene into two major subfamilies: one comprising the genes for mammalian liver FABP and gastrotropin, the other comprising the genes for mammalian cellular retinol‐binding proteins I and II, cellular retinoic‐acid‐binding protein myelin P2 protein, adipocyte FABP, heart FABP and shark liver FABP, the latter having diverged from the ancestral gene that ultimately gave rise to the present day mammalian heart‐FABP, adipocyte FABP and myelin P2 protein sequences. The sequence for intestinal FABP from the rat could be assigned to either subfamily, depending on the approach used for phylogenetic tree construction, but clearly diverged at a relatively early evolutionary time point. Indeed, sequences proximately ancestral or closely related to mammalian intestinal FABP, liver FABP, gastrotropin and the retinoid‐binding group of proteins appear to have arisen prior to the divergence of shark liver FABP and should therefore also be present in elasmobranchs. The presence in shark liver of an FABP which differs substantially in primary structure from mammalian liver FABP, while being closely related to the FABP expressed in mammalian heart muscle, peripheral nerve myelin and adipocytes, opens a further dimension regarding the question of the existence of structure‐dependent and tissue‐specific specialization of FABP function in lipid metabolism.
Interaction between the cell‐cycle‐control proteins p34cdc2 and p9CKShs2
Authors: AZZI Lyamine , MEIJER Laurent , REED Steven I. , PIDIKITI Ravidernath , TUNG H. Y. Lim
Published: March 2005

A universal intracellular factor, the ‘M‐phase‐promoting factor’ (MPF), displaying histone H1 kinase activity and constituted of at least two subunits, p34cdc2 and cyclin Bcdc13, triggers the G2→M transition of the cell cycle in all organisms. The yeast p13suc1 and p18cks1 subunits and their functionally interchangeable human homologues, p9CKShs1 and p9CKShs2, directly interact with p34cdc2 and may actually be part of the MPF complex. We have chemically synthesized p9CKShs2 and several of its peptide domains in order to investigate the binding of p9CKShs2 and p34cdc2. Several arguments support the hypothesis that the N‐terminal half (peptide B) and the C‐terminal half (peptide E) each contain a p34cdc2‐binding site and that these two binding domains cooperate in establishing a stable p9CKShs2‐ p34cdc2 complex: (a) only the combination of peptides B + E, and not B or E alone, is able to elute the cdc2 kinase from p9CKShs1‐Sepharose beads; (b) only immobilized peptides B + E, and not immobilized B or E, bind the cdc2 kinase; (c) only the peptides B + E combination, and not B or E alone, can compete with p9CKShs1 for cdc2 kinase binding; (d) only when supplemented with E or B free peptide does the cdc2 kinase bind to B‐ or E‐Sepharose beads, respectively. No binding occurs in the absence of free peptide. This additivity cannot be attributed to the formation of a B‐E complex mimicking the full‐length p9CKShs2. The cyclin B subunit is not required for the formation of the p9CKShs2‐p34cdc2 complex through these two binding domains. The implications of the existence of two cooperative p34cdc2‐binding domains in p9CKShs2 on the structure of the active M‐phase‐specific kinase is discussed.
1‐Deoxy‐1‐phosphatidylethanolamino‐lactitol‐type neoglycolipids serve as acceptors for sialyltransferases from rat liver golgi vesicles
Authors: POHLENTZ Gottfried , SCHLEMM Susanne , EGGE Heinz
Published: March 2005

1‐Deoxy‐1‐phosphatidylethanolamino‐lactitols (LacPtdEtns), 1‐deoxy‐1‐phosphatidylethanol‐amino‐sialyllactitols (NeuAcLacPtdEtns) and their corresponding N‐acetylated derivatives were synthesized and characterized by fast‐atom‐bombardment mass spectrometry (FAB MS). The neoglycolipids were used as acceptors for sialyltransferases from rat liver Golgi vesicles. Sialylation rates were as good as or even better than those obtained with the corresponding authentic acceptors lactosylceramide (LacCer) and ganglioside GM3. The sialylation of LacPtdEtns and NeuAcLacPtdEtns yielded sialyl and disialyl compounds, respectively, as shown by FAB MS analysis of the reaction products. The results of competition experiments indicate that the neoglycolipids and the authentic acceptors are sialylated by the same sialyltransferases.
Engineering mammalian aspartyl‐tRNA synthetase to probe structural features mediating its association with the multisynthetase complex
Authors: MIRANDE Marc , LAZARD Myriam , MARTINEZ Ricardo , LATREILLE Marie‐Thérèse
Published: March 2005

