bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and expression of γ‐glutamyl transpeptidase from isolated porcine brain capillaries
Authors: PAPANDRIKOPOULOU Anastasia , FREY Andreas , GASSEN Hans G.
Published: March 2005

In order to understand the mechanisms regulating the blood‐brain barrier we isolated a cDNA clone for γ‐glutamyl transpeptidase from purified porcine brain capillaries by cross‐species DNA hybridization using a DNA fragment from a rat kidney cDNA clone. The sequence of the porcine γ‐glutamyl transpeptidase cDNA shows 84% identity in the coding region to the sequence of human placenta γ‐glutamyl transpeptidase cDNA. The derived protein sequences of the human and porcine γ‐glutamyl transpeptidase are 98% similar. Alignment of the porcine and the published rat γ‐glutamyl transpeptidase cDNA sequences revealed an average identity of 79%, except for a small region of 194 base pairs containing a high number of mismatches. After four nucleotide changes within this region of the published rat cDNA sequence, which correct two frame shifts, the derived protein sequences of the rat and the porcine γ‐glutamyl transpeptidase show 97% similarity. The corrected sequence, which includes a region of 65 amino acids, contains additional N‐glycosylation sites as well as a hydrophobic protein domain that might represent a second membrane‐spanning domain. Tissue‐specific expression and diversity of the γ‐glutamyl transpeptidase mRNA transcripts in various porcine and human tissues were analysed by Northern blot hybridization. The 2.2‐kb transcript of brain γ‐glutamyl transpeptidase is mainly expressed in the endothelial cells of brain microvessels, in agreement with previous data that showed the brain protein being localized within the blood‐brain barrier.
Molecular forms of acetylcholinesterase in two sublines of human erythroleukemia K562 cells
Authors: TOUTANT Jean‐Pierre , RICHARDS Michael K. , KRALL Jennifer A. , ROSENBERRY Terrone L.
Published: March 2005

Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N‐ethylmaleimide was required to preserve the G2a form.

In Triton X‐100 extracts of the subline K562‐243, phosphatidylinositol‐specific phospholipase C (PtdIns‐PLC) from Bacillus thuringiensis converted most of the G2a AChE into a hydrophilic dimer (G2h), indicating that the G2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562‐243 cells with PtdIns‐PLC released approximately 60% of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns‐PLC was not obtained in extracts or intact cells of the subline K562‐48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562‐48 AChE resembles the G2a form of human erythrocyte AChE, which is resistant to PtdIns‐PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766–18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns‐PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503–508].

In both K562 sublines, sialidase decreased the migration of the G2a form but not of the G1a form of AChE. G1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans‐Golgi apparatus. The sialidase‐resistant G1a molecules were also resistant to PtdIns‐PLC digestion; possible explanations for this resistance are presented.
Arginyl groups involved in the binding of Anabaena ferredoxin–NADP+ reductase to NADP+ and to ferredoxin
Authors: SANCHO Javier , MEDINA Milagros , GÓMEZ‐MORENO Carlos
Published: March 2005

Chemical modification of ferredoxin–NADP+ reductase from the cyanobacteria Anabaena has been performed using the α‐dicarbonyl reagent phenylglyoxal. Inactivation of both the diaphorase and cytochrome‐c reductase activities, characteristic of the enzyme, indicates the involvement of one or more arginyl residues in the catalytic process of the enzyme. The determination of the rate constants for the inactivation process under different conditions, including those in which substrates, NADP+ and ferredoxin, as well as other NADP+ analogs were present, indicates the involvement of two different groups in the inactivation process, one that reacts very rapidly with the reagent (kobs= 8.3 M−1 min−1) and is responsible for the binding of NADP+, and a second less reactive group (kobs= 0.9 M−1 min−1), that is involved in the binding of ferredoxin. Radioactive labeling of the enzyme with [14C]phenylglyoxal confirms that two groups are modified while amino acid analysis of the modified protein indicates that the modified groups are arginine residues. The identification of the amino acid residues involved in binding and catalysis of the substrates of ferredoxin–NADP+ reductase will help to elucidate the mechanism of the reaction catalyzed by this important enzyme.
Enzyme kinetics in reversed micelles
Authors: TYRAKOWSKA Bozena , VERHAERT Raymond M. D. , HILHORST Riet , VEEGER Cees
Published: March 2005

The kinetic parameters of 20β‐hydroxysteroid dehydrogenase were determined in aqueous solutions and in reversed micellar media composed with either an anionic, a cationic or a nonionic surfactant, at low and at high ionic strength. The velocity data were analysed in two ways: first by extrapolation to infinite concentrations of both substrates to determine ‘apparent’ Michaelis constants and ν values, and secondly by comparison to reaction rates calculated using the model presented (see first of this series of papers in this issue of the journal). Data analysis according to the first method reveals some differences in the kinetic parameters in reversed micelles as compared to those in aqueous solution, though the kinetic parameters of the enzyme seem not to be much affected by enclosure in reversed micelles. It is shown that the changes that do occur are not caused by a shift of the intramicellar pH or by electrostatic interactions between the enzyme and the surfactant head groups. Interpretation of the data using the second method assumes that the enzyme is not affected by the enclosure in reversed micelles, and that deviations with respect to the aqueous parameters are caused by exchange phenomena between distinct aqueous droplets in the organic phase and by a high effective intramicellar substrate concentration. This model is able to predict reaction rates that agree rather well with experimentally determined rates and explains why the enzyme mechanism in reversed micelles is, at all progesterone concentrations used, the same as observed at high progesterone concentrations in aqueous solution. Furthermore it clarifies the occurrence of substrate inhibition in sodium‐di(ethylhexyl)sulphosuccinate‐reversed micelles and the observed low activity in Triton‐reversed micelles, as arising from the high partition coefficient of progesterone and the slow rate of diffusion of progesterone into the reversed micelles. From these results, and those reported for enoate reductase (see preceding paper in this issue of the journal) it can be concluded that the theory presented before (see first of this series of papers in this issue of the journal) offers a good explanation for the observed kinetic behaviour in reversed micelles, and emphasizes the importance of exchange processes between micelles.
Oligosaccharides at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus
Authors: GEYER Rudolf , DABROWSKI Janusz , DABROWSKI Ursula , LINDER Dietmar , SCHLÜTER Michael , SCHOTT Hans‐Henning , STIRM Stephan
Published: March 2005

