bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and structure of two novel 6‐kDa dimeric peptides from the copora cardiaca of the insect Locusta migratoria
Authors: HIETTER Hélène , LUU Bang , GOLTZENE Francine , ZACHARY Daniel , HOFFMANN Jules , VAN DORSSELAER Alain
Published: March 2005

We have isolated two major 6‐kDa peptides from extracts of corpora cardiaca of adult females of Locusta migratoria. These peptides have been characterized by peptide sequencing and liquid secondary‐ion mass spectrometry. They are structurally related dimers, one (6278.5 Da) being a homodimer (A‐A chains), the other (6280.5 Da) being a heterodimer (A‐B chains). A 60% similarity exists between the A and B chains. Both peptides have been chemically synthesized and the synthetic compounds appeared to be identical to the native ones. Polyclonal antibodies raised against each of these peptides demonstrated that they were contained within the secretory granules of the intrinsic cells of the glandular lobes of the corpora cardiaca. The physiological significance of these two peptides is unknown but, using the synthetic peptides, we are currently probing their biological role.
Structures of O‐glycosidically linked oligosaccharides isolated from human meconium glycoproteins
Authors: CAPON Calliope , LEROY Yves , WIERUSZESKI Jean‐Michel , RICART Guy , STRECKER Gérard , MONTREUIL Jean , FOURNET Bernard
Published: March 2005

The structure of major O‐glycosidicall linked neutral and acidic oligosaccharides isolated from human meconium glycoproteins were established. Neutral and acidic oligosaccharides were released by alkaline borohydride treatment, purified by Biogel P‐6 and fractionated by high‐performance liquid chromatography. This approach resulted in 50 neutral and 30 acidic oligosaccharides. The present study reports the primary stuctural analysis of five neutral oligosaccharides, ten monosialylated oligosaccharides, one monosialylated monosulfated oligosaccharide and three disialylated oligosaccharides, by permethylation, fast‐atom‐bombardment mass spectrometry analysis and 400‐MHz 1H‐NMR spectroscopy.

The following structure have not been described previously:
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Identification of 3‐deoxy‐manno‐2‐octulosonic acid, 3‐deoxy‐5‐O‐methyl‐manno‐2‐octulosonic acid and 3‐deoxy‐lyxo‐2‐heptulosaric acid in the cell wall (theca) of the green alga Tetraselmis striata Butcher (Prasinophyceae)
Authors: BECKER Burkhard , HÅRD Karl , MELKONIAN Michael , KAMERLING Johannis P. , VLIEGENTHART Johannes F. G.
Published: March 2005

The main constituent of the cell wall complex carbohydrate of the scaly green alga Tetraselmis striata Butcher is shown to be 3‐deoxy‐manno‐2‐octulosonic acid (42%). In addition two other 2‐keto‐sugar acids are present, namely, 3‐deoxy‐5‐O‐methyl‐manno‐2‐octulosonic acid (7%), the first methylated derivative of 3‐deoxy‐manno‐2‐octulosonic acid found in nature, and 3‐deoxy‐lyxo‐2‐heptulosaric acid (11%). The characterization of the three 2‐keto‐sugar acids has been carried out on the corresponding methyl ester methyl glycosides using GLC‐MS and 500‐MHz 1H‐NMR spectroscopy, and on the corresponding reduced alditol acetates using GLC‐MS. Other monosaccharides occurring in the cell wall are d‐galacturonic acid (14%), d‐galactose (4%), d‐gulose (2%), d‐glucose (1%) and l‐arabinose (1%).
Correlation of pause sites in MDV‐1 RNA replication with kinetic refolding of the growing chain
Authors: FERNÁNDEZ Ariel
Published: March 2005

We consider a template‐instructed MDV‐1 RNA replication catalyzed by Qß replicase. The relaxation of the effective binding of the growing chain to the enzyme is simulated, considering a Markov process where refolding events in the chain are concomitant with chain elongation events. When realistic transition probabilities are considered, it becomes evident that the pause sites in replication are due to relaxation of the binding by folding of the growing chain.
Endothelin binding sites in porcine‐aortic and rat lung membranes
Authors: KANSE Sandip M. , GHATEI Mohammad A. , BLOOM Stephen R.
Published: March 2005

High‐affinity binding sites for endothelin were identified on porcine aortic and rat lung membranes. Interaction of 125I‐labelled endothelin with its binding site was specific, saturable, time‐and temperature‐dependent but dissociation of receptor‐bound ligand was minimal. Maximal binding was observed at pH 7.0 in porcine aorta and at pH 3.1 in the rat lung. Treatment of membranes with trypsin destroyed the binding site in both tissues. Porcine endothelin showed a higher affinity for receptors in both tissues compared to rat endothelin. Vasoactive peptides and Ca2+ channel antagonists did not interact with this site suggesting high specificity of binding. Analysis of saturation binding showed that the number of binding sites was 1250 ± 104 and 1650 ± 170 fmol/mg protein and the affinity of binding sites was 0.47 ± 0.15 and 0.16 ± 0.07 nM in the aorta and the lungs respectively (n= 5). Presence of protease inhibitors did not alter binding suggesting that the label was stable under the incubation conditions. This was further confirmed by HPLC. Removal of the endothelium from the aorta did not change the binding characteristics of this tissue. Ca2+ and Mg2+ ions caused an increase in binding by increasing the affinity. Binding was completely abolished in the presence of Triton and dithiothreitol. The binding sites identified in this study may be responsible for the actions of endothelin in the aorta and the lung.
Phosphorylation/dephosphorylation of the β light chain of clathrin from rat liver coated vesicles
Authors: LOEB Jacques E. , CANTOURNET Benoit , VARTANIAN Jean‐Pierre , GORIS Jozef , MERLEVEDE Wilfried
Published: March 2005

The phosphorylation in vitro, on serine residues by endogenous casein kinase 2, of the clathrin β light chain (33 kDa) of rat liver coated vesicles requires the presence of poly(l‐lysine) which acts through binding to the β light chain. The phosphorylation of other proteins is also increased in the presence of poly(l‐lysine) and casein kinase 2. In contrast, the phosphorylation of the upper band of the 50‐kDa protein doublet from rat liver coated vesicles is inhibited.

