bluuebunny/crossref_metadata_2025 huggingface dataset:
Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylase
Authors: Solstad Therese , Carvalho Raquel N. , Andersen Ole A. , Waidelich Dietmar , Flatmark Torgeir
Published: March 2003

Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302–6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N‐terminal autoregulatory sequence (residues 19–33) of wt‐hPAH, are among the susceptible residues. First, on MALDI‐TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28‐residue peptide, L15–K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse‐phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28‐residue peptide by its methylation by protein‐l‐isoaspartic acid O‐methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 °C of the phosphorylated form (at Ser16) of this 28‐residue peptide, a time‐dependent mobility shift from tR≈ 34 min to ≈ 31 min (i.e., to a more hydrophilic position) was observed on reverse‐phase chromatography, and the recovery of the tR≈ 34 min species decreased with a biphasic time‐course with t0.5‐values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence‐controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half‐time of ≈ 1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 °C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen‐bond network. Deamidation of Asn32 introduces a negative charge and a partial β‐isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site‐directed mutagenesis, and the mutant form (Asn32→Asp) revealed a 1.7‐fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L‐Phe binding as well as substrate inhibition.
Cloning, purification and characterization of DNA polymerase β from Xenopus laevis
Authors: Reichenberger Susanne , Pfeiffer Petra
Published: March 2003

Double‐strand breaks in the DNA of vertebrate cells are joined by mechanisms of non‐homologous DNA‐end joining (NEJ). In extracts from Xenopus eggs, NEJ is inhibited by dideoxynucleotides, indicating a possible involvement of DNA polymerase β (Polβ). Since some types of NEJ products were shown to be formed in vitro by prokaryotic DNA polymerases lacking exonuclease activity, we were interested in whether Polβ alone would be capable of catalyzing NEJ reactions. Therefore we have cloned the full‐length cDNA of the Xenopus laevis Polβ. The cDNA, predicting a highly conserved 39‐kDa protein of 334 amino acids, was tagged with six histidine residues at its N‐terminus for overexpression in Escherichia coli, purified to near homogeneity, and shown to have the same catalytic properties as the previously cloned rat and human enzymes. Using oligonucleotides as substrates we show that the recombinant Xenopus Polβ adds single untemplated nucleotides to blunt ends. However, under conditions that permit efficient NEJ in Xenopus egg extracts, Polβ does not form those types of NEJ products formed by the prokaryotic polymerases indicating that Polβ alone is not able to mediate the complex NEJ process in vitro. Using substrates with 3′ protruding single strands of increasing length (6−16 nucleotides) we show that Polβ initiates fill‐in DNA synthesis on fold‐back structures formed by the longest 3′ protruding stand. This unusual feature of β‐type polymerases requires that the loop of the fold‐back structure consists of at least six bases and the stem be paired by at least 2bp to facilitate priming of DNA synthesis.
The interactions between highly de‐N‐acetylated chitosans and lysozyme from chicken egg white studied by 1H‐NMR spectroscopy
Authors: Kristiansen Are , Vårum Kjell M. , Grasdalen Hans
Published: March 2003

We have investigated the binding interactions between highly de‐N‐acetylated chitosans and lysozyme from chicken egg white by one‐dimensional and two‐dimensional 1H‐NMR spectroscopy. A fully de‐N‐acetylated chitosan (fraction of N‐acetylated units, FA < 0.001) induced no observable changes in the 1H chemical shifts of lysozyme. However, a chitosan with FA = 0.04, where the N‐acetylated units are predominantly surrounded by de‐N‐acetylated units (a monoacetylated sequence), induced significant shifts of several lysozyme resonances, demonstrating a specific interaction between lysozyme and de‐N‐acetylated units in the chitosan. The interaction between the two positively charged molecules increased with increasing ionic strength, as expected. The dissociation constant (Kd) between lysozyme and the monoacetylated sequence was strongly dependent on pH* (pH measured in D2O), with Kd = 0.02 ± 0.01 mM at pH* 6.0, Kd = 0.11 ± 0.02 mM at pH* 4.5, and Kd≈2 mM at pH* 3, suggesting that electrostatic forces contribute to the observed binding. The complex was strikingly stable, with bound lifetimes in the range of 10−25 ms at pH* 4.5 and 328−300 K. Most lysozyme resonances that were affected by the binding were assigned, and we suggest that the monoacetylated chitosan sequence binds to the active site cleft of lysozyme with the N‐acetylated unit in subsite C. Assuming this binding mode, we have discussed the contributions in energetic terms from individual subsites of lysozyme towards binding of N‐acetylated and de‐N‐acetylated units.
Cycloheximide sensitivity of orotic acid biosynthesis induced by ammonia and glycine administration
Authors: Vasudevan Shanthi , Laconi Ezio , Rao Prema M. , Rajalakshmi Srinivasan , Sarma Dittakavi S. R. , La Piana Gianluigi , Fransvea Emilia , Marzulli Domenico , Lofrumento Nicola E.
Published: March 2003