Aspartyl‐tRNA synthetase from higher eukaryotes is a component of a multienzyme complex comprising nine aminoacyl‐tRNA synthetases. The cDNA encoding cytoplasmic rat liver aspartyl‐tRNA synthetase was previously cloned and sequenced. This work reports the identification of structural features responsible for its association within the multisynthetase complex. Mutant and chimeric proteins have been expressed in mammalian cells and their structural behavior analyzed. A wild‐type rat liver aspartyl‐tRNA synthetase, expressed in Chinese hamster ovary (CHO) cells, associates within the complex from CHO cells, whereas a mutant enzyme with a deletion of 34 amino acids from its amino‐terminal extremity does not. A chimeric enzyme, made of the amino‐terminal moiety of rat liver aspartyl‐tRNA synthetase fused to the catalytic domain of yeast lysyl‐tRNA synthetase, has been expressed in Lys‐101 cells, a CHO cell line with a temperature‐sensitive lysyl‐tRNA synthetase. The fusion protein is stable in vivo, does not associate within the multisynthetase complex and cannot restore normal growth of the mutant cells. These results establish that the 3.7‐kDa amino‐terminal moiety of mammalian aspartyl‐tRNA synthetase mediates its association with the other components of the complex. In addition, the finding that yeast lysyl‐tRNA synthetase cannot replace the aspartyl‐tRNA synthetase component of the mammalian complex, indicates that interactions between neighbouring enzymes also play a prominent role in stabilization of this multienzyme structure and strengthened the view that the multisynthetase complex is a discrete entity with a well‐defined structural organization.
Three‐dimensional 15N‐1H‐1H and 15N‐13C‐1H nuclear‐magnetic resonance studies of HPr a central component of the phosphoenolpyruvate‐dependent phosphotransferase system from Escherichia coli
Authors: van NULAND Nico A. J. , van DIJK Alard A. , DIJKSTRA Klaas , van HOESEL Frans H. J. , SCHEEK Ruud M. , ROBILLARD George T.
Published: March 2005

We have performed three‐dimensional NMR studies on a central component of the phosphoenolpyruvate‐dependent phosphotransferase system of Escherichia coli, denoted as HPr. The protein was uniformly enriched with 15N and 13C to overcome spectral overlap. Complete assignments were obtained for the backbone 1H, 15N and 13C resonances, using three‐dimensional heteronuclear 1H NOE 1H‐15N multiple‐quantum coherence spectroscopy (3D‐NOESY‐HMQC) and three‐dimensional heteronuclear total correlation 1H‐15N multiple‐quantum coherence spectroscopy (3D‐TOCSY‐HMQC) experiments on 15N‐enriched HPr and an additional three‐dimensional triple‐resonance 1HN‐15N‐13Cα correlation spectroscopy (HNCA) experiment on 13C, 15N‐enriched HPr. Many of the sequential backbone 1H assignments, as derived from two‐dimensional NMR studies [Klevit, R. E., Drobny, G. P. & Waygood, E. B. (1986) Biochemistry 25, 7760–7769], were corrected. Almost all discrepancies are in the helical regions, leaving the published antiparallel β‐sheet topology almost completely intact.
The sequence location of the actin metal
Authors: UE Kathleen , MUHLRAD Andras , EDMONDS Charles G. , BIVIN Donald , CLARK Astrid , PIECHOWSKI William V. , MORALES Manuel F.
Published: March 2005