Rat liver coated vesicles display a protein phosphatase activity which preferentially dephosphorylates clathrin β light chain. This activity is different from the protein phosphatase which dephosphorylates the 50‐kDa protein. This enzyme seems to be unrelated to the ATP/Mg‐dependent protein phosphatase, or the polycation‐stimulated protein phosphatases, which dephosphorylate the 50‐kDa protein and β light chain very efficiently, but with a different specificity. After dissociation of coated vesicles the β‐light‐chain phosphatase activity is recovered in the membrane fraction. This phosphatase activity is inhibited by 50 μM orthovanadate and 5 mM p‐nitrophenyl phosphate but not by 10 mM EDTA.
Ethanol‐induced inhibition of leukotriene degradation by ω‐oxidation
Authors: BAUMERT Thomas , HUBER Michael , MAYER Doris , KEPPLER Dietrich
Published: March 2005

ω‐Oxidation of leukotrienes is a major pathway in the degradation and inactivation of these proinflammatory mediators. Ethanol inhibited this process in vivo and in vitro. In rat liver in vivo the catabolism of LTE4 to ω‐carboxylated leukotrienes was inhibited by 57% by an ethanol dose of 25 mmol/kg body mass administered intragastrically. The site of inhibition was the oxidation of ω‐hydroxy‐N‐acetyl‐LTE4 to ω‐carboxy‐N‐acetyl‐LTE4 resulting in an accumulation of ω‐hydroxy‐LTE4 and of N‐acetyl‐LTE4. Analogous results were obtained for the oxidative degradation of LTB4 and ω‐hydroxy‐LTB4 in rat hepatocyte suspensions. Ethanol, at a concentration of 12.5 mmol/l (0.07%; by vol.), caused 68% inhibition of the oxidation of ω‐hydroxy‐LTB4 leading to an accumulation of the biologically active LTB4 as well as of ω‐hydroxy‐LTB4. 4‐Methylpyrazole, an inhibitor of cytosolic alcohol dehydrogenase, at a concentration of 23 μmol/l inhibited the oxidation of ω‐hydroxy‐LTB4 by 50% in hepatocyte suspensions. The conversion of ω‐hydroxy‐LTB4 to ω‐carboxy‐LTB4 by rat and human liver cytosol was inhibited by ethanol with half maximal concentrations of 100 μmol/l and 300 μmol/l, respectively. Our measurements indicate that direct interference by ethanol of the ω‐oxidation of leukotrienes as well as an increased NADH/NAD+ ratio induced by ethanol led to the inhibition of leukotriene degradation. The impairment of leukotriene inactivation in the liver by ethanol may contribute to the development of the inflammatory reaction in acute alcoholic liver disease.
Effect of spermine on the efficiency and fidelity of the codon‐specific binding of tRNA to the ribosomes
Authors: NARANDA Tatjana , KUČCN Željko
Published: March 2005

Binding of the yeast Tyr‐tRNA and Phe‐tRNA to the A site, and the binding of their acetyl derivatives to the P site of poly(U11,A)‐programmed Escherichia coli ribosomes was studied. Spermine stimulated the rate of binding of both tRNAs at least threefold, enabling more than 90% final saturation of both ribosomal binding sites. The effect is observed when the tRNAs, but not ribosomes or poly(U11,A), are preincubated with polyamine. Regardless of the binding site, optimal saturation was reached at spermine/tRNA molar ratios of 3 for tRNAPhe and 5 for tRNATyr. The same low spermine/tRNA ratios were previously reported to stabilize the conformation of these tRNAs in solution. On the other hand, the messenger‐free, EF‐Tu‐ and EF‐G‐dependent polymerization of lysine from E. coli Lys‐tRNA is drastically reduced, while the poly(A)‐directed polymerization is stimulated by spermine through a wide range of Mg2+ concentrations. Misreading of UUU codons as isoleucine, assayed by the A‐site binding of E. coli, Ile‐tRNA, is also inhibited by spermine. All these results demonstrate that spermine increases the efficiency and accuracy of a series of macromolecular interactions leading to the correct incorporation of an amino acid into protein, at the same time preventing some unspecific or erroneous interactions. From the analogy with its known strucutral effects, it can be inferred that spermine does so by conferring on the tRNA a specific biologically functional conformation.
Biochemical and theoretical approach to localization of metal‐ion‐binding sites in the actin primary structure
Authors: STRZELECKA‐GOŁLASZEWSKA Hanna , BOGUTA Grzegorz , ZMORZYŃSKI Stainslaw , MORACZEWSKA Joanna
Published: March 2005

The number of Ca2+ ions bound at sites other than the single high‐affinity site in CaCl2‐induced polymers of rabbit skeletal muscle, chicken gizzard, and bovine aorta actin was determined. The polymer of skeletal muscle and aorta actin contained 4 mol Ca2+/mol, whereas gizzard actin only 3 mol weakly bound Ca2+/mol monomer. This difference correlates with the deletion in smooth muscle γ‐actin of one out of four NH2‐terminal acidic residues typical of skeletal and smooth muscle α‐actin isoforms, suggesting that this additional acidic residue in α‐actins is involved in the weak binding of cations which is essential for polymerization. This experimental result, as well as a theoretical analysis of the actin primary structure, argue against the implication of the NH2‐terminal acidic residues in the high‐affinity site for divalent cation.