Administration of either ammonia or glycine to both rats and mice results in an increased synthesis in the liver and urinary excretion of orotic acid. The two most relevant observations obtained are that carbamoyl phosphate synthesized inside the mitochondria is involved in the increased synthesis of orotic acid and that this latter process is almost completely abolished by cycloheximide and actinomycin D, inhibitors of protein and RNA synthesis. Orotic acid synthesis could be controlled by an induction‐suppression mechanism. Inhibition of synthesis of excess orotic acid brought about by N‐(phosphonacetyl)‐
L‐aspartic acid but not by acivicin, suggests that glutamine‐dependent cytosolic synthesis of carbamoyl phosphate, is not involved. Administration of ornithine together with glycine completely suppressed the synthesis of orotic acid, but promoted a twofold increase of urea excretion. The concentration of ornithine rather than that of carbamoyl phosphate or the activity of the enzymes involved, may represent a limiting factor controlling both the flux of ammonia in the urea cycle and the availability of mitochondrial carbamoyl phosphate for orotic acid synthesis. Two enzymes have been found to be induced by glycine : ornithine decarboxylase and aspartate transcarbamoylase (aspartate carbamoyltransferase). Both enzymes may contribute to the increase in orotic acid synthesis, aspartate transcarbamoylase more directly and ornithine decarboxylase by lowering the ornithine concentration. Ornithine decarboxylase activity was completely suppressed but that of aspartate transcarbamoylase was further increased by cycloheximide treatment. Inhibition of orotic acid biosynthesis by cycloheximide appears to be the result of a decreased availability in the cytosol of carbamoyl phosphate synthesized inside the mitochondria.
Equus caballus gelsolin
Authors: Koepf Edward K. , Hewitt Jeff , Vo Hung , Macgillivray Ross T. A. , Burtnick Leslie D.
Published: March 2003

We have generated and characterized the cDNA from equine smooth muscle that encodes gelsolin, an actin‐modulating protein. Overlapping cDNA clones synthesized by the reverse transcriptase/polymerase chain reaction and clones isolated from a horse genomic library provided the complete primary structure for the intracellular isoform of gelsolin, while cDNA complemented with protein sequence data produced the full‐length primary transcript of the gelsolin isoform found circulating in equine plasma. The deduced amino acid sequences of the intracellular and secreted versions of equine gelsolin infer polypeptides of 731 and 755 residues with apparent molecular masses of 80.7 kDa and 83.2 kDa, respectively. Multiple sequence alignment analysis of equine, human, porcine, and murine orthologs of gelsolin demonstrates prominent similarities among all of these proteins, with the horse and human molecules exhibiting the largest degree of likeness with respect to polypeptide length and overall sequence composition. Both horse and human plasma gelsolins are comprised of 755 amino acids with 94 % of the residues identical, while the degree of sequence identity in the shorter (731 residues) cytoplasmic gelsolins is 95 %. Analysis of the sequences and structures of the six related domains that comprise gelsolin emphasizes the strong correlation that exists between primary structural conservation among mammalian gelsolins and maintenance of the three‐dimensional domain fold characteristic of members of this protein family.
Identification of structural elements of subunit β of human protein kinase CK2 participating in tight physical α−β intersubunit contacts directly adjacent to a surface‐oriented region
Authors: Krehan Andreas , Meggio Flavio , Pipkorn Rüdiger , Pinna Lorenzo A. , Pyerin Walter
Published: March 2003

Sites essential for tight physical intersubunit interaction in protein kinase CK2, a tetramer composed of two catalytic (α and/or α′) and two regulatory subunits (β), have been assigned to the C‐terminal part of subunit β. Mutational analysis suggests region 171−181 of β to be one of these but this is not consistent with the observation of coprecipitation of catalytic subunits by antibodies directed specifically to this β segment which indicates that this region is accessible to antibodies even if the β subunit is associated with the α subunit. In an attempt to clarify the apparent contradiction, we have subdivided β‐(155−181)‐peptide, which includes the fragment of β and that both binds to catalytic subunits and stimulates kinase activity, into six more or less overlapping peptides with a length of 9−16 amino acid residues and performed peptide competition and α subunit binding assays. The kinase‐stimulating effect of β‐(155−181)‐peptide was counteracted significantly by β‐(162−175)‐peptide while the others had less or no effect. In an ELISA assay with the peptides covalently bound to wells via a C10 spacer arm, binding of α occurred besides β‐(155−181)‐peptide only with β‐(162−175)‐peptide and β‐(165−175)‐peptide. The data provide the first evidence that the contact and the stimulation competences of subunit β represent separable functions and suggest further that a tight physical contact to catalytic subunits is located at position 162−175 consisting of a hydrophobic stretch (position 162−171) and a hydrophilic binding motif, PEY (position 172−174), with the adjoining downstream part allowing for surface exposure and antibody binding. The presence of several Pro residues within that region might relate to the structural basis for both the α−β interaction and the surface orientation.
The 3′‐untranslated region of the urokinase gene enhances the expression of chimeric genes in cultured cells and correlates with specific brain expression in transgenic mice
Authors: Smicun Yoel , Kopf Eliezer , Miskin Ruth
Published: March 2003

Transgenic mice were previously described, carrying the cDNA of the human or murine protease urokinase‐type plasminogen activator (uPA) while linked to the cell‐specific promoter of the αA‐crystallin gene. Surprisingly, these mice produced transgenic uPA in an ectopic manner specifically in the brain. Here we tested the possibility that this ectopic expression could have been contributed primarily by the uPA transgenic moiety. Several experimental approaches have been used. (a) Constructs consisting of uPA cDNA linked to the cell‐specific promoters of the αA‐crystallin or insulin genes yielded active uPA after transfection into cells where these promoters are thought to be inactive. (b) When reporter genes were inserted into these constructs between the promoter and the cDNA, the cDNA enhanced the chimeric reporter expression 5−50‐fold. This effect was obtained upon stable or transient construct transfection into four different cell types. (c) Reporter enhancement also took place in the presence of the homologous uPA gene promoter. (d) Mapping of the cDNA through deletion‐substitution analysis has detected fragments mediating positive or negative effects on reporter expression, all fragments residing in the 3′‐untranslated region (3′UTR) of the uPA gene that was included in the cDNA. Some fragments exhibited cell‐specific effects. One fragment (2002/2187) behaved like a classical transcriptional enhancer, enhancing reporter expression from different positions and orientations. (e) Transgenic mice have now been generated that carry a transgene consisting of the αA‐crystallin promoter, the luciferase reporter gene and mouse uPA cDNA. Among four transgenic lines producing luciferase activity in the eye lens, three lines exhibited ectopic luciferase activity exclusively in the brain, where luciferase mRNA was localized through in situ hybridization.

