bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of mitochondrial swelling and calcium on phosphate‐activated glutaminase in pig renal mitochondria
Authors: KVAMME Elling , ROBERG Bjørg , TORGNER Ingeborg Aa. 
Published: March 2005

The effects of mitochondrial swelling and calcium have been used to study the possible function of the glutamine transporter in regulating glutamine hydrolysis. Salt‐induced swelling of pig renal mitochondria and an iso‐osmotic mixed salt solution and swelling caused by reducing the osmolarity of the incubation medium, are accompanied by activation of glutamine hydrolysis. Regulation of the glutaminase activity by salt‐induced mitochondrial swelling is likely to have physiological importance, similar to the regulation of hepatic glutaminase by changing the matrix volume, that has been described by others.0.1 – 1.0 mM calcium stimulates glutamine hydrolysis and the calcium activation curve follows Michaelis‐Menten kinetics. The calcium activation is reversible, it is unaffected by phosphate, high glutamine and mitochondrial calcium uptake, as well as by sonication and the activation is calmodulin independent. The calcium activation is additive to that of swelling.Similar to calcium, hypo‐osmotic swelling mainly increases the apparent Vmax for glutamine, whereas the apparent Km is little changed, indicating that the effects are primarily on the phosphate‐activated glutaminase itself rather than on the glutamine transporter. Furthermore, calcium which activates glutamine hydrolysis, inhibits glutamine uptake into the mitochondria and so does alanine having no effect on glutamine hydrolysis. Therefore, it is indicative that glutamine transport is not rate limiting for glutamine hydrolysis.
Ca2+‐dependent phospholipid and arachidonic acid binding by the placental annexins VI and IV
Authors: EDWARDS Helena C. , CRUMPTON Michael J. 
Published: March 2005

Using an assay system in which phospholipids were immobilised on phenyl‐Sepharose, we examined the affinities of the placental annexins VI and IV for binding to specific phospholipids over a range of Ca2+ concentrations. Purified annexin VI showed half‐maximal binding to phosphatidylserine, phosphatidylethanol‐amine and phosphatidylinositol at Ca2+ concentrations of 0.6, 0.4 and 3.5 μM, respectively, compared to values of 4.5, 4.5 and 20 μM Ca2+, respectively for purified annexin IV. These values did not change significantly in the presence of other proteins from the family. Neither annexin VI or IV bound to phosphatidylinositol bisphosphate and phosphatidylcholine, even at millimolar concentrations of Ca2+. However, both proteins bound to arachidonic acid, oleic acid and palmitic acid in a Ca2+‐dependent manner, using the same assay system. The level of binding for both proteins was significantly increased when mixtures of phosphatidylcholine and arachidonic acid were examined. A dose‐dependent inhibition of phospholipase A2 by both annexins VI and IV, at millimolar concentrations of Ca2+ was observed when phosphatidylcholine liposomes were used as a substrate.These results raise questions about the interpretation of experiments in which the release of arachidonic acid is used as a measure of lipase activity, and of the validity of the substrate‐depletion model for the inhibition of phospholipases by the annexins.
Isolation and structural characterization of novel neutral oligosaccharide‐alditols from respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis
Authors: van KUIK J. Albert , de WAARD Pieter , VLIEGENTHART Johannes F. G. , KLEIN André , CARNOY Christophe , LAMBLIN Geneviève , ROUSSEL Philippe 
Published: March 2005

Oligosaccharide‐alditols derived from the respiratory mucins of a patient suffering from bronchiectasis were separated by HPLC into 46 fractions. The structures of 11 oligosaccharides with GlcNAcβ(1→3)Galβ(1→4)‐GlcNAcβ(1→6)GalNAc‐ol in common have been established (preceding paper in this journal). In this second part the structures of 12 oligosaccharides were established 8 of which have not been described before. Of the 12 compounds, 6 possess the GlcNAcβ(1→3)[Galβ(1→4)GlcNAcβ(1→6)]Galβ(1→3)GalNAc‐ol element:
image
The other compounds, that could not be classified on basis of a common structural element, are:
image
Purification, characterization and some properties of diacetyl(acetoin) reductase from Enterobacter aerogenes
Authors: CARBALLO Javier , MARTIN Roberto , BERNARDO Ana , GONZALEZ Josefa 
Published: March 2005