The analysis of the actin primary structure aimed at identification of sequences resembling the known Ca2+‐binding patterns has revealed the absence of an EF‐hand Ca2+ ‐binding site. The best match was obtained between the sequence of the 292–301/segment and that of Ca2+ site in lectins. However, in the light of experimental data discussed, it is more plausible that the actual high‐affinity Ca2+ site in actin involves sequentially distant residues from the NH2‐ and COOH‐terminal portions of the polypeptide chain.
Glutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A2
Authors: van den BERGH Carel J. , BEKKERS Augustinus C. A. P. A. , VERHEIJ Hubertus M. , de HAAS Gerard H.
Published: March 2005

In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium‐binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam‐Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387–5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site‐directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66→Asn, Glu71→Asn and Glu92→Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low‐affinity calcium binding.
Nitrilase of Rhodococcus rhodochrous J1
Authors: KOBAYASHI Michihiko , NAGASAWA Toru , YAMADA Hideaki
Published: March 2005

Nitrilase was purified from an extract of isovaleronitrile‐induced cells of Rhodococcus rhodochrous J1 in seven steps. In the last step, the enzyme was crystallized by adding ammonium sulfate. The crystallized enzyme appeared to be homogeneous by polyacrylamide electrophoresis, ampholyte electrofocusing and double immunodiffusion in agarose. The enzyme has a molecular mass of about 78 kDa and consists of two subunits identical in molecular mass. The purified enzyme exhibits a pH optimum of 7.6 and a temperature optimum of 45°C. The enzyme catalyzed stoichiometrically the hydrolysis of benzonitrile to benzoic acid and ammonia, and no formation of amide was detected. The enzyme required thiol compounds such as dithiothreitol, l‐cysteine or reduced glutathione to exhibit maximum activity. The enzyme was specific for nitrile groups attached to an aromatic or heteroaromatic ring, e.g. benzonitrile, 3‐chlorobenzonitrile, 4‐tolunitrile, 2‐furonitrile and 2‐thiophenecarbonitrile. The comparison of the properties of the enzyme with other nitrilases and nitrile hydratases has been also discussed.
Spectroscopic characterization of the nickel and iron‐sulphur clusters of hydrogenase from the purple photosynthetic bacterium Thiocapsa roseopersicina
Authors: CAMMACK Richard , BAGYINKA Csaba , KOVACS Kornel L.
Published: March 2005

The thermostable hydrogenase from Thiocapsa roseopersicina was examined by low‐temperature ESR spectroscopy. Two types of signals were detected, from an oxidized iron‐sulphur cluster and a nickel centre (Ni‐A). In the oxidized protein additional signals were observed due to spin‐spin interaction between the two paramagnetic centres. This interaction could be reversibly abolished by reduction to a redox potential below 105 mV. This implies that an additional redox centre is involved in the interaction, for which an Fe3+ ion is suggested. Reduction with hydrogen induced a second type of nickel ESR signal (Ni‐C), corresponding to an intermediate redox state seen in other nickel hydrogenases. The Ni‐C species was light‐sensitive at cryogenic temperatures. At temperatures near to 4.2 K the Ni‐C signal showed evidence of interaction with another paramagnetic centre, presumably a second iron‐sulphur cluster. On reoxidation a signal due to a third Ni(III) species, Ni‐B, increased in amplitude. These results establish that metal centres in the hydrogenase from T. roseopersicina are closely similar to those of the well‐studied hydrogenase from Chromatium vinosum.
Monoclonal antibodies to protein kinase Cγ
Authors: CAZAUBON Sylvie , MARAIS Richard , PARKER Peter , STROSBERG A. Donny
Published: March 2005

Four monoclonal antibodies (mAbs), derived from high‐responder Biozzi mice immunized with purified protein kinase C, were selected by ELISA and further characterized by immunoblot: 3G12, 5A2, 36G9 are of isotype γ1, K and 15G4 is of isotype γ2b,K. Competition analysis between 15G4 and the three other mAbs showed that 15G4 and 3G12 are directed against either the same or overlapping epitope(s). All four mAbs are specific of the bovine γ isoform of protein kinase C and cross‐react with protein kinase Cγ from a variety of animal species.

Immunoblot analysis of protein kinase C tryptic fragments revealed that the mAbs recognize the regulatory domain and not the catalytic domain. Two of the mAbs, 36G9 and 5A2, inhibit protein kinase Cγ cofactor‐dependent activity (80% and 50% respectively). Consistent with the epitope mapping, none of mAbs inhibit the cofactor‐independent catalytic activity of protein kinase Cγ. Competition analysis between these mAbs and phosphatidylserine, 12‐O‐tetradecanoylphorbol 13‐acetate and Ca2+ showed that 36G9 and 5A2 block cofactor binding to protein kinase Cγ. These four mAbs thus interact with at least three distinct epitopes in the regulatory domain of protein kinase Cγ.
Terminal β‐d‐galactofuranosyl epitopes recognized by antibodies that inhibit Trypanosoma cruzi internalization into mammalian cells
Authors: de ARRUDA Monika V. , COLLI Walter , ZINGALES Bianca
Published: March 2005

Polyclonal antisera to 80–90‐kDa and to 50–60‐kDa polypeptides of tissue‐culture trypomastigotes which inhibit the interiorization of trypomastigotes of the Y strain (up to 70%) and of metacyclic trypomastigotes of the CL‐14 clone (up to 50%) in cultured mammalian cells, were obtained. Both sera immunoprecipitate surface polypeptides of 90 kDa, 80 kDa, 72 kDa and 58 kDa in trypomastigotes and of 80 kDa, 77 kDa and 74 kDa in metacyclic trypomastigotes. These antigens are glycoproteins with affinity for concanavalin A. The antibodies (IgG class) of the inhibitory sera are mainly directed against carbohydrate epitopes, which were identified as being β‐d‐galactofuranosyl units by radioimmunoinhibition assays. The direct involvement of the β‐d‐galactofuranosyl unit in the process of parasite infection was verified using the synthetic disaccharide β‐d‐galactofuranose (1→3)‐α‐d‐mannopyranose which promoted, at 1 mg/ml concentration, 50% inhibition of internalization.
Insulin antagonism of dexamethasone induction of tyrosine aminotransferase in cultured fetal hepatocytes
Authors: CAKE Max H. , HO Kevin K. W. , SHELLY Leslie , MILWARD Elizabeth , YEOH George C. T.
Published: March 2005