From these results we conclude that the 3′UTR of the uPA gene contains sequences capable of exerting variable effects on gene expression, including transcriptional enhancement. In addition, uPA cDNA correlates with transgenic brain expression. Therefore, we suggest that the 3′UTR of the uPA gene is involved in brain expression of the transgenes containing uPA cDNA as well as of the normal uPA gene.
Purification and characterization of retinyl ester hydrolase as a member of the non‐specific carboxylesterase supergene family
Authors: Schindler Rainer , Mentlein Rolf , Feldheim Walter
Published: March 2003

Hepatic retinyl ester hydrolase (REH) activity was isolated from porcine and human liver and characterized, and some of its properties were compared with those of other retinyl‐ester‐splitting enzymes. Sequence analysis revealed that the REH proteins are structurally similar to non‐specific carboxylesterases and distinct from bile salt‐activated lipases and cholesterol esterases. Pig REH, a 64‐kDa protein, hydrolyzed retinyl palmitate at a rate of 595 nmol h−1 mg−1 protein in the presence of 100 mM Chaps with an apparent Km value for retinyl palmitate of 27.5 μM. The pH optimum was 7.0−9.2. Its human counterpart has a molecular mass of 65 kDa and a pH optimum near 6.5. In the presence of Chaps, pig REH activity was stimulated up to 1.7‐fold by various non‐ionic detergents. The ranking order of retinyl palmitate cleavage initiated by the stimuli was n‐dodecylglucoside > octanoyl‐N‐methylglucamide > n‐octyglucoside > n‐dodecylmaltoside > Triton X‐114 > Triton X‐100. Porcine REH was effectively inhibited by α‐tocopherol and bis‐(4‐nitrophenyl) phosphate [(Np)2P].

The structural, immunological and catalytic features, pH dependence, and the effect of (Np)2P on enzyme activity of pig REH are similar to those reported for the non‐specific carboxylesterase ES‐4. However, ES‐4 differed from REH in molecular mass and the requirement of Chaps or Chaps‐like detergents as cofactor. Judging from these results, pig REH may be a non‐specific carboxylesterase isoform.
The effect of acylated polyamine derivatives on polyamine uptake mechanism, cell growth, and polyamine pools in Escherichia coli, and the pursuit of structure/activity relationships
Authors: Karahalios Panagiotis , Mamos Petros , Vynios Dimitrios H. , Papaioannou Dionissios , Kalpaxis Dimitrios L.
Published: March 2003

Two acetyl analogues of spermidine and five analogues of spermine were used to determine the structural specificity of the polyamine transport system in Escherichia coli by measuring their ability to compete with [14C]putrescine or [14C]spermine for uptake, as well as to inhibit cell growth, and, finally, to affect the intracellular polyamine pools. Spermine uptake follows simple Michaelis‐Menten kinetics (Kt = 24.58 ± 2.24 μM). In contrast, the putrescine uptake system involves two saturable Michaelis‐Menten carriers exhibiting different affinity towards putrescine (Kt = 3.63 ± 0.43 μM, .cf2.K.cf2..cf1..es.cf1.′.rb.eit.rb = 0.61 ± 0.10 μM). From the Ki values, it is inferred that N1‐5‐amino‐2‐nitrobenzoylspermine is the most effective competitive inhibitor followed by N1‐acetylspermine, and then N1,N12‐diacetylspermine. N1‐acetylspermidine and N8‐acetylspermidine also inhibit competitively the uptake of spermine, the latter being the most effective inhibitor. In addition, the above‐mentioned analogues inhibit identically one of the carriers of putrescine uptake, suggesting the existence of a common transporter for both putrescine and spermine. The order of analogue potency, regarding the other carrier of putrescine is as follows : N1,N12‐diacetylspermine ≅N1‐5‐amino‐2‐nitro‐benzoylspermine > N1‐acetylspermine. Both N1‐acetylspermidine (Ki = 753 ± 25 μM, .cf2.K.cf2..cf1..es.cf1.′.cf5..rb.eii.rb = 128 ± 5 μM) and N 8‐acetylspermidine (Ki = 22.4 ± 0.4 μM, .cf2.K.cf2..cf1..es.cf1.′.rb.eii.rb = 279 ± 3 μM) also cause competitive inhibition of putrescine uptake, however with inverse affinity towards the putrescine carriers. Neither N4,N 9‐diacetylspermine, nor N1,N 4‐bis(β‐alanyl)diaminobutane affect the uptake of any polyamine. Interestingly, none of the acetyl analogues of spermine has a measurable effect on cell growth and cellular polyamine pools, although some of them are accumulated in cells. Based on these findings, the relative significance of the primary and secondary amines and of the chain flexibility as determinants of cellular uptake are discussed.
Effects of adrenal steroid hormones on mitochondrial maturation during the late fetal period
Authors: Prieur Béatrice , Bismuth Jeanne , Delaval Evelyne
Published: March 2003