Glycoprotein 71 from Friend murine leukemia virus was digested with proteases and the glycopeptides obtained were isolated and assigned, by amino acid sequencing, to the eight N‐glycosylated asparagines in the molecule; only Asn334 and Asn341 could not be separated. The oligosaccharides liberated from each glycopeptide by endo‐β‐N‐acetylglucosaminidase H, or by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F, were fractionated and subjected to structural analysis by one‐ and two‐dimensional 1H NMR, as well as by methylation/gas‐liquid‐chromatography/mass‐fragmentography. At each glycosylation site, the substituents were found to be heterogeneous including, at Asn334/341 and Asn410, substitution by different classes of N‐glycans: oligomannosidic oligosaccharides, mainly Manα1→6(Manα1 →3)Manα1 →6(Manα1 →3)Manβ1 →4Glc‐NAcβ1 →4GlcNAcβ1 →, were detected at Asn168, Asn334/341 and Asn410. Hybrid species, partially sialylated, intersected and (proximally) funcosylated Manα1 →6(Manα1 →3)Manα1 →6 and Manα1 →3Manα1 →6(Galβ1 →4GlcNAcβ1 →2Manα1 →3)Manβ1 →4GlcNAcβ1 →4GlcNAcβ1 →, were found at Asn12, as previously published [Schlüter, M., Linder, D., Geyer, R., Hunsmann, H., Schneider, J. & Stirm, S. (1984) FEBS Lett. 169, 194–198] and at Asn334/341. N‐Acetyllactosaminic glycans, mainly partially intersected and fucosylated NeuAcα2 →3 or Galα1 →3Galβ1 →4GlcNAcβ1 →2Manα1 →6(NeuAcα2 →6 or NeuAcα2 →3Gal‐β1 →4GlcNAcβ1 →2Manα1 →3)Manβ1 →4GlcNAcβ1 →4GlcNAcβ1 → with some bifurcation at →6Manα1 → 6, were obtained from Asn266, Asn302, Asn334/341, Asn374 and Asn410. In addition, Thr268, Thr277, Thr279, Thr304/309, as well as Ser273 and Ser275, were found to be O‐glycosidically substituted by Galβ1 →3GalNAcα1 →, monosialylated or disialylated at position 3 of Gal or/and position 6 of GalNAc.
Inhibition of leukotriene ω‐oxidation by isonicotinic acid hydrazide (isoniazid)
Authors: PARTHÉ Sylvia , HAGMANN Wolfgang
Published: March 2005

Metabolism of leukotrienes via ω‐oxidation represents a major degradative and inactivating pathway of these biologically active icosanoids. Isonicotinic acid hydrazide (isoniazid) inhibited this process in rats in vivo, in the isolated perfused rat liver, and in hepatic microsomes. The in vivo catabolism of leukotriene E4 via N‐acetyl‐leukotriene E4 to its ω‐oxidized metabolites was inhibited by 50% or 71% using single intravenous isoniazid doses of 0.6 mmol or 1.0 mmol/kg body mass, respectively. Isoniazid interfered with leukotriene catabolism at the initial ω‐oxidation step, resulting in an accumulation of N‐acetyl‐leukotriene E4. Analogous although weaker inhibition of leukotriene ω‐oxidation in vivo was observed by pretreatment with isonicotinic acid 2‐isopropylhydrazide and monoacetyl hydrazine. In the isolated perfused liver, isoniazid at concentrations varying over 0.2–10 mM decreased the ω‐oxidation of cysteinyl leukotrienes dose‐dependently by up to 94%. ω‐Oxidation of both leukotriene E4 and leukotriene B4 by rat liver microsomes was inhibited by isoniazid, isonicotinic acid 2‐isopropylhydrazide, and monoacetyl hydrazine with half‐maximal concentrations in the range of 5–15 mM. Our measurements indicate that the impairment of leukotriene ω‐oxidation by isoniazid involves both cytochrome‐P450‐dependent enzyme systems responsible for ω‐oxidation of leukotriene E4 and leukotriene B4. In effect, under isoniazid treatment one can expect a prolongation of the proinflammatory actions of endogenously produced leukotrienes.
Affinity labelling of endothelin receptor and characterization of solubilized endothelin–endothelin‐receptor complex
Authors: MIYAZAKI Hitoshi , KONDOH Motohiro , WATANABE Hirotoshi , MASUDA Yasushi , MURAKAMI Kazuo , TAKAHASHI Masami , YANAGISAWA Masashi , KIMURA Sadao , GOTO Katsutoshi , MASAKI Tomoh
Published: March 2005

Chick cardiac membranes were affinity labelled by cross‐linking to membrane‐bound 125I‐endothelin‐1 with disuccinimidyl tartarate. SDS/PAGE and autoradiographic analysis of the 125I‐endothelin‐1‐labelled material in the presence or absence of 2‐mercaptoethanol revealed one major labelled band, corresponding to a molecular mass of 53 kDa, whose appearance was dose‐dependently inhibited by the addition of unlabelled endothelin‐1 (1–100 nM). Subtracting the molecular mass of 125I‐endothelin‐1 and disuccinimidyl tartarate, the binding protein appeared to have a molecular mass of 50 kDa.