Previous studies have shown that insulin depresses the induction of tyrosine aminotransferase by glucocorticoids in cultured fetal rat hepatocytes. However, the site at which this inhibitory effect is exerted was not elucidated, since only enzyme activity was determined in such studies. Therefore, the effet of insulin on tyrosine aminotransferase synthesis, the level of its mRNA as well as the rate of transcription of the gene in isolated nuclei have been determined. The results obtained indicate that in cultures exposed to dexamethasone, Bt2cAMP, insulin and combinations of these additives, there is an excellent correlation between the enzyme activity, enzyme synthesis and the level of mRNA. Run‐on transcription experiments indicate that the reduction in the level of mRNA by insulin in dexamethasone‐supplemented cultures is the result of a diminished rate of gene transcription.
Spectral and kinetic studies of phosphate and magnesium ion binding to yeast inorganic pyrophosphatase
Authors: SMIRNOVA Iruna N. , SHESTAKOV Alexander S. , DUBNOVA Elena B. , BAYKOV Alexander A.
Published: March 2005

Inorganic pyrophosphatase must bind two phosphate molecules in order to catalyze pyrophosphate synthesis. In this report it is shown that Pi causes marked effect on the absorption spectrum of baker's yeast inorganic pyrophosphatase and this effect can be used to analyze Pi binding to this enzyme. A series of absorbance versus Pi concentration curves in the presence of 0.5–20mM free Mg2+ were obtained at pH 7.2 and computer‐fitted to 19 models. The dissociation constant of magnesium phosphate (8.5 ± 0.4 mM) used in this analysis was measured with a Mg2+ ‐sensitive electrode. The best model implies successive binding of two magnesium phosphate molecules or random‐order binding of magnesium phosphate and free phosphate molecules. The first route predominates at physiological concentrations of Mg2+. The Pi‐inhibition pattern of pyrophosphate hydolysis confirmed that Pi adds to the active site and provided further evidence for the existence of an activating Pi‐binding site. The possibility is raised that the pathways of pyrophosphate synthesis and hydrolysis by inorganic pyrophosphatase may differ in the sense that the binding of the fourth metal ion/subunit may facilitate the synthesis and inhibit the hydrolysis.
Primary structure of twenty three neutral and monosialylated oligosaccharides O‐glycosidically linked to the human secretory immunoglobulin A hinge region determined by a combinationof permethylation analysis and 400‐MHz 1H‐NMR spectroscopy
Authors: PIERCE‐CRÉTEL Annick , DECOTTIGNIES Jean‐Pierre , WIERUSZESKI Jean‐Michel , STRECKER Gérard , MONTREUIL Jean , SPIK Geneviève
Published: March 2005

Sialooligosaccharide and asialooligosaccharide alditols, derived from the human milk secretory immunoglobulin A hinge region, have been purified by HPLC using, successively, an amino‐bonded silica column and an octadecyl‐bonded silica column. Their primary structures were completely resolved by applying a combination of sugar analysis, methylation mass spectrometry and 400‐MHz 1H‐NMR spectroscopy. In the present report, twenty three oligosaccharide alditols are described and all possess a type two core consisting of the branched trisaccharide: Gal(β1–3)[GlcNAc(β1–6)]GalNAc‐ol. The elongation of this core arises by chain lengthening only on the GlcNAc(β1–6) branch for the sialylated compounds, leading to a dodecasaccharide, and on both branches for the neutral compounds leading to a nonasaccharide. Sixteen of the described oligosaccharide structures are original. Moreover, some of the fucosylated structures were found to support Lea, Leb and X blood group determinants.
Human gastric lipase
Authors: BERNBÄCK Stefan , BLÄCKBERG Lars
Published: March 2005

Human gastric lipase subjected to limited tryptic proteolysis lost its ability to hydrolyze emulsified long‐chain triacylglycerol. Activity against a water‐soluble substrate was however retained, indicating that proteolysis did not affect the active site. Sequence analysis revealed that trypsin specifically cleaved the linkage between lysine‐4 and leucine‐5. This cleavage rendered the enzyme unable to bind to emulsified triacylglycerol particles, e.g. human milk fat globules. We suggest that the N‐terminal tetrapeptide, in particular lysine‐4, is essential for the binding of human gastric lipase to lipid/water interfaces, and hence, for its physiological function.
Alternative occupancy of a dual ribosomal binding site by mRNA affected by translation initiation factors
Authors: CANONACO Maria A. , GUALERZI Claudio O. , PON Cynthia L.
Published: March 2005

The interaction between Escherichia coli 30S ribosomal subunits and mRNAs, and the effect of the initiation factors on this process, have been studied using MS2 RNA, polyribonucleotides and model mRNAs encoded by synthetic genes. The interactions were analyzed by gel filtration, by sucrose gradient centrifugation and by competition for ribosome binding between the various mRNAs and a Shine‐Dalgarno deoxyoctanucleotide. It was found that the initiation factors do not significantly affect the Shine‐Dalgarno interaction nor the apparent Ka values of the 30S‐subunit – mRNA binary complexes, but influence the positioning of the mRNAs on the 30S subunit with respect to the Shine‐Dalgarno octanucleotide. The results suggest that, in the absence of initiation factors, the mRNA occupies a ribosomal “stand‐by” site which is close to or includes the region where the Shine‐Dalgarno interaction takes place; in the presence of the factors, the mRNA is shifted away from the stand‐by site, towards another ribosomal site with similar affinity for the mRNA. This shift does not require the presence of fMet‐tRNA and, depending upon the type of mRNA, is mediated by IF‐2 and/or IF‐3.
Direct electron transfer of redox proteins at the bare glassy carbon electrode
Authors: HAGEN Wilfred R.
Published: March 2005