In the present work, we described the perinatal changes in mitochondrial maturation that contribute to metabolic development in the rat kidney. We focused on cytochrome‐c oxidase activity and gene expression from the last three days of gestation to one day after birth. The role of adrenal steroids in the development of cytochrome‐c oxidase expression and of mitochondrial DNA content was also investigated by studying the effects of fetal adrenalectomy. During the perinatal period, the developmental pattern of the cytochrome‐c oxidase enzymatic complex exhibited parallel increases in transcript levels, protein content and enzyme activity, suggesting a transcriptional regulation of this enzyme. Adrenalectomy led to a decrease in fetal kidney cytochrome‐c oxidase messengers and mtDNA content while administration of dexamethasone restored normal levels. In contrast, mtDNA content was unchanged in liver and heart after adrenalectomy whereas levels of cytochrome‐c oxidase transcripts were controlled by adrenal steroids in liver but not in heart. These results indicate that adrenal steroid hormones contribute to the regulation of perinatal maturation of mitochondria in rat kidney and that these hormones are involved in the fetal mitochondrial biogenesis in a tissue‐specific manner.
Analysis of stress in the active site of myosin accompanied by conformational changes in transient state intermediate complexes using photoaffinity labeling and 19F‐NMR spectroscopy
Authors: Maruta Shinsaku , Henry Gillian D. , Ohki Takashi , Kambara Taketoshi , Sykes Brian D. , Ikebe Mitsuo
Published: March 2003

Myosin forms stable ternary complexes with ADP and the phosphate analogues, fluoroaluminate (AlF.es−.rb.ei4.rb), fluoroberyllate (BeFn) or orthovanadate (Vi); these ternary complexes mimic transient intermediates in the myosin ATPase cycle. Moreover, we previously demonstrated that these complexes may mimic different myosin ATPase reaction intermediates corresponding to separate steps in the cross‐bridge cycle [Maruta, S., Henry, G. D., Sykes, B. D. & Ikebe, M. (1993) J. Biol. Chem. 268, 7093−7100]. Park et al. suggested that the changing conformation of ATP during hydrolysis stresses the active site of myosin subfragment‐1 (S‐1) through protein‐nucleotide contacts at the γ‐phosphate and nucleotide base, and the stress‐induced strain in the cross‐bridge may be the mechanism by which energy in ATP is transferred to the myosin structure [Park, S., Ajtai, K. & Burghardt, T. P. (1997) Biochemistry 36, 3368−3372]. In the present study, the photoactive ADP analogue, 3′‐O‐(N‐methylanthraniloyl)‐2‐azido‐ADP (Mant‐2‐N3‐ADP), and the 19F‐labeled ADP analogue, 2‐[(trifluoromethylnitrophenyl)aminoethyl]diphosphate, were employed to examine conformational differences in protein‐nucleotide contact in the ATP‐binding site that may correlate with energy transduction. Mant‐2‐N3‐ADP was trapped within the active site of skeletal and smooth muscle myosin in the presence of AlF.es−.rb.ei4.rb, BeFn or Vi. For both skeletal and smooth muscle myosins, trapped Mant‐2‐N3‐ADP was covalently linked to the 25‐kDa N‐terminal fragment of S‐1 of both myosin/Mant‐2‐N3‐ADP/AlF.es−.rb.ei4.rb and BeFn complexes, presumably at Trp130. However, the efficiency of the incorporation was much higher for skeletal than for smooth muscle myosin suggesting that the conformations of the adenine‐binding pockets of the two myosins are somewhat different. Although the amount of Mant‐2‐N3‐ADP trapped in the presence of AlF.es−.rb.ei4.rb and BeFn was the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeFn complex than for AlF.es−.rb.ei4.rb complex. The 19F‐NMR spectra of the bound 2‐[(trifluoromethylnitrophenyl)aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF.es−.rb.ei4.rb, BeFn or Vi showed small but distinguishable differences. Taken together, these results indicate that there is some variation in the protein‐nucleotide contacts at the nucleotide base among the ternary complexes studied, and these differences mimic separate steps occurring transiently during the contractile cycle.
A fully active FAD‐containing precursor remains folded up to its translocation across the chloroplast membranes
Authors: Ottado Jorgelina , Ceccarelli Eduardo A.
Published: March 2003

The cytosolic and two recombinant precursors, containing 10 and 30 amino acid spacers between the transit peptide and the mature region of the chloroplast flavoprotein ferredoxin‐NADP+ reductase (FNR), were expressed in Escherichia coli cells. These proteins were purified rendering fully active precursors that contained bound FAD. Neither the transit peptide nor the spacers affected the formation of the tightly folded enzyme structure. Protease treatment of the folded precursors resulted in a rapid removal of the transit sequence, rendering an enzymatically active resistant core, even at high protease concentration. All three preproteins could be efficiently imported by isolated pea chloroplasts. Addition of the enzyme substrate NADP+ to the import medium slightly decreased the polypeptide translocation. The precursor bound to isolated chloroplasts in the presence or absence of leaf extracts was as resistant to proteolysis as the folded precursor in solution. In contrast, the FNR precursor unfolded by urea was rapidly digested even at the lowest protease concentration. Together, our results indicate that precursor unfolding may take place during translocation but not during binding to chloroplast envelopes or by interaction with leaf extract soluble factors, and that this process is independent of the distance between the transit peptide and the folded mature region of the protein.
Lys42 and Ser42 variants of p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens reveal that Arg42 is essential for NADPH binding
Authors: Eppink Michel H. M. , Schreuder Herman A. , Van Berkel Willem J. H.
Published: March 2003