To investigate further the molecular properties of endothelin receptor, the 125I‐endothelin‐1–endothelin‐receptor complex was solubilized from chick cardiac membranes using the detergent digitonin. Sucrose gradient sedimentation of the solubilized complex indicated a sedimentation coefficient of 13 S, whereas the complex of (+)‐[3H]PN200–110, a dihydropyridine derivative, and dihydropyridine‐sensitive Ca2+ channels sedimented at 22 S. A monoclonal antibody raised against dihydropyridine‐sensitive Ca2+ channels from the chick brain did not immunoprecipitate the 125I‐endothelin‐1–endothelin‐receptor complex. These data suggest that endothelin receptor is clearly distinct from dihydropyridine‐sensitive Ca2+ channels and endothelin has its own specific 50‐kDa receptor.
Soluble hydrogenase of Anabaena cylindrica
Authors: EWART Gary D. , REED Ken C. , SMITH Geoffrey D.
Published: March 2005

A gene potentially encoding a subunit of the soluble hydrogenase of Anabaena cylindrica was isolated from a genomic library by screening with a set of redundant oligonucleotides, the sequence of which was deduced from the amino acid sequence of the purified hydrogenase subunit that catalyses tritium exchange. The nucleotide sequence of the potential gene was determined from two overlapping DNA fragments spanning 7237 bp of the A. cylindrica genome. The region sequenced contained an open reading frame encoding a protein of 383 amino acids with a predicted molecular mass of 41 108 Da. The NH2‐terminal amino acid sequence of the purified enzyme, determined by Edman degradation, corresponds exactly with that deduced from the nucleic acid sequence.

No significant amino acid or nucleotide similarity is evident between this gene and the periplasmic hydrogenases from three species of Desulfovibrio (D. vulgaris, D. baculatus and D. gigas), or with the membrane‐bound ‘uptake’ hydrogenases of Bradyrhizobium japonicum and Rhodobacter capsulatus. This suggests that the soluble enzyme from cyanobacteria represents a discrete class of hydrogenase.

The gene encoding the second subunit (m= 50 kDa) of the soluble hydrogenase, which is required for the catalysis of hydrogen production from dithionite‐reduced methyl viologen [Ewart, G. D. & Smith, G. D. (1989) Arch. Biochem. Biophys. 268, 327–337], apparently comprises a separate transcription unit since it appears not to be located adjacent to that for the 42‐kDa subunit.
Amino acid sequences around the pyridoxal‐5′‐phosphate‐binding sites of phenol hydroxylase
Authors: SEJLITZ Torsten , WERNSTEDT Christer , ENGSTRÖM Åke , NEUJAHR Halina Y.
Published: March 2005

Phenol hydroxylase was labelled with pyridoxal 5′‐phosphate. A radioactive label was introduced by using sodium boro[3H]hydride to reduce the initially formed Schiff's base. The labelled enzyme was digested with Staphylococcus V8 protease. Labelled peptides were isolated and their sequences were determined. The label could be located to three different lysyl residues. Sequence similarities with the known structures of p‐hydroxybenzoate hydroxylase and glutathione reductase are discussed. The positions of the labelled sequences, relative to the bound ligands at the active site, are proposed on the basis of such sequence similarities.
Aspartate aminotransferase with the pyridoxal‐5′‐phosphate‐binding lysine residue replaced by histidine retains partial catalytic competence
Authors: ZIAK Martin , JAUSSI Rolf , GEHRING Heinz , CHRISTEN Philipp
Published: March 2005

The active site residue lysine 258 of chicken mitochondrial aspartate aminotransferase was replaced with a histidine residue by means of site‐directed mutagenesis. The mutant protein was expressed in Escherichia coli and purified to homogeneity. Addition of 2‐oxoglutarate to its pyridoxamine form changed the coenzyme absorption spectrum (λmax= 330 nm) to that of the pyridoxal form (λmax= 330/392 nm). The rate of this half‐reaction of transamination (kcat= 4.0 × 10−4 s−1) is five orders of magnitude slower than that of the wild‐type enzyme. However, the reverse half‐reaction, initiated by addition of aspartate or glutamate to the pyridoxal form of the mutant enzyme, is only three orders of magnitude slower than that of the wild‐type enzyme, kmax of the observable rate‐limiting elementary step, i.e. the conversion of the external aldimine to the pyridoxamine form, being 7.0 × 10−2 s−1. Aspartate aminotransferase (Lys258 His) thus represents a pyridoxal‐5′‐phosphate‐dependent enzyme with significant catalytic competence without an active site lysine residue. Apparently, covalent binding of the coenzyme, i.e. the internal aldimine linkage, is not essential for the enzymic transamination reaction. and a histidine residue can to some extent substitute for lysine 258 which is assumed to act as proton donor/acceptor in the aldimine‐ketimine tautomerization.
Metal‐ion‐governed molecular recognition: extent of intramolecular stack formation in mixed‐ligand–copper(II) complexes containing a heteroaromatic N base and an adenosine monophosphate (2′AMP, 3′AMP, or 5′AMP)
Authors: MASSOUD Salah S. , TRIBOLET Roger , SIGEL Helmut
Published: March 2005