A simple system is presented for the microscale, direct voltammetry of redox proteins, typically 25 μg, in the absence of mediators and/or modifiers. The sample consists of a droplet of anaerobic solution laid onto an oversized disc of nitric‐cid‐pretreated glassy carbon as the working electrode. Very reproducible, Nernstian responses are obtained with horse heart cytochrome c. The midpoint potential Em (pH 7.0) is dependent on the ionic strength, ranging from +293 mV in 1 mM potassium phosphate to +266 mV in 0.1 M phosphate. At fixed buffer and cytochrome concentrations the magnitude of the voltammetric response is found to be independent of pH over six pH units around neutrality. It is suggested that the response of the present system is not complicated by pH‐dependent properties of the electrode surface around physiological pH and, therefore, that the use of this system is practical in biochemically oriented studies. Direct, quasi‐reversible responses have also been obtained at pH 7.0 (5 mM phosphate) from Desulfovibrio vulgaris. Hildenborough strain, tetraheme cytochrome c3 (pI = 10.0 at 4°C; 3×Em=−0.32 mV, Em=−0.26 V), and cytochrome c553 (pI = 9.3; Em=+60 mV), and from Megasphaera elsdenü rubredoxin (pI ≃ 3; Em=−42 mV) and 2[4F–‐4S] ferredoxin (pI ≃ 3; Em=−353 mV). The latter protein adsorbs onto the glassy carbon surface, thus forming a system with possible applications in the electrochemical study of ferredoxin‐linked enzymes.
Impact of altered protein structures on the intracellular traffic of a mutated vasopressin precursor from Brattleboro rats
Authors: SCHMALE Hartwig , BOROWIAK Bernhard , HOLTGREVE‐GREZ Heidi , RICHTER Dietmar
Published: March 2005

The rat vasopressin precursor, synthesized in the reticulocyte lysate system under the direction of in vitro transcribed mRNA, is processed and correctly delivered to the lumen of added microsomal vesicles. Translation of mRNA for the mutant (Brattleboro) vasopressin precursor which lacks a translational stop codon as a consequence of a frame‐shift mutation, gives rise to a mutated protein (B‐mutant precursor) with a C‐terminal poly(lysine) sequence encoded by the poly(A) tail. Upon addition of microsomal membranes, the mutated precursor has access to the lumen of the vesicles as indicated by removal of the signal peptide; however, the C‐terminal part with the poly(lysine) tail remains outside the vesicles as shown by its sensitivity to proteinase K. When a modified RNA, including a stop codon located similarly to that found in the cDNA encoding the normal precursor, is translated in the presence of microsomal membranes, the resulting product (S‐mutant precursor) is refractory to proteolysis by exogenously added proteinase K. Analysis of the microsomal membranes indicates, however, that the C‐terminus of the S‐mutant precursor is still anchored within membranes. For studying the intracellular transport of the mutated precursor Xenopus laevis oocytes were injected with various RNA constructs. To monitor the transport steps from the endoplasmic reticulum to the Golgi compartment an RNA encoding a glycosylation site within the S‐mutant precursor sequence was constructed. The resulting GS‐mutant precursor is synthesized in the oocyte but not secreted into the incubation medium, completely in contrast to the normal vasopressin precursor which can be detected in the incubation bath 4 h after injection of the respective RNA. The sensitivity of the GS‐mutant precursor carbohydrate side chain to endoglycosidase H treatment suggests that the mutated precursor does not reach the Golgi apparatus.
Expression of muscle‐specific proteins necessary to regulate translation of the mRNA for a 40‐kDa housekeeping polypeptide in rat L6 cells
Authors: PRAMANIK Saroj K. , BAG Jnanankur
Published: March 2005

Translation of the mRNA for a housekeeping polypeptide of 40 kDa (P40) was found to be regulated under a variety of conditions in rat L6 cells. This mRNA was translated in the proliferating myoblasts but not in the non‐proliferating myotubes. A number of chemicals, such as dimethyl sulfoxide, sodium butyrate and aphidicolin, were used to prevent expression of muscle‐specific genes in mitogen‐poor differentiation medium. In the absence of any detectable accumulation of muscle‐specific α‐actin mRNA, the P40 mRNA remained in the translated state. A fourth chemical, EGTA, a known inhibitor of fusion of muscle cells, blocked translation of muscle specific actin and tropomyosin mRNAs. On the other hand, it showed no effect on the translation of P40 mRNA. Addition of Ca2+ to the EGTA‐treated cultures, however, almost completely reversed the block of translation of actin tropomyosin mRNAs within four days. Concomitant to Ca2+ reversal of the translational block of muscle mRNAs, P40 mRNA entered the non‐translated state. An inverse relationship, therefore, was observed between the translation of housekeeping P40 mRNA and muscle‐specific mRNAs.

The ability to mimic in vivo regulation of P40 mRNA translation was examined in mRNA‐dependent micrococcal‐nuclease‐treated homologous cell‐free extracts. The extracts from myoblasts and myotubes were able to translate P40 mRNA. Furthermore, ribosomes of both myoblast and myotube extracts containing endogenous mRNAs were also able to bind to P40 and actin mRNAs. Myotube extract, however, showed a lower binding ability to P40 mRNA than to the actin mRNA. The ability of ribosomes of myotube extract to bind P40 mRNA was somewhat enhanced by addition of proteins derived from washing thdse ribosomes with a high‐ionec‐strength buffer. In order to elucidate the role of interaction between mRAN and proteins in translational control of P40 mRNA, the polypeptide complements of polysomal and free P40 mRNA‐protein(mRNA) complexes were also examined. Hybrid selection of polysomal and free P40 mRNP complexes followed the covalent joining of mNA and protein moieties of mRNP complexes by ultraviolet irradiation of rat L6 cells. Analysis of buoyant densities of these complexes showed that free P40 mRNP had slightly less protein then polysomal P40 mRNP. Furthermore, analysis of the polypeptide complements of both free and polysomal P40 mRNA complexes showed that they were composed of identical polypeptides. The only detectable difference between the polypeptide complements of these complexes was that two polypeptides of 72 kDa and 55 kDa were more abundant in the polysomal P40 mRNP than free P40 mRNP.
Purification of cytochrome‐c oxidase retaining its pulsed form
Authors: BRANDT Ulrich , SCHÄGGER Hermann , von JAGOW Gebhard
Published: March 2005