We have incorporated Fe2+ into the high‐affinity metal‐ion‐binding site of actin. By supplying the system with oxygen from air and a reductant (dithiothreitol or ascorbate), we have induced freeradical generation, with the intent of causing peptide cleavage at the metal‐ion‐binding site. By analysis of the resulting fragments from actin in the F‐form, we have deduced that cuts occurred at positions 159–160 and 301–302 (at the latter location we could not be sure if more than one cut occurred). We considered that these two cuts occurred in the chain strand coursing from the outer to the inner domain and vice‐versa. Our results harmonize very well with the recently reported atomic structure of actin [Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F. & Holmes, K. C. (1990) Nature 347, 37–44] and remove ambiguities that had remained in the structure. The results partly bear out the homology‐based prediction of Strzelecka‐Golaszewska et al. [Strzelecka‐Golaszewska, H., Boguta, G., Zmorzynshi, S. & Moraczcwska, J. (1989) Eur. J. Biochem. 182, 299–305].
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).
Endotoxin stimulation of liver parenchymal cell phosphofructokinase activity requires nonparenchymal cells
Authors: MILLER Bonnie C. , UYEDA Kosaku , COTTAM G. Larry
Published: March 2005

The rate of carbohydrate flux through phosphofructokinase (measured as the rate of [3‐3H]glucose detritiation) was increased fourfold in rat liver parenchymal cells incubated with conditioned medium from lipopolysaccharide‐stimulated adherent liver non‐parenchymal cells. The rate was not affected in parenchymal cells incubated either with lipopolysaccharide directly or with conditioned medium from non‐stimulated non‐parenchymal cells. The stimulation of carbohydrate flux through phosphofructokinase by conditioned medium was not duplicated by peptide cytokines known to be released by lipopolysaccharide‐activated liver non‐parenchymal cells (interleukin‐1, interleukin‐6, tumor necrosis factor‐α, and transforming growth factor‐β) or platelet activating factor. Furthermore, formation of the active conditioned medium was not prevented by inclusion of cycloheximide or dexamethasone to inhibit cytokine synthesis, or indomethacin or BW755c to inhibit arachidonic acid metabolism, during lipopolysaccharide‐stimulation of the non‐parenchymal cells. The results indicate that intercellular communication between lipopolysaccharide‐stimulated liver non‐parenchymal cells and parenchymal cells by soluble mediators is responsible for the stimulation of liver phosphofructokinase activity during endotoxin‐induced shock. Studies to isolate and identify the factor(s) in the conditioned medium are currently in progress.
1H‐NMR studies on d(GCTTAAGC)2 and its complex with berenil
Authors: HU Sungho , WEISZ Klaus , JAMES Thomas L. , SHAFER Richard H.
Published: March 2005

Two‐dimensional (2D) 1H‐NMR spectroscopy has been used to analyze the structure of d(GCTTAAGC)2 and its interaction with berenil in solution. Nuclear Overhauser enhancement connectivities enabled sequential assignments of nearly all proton resonances in the self‐complementary octamer duplex and demonstrated that the oligonucleotide is primarily in a B‐type conformation. No major conformational changes were observed by the addition of berenil, but proton resonances of the two adenosine nucleotides shifted substantially. Intermolecular nuclear Overhauser effects between berenil and the DNA duplex revealed that the drug binds via the minor groove of d(GCTTAAGC)2 in the A·T‐base‐pair region. At 18°C the twofold symmetry of the duplex is preserved on berenil binding. However, strongly shifted proton resonances broadened significantly. A model is proposed for the berenil‐d(GCTTAAGC)2 complex involving fast exchange of berenil between two equivalent symmetry‐related binding sites, which span the 5′‐TAA‐3′ region and are asymmetrically disposed with respect to the dyad axis of the duplex. These results are compared with previous studies on the berenil‐d(GCAATTGC)2 complex.
Cis‐diol dehydrogenases encoded by the TOL pWW0 plasmid xylL gene and the Acinetobacter calcoaceticus chromosomal benD gene are members of the short‐chain alcohol dehydrogenase superfamily
Authors: NEIDLE Ellen , HARTNETT Christopher , ORNSTON L. Nicholas , BAIROCH Amos , REKIK Monique , HARAYAMA Shigeaki
Published: March 2005

In the aerobic degradation of benzoate by bacteria, benzoate is first dihydroxylated is a ring‐hydroxylating dioxygenase to form a cis‐diol (1,2‐dihydroxycohexa‐3,4‐diene carboxylate) which is subsequently transformed to a catechol by an NAD+‐dependent cis‐diol dehydrogenase. The structural gene for this dehydrogenase, encoded on TOL plasmid pWW0 of Pseudomonas putida (xylL) and that encoded on the chromosome of Acinetobacter calcoaceticus (benD), were sequenced. They encode polypeptides of about 28 kDa in size. These proteins are similar to each other, exhibiting 58% sequence identity. They are also similar to other proteins of at least 20 different functions, which are members of the short‐chain alcohol dehydrogenase family. The alignment of these proteins suggest two amino acids, lysine and tyrosine, as catalytically important residues.
Expression of the Saccharomyces cerevisiae Kex2p endoprotease in insect cells
Authors: GERMAIN Doris , VERNET Thierry , BOILEAU Guy , THOMAS David Y.
Published: March 2005