Stability constants of mixed‐ligand Cu(Arm)(AMP) complexes [where Arm = 2,2′‐bipyridyl (Bpy) or 1,10phenanthroline (Phen) and AMP2−= 2′AMP2−, 3′AMP2− or 5′AMP2−] were determined by potentiometric pH titrations in aqueous solution at I= 0.1 M (NaNO3) and 25°C. The ternary Cu(Arm)(AMP) complexes are more stable than corresponding Cu(Arm)(R‐MP) complexes, where R‐MP2− represents a phosphate monoester with a group R that is unable to participate in any kind of interaction within the complexes as, for example, D‐ribose 5′‐monophosphate. This increased stability is attributed, in agreement with previous results, to intramolecular stack formation in the Cu(Arm)(AMP) complexes between the purine residue of the AMPs and the aromatic rings of Bpy or Phen. Based on correlation lines (previously obtained from log K versus pKa plots) for Cu(Arm)(R‐MP) complexes without a ligand‐ligand interaction, a quantitative evaluation was carried out. The degree of formation of the species with the intramolecular stacks increases for the Cu(Arm)(AMP) complexes in the series: 3′AMP2− < 5′AMP2− < 2′AMP2−; e.g. in Cu(Bpy)(3′AMP) the stack reaches a formation degree of 45 ± 11% and in Cu(Bpy)(2′AMP) one of 96.1 ± 0.7% is obtained. It must be emphasized that these differences are due to the different steric orientations of the bridging metal ion, which result from the varying position of the phosphate group on the ribose ring. As shown by 1H‐NMR shift measurements, there is no significant effect of the position of the phosphate group on the stability of the binary (Phen)(AMP)2− adducts (K∼ 36 M−1 in D2O); such an effect is seen only if a metal‐ion bridge is formed between the moieties forming the stack, i.e. metal‐ion coordination imposes individual properties on the AMPs. By also taking into account some recent results on other nucleoside 5′‐monophosphate complexes, the following trend for an increasing stacking tendency of the nucleic base moieties can be established: uracil ≲ cytosine ≲ thymine ≪ adenine < 7‐deazaadenine. Some additional conclusions of general importance are given and the relevance of the results with regard to bio‐systems is indicated.
Antigenic sites on cytochrome c2 from Rhodospirillum rubrum
Authors: SAAD Bashar , BOSSHARD Hans Rudolf
Published: March 2005

The antigenic determinants for three monoclonal antibodies against cytochrome c2 from Rhodospirillum rubrum were partially characterized by differential chemical modification of free and antibody‐bound cytochrome c2 and by cross‐reactivity analysis with different antigens. Circular dichroism spectroscopy was used to probe the effect of antibody binding on the conformation of cytochrome c2. The binding of two antibodies was strongly dependent on the native folding of the antigen. The first antibody bound to a determinant around the exposed heme edge on the ‘front side’ of the molecule which is not antigenic in mitochondrial cytochrome c2. Binding of this antibody to cytochrome c increased the induced CD of the ferric heme in a manner similar to that observed previously when mitochondrial cytochrome‐c oxidase bound to the front side of cytochrome c. This observation points to a subtle conformational adaptation of the antigen induced by the antibody. The determinant for the second antibody, which also affected the heme CD spectrum of the antigen, was on a polypeptide loop where cytochrome c2 differs from mitochondrial cytochrome c by an eight‐residue insertion. The third antibody, which did not induce a change in CD, bound to a sequential determinant near the amino end of cytochrome c2. Only this antibody cross‐reacted with isolated cytochrome‐c‐derived peptides and with apo‐cytochrome c2.

A preliminary analysis of the polyclonal immune response of five rats against cytochrome c2 indicates that, unlike in eukaryotic cytochrome c, antigenic determinants are distributed over the whole polypeptide chain of the prokaryotic immunogen.
Calcium‐ionophore‐induced formation of platelet‐activating factor and leukotrienes by horse eosinophils: a comparative study
Authors: ASMIS Reto , JÖRG Andreas
Published: March 2005

Horse eosinophils preincubated with 3H‐labelled acetate and stimulated with the Ca2+ ionophores ionomycin or A23187 form a radioactive compound, which we have shown to be 1‐O‐alkyl‐2‐[3H]acetyl‐sn‐glycero‐3‐phosphocholine (platelet‐activating factor). We could detect no 1‐O‐acyl‐2‐[3H]acetyl‐glycero‐3‐phosphocholine in the radioactive fraction. The formation of platelet‐activating factor was strongly correlated to the generation of leukotriene C4, the main arachidonate metabolite in horse eosinophils, suggesting that platelet‐activating factor and leukotriene C4 have a common precursor pool (1‐O‐alkyl‐2‐arachidonyl‐glycero‐3‐phosphocholine) and a common regulation of synthesis. Even though both ionomycin and A23187 are described as Ca2+ ionophores, they have a series of significantly different effects on the eosinophils with respect to formation of platelet‐activating factor and leukotriene C4. While A23187 induces an asymptotic maximum of mediator formation at concentrations higher than 20 μM, ionomycin expressed a narrow optimum at 2μM. The effects of exogenous pH on the release of mediators also differ strongly between the two ionophores: for A23187 the effects are the same for both mediators but when ionomycin is used as stimulant, generation of platelet‐activating factor and leukotriene C4 are affected differently.
Reduced transcription of mitochondrial DNA in the senescent rat
Authors: GADALETA Maria Nicola , PETRUZZELLA Vittoria , RENIS Marcella , FRACASSO Flavio , CANTATORE Palmiro
Published: March 2005