A new purification procedure for cytochrome‐c oxidase from bovine heart mitochondria is described. The enzyme was purified by selective solubilization in Triton X‐100 and subsequent hydroxyapatite and gel chromatography. The preparation was highly pure and active. The subunit composition and steady‐state kinetics were found to be the same as thoes reported for other preparations.

In contrast to most of the previously published protocols the method presented here resulted in a preparation which had a rapid intramolecular electron transfer from cytochrome a to cytochrome a3, i.e. it was found to have retained its pulsed state. This correlated with monoexponential cyanide‐binding kinetics.

The formation of resting kinetics biphasic cyanide‐binding kinetics was shown to be induced by a short incubation at pH 5.0.
Catalytic properties of thermophilic lactate dehydrogenase and halophilic malate dehydrogenase at high temperature and low water activity
Authors: HECHT Katrin , WRBA Alexander , JAENICKE Rainer
Published: March 2005

Thermophilic lactate dehydrogenases from Thermotoga maritima and Bacillus sterothermophilus are stable up to temperature limits close to the optimum growth temperature of their parent organisms. Their catalytic properties are anomalous in that Km shows a drastic increase with increasing temperature. At low temperatures, the effect levels off.

Extreme halophilic malate dehydrogenase from Halobacterium marismortui exhibits a similar anomaly. Increasing salt concentration (NaCl) leads to an optmum curve for Km, oxaloacetate while Km, nadh remains constant. Previous claims that the activity of halophilic malate dehydrogenase shows a maximum at 1.25 M NaCl are caused by limiting substrate concentration; at substrate saturation, specific activity of halophilic malate dehydrogenase reaches a constant value at ionic strengths I 1 M. Non‐halophilic (mitochondrial) malate dehydrogenase shows Km characteristics similar to those observed for the halophilic enzyme. The drastic decrease in specific activity of the mitochondrial enzyme at elevated salt concentration is caused by the salt‐induced increase in rigidity of the enzyme, rather than gross structural changes.
The effect of myosin light chain phosphorylation and Mg2+ on the conformation of myosin in thick filaments of glycerinated fibers of rabbit skeletal muscle
Authors: BOROVIKOV Yurii S. , LEVITSKY Dmitrii I.
Published: March 2005

It has been shown by polarization microfluorimetry that phosphorylation of myosin light chain 2, in stretched single glycerinated fibers of rabbit skeletal muscle, results in changes in polarized fluorescence anisotropy of both the tryptophan residues of myosin molecules and the fluorescent label, N‐iodoacetyl‐N′‐(5‐sulfo‐1‐naphthyl)ethylenediamine, associated with the fast‐reacting thiol group in myosin heads. These changes are also dependent on the presence or absence of Mg2+ in the medium: they are most pronouced in the presence of 5 mM MgCl2. It is assumed that both Mg2+ binding to myosin and phosphorylation of light chain 2 associated with myosin heads induce structural changes in myosin filaments of muscle fibres which are expressed as changes in the orientation of myosin heads and in the conformation of myosin rods.
Dual Mg2+ control of formyl‐peptide‐receptor–G‐protein interaction in HL 60 cells
Authors: GIERSCHIK Peter , STEISSLINGER Marita , SIDIROPOULOS Dimitrios , HERRMANN Eva , JAKOBS Karl H.
Published: March 2005

In neutrophils and several other phagocytic cell types, a pertussis‐ and cholera‐toxin‐sensitive form of the guanine‐nucleotide‐binding protein (G‐protein) Gp couples receptors for N‐formylmethionine‐containing chemotactic peptides to stimulation of phospholipase C. Using membranes of myeloid differentiated HL 60 cells, we have examined the role of Mg2+ and guanine nucleotides in regulating (a) the interaction of the formyl‐peptide receptor with the chemotatic agonist N‐formylmethionyl‐leucyl‐phenylalanine (fMet‐Leu‐Phe) and (b) the receptor‐mediated activation of Gp. Mg2+ markedly enhanced the number of receptors with high affinity for the radiolabeled oligopeptide fMet‐Leu‐[3H]Phe. At the same time, Mg2+ largely increased the potency of guanosine‐5′‐(3‐O‐thio)triphosphate, but not of GDP or guanosine‐5′‐(2‐O‐thio)diphosphate, to inhibit binding of the peptide. Comparison of the potency of Mg2+ in eliciting these two effects and analysis of the specificities of the relevant divalent cation sites revealed that Mg2+ interacts with at least two independent sites on the receptor‐Gp complex. One site is specific for Mg2+ and exhibits affinity in the micromolar range, the other site interacts with millimolar concentrations of several divalent cations in a non‐selective fashion. It is suggested that the former site is located on Gp and that interaction of Mg2+ with this site is necessary for the receptor‐mediated G‐protein activation, whereas interaction of divalent cations with the latter site is necessary for high affinity agonist binding. The regulation of the formyl‐peptide receptor binding properties by guanine nucleotides is independent of Gp activation, since inhibition of peptide binding is achieved by addition of both guanine nucleoside diphosphates and triphosphates and is readily seen both in the presence and in the absence of Mg2+. The latter finding, together with the observation that, at micromolar concentrations of Mg2+, high‐affinity GTPase activity is stimulated by fMet‐Leu‐Phe primarily via low affinity receptors, suggests that, contrary to widely held opinions, (a) divalent cations are not required for a functional receptor–G‐protein interaction and (b) high‐affinity agonist binding is not a prerequisite for the receptor‐mediated activation of the G‐protein.
Inhibition of NADPH oxidase by aminoacyl chloromethane protease inhibitors in phorbol‐ester‐stimulated human neutrophils: a reinvestigation
Authors: CONSEILLER Emmanuel C. , LEDERER Florence
Published: March 2005