The conserved Arg42 of the flavoprotein p‐hydroxybenzoate hydroxylase is located at the entrance of the active site in a loop between helix H2 and sheet E1 of the FAD‐binding domain. Replacement of Arg42 by Lys or Ser decreases the turnover rate of p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens by more than two orders of magnitude. Rapid reaction kinetics show that the low activity of the Arg42 variants results from impaired binding of NADPH. In contrast to an earlier conclusion drawn for p‐hydroxybenzoate hydroxylase from Acinetobacter calcoaceticus, substitution of Arg42 with Ser42 in the enzyme from P. fluorescens hardly disturbs the binding of FAD. Crystals of [Lys42]p‐hydroxybenzoate hydroxylase complexed with 4‐hydroxybenzoate diffract to 0.22‐nm resolution. The structure of the Lys42 variant is virtually indistinguishable from the native enzyme with the flavin ring occupying the interior position within the active site. Lys42 in the mutant structure interacts indirectly via a solvent molecule with the 3‐OH of the adenosine ribose moiety of FAD. Substrate perturbation difference spectra suggest that the Arg42 replacements influence the solvent accessibility of the flavin ring in the oxidized enzyme. In spite of this, the Arg42 variants fully couple enzyme reduction to substrate hydroxylation. Sequence‐comparison studies suggest that Arg42 is involved in binding of the 2′‐phosphoadenosine moiety of NADPH.
Carnitine palmitoyltransferase II specificity towards β‐oxidation intermediates
Authors: Ventura Fátima V. , Ijlst Lodewijk , Ruiter Jos , Ofman Rob , Costa Catarina G. , Jakobs Cornelis , Duran Marinus , De Almeida Isabel Tavares , Bieber Loran L. , Wanders Ronald J. A.
Published: March 2003

Using isolated rat liver mitochondria, in the absence or presence of malonyl‐CoA (an inhibitor of carnitine palmitoyltransferase I), we have found that carnitine palmitoyltransferase II (CPT II) is active with palmitoyl‐CoA as well as with its β‐oxidation intermediates. A partially purified CPT II fraction from rat liver mitochondria was shown to be able to convert 3‐hydroxypalmitoyl‐CoA to 3‐hydroxypalmitoylcarnitine, which could be identified by fast‐atom‐bombardment mass spectrometry. This apparent broad specificity of CPT II was further evaluated by kinetic studies using purified CPT II. It was found that CPT II readily accepts 3‐oxopalmitoyl‐CoA, palmitoyl‐CoA, 3‐hydroxypalmitoyl‐CoA and 2,3‐unsaturated palmitoyl‐CoA as substrates with decreasing order of affinity. The apparent Vmax values found for the first three compounds were of the same order of magnitude; the 2,3‐unsaturated acyl‐CoA was the poorest substrate. Kinetic studies with purified CPT II showed 3‐hydroxypalmitoyl‐CoA to have the lowest K0.5 value (20 ± 6 μM) of all the CoA esters studied; the highest K0.5 value (65 ± 17 μM) was found for the 3‐oxo intermediate.

These findings support the hypothesis that CPT II is involved in the export of toxic long‐chain acyl‐CoA esters from the mitochondria by first converting them into the corresponding carnitine esters, followed by transport out of the mitochondria and subsequently out of the cell.
A bifunctional hybrid molecule of the amino‐terminal fragment of urokinase and domain II of bikunin efficiently inhibits tumor cell invasion and metastasis
Authors: Kobayashi Hiroshi , Sugino Dan , She Ming Yu , Ohi Hidekazu , Hirashima Yasuyuki , Shinohara Hiromitsu , Fujie Michio , Shibata Kiyoshi , Terao Toshihiko
Published: March 2003

Urinary trypsin inhibitor (UTI) inhibits efficiently tumor cell invasion and the formation of metastasis. The anti‐metastatic effect is dependent on the COOH‐terminal domain II of UTI [UTI‐(78−136)‐peptide]. To develop a molecule that binds with high affinity to the urokinase (uPA) receptor (uPAR) on tumor cell surfaces, a bifunctional hybrid molecule [uPA‐(1−134)‐UTI‐(78−136)] consisting of the uPAR‐binding NH2‐terminal fragment [UTI‐(78−136)‐peptide] of uPA at the NH2‐terminus of UTI‐(78−136)‐peptide was produced in Escherichia coli by genetic engineering. The purified hybrid protein inhibited trypsin and plasmin 2−3‐fold less effectively than UTI‐(78−136)‐peptide and was found to bind to human tumor cells via uPAR, which was confirmed by cell binding and competition experiments. Using a modified Boyden chamber and an artificial basement membrane, Matrigel, it was found that the hybrid protein is very effective at inhibiting invasion by uPAR‐expressing human tumor cells. Sensitivities of tumor cells towards the anti‐invasive effect of uPA‐(1−134)‐UTI‐(78−136) correlated with the density of uPAR on human tumor cells. Furthermore, in the spontaneous metastasis model, the hybrid protein inhibited the formation of lung and/or lymphatic metastasis by human ovarian carcinoma and choriocarcinoma cells. The hybrid protein was much more effective than uPA‐(1−134)‐peptide, UTI‐(78−136)‐peptide, or UTI. We conclude that this approach extends the possibility of applying recombinant protein for therapeutic use in inhibition of human tumor cell metastasis.
Carbon‐flux distribution in the central metabolic pathways of Corynebacterium glutamicum during growth on fructose
Authors: Dominguez Hélène , Rollin Catherine , Guyonvarch Armel , Guerquin‐Kern Jean‐Luc , Cocaign‐Bousquet Muriel , Lindley Nicholas D.
Published: March 2003