A quantitative study on the effect of senescence on mitochondrial DNA expression has been carried out by measuring the levels of the 12S rRNA and of the mRNA for the subunit I of cytochrome oxidase in several tissues of adult and senescent rats. The concentration of both RNA species/mitochondrial DNA molecule is significantly reduced in senescent brain and heart, as opposed to the respective adult tissues. No appreciable variation occurs in the liver. A 1‐h pretreatment with acetyl‐L‐carnitine brings back the level of senescent brain and heart transcripts to that of adult tissues. The same treatment of adult rats does not cause significant changes in mitochondrial RNA content. These results suggest that the age‐dependent impairment of both heavy‐strand mitochondrial DNA transcription units is related to altered environmental conditions which acetyl‐L‐carnitine, a substance which acts by stimulating, directly or indirectly, the energy metabolism, is able to remove.
A factor known to bind to the endogenous Ig heavy chain enhancer only in lymphocytes is a ubiquitously active transcription factor
Authors: ELMALEH Nadav , MATTHIAS Patrick , SCHAFFNER Walter
Published: March 2005

The transcriptional enhancer located in the first intron of the immunoglobulin heavy chain constant region is a major determinant of B‐cell‐specific expression of immunoglobulin genes. Like other enhancers, the Ig heavy chain enhancer contains several short sequence motifs that bind specific transcription factors. Each binding site contributes to the overall activity of the enhancer, however no single element seems absolutely required for activity. For a better understanding of the Ig heavy chain enhancer components, we have cloned and analyzed individual sequence elements. We find that the factor that binds to the E3 enhancer motif, CATGTGGC, is a ubiquitous transcription factor. It is present in an active form in both B cells and non‐B cells, where it can mediate transcriptional activation in vitro and in vivo. However, despite its ability to activate transcription of a transfected reporter gene, the factor is apparently unable to bind to the endogenous Ig heavy chain enhancer in non‐lymphoid cells: In previous experiments by others, the characteristic in vivo footprint of this factor, designated NF‐μE3, was detected in B cells but not in non‐B cells. From this and other findings the picture emerges that there are at least three categories of factors which mediate cell‐type‐specific transcription in B lymphocytes: (a) cell‐specific factors such as Oct‐2A and Oct‐2B that are not expressed in most other cell types; (b) ubiquitous factors such as NF‐κB that are constitutively active in B cells but are sequestered in an inactive form in other cells; (c) ubiquitously active factors, exemplified by the one binding to the E3 sequence motif. This factor is present in an active form in a variety of cell types but is apparently unable to bind to the endogenous Ig heavy chain enhancer in non‐B cells, perhaps due to a non‐permissive chromatin structure of the Ig heavy chain locus.
The ribosomal domain of the bacterial release factors
Authors: TATE Warren P. , KASTNER Berthold , EDGAR Christina D. , McCAUGHAN Kim K. , TIMMS Kirsten M. , TROTMAN Clive N. A. , STOFFLER‐MEILICKE Marina , STOFFLER Georg , NAG Bishwajit , TRAUT Robert R.
Published: March 2005

Polyclonal antibodies (pAb 1–73 and pAb 26–120) have been raised against both an N‐terminal fragment of Escherichia coli ribosomal protein L7/L12 (amino acids 1–73), and a fragment lacking part of the N‐terminal domain (amino acids 26–120).

Only pAb 26–120 inhibited release‐factor‐dependent in vitro termination functions on the ribosome. This antibody binds over the length of the stalk of the large subunit of the ribosome as determined by immune electron microscopy, thereby not distinguishing between the C‐terminal domains of the two L7/L12 dimers, those in the stalk or those in the body of the subunit.

A monoclonal antibody against an epitope of the C‐terminal two thirds of the protein (mAb 74–120), which binds both to the distal tip of the stalk as well as to a region at its base, reflecting the positions of the two dimers is strongly inhibitory of release factor function.

A monoclonal antibody against an epitope of the N‐terminal fragment of L7/L12 (mAb 1–73), previously shown to remove the dimer of L7/L12 in the 50S subunit stalk but still bind to the body of the particle, partially inhibited release‐factor‐mediated events.

The mAb 74–120 inhibited in vitro termination with a similar profile when the stalk dimer of L7/L12 was removed with mAb 1–73, indicating that the body L7/L12 dimer, and in particular its C‐terminal domains, are important for release factor/ribosome interaction.

The two release factors have subtle differences in their binding domains with respect to L7/L12.
Pertussis toxin and H‐7 distinguish mechanisms involved in eicosanoid release from lipopolysaccharide‐primed macrophages
Authors: MATSUNAGA Akira , MILLER Bonnie C. , COTTAM G. Larry
Published: March 2005

Release of eicosanoids is an important response of macrophages to inflammation and bacterial infection. At low concentrations, bacterial lipopolysaccharide (1–2 μg/ml) fails to stimulate eicosanoid release in resident peritoneal macrophages but primes the macrophages for a greatly enhanced release of eicosanoids on stimulation with the calcium ionophore A23187 (0.1 μM) or with phorbol 12‐myristate 13‐acetate (50 nM), an activator of protein kinase C. Incubation of macrophages with Bordetella pertussis toxin, prior to priming with lipopolysaccharide, inhibited the release of both cyclooxygenase and lipoxygenase products upon A23187 stimulation. Pertussis toxin treatment of macrophages had no effect on eicosanoid release when the stimulus was phorbol 12‐myristate 13‐acetate. The presence of 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H‐7), an effective inhibitor of protein kinase C, during lipopolysaccharide priming and subsequent stimulation significantly inhibited eicosanoid release when phorbol 12‐myristate 13‐acetate was the stimulus, but did not affect eicosanoid release stimulated by A23187. Based on these results, at least two mechanisms, distinguished by apparent differences in sensitivity to pertussis‐toxin‐sensitive, guanine‐nucleotide‐binding proteins and protein kinase C, are involved in eicosanoid secretion by lipopolysaccharide‐activated macrophages in response to A23187 and phorbol 12‐myristate 13‐acetate.
Characterization of the nickel‐iron periplasmic hydrogenase from Desulfovibrio fructosovorans
Authors: HATCHIKIAN Claude E. , TRAORE Alfred S. , FERNANDEZ Victor M. , CAMMACK Richard
Published: March 2005