Superoxide anion production by polymorphonuclear leukocytes stimulated with phorbol 12‐myristate 13‐acetate is known to be inhibited by a number of inhibitors and substrates of serine proteases, in particular by tosylphenylalanylchloromethane (TosPheCH2Cl) and to a lesser extent by tosyllysylchloromethane (TosLysCH2Cl). We have reinvestigated the characteristics of this inhibition, in view of the fact that other serine protease inhibitors with similar specificities, phenylmethanesulfonyl fluoride and leupeptin, were without effect. We found that the inhibition of phorbol‐ester‐induced superoxide production after cell preincubation with the chloromethanes followed saturation kinetics, with Kinact and kinact values of 100 μM and 31 min−1 for TosPheCH2Cl and 2 mM and 18 min−1 for TosLysCH2Cl. We also showed that the two compounds, which can inhibit protein kinase C in vitro, inhibited neither its activity in vivo, nor its translocation induced by phorbol myristate acetate. Furthermore the intracellular non‐protein sulfhydryl group content was not affected by the treatment with the chloromethanes. Finally, addition of the inhibitors to stimulated cells also led to a time‐dependent, concentration‐dependent inhibition of superoxide production. Altogether, our results suggest that the chloromethane target is neither a protease nor protein kinase C and is not involved in NADPH oxidase activation, but rather in maintainance of its activity. The possible identity of this protein is discussed.
Differential effect of hemin‐controlled eIF‐2α kinases from mouse erythroleukemia cells on protein synthesis
Authors: SARRE Thomas F. , HERMANN Marita , BADER Michael
Published: March 2005

Cultured mouse erytholeukemia (MEL) cells can be induced to erythroid differentiation by a variety of chemical agents. This differentiation process is marked by the onset of globin mRNA and hemoglobin synthesis. In rabbit reticulocytes, globin synthesis is regulated by a hemin‐controlled translational inhibitor (HCI) which acts via phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF‐2). From both uniduced and induced MEL cells, hemin‐controlled eIF‐2α kinases have been partially purified. They resemble HCI with respect to their chromatographic behaviour and their sensitivity towards physiological concentrations of hemin (5–10 μM). Further purification on phosphocellulose, however, reveals that the eIF‐2α kinase from uninduced MEL cells is chromatographically distinct from HCI, whilst the eIF‐2α kinase activity from induced MEL cells represents a mixture of the former and the HCI‐type eIF‐2α kinase. The latter inhibits protein synthesis in a fractionated system from rabbit reticulocytes which is free of, but sensitive to, HCI, whereas the eIF‐2α kinase from uninduced MEL cells does not show any inhibitory activity. This observation is supported by the finding that induced MEL cells respond in vivo to iron depletion with a shut‐off of protein synthesis (as do rabbit reticulocytes), whilst uninduced MEL cells do not.
Transcription of the αA‐globin gene of the duck
Authors: WEINGART Sabine , SOMMER Ursula , GERHOLD Helmut , SEIFART Klaus H.
Published: March 2005

A homologous in vitro transcription system was developed in which the αA‐globin gene of the duck was faithfully transcribed. Whole‐cell extracts from duck erythrocytes were separated into fractions A, B, C and D by consecutive elution from phosphocellulose columns and were individually reconstituted in run‐off transcription assays. Fractions A, C and D were required to achieve faithful initiation on the αA‐globin gene. The latter fractions were mutually interchangeable with comparable fractions from HeLa cells. A fourth fraction, B, was not required but enhanced‐basal transcription when reconstituted with fractions A, C and D or a very low amount of HeLa whole‐cell extract which by itself did not yield a detectable signal. Fraction B from duck erythrocytes was further purified by chromatography on DEAE‐Sephadex and was shown to contain two trans‐acting factors. One of these differentially acts on the αA‐globin gene of the duck. The other component from duck erythrocytes surprisingly resembles the upstream stimulatory factor, previously isolated from HeLa cells. This latter protein binds to and trans‐activates the adenovirus 2 major late promoter, but is not involved in the transcription of the αA‐globin gene.
Preliminary assignments of the aromatic and some methyl group resonances of the 1H‐NMR spectrum of the oxidized form of uteroglobin
Authors: JAMIN Nadège , ROY Pierre , FRIDLANSKY Françoise , DELEPIERRE Muriel , MILGROM Edwin , ROQUES Bernard P. , MORNON Jean‐Paul
Published: March 2005

Two‐dimensional NMR methods have been used to assign aromatic and methyl group resonances in the 1H‐NMR spectrum of oxidized uteroglobin. Assignments to specific amino acids are based on X‐ray‐determined structures of two crystal forms (C2221 and P21) and on an energy‐minimized X‐ray structure of the C2221 form of uteroglobin. These preliminary assignments are sufficient to probe the interaction of oxidized uteroglobin with progesterone in solution. The protein global structure is unmodified but some direct or indirect conformational changes are induced in the H1H4(H1′H4′) pockets and close to Phe28 by progesterone.
Mitogen‐responsive S6 kinase
Authors: LAWEN Alfons , BURGER Matthias , MARTINI Oskar H. W.
Published: March 2005