The pheromone‐processing Kex2p endoprotease of Saccharomyces cerevisiae has been difficult to characterize due to its low level of expression in yeast cells. To overcome this problem, we have overexpressed Kex2p using the baculovirus/insect cell expression system. Spodoptera frugiperda Sf9 insect cells infected with a recombinant baculovirus, containing the complete KEX2 gene which encodes the Kex2p protease (814 amino acids), accumulate an 120‐kDa functional form of the enzyme. The inhibition profile of the insect‐cell‐derived endoprotease is similar to that of the yeast enzyme. The recombinant infected insect cells also secrete into the medium about half of the total Kex2p activity produced. Deleting the carboxyl‐terminal tail and the transmembrane domain of Kex2p (Kex2Δp, 666 amino acids) does not measurably interfere with the enzyme characteristics and results in the secretion of up to 90% of the total enzyme activity. The truncated form, Kex2Δp, of the endoprotease accumulates in the cell supernatant to 6.7 × 105 U/l. The molecular mass of the secreted forms for both the wild‐type Kex2p and Kex2Δp is the same (70 kDa) and is 50‐kDa lower than the intracellular form. This result implicates a processing event which gives rise to shorter extracellular forms of both the wild‐type Kex2p and Kex2Δp and which trims their carboxy termini upsteam of amino acid 666. This processing event requires the integrity of the Ser385 of the Kex2p active site.
Sequence‐specific NMR assignments of the trp repressor from Escherichia coli using three‐dimensional 15N/1H heteronuclear techniques
Authors: BORDEN Katherine L. B. , BAUER Christopher J. , FRENKIEL Thomas A. , BECKMANN Pamela , LANE Andrew N.
Published: March 2005

Sequence‐specific 15N and 1H assignments for the trp holorepressor from Escherichia coli are reported. The trp repressor consists of two identical 107‐residue subunits which are highly helical in the crystal state [Schevitz, R., Otwinowski, Z., Joachimiak, A., Lawson, C. L. & Sigler, P. B. (1985) Nature 317, 782–786]. The high helical content and the relatively large size of the protein (Mr= 25000) make it difficult to assign even the main‐chain resonances by conventional homonuclear two‐dimensional NMR methods. However, we have now assigned the main‐chain resonances of 94% of the residues by using three‐dimensional 15N/1H heteronuclear experiments on a sample of protein uniformly labelled with 15N. The additional resolution obtained by spreading out the signals into three dimensions proved indispensable in making these assignments. In particular, we have been able to resolve signals from residues in the N‐terminal region of the A helix for the first time in solution. The observed NOE results confirm that the repressor is highly helical in solution, and contains no extended chain conformations.
Characterization of a recombinant bifunctional enzyme, galactose dehydrogenase/bacterial luciferase, displaying an improved bioluminescence in a three‐enzyme system
Authors: LINDBLADH Christer , PERSSON Mats , BÜLOW Leif , MOSBACH Klaus
Published: March 2005

The two structural genes encoding galactose dehydrogenase (Pseudomonas fluorescens) and the β subunit of luciferase (Vibrio harveyi) were fused in‐frame in order to prepare and subsequently characterize an artificial bifunctional enzyme complex. This hybrid enzyme exhibited both galactose dehydrogenase activity and bioluminescence when expressed in Escherichia coli together with the α subunit of luciferase. The purified conjugate was used to study possible proximity effects in a sequential three‐enzyme reaction with the bifunctional enzyme catalyzing the first and the last reaction. The intermediate enzyme, diaphorase, was added separately. The engineered enzyme system, comprising the galactose dehydrogenase/luciferase conjugate, could display a twofold higher bioluminescence in the overall enzyme reaction compared to a corresponding reference system with separate native enzymes. The increased bioluminescence obtained for the engineered enzyme system is proposed to be due to an improved organization of the enzymes in solution.