The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa. The absorption spectrum of the enzyme is characteristic of an iron‐sulfur protein and its absorption coefficients at 400 and 280 nm are 50 and 180 mM−1 cm−1, respectively. D. fructosovorans hydrogenase contains 11 ± 1 iron atoms, 0.9 ± 0.15 nickel atom and 12 ± 1 acid‐labile sulfur atoms/molecule but does not contain selenium. The amino acid composition of the protein and of its subunits, as well as the N‐terminal sequences of the small and large subunits, have been determined. The cysteine residues of the protein are distributed between the large (9 residues) and the small subunits (11 residues). Electron spin resonance (ESR) properties of the enzyme are consistent with the presence of nickel(III), [3Fe–4S] and [4Fe–4S] clusters. The hydrogenase of D. fructosovorans isolated under aerobic conditions required an incubation with hydrogen or other reductants in order to express its full catalytic activity. H2 uptake and H2 evolution activities doubled after a 3‐h incubation under reducing conditions. Comparison with the (NiFe) hydrogenase from D. gigas shows great structural similarities between the two proteins. However, there are significant differences between the catalytic properties of the two enzymes which can be related to the respective state of their nickel atom. ESR showed a higher proportion of the Ni‐B species (g= 2.33, 2.16, 2.01) which can be related to a more facile conversion to the ready state. The periplasmic location of the enzyme and the presence of hydrogenase activity in other cellular compartments are discussed in relation to the ability of D. fructosovorans to participate actively in interspecies hydrogen transfer.
Characterisation of the asparagine‐linked oligosaccharides from Trypanosoma brucei type‐I variant surface glycoproteins
Authors: ZAMZE Susanne E. , WOOTEN E. Wrenn , ASHFORD David A. , FERGUSON Michael A. J. , DWEK Raymond A. , RADEMACHER Thomas W.
Published: March 2005

The complete primary structures of the Asn‐linked oligosaccharides from the conserved glycosylation site of the type‐I variant surface glycoproteins of Trypanosoma brucei MITat 1.4 and MITat 1.6 were determined using a combination of exoglycosidase digestions, permethylation analysis, acetolysis and 1H NMR. Both variants contained almost exclusively oligomannose‐type oligosaccharides, identical in structure to those of mammalian glycoproteins. The oligosaccharides ranged in size from (Man)9(GlcNAc)2 to (Man)5(GlcNAc)2. The relative abundance of each component was similar in both variants. The major components were (Man)8(GlcNAc)2 and (Man)7(GlcNAc)2 with slightly less (Man)9(GlcNAc)2 and (Man)6(GlcNAc)2 and much less (Man)5(GlcNAc)2. Both variants also contained the same structural isomers. The close similarity of the oligomannose series indicates identical processing at the conserved site in both variants.
Dictyopterin, 6‐(d‐threo‐1,2‐dihydroxypropyl)‐pterin, a new natural isomer of l‐biopterin
Authors: KLEIN Roger , THIERY Richard , TATISCHEFF Irene
Published: March 2005

A major pterin was isolated by reverse‐phase high‐performance liquid chromatography from cellular extract of vegetative cells of Dictyostelium discoideum after perchloric deproteinization and oxidation with acidic iodine. This compound was characterized by its chromatographic behavior, its absorption and fluorescence properties, by its oxidation product with alkaline permanganate, by secondary ion mass spectrometry and by circular dichroism. The final identification was obtained by comparison with authentic materials. It is concluded that the major pterin of D. discoideum is the compound 6‐(D‐threo‐1,2‐dihydroxypropyl)‐pterin. The name dictyopterin is proposed for this new natural isomer of L‐biopterin.
Expression of guinea‐pig liver transglutaminase cDNA in Escherichia coli
Authors: IKURA Koji , TSUCHIYA Yoichi , SASAKI Ryuzo , CHIBA Hideo
Published: March 2005

Transglutaminases (EC 2.3.2.13) catalyze the formation of g‐(γ‐glutamyl)lysine cross‐links and the substitution of a variety of primary amines for the γ‐carboxamide groups of protein‐bound glutamine residues. These enzymes are involved in many biological phenomena. Transglutaminase reactions also have been shown to be suitable for applied enzymology. In this study, as a first step of studies to elucidate the structure/function relationship of transglutaminase, we constructed an expression plasmid, pKTG1, containing a cDNA of guinea‐pig liver transglutaminase between the NcoI and PstI sites of an expression vector, pKK233–2, and produced the liver transglutaminase as an unfused protein in Escherichia coli. The purified recombinant enzyme was indistinguishable from natural liver transglutaminase in some structural properties such as molecular mass, amino acid composition, and amino‐ and carboxyl‐terminal sequences. However, the α‐amino group of the amino‐terminal alanine residue of the recombinant transglutaminase was not acetylated as was that of the natural enzyme. Comparison of the recombinant enzyme with the natural one did not indicate significant differences in specific activity and apparent Km values for substrates in the histamine incorporation into acetyl αs1‐casein. The sensitivity to activation by Ca2+ and the rate of catalyzed protein cross‐linking were also similar between recombinant and natural transglutaminases. These results indicated that the Nα‐acetyl group in natural liver transglutaminase has not a particular role in the catalytic function of this enzyme.
Expression of completely γ‐carboxylated and β‐hydroxylated recombinant human vitamin‐K‐dependent protein S with full biological activity
Authors: MALM Johan , COHEN Edward , DACKOWSKI William , DAHLBÄCK Björn , WYDRO Robert
Published: March 2005