Growth of Corynebacterium glutamicum on fructose was significantly less than that obtained on glucose, despite similar rates of substrate uptake. This was in part due to the production of overflow metabolites (dihydroxyacetone and lactate) but also to the increased production of CO2 during growth on fructose. These differences in carbon‐metabolite accumulation are indicative of a different pattern of carbon‐flux distribution through the central metabolic pathways. Growth on glucose has been previously shown to involve a high flux (> 50 % of total glucose consumption) via the pentose pathway to generate anabolic reducing equivalents. NMR analysis of carbon‐isotope distribution patterns of the glutamate pool after growth on 1‐13C‐ or 6‐13C‐enriched fructose indicates that the contribution of the pentose pathway is significantly diminished during exponential growth on fructose with glycolysis being the predominant pathway (80 % of total fructose consumption). The increased flux through glycolysis during growth on fructose is associated with an increased NADH/NAD+ ratio susceptible to inhibit both glyceraldehyde‐3‐phosphate dehydrogenase and pyruvate dehydrogenase, and provoking the overflow of metabolites derived from the substrates of these two enzymes. The biomass yield observed experimentally is higher than can be estimated from the apparent quantity of NADPH associated with the pentose pathway and the flux through isocitrate dehydrogenase, suggesting an additional reaction yielding NADPH. This may involve a modified tricarboxylic acid cycle involving malic enzyme, expressed to significantly higher levels during growth on fructose than on glucose, and a pyruvate carboxylating anaplerotic enzyme.
Sequence and structure of the human 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase heart isoform gene (PFKFB2)
Authors: Heine‐Suñer Damián , Díaz‐Guillén Miguel Angel , Lange Alex J. , Rodríguez de Córdoba Santiago
Published: March 2003

6‐Phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFK‐2/FBPase‐2) is a bifunctional enzyme that catalyzes the synthesis and degradation of Fru‐2,6‐P2, a key regulator of glycolysis. In mammals, several genes have been found to code for different PFK‐2/FBPase‐2 isoforms that differ in tissue distribution and enzymatic activities. In the present study, we report the characterization of the PFK‐2/FBPase‐2 heart isoform gene in humans (PFKFB2), including a full analysis of repetitive sequences and potential transcription binding sites. The genomic sequence of the PFKFB2 gene spans 22 485 bp and contains 15 exons. Heart cDNA analysis shows that PFKFB2 codes for a protein of 505 amino acids with a deduced molecular mass of 58 849 Da. Comparison of the human PFKFB2 gene to the homologous genes in rat and ox outlines a significant conservation of the intron‐exon structure, sequence of 5′ and 3′ flanking regions, and simple sequence repetitive element positions. Most important, the human heart PFK‐2/FBPase‐2 protein was found to retain all the important regulatory sites, as well as the catalytic and substrate binding sites identified in the rat and bovine heart isoforms, suggesting that the human enzyme is regulated in a manner similar to that observed in these organisms.
Two distinct structures of α‐conotoxin GI in aqueous solution
Authors: Maslennikov Innokenty V. , Sobol Alexander G. , Gladky Konstantin V. , Lugovskoy Alexey A. , Ostrovsky Andrey G. , Tsetlin Victor I. , Ivanov Vadim T. , Arseniev Alexander S.
Published: March 2003

The detailed analysis of conformational space of α‐conotoxin GI in aqueous solution has been performed on the basis of two‐dimensional NMR spectroscopy data using multiconformational approach. As the result, two topologically distinct interconvertible sets of GI conformations (populations of 78  % and 22  %) have been found. A common feature of the two sets is the Asn4‐Cys7 β‐turn. The Gly8 to Tyr11 region has a structure of right‐handed helical turn in the major set and two sequential bends in the minor one. N‐terminus and C‐terminus also have different orientations, anti‐parallel in the major conformational set and parallel in the minor one. An average pairwise rmsd for backbone heavy atoms is 0.56 Å in the major set, 0.23 Å in the minor, and 1.85 Å between the structures of the two sets. The X‐ray structure of GI [Guddat, L. W., Martin, J. A., Shan, L., Edmundson, A. B. & Gray, W. R. (1996) Biochemistry 35, 11 329−11 335] has the same folding pattern as the major NMR set, the average backbone rmsd between the two structures being 0.77 Å.
Suppression of decorin expression and partial induction of anchorage‐independent growth by the v‐src oncogene in human fibroblasts
Authors: Kolettas Evangelos , Rosenberger Robert F.
Published: March 2003

To determine its role in cell transformations, the v‐src oncogene was introduced into the human foetal diploid fibroblasts MRC‐5 and into MRC‐5V1, a simian virus 40 (SV40)‐transformed cell line derived from them. Infected cells were found to contain stably integrated intact proviruses, as determined by Southern blot analysis. Although highly expressed, v‐src did not change the morphology or growth patterns of MRC‐5 cells and failed to induce foci or alter their saturation densities. However, overexpression of v‐src reduced the plating efficiencies of MRC‐5 and induced anchorage‐independent growth in a low but significant number of cells. Northern blot analysis showed that v‐src selectively abolished the expression of decorin, a small dermatan/chondroitin sulphate proteoglycan that interacts with extracellular‐matrix components and modulates collagen‐fibril formation and the activity of transforming growth factor (TGF) β1. Addition of herbimycin A, a potent pp60src tyrosine‐kinase inhibitor, resulted in the reexpression of decorin in MRC‐5 carrying v‐src. There were no changes in the expressions of fibronectin, procollagen type I, or tissue plasminogen activator, an activator of extracellular‐matrix‐degrading enzymes. Moreover, v‐src did not alter the expressions of the epidermal‐growth‐factor receptor or TGFβ1 or reduce the growth‐factor requirements of MRC‐5 fibroblasts. MRC‐5 and MRC‐5V1 expressing v‐src remained non‐tumourigenic when injected into nude mice. Constitutive expression of v‐src did not alter the mRNA levels of c‐jun and junB, suggesting that the effects of the oncogene are not mediated by AP‐1. Decorin gene expression has been shown previously to be maximal in quiescent cells and virtually absent in transformed ones. Our data indicate that the ability to synthesise decorin can be suppressed in human fibroblasts without their becoming transformed, and that the relations between decorin synthesis and growth controls need further clarification.
NMR studies on the structure/function correlations of T‐cell‐epitope analogs from pertussis toxin
Authors: Scarselli Maria , Esposito Gennaro , De Magistris M. Teresa , Domenighini Mario , Rappuoli Rino , Burroni Gigliola , Bernini Andrea , Niccolai Neri
Published: March 2003