Many cell lines respond to mitogenic stimuli (serum, growth factors) with rapid phosphorylation of the ribosomal protein S6 at several serine sites. We have tried to identify the protein kinase(s) mediating this effect of growth stimuli. Examining post‐DEAE chromatography fractions of S49 kin− cell extracts, we could detect a highly active effector‐independent S6 kinase with specificity for serine residues. The study was extended to the presumably homologous human enzyme, using HeLa S3 cells as model system. Activity yields increased up to sevenfold when exhausted HeLa cells were supplied with fresh medium plus serum. The enzyme uses ATP, not GTP, as cosubstrate, 40‐S or 80‐S (reassociated from subunits) ribosomal particles being substrate. The optimal K+ concentration, measured at 3 mM Mg2+, is 35 mM. Under optimized assay conditions S6 phosphorylation proceeded faster in vitro than it appeared to do in vivo. The apparent Mr of the enzyme, as estimated by gel filtration on Sephadex G‐100, is 56 000 (determination in the presence of 200 mM KCl in 25 mM phosphate buffer). Tighter binding to DEAE‐Sephacel and higher specificity for S6 distinguishes this enzyme from the following S6‐phosphorylating protein kinases: protein kinase C, protease‐activated kinase II, histone‐4 phosphotransferase and an enzyme with the properties of casein kinase I. In published summaries of observations shown here and in a follow‐up study with chick embryo fibroblasts, the enzyme(s) has been referred to as mitogen‐responsive S6 kinase(s) [Martini, O. H. W. and Lawen, A. (1985) in Hormones and cell regulation (Dumont, J. E., Hamprecht, B. and Nunez, J., eds) vol. 9, pp. 411–412. Elsevier Company, North‐Holland, Amsterdam; Lawen, A. and Martini, O. H. W. (1985) FEBS Lett. 185, 272–276].
Properties of vasoactive‐intestinal‐peptide receptors and β‐adrenoceptors in the murine radiation leukemia‐virus‐induced lymphoma cell line BL/VL3
Authors: ABELLO Jacques , DAMIEN Catherine , DE NEEF Philippe , TASTENOY Michèle , HOOGHE Robert , ROBBERECHT Patrick , CHRISTOPHE Jean
Published: March 2005

Based on radioligand binding and adenylate cyclase activation, functional receptors to vasoactive intestinal peptide(VIP)/helodermin, were shown to coexist with β2‐adrenoceptors and prostaglandin receptors in membranes from a cultured cloned BL/VL3 cell line of murine T‐cell lymphoma induced by a radiation leukemia virus.

The relative potency of VIP‐related peptides to stimulate adenylate cyclase activity was: helodermin > VIP > peptide histidine isoleucinamide. Five VIP analogs inhibited 125I‐iodo‐VIP binding and stimulated adenylate cyclase activity, their decreasing order of potency being: VIP > [D‐Asp3]VIP > [D‐Ser2]VIP > [D‐Ala4]VIP = [D‐His1]VIP = [D‐Phe2]VIP. [D‐Phe2]VIP acted as a partial agonist (with an intrinsic activity of 0.1 as compared to that of VIP = 1.0) and competitively inhibited helodermin‐ and VIP‐stimulated adenylate cyclase activity with a similar Ki (0.07–0.10 μM). These data suggest the existence, in this murine T‐cell lymphoma, of VIP receptors of the ‘helodermin‐preferring’ subtype that are coupled to adenylate cyclase.
Homologous and heterologous regulation of the helodermin/vasoactive‐intestinal‐peptide response in the murine radiation leukemia‐virus‐induced lymphoma cell line BL/VL3
Authors: ABELLO Jacques , DAMIEN Catherine , ROBBERECHT Patrick , HOOGHE Robert , VANDERMEERS André , VANDERMEERS‐PIRET Marie‐Claire , CHRISTOPHE Jean
Published: March 2005

Functional vasoactive intestinal peptide (VIP)/helodermin receptors and β2‐adrenoceptors coexist in membranes from a cultured cloned BL/VL3 cell line of murine T‐cell lymphoma induced by a radiation leukemia virus (see preceding paper in this journal).

Short‐term (5–30 min) exposures of BL/VL3 cells to VIP or isoproterenol induced both homologous and heterologous desensitization. The potency of VIP and isoproterenol to desensitize was similar to their potency to occupy receptors and activate adenylate cyclase.

Long‐term (16‐h) exposure of BL/VL3 cells to VIP induced homologous down regulation only, whereas isoproterenol induced both homologous and heterologous down regulation. The potency of VIP, peptide histidine isoleucinamide, helodermin, helospectin, and [D‐Phe2]VIP on the one hand, and of isoproterenol on the other hand, to decrease homologous responses was comparable to their potency for receptor occupancy and adenylate cyclase activation.
Purification and characterization of spermidine N1‐acetyltransferase from chick duodenum
Authors: SHINKI Toshimasa , SUDA Tatsuo
Published: March 2005

We have reported that spermidine N1‐acetyltransferase has a larger role than ornithine decarboxylase in putrescine synthesis in chick duodenum induced by 1α,25‐dihydroxycholecalciferol (calcitriol) [Shinki, T., Kadofuku, T., Sato, T. and Suda, T. (1986) J. Biol. Chem. 261, 11 712–11 716]. In the present study, spermidine N1‐acetyltransferase was purified from the duodenal cytosol of calcitriol‐treated chicks to homogeneity judged by SDS/polyacrylamide gel electrophoresis. The purified enzyme converted spermidine only to N1‐acetylspermidine. The apparent molecular mass of the purified spermidine N1‐acetyltransferase was found to be 36 kDa by gel filtration on Sephacryl S‐200 and 18 kDa by SDS polyacrylamide gel electrophoresis. When duodenal crude 105000 x g extracts were directly applied to a Sephacryl S‐200 column without prior purification, three peaks with spermidine N1‐acetyltransferase activity appeared. The first peak was in the void volume, the second peak was in the fraction corresponding to an apparent molecular mass of 70 kDa, and the third peak was in the fraction corresponding to 36 kDa. These results suggest that spermidine N1‐acetyltransferase exists as a dimer of the 18 kDa subunits and is stabilized in (a) form(s) bound to other components or proteins in intact cells.