Human anticoagulant vitamin‐K‐dependent protein S was expressed in mouse C127 cells using a bovine papilloma virus vector system. A full‐length cDNA construct was introduced into the vector in the 5′ untranslated region of the mouse metallothionein‐I gene. Transfected cells expressed approximately 10 μg/ml of the recombinant protein which was purified by ion‐exchange chromatography followed by affinity chromatography using Ca2+‐dependent monoclonal antibodies against the region of protein S containing 4‐carboxyglutamic acid. Recombinant protein S was structurally and functionally similar to protein S purified from plasma. On SDS/polyacrylamide‐gel electrophoresis recombinant protein S had a slightly higher molecular mass than plasma protein S. After treatment with endoglycosidase F, the proteins comigrated suggesting the observed molecular mass difference to be due to alterations in the N‐linked carbohydrate side chains. Recombinant and plasma protein S demonstrated identical amino‐terminal sequences, similar amino acid composition and number of 4‐carboxyglutamyl and 3‐hydroxyaspartyl/asparaginyl residues. Recombinant protein S had the same affinity for Ca2+ as protein S from plasma and the two proteins had the same activated protein C cofactor activity in a functional assay. In addition, both forms of protein S formed complexes with C4b‐binding protein with the same apparent Kd. Protein S is the most extensively post‐translationally modified vitamin‐K‐dependent protein, and all the modifications were carried out in the recombinant DNA system yielding a recombinant protein S with full biological activity.
Structural analysis of p36, a Ca2+/lipid‐binding protein of the annexin family, by proteolysis and chemical fragmentation
Authors: JOHNSSON Nils , WEBER Klaus
Published: March 2005

Limited proteolysis of the core domain of the 36‐kDa protein p36 by trypsin gives a first insight into the structural organization of the four annexin repeats. Trypsin opens only a single peptide bond, situated between residues 204 and 205. The two fragments (of 20 kDa and 15 kDa), each containing two annexin repeats, remain as a tight complex (nicked core), which binds phospholipids in a Ca2+‐dependent manner. After denaturation by 9 M urea, the nicked core is again formed upon renaturation provided both fragments present. If the fragments are separated by chromatography in urea prior to renaturation, they show different behaviour. The 15‐kDa C‐terminal repeats aggregate, while the 20‐kDa N‐terminal repeats stay in solution. In comparison to p36, fragments with two (20‐kDa fragment)or one(N‐terminal CNBr fragment)annexin repeats show a conformational alteration in CD spectroscopy and hydrodynamics and display an increased susceptibility to proteases. In line with these differences, their Ca2+‐dependent affinity to phospholipids is more than 10–20‐fold decreased. Thus the four annexin repeats form together an integrated domain with multiple contacts between the repeats. Although stable derivatives with less than four repeats can be obtained, their Ca2+/phospholipid binding affinities are noticeably reduced.
Mapping of glucose and glucose‐6‐phosphate binding sites on bovine brain hexokinase
Authors: JARORI Gotam K. , IYER Sujata B. , KASTURI Sitapati R. , KENKARE Umakant W.
Published: March 2005

Inhibition of bovine brain hexokinase by its product, glucose 6‐phosphate, is considered to be a major regulatory step in controlling the glycolytic flux in the brain. Investigations on the molecular basis of this regulation, i.e. allosteric or product inhibition, have led to various proposals. Here, we attempt to resolve this issue by ascertaining the location of the binding sites for glucose and glucose 6‐phosphate on the enzyme with respect to a divalent‐cation‐binding site characterized previously[Jarori, G. K., Kasturi, S. R. & Kenkare, U. W.(1981) Arch. Biochem. Biophys. 211, 258–268]. The paramagnetic effect of enzyme‐bound Mn(II) on the spinlattice relaxation rates (T−11) of ligand nuclei (1H and31P)in E. Mn(II). Gle and E. Mn(II). Glc66 complexes have been measured. The paramagnetic effect of Mn(II) on the proton relaxation rates of C1‐Hα, C1‐Hβ and C2‐Hβ of glucose in the E · Mn(II) · Glc complex was measured at 270 MHz and 500 MHz. The temperature dependence of these rates was also studied in the range of 5–30°C at 500 MHz. The ligand nuclear relaxation rates in E · Mn(II) · Glc are field‐dependent and the Arrhenius plot yields an activation energy (ΔE) of 16.7–20.9 kJ/mol. Similar measurements have also been carried out on C1‐Hα, C1‐Hβ and C6‐31P at 270 MHz (1H and 202.5 MHz (31P) for the E · Mn(II) · Glc6P complex. The temperature dependence of 31P relaxation rates in this complex was measured in the range 5–30°C, which yielded Δ= 9.2 kJ/mol. The electron‐nuclear dipolar correlation time (τc), determined from the field‐dependent measurements of proton relaxation rates in the E · Mn(II)· Gle complex, is 0.22–1.27 ns. The distances determined between Mn(II) and C1‐H of glucose and glucose 6‐phosphate are ∼ 1.1 nm and ∼ 0.8 nm, respectively. These data, considered together with our recent results [Mehta, A., Jarori, G. K. & Kenkare. U. M.(1988) J. Biol. Chem. 263, 15492–15498], suggest that glucose and glucose 6‐phosphate may bind to very nearly the same region of the enzyme.