A synthetic tridecapeptide, corresponding to the 30−42 fragment of the S1 subunit of pertussis toxin, has been structurally characterised by using NMR spectroscopy. The molecule corresponds to a T‐cell epitope of the bacterial toxin which has been extensively analysed with the alanine scanning approach to check the relevance of each residue for the biological activity of the peptide. Five of these Ala‐substituted analogs have also been spectroscopically studied. In the experimental conditions used, different extents of helicity were found for the six peptides in a way which cannot be related to their capabilities of of binding to major histocompatibility complex (MHC) class II and inducing T‐cell proliferation. Backbone flexibility around helical transient conformations seems to constitute the structural intermediate step between the structure of the corresponding sequence within the parental protein and in the MHC class II complex. A model of the latter complex, which accounts for the different biological activities of the analogs, is proposed.
Subtypes of odorant‐binding proteins
Authors: Löbel Dietrich , Marchese Silvana , Krieger Jürgen , Pelosi Paolo , Breer Heinz
Published: March 2003

Odorant‐binding proteins (OBP) in the mucus of the olfactory epithelium are thought to transfer the hydrophobic odorous compounds through the aqueous barrier towards the chemo‐sensory cells. To evaluate their binding properties, two distinct OBP subtypes of the rat were expressed as N‐terminal His‐tagged fusion proteins in Escherichia coli, thus allowing an efficient purification. Based on gel chromatography and CD spectroscopy analysis the recombinant OBP subtypes seem to share several structural features with other members of the lipocalin family. Approaches to elucidate whether heterologous expressed OBPs interact with odorous compounds revealed that OBP1 specifically binds 2‐[3H]‐isobutyl‐3‐methoxypyrazine whereas OBP2 did not shown any specific binding to this compound. In contrast, the chromophore 1‐anilinonaphthalene 8‐sulfonic acid (1,8‐ANS) specifically interacted with OBP2 but not with OBP1. Displacement experiments monitored by the relative fluorescence intensity revealed that fatty acids with appropriate chain length act as efficient competitors. Some odorous compounds, notably lilial (p‐tert‐butyl‐α‐methyl dihydrocinnamic aldehyde) and citralva (3,7‐dimethyl‐2,6‐octadienenitrile), also displaced efficiently the chromophore, whereas pyrazine derivatives including 2‐isobutyl‐3‐methoxypyrazine and other odorants did not. These results indicate that rat OBPs have distinct ligand specificities.
The Streptomyces tendae Tü901	L‐lysine 2‐aminotransferase catalyzes the initial reaction in nikkomycin D biosynthesis
Authors: Bruntner Christina , Bormann Christiane
Published: March 2003

Protein P8 was previously identified as a putative nikkomycin biosynthesis protein. The gene (nikC) encoding protein P8 was cloned from the Streptomyces tendae Tü901 nikkomycin gene cluster and sequenced. The nikC gene was inactivated by inserting a kanamycin resistance cassette; the mutant did not produce the biologically active nikkomycins I, J, X, and Z, but accumulated the nucleoside moieties nikkomycins CX and CZ. The mutant was complemented to nikkomycin production (I, J, X, Z) by nikC expressed from the mel promoter of the vector pIJ702. Furthermore, the nikkomycin‐negative phenotype was reversed by the addition of picolinic acid, a precursor of the peptidyl moiety of nikkomycins (nikkomycin D), into the culture medium. The nikC gene was expressed in Escherichia coli and identified and characterized at the enzyme level. NikC encodes an
L‐lysine 2‐aminotransferase, and the activity was exclusively detected in nikkomycin producers and its presence correlated to nikkomycin production. The nikC‐inactivated mutant grew with
L‐lysine as sole source of nitrogen and carbon, indicating that
L‐lysine 2‐aminotransferase is not required for lysine catabolism. Our results identified the nikC‐encoded
L‐lysine 2‐aminotransferase as the nikkomycin biosynthetic enzyme that catalyzes the initial reaction in nikkomycin D biosynthesis. The NikC protein belongs to a novel family of pyridoxamine or pyridoxal‐phosphate‐dependent dehydrases and aminotransferases, some of which are involved in dideoxy‐ and deoxy‐aminosugar biosynthesis.
Control by ATP and ADP of voltage‐induced mammalian‐cell‐membrane permeabilization, gene transfer and resulting expression
Authors: Rols Marie‐Pierre , Delteil Christine , Golzio Muriel , Teissié Justin
Published: March 2003

We have permeabilized and transfected mammalian cells by transient alteration of their native transmembrane electrical potential difference. Chinese hamster ovary cells were chosen as a model in order to study the electropermeabilization and electrotransfection processes. Propidium iodide was used to monitor permeabilization. A plasmid carrying the β‐galactosidase gene was used to follow direct gene transfer and expression by determining transient expression of the electrotransfered activity at the single‐cell level. The effect of nucleotides on cell permeabilization and transfection was studied by altering the cytosolic ATP and ADP contents of cells either during the pulsation or during the period following it.

Permeabilization and transfection are not regulated in the same way by the ATP and ADP levels. The permeabilization efficiency remains unaffected. Cell viability and the transfection yield are dramatically affected. While ADP is involved in the step of DNA transfer across the electropermeabilized plasma membrane, ATP controls other steps (cytoplasmic DNA migration towards the nucleus, expression). Our results prove, firstly, that membrane resealing is required but is not sufficient to preserve cell viability and, secondly, that transfection is a cell‐mediated process and not only an electrophoretic step driven by the external field.
Purification of a 58‐kDa protein (ER58) from monkey liver microsomes and comparison with protein‐disulfide isomerase
Authors: Bonfils Claude
Published: March 2003

A 58‐kDa protein (ER58) was purified from monkey liver to apparent homogeneity. It accounts for more than 3  % of microsomal proteins and is highly conserved among several mammalian species. The amino acid compositions of the N‐terminal part and that of two internal peptide fragments present strong similarities with the sequence ascribed to phospholipase C‐α. Numerous proteins exhibiting a high similarity with this sequence have been isolated by other investigators. Their biological function is controversial. Our purified protein is not active as a phosphatidylinositol‐specific phospholipase C, protease or carnitine acyl transferase. Although less efficient than authentic protein‐disulfide isomerase, ER58 catalyses the glutathione‐dependent reduction of insulin and the reorganization of disulfide bonds of randomly oxidized (scrambled) ribonuclease in reducing conditions. In contrast, ER58 is devoid of oxidizing activity on thiol groups of reduced proteins. Many studies suggest that the proteins bearing the phospholipase C‐α sequence could be considered as protein‐disulfide isomerase isozymes. Our results indicate that ER58 is not totally similar to protein‐disulfide isomerase in performing thiol :protein‐disulfide oxidoreductase reactions and suggest that the two proteins may exert distinct cellular functions.
Apoptosis signaling by death receptors
Authors: Schulze‐Osthoff Klaus , Ferrari Davide , Los Marek , Wesselborg Sebastian , Peter Marcus E.
Published: March 2003

Death receptors have been recently identified as a subgroup of the TNF‐receptor superfamily with a predominant function in induction of apoptosis. The receptors are characterized by an intracellular region, called the death domain, which is required for the transmission of the cytotoxic signal. Currently, five different such death receptors are known including tumor necrosis factor (TNF) receptor‐1, CD95 (Fas/APO‐1), TNF‐receptor‐related apoptosis‐mediated protein (TRAMP) and TNF‐related apoptosis‐inducing ligand (TRAIL) receptor‐1 and ‐2. The signaling pathways by which these receptors induce apoptosis are rather similar. Ligand binding induces receptor oligomerization, followed by the recruitment of an adaptor protein to the death domain through homophilic interaction. The adaptor protein then binds a proximal caspase, thereby connecting receptor signaling to the apoptotic effector machinery. In addition, further pathways have been linked to death receptor‐mediated apoptosis, such as sphingomyelinases, JNK kinases and oxidative stress. These pro‐apoptotic signals are counteracted by several mechanisms which inhibit apoptosis at different levels. This review summarizes the current and rapidly expanding knowledge about the biological functions of death receptors and the mechanisms to trigger or to counteract cell death.
Identification of a human 17p‐located cDNA encoding a protein of the Snf2‐like helicase family
Authors: Aubry Florence , Mattéi Marie‐Geneviève , Galibert Francis
Published: March 2003

Following immunoscreening, we have cloned and sequenced a human cDNA encoding a novel member of the expanding helicase family. The deduced protein, designated hZFH (human zinc‐finger helicase), contains the seven domains conserved among the helicase superfamily II and four potential zinc‐fingers motifs. In particular, hZFH shows significant similarity to some proteins of the Snf2‐like family, known to act as transcriptional regulators for multiples genes. Furthermore, hZFH has 68.5 % identity to a human Mi‐2 autoantigen to which autoantibodies are produced by a subgroup of patients affected by dermatomyositis. Northern‐blot analyses have revealed several hZFH mRNAs with quantitative differences in various human tissues. One alternative splice site of hZFH mRNA was demonstrated and others were predicted. We also report the chromosomal localization of gene hZFH to locus 17p13‐17p12 by in situ hybridization. Thus, this novel gene appears as a candidate for several malignant and genetic diseases associated with this region of the genome. The combination of these features suggests that hZFH plays an important role in gene regulation.
Interaction of a menaquinol binding site with the [3Fe‐4S] cluster of Escherichia coli fumarate reductase
Authors: Rothery Richard A. , Weiner Joel H.
Published: March 2003

We have used EPR to study the interaction between the [3Fe‐4S] FR3 cluster and a menaquinol (MKH2) binding site of Escherichia coli fumarate reductase (FrdABCD). The MKH2 analog 2‐n‐heptyl‐4‐hydroxyquinoline N‐oxide (HOQNO) binds to FrdABCD and elicits a significant change in the EPR lineshape of the FR3 cluster of FrdB. In a mutant of FrdABCD in which His82 of FrdC is changed to Arg, this HOQNO effect is eliminated. The HOQNO effect can also be eliminated by reaction of membranes with the imidazole‐reactive reagent ethoxyformic anhydride. Potentiometric titrations indicate that neither HOQNO nor [Arg82]FrdC have a detectable effect on the midpoint potential (Em,7) of FR3. Quinol :fumarate oxidoreductase activity assays confirm that HOQNO and [Arg82]FrdC both block MKH2 oxidation by FrdABCD. Fluorescence spectroscopy was used to quantitate HOQNO binding to FrdABCD; approximately 1.30 binding sites were detected/FR3 cluster. Overall, the data support a quinol‐binding model for FrdABCD in which one site has a close functional relationship with the FR3 cluster.