The structure of the binary Glc6P· Mn(II) complex has also been determined. The phosphoryl group of the sugar phosphate forms a first co‐ordination complex with the cation. However, on the enzyme, the phosphoryl group is located at a distance of ∼ 0.5–0.6 nm from the cation.
Novel hyperglycosylated weak gelatin‐binding fibronectin from human fetal placenta
Authors: ZHU Betty C. R. , LAINE Roger A.
Published: March 2005

A novel hyperglycosylated fraction of human term fetal placental fibronectin was detected by long‐term affinity binding to gelatin‐Sepharose. An 18‐h batch‐wise gelatin‐binding step was necessary to obtain a very low‐affinity binding fraction, characterized by especially high N‐acetylglucosamine and galactose content, and diffuse, poorly stained Coomassie bands on SDS/polyacrylamide electrophoretograms. The presence of a high proportion of long 7 – 10‐kDa poly(N‐acetyllactosamine)‐containing N‐linked carbohydrate chains was confirmed by their gel permeation behavior, susceptibility to endo‐β‐galactosidase and by methylation analysis. Our previous results suggest that 4.5 – 7‐kDa poly(N‐acetyllactosamine) structures reduce the binding of fibronectin and its chymotryptic Ala260–Trp599 subdomain GB44 to gelatin [Zhu, B. C. R. & Laine, R. A.(1985) J. Biol. Chem. 260, 4041–3045]. Based on a gradient of urea used to dissociate gelatin‐bound GB44, in the present study, fractions containing the novel 7–10‐kDa carbohydrates showed significantly weaker binding to gelatin. Weak gelatin‐binding characteristics of this novel hyperglycosylated fraction suggest that extended poly(N‐acetyllactosamine) N‐linked chains can significantly weaken heterotropic binding functions of fetal glycoproteins. The combined properties of weak Coomassie staining and weak gelatin binding have caused the novel hyperglycosylated fibronectin to be overlooked in previous investigations.
Infrared spectroscopic studies of detergent‐solubilized uncoupling protein from brown‐adipose‐tissue mitochondria
Authors: RIAL Eduardo , MUGA Arturo , VALPUESTA José M. , ARRONDO José‐Luis R. , GOÑI Félix M.
Published: March 2005

The uncoupling protein of brown‐adipose‐tissue mitochondria has been purified in the form of micelles with lipid and reduced Triton X‐100. This surfactant has the advantage over conventional Triton X‐100, that it does not interfere with amide bands in infrared spectra. The structure of the uncoupling protein in micellar form has been examined by Fourier‐transform infrared spectroscopy (FTIR). In order to decompose the amide I contour into its components, band‐narrowing (Fourier derivation and deconvolution) and band‐decomposition techniques have been used. Combining data from spectra taken in H2O and 2H2O media, the following percentage distribution of secondary structure patterns has been obtained: 50%α‐helix, 28–30%β‐structure; 13–15%β‐turns and 7% unordered. Thermal denaturation of the uncoupling protein has also been monitored by FTIR. In acordance with previous observations of different proteins, thermal denaturation is marked by a shift in the amide I maximum and the appearance of two new peaks in 1H2O, at around 1620 cm−1 and 1685 cm−1. Denaturation occurs in the 40–50°C temperature range, in agreement with studies of GDP‐binding capacity. Cooling down the thermally denatured protein produces a new change in its secondary structure; however, the original conformation is not restored. The uncoupling protein possesses a nucleotide‐binding site. On addition of GDP, small changes in protein conformation occur, attributable to changes in tertiary structure. However, no detectable effects are seen in the presence or absence of the other physiological regulators, the free fatty acids. The uncoupling protein shares important similarities in its primary structure with other anion carriers of the mitochondrial membrane: one of these, the adenine‐nucleotide translocator, has been used in a comparative study, applying the same FTIR techniques described above for the uncoupling protein. Both proteins have a similar proportion of αhelix, probably corresponding to the segments spanning the membrane, but the conformation of the polar domains appears to differ.
Identification of a cholesterol‐regulated 180‐kDA microsomal protein in rat hepatocytes
Authors: HARO Diego , MARRERO Pedro F. , AYTÉ Jose , HEGARDT Fausto G.
Published: March 2005

The immunoprecipitation by antibodies to 3‐hydroxy‐3‐methylglutry‐coenzyme A (HMG‐CoA) reductase of extracts of [35S]methionine‐pulse‐labelled isolated hepatocytes, followed by electrophoresis and fluorography, showed the presence not only of 97‐kDa HMG‐CoA reductase, but also of another protein of 180 kDa. Boiling the immunoprecipitates both in the presence and in the absence of 2‐mercaptoethanol, followed by SDS/polyacrylamide gel electrophoresis both in the presence and in the absence of 8 M urea, was not found to change the ratio of 180‐kDa/97‐kDa proteins. These facts suggest that the 180‐kDa protein is not an aggregated form of HMG‐CoA reductase. A different batch of antibodies obtained from a newly purified HMG‐CoA reductase fully titrated the reductase activity, but did not immunoprecipitate the 180‐kDa protein, showing that there is no cross‐reactivity between these proteins. The 180‐kDa polypeptide is a glycoprotein of N‐linked high‐mannose oligosaccharide chains, which is not processed on the Golgi system. The apparent molecular mass of the carbohydrate is 16 kDa.

The incubation of rat hepatocytes with sterols produces, on the one hand, a decrease in the rate of synthesis, and on the other hand, an acceleration in the turnover rate of the 180‐kDa protein. In addition, mevalonate is known to decrease its rate of synthesis. The carbohydrate‐free 164‐kDa protein was found to degrade only a tenth as fast as the glycoprotein and, furthermore, the degradation was no longer accelerated by sterols. These results support the notion that the 180‐kDa protein is not a modified form of 97‐kDa reductase, but probably a different protein related to cholesterol metabolism. and also that the N‐linked, high‐mannose chains. Which are bound to the glycoprotein, are required for rapid and controlled degradation of the protein.