A new method, faster, milder and more efficient than the one previously described [Bryn, K., Hetland, O. & Stormer, F. C. (1971) Eur. J. Biochem. 18, 116–119], for purification of diacetyl(acetoin) reductase from Enterobacter aerogenes is proposed. The experiments carried out with the electrophoretically pure preparations obtained by this procedure show that the enzyme (a) produces l‐glycols from the corresponding l‐α‐hydroxycarbonyls by reversible reduction of their oxo groups and also reduces the oxo group of uncharged α‐dicarbonyls converting them into l‐α‐hydroxycarbonyls, and (b) is specific for NAD. This is a new enzyme for which we suggest the systematic name of l‐glycol:NAD+ oxidoreductase and the recommended name of l‐glycol dehydrogenase(NAD).The molecular mass, pI, affinity for substrates and pH profiles of this enzyme are also described.
Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617
Authors: ARNAUD Sylvain , MALATESTA Francesco , GUIGLIARELLI Bruno , GAYDA Jean‐Pierre , BERTRAND Patrick , MIRAGLIO Roger , DENIS Michel 
Published: March 2005

The aerobic respiratory system of the hydrocarbonoclastic marine bacterium Pseudomonas nautica 617 ends with a single terminal oxidase. It is a heme‐containing membranous protein which has been demonstrated only to reduce molecular oxygen to hydrogen peroxide [Denis, M., Arnaud S. & Malatesta, F. (1989) FEBS Lett. 247, 475–479]. The purification of this oxidase was achieved in a single step through by DEAE‐Trisacryl chromatography. SDS/PAGE showed the presence of four subunits. The pI was found to be 4.45 and a Mr of 130000 was determined by gel filtration.The amino acid composition of the purified terminal oxidase has been determined. About 52% of the residues are hydrophobic, strengthening the membranous nature of this bacterial oxidase. Room temperature optical spectra are typical of heme b with a 560‐nm band for the reduced form in the α range. The prosthetic group is made of two hemes b, one high‐spin (S= 5/2, g⊥= 5.9, g∥∼ 2.0), the other low‐spin (S= 1/2, gz= 2.94, gy= 2.27). No other metal centre was detected by EPR. The two hemes remained unresolved in optical spectra, even at low temperature, and throughout redox titration. They behaved potentiometrically like a one‐electron, single redox couple, with Em= 87 ± 10 mV at pH 7.2 and 293 K. The purified oxidase did not oxidize ferrocytochrome c, but displayed quinol oxidase activity both with the native quinone (2419 nmol O2· min−1· mg protein−1 and commercially available coenzyme (101.74 nmol O2· min−1· mg protein−1). Exposure of the reduced enzyme to CO induced the collapse of α and β bands as occurred during reoxidation. In contrast, NaCN and NaN3 fully inhibited the oxidase activity. Results are discussed with respect to other purified quinol oxidases.
N‐(Phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5‐enolpyruvylshikimate‐3‐phosphate synthase
Authors: REINBOTHE Steffen , NELLES Axel , PARTHIER Benno 
Published: March 2005

Photoautotrophic cells of Euglena gracilis can be adapted to N‐(phosphonomethyl)glycine (glyphosate) by cultivation in media with progressively higher concentrations of the herbicide. Two different mechanisms of tolerance to the herbicide were observed. One is characterized by the overproduction and 40‐fold accumulation of the target enzyme, 5‐enolpyruvylshikimate‐3‐phosphate synthase, in cells adapted to 6 mM N‐(phosphonomethyl)glycine. The other is connected with a herbicide‐insensitive enzyme. No evidence was obtained for the involvement of the putative multifunctional arom protein previously reported to be involved in the biosynthesis of aromatic amino acids in Euglena. Cells adapted to N‐(phosphonomethyl)glycine excreted shikimate and shikimate 3‐phosphate into the medium; the amounts depended on the actual concentration of the herbicide. Two‐dimensional gel electrophoresis and determination of 5‐enolpyruvylshikimate‐3‐phosphate synthase activity in crude extracts, as well as after separation by non‐denaturing gel electrophoresis, revealed that the overproduction of the enzyme in adapted cells correlates with the accumulation of a 59‐kDa protein. Overproduction of this 59‐kDa protein resulted from a selectively increased level of a mRNA coding for a 64.5‐kDa polypeptide which appeared in adapted cells, as shown by cell‐free translation in the wheat germ system.In contrast to this quantitative, adaptive type of tolerance, the second mechanism causing tolerance to N‐(phosphonomethyl)glycine in the Euglena cell line NR 6/50 was probably related to a qualitatively altered 5‐enolpyruvylshikimate‐3‐phosphate synthase, which could not be inhibited by even 2 mM N‐(phosphonomethyl)glycine in vitro. In agreement with this observation, the putatively mutated cell line excreted neither shikimate nor shikimate 3‐phosphate into the growth medium containing N‐(phosphonomethyl)glycine, even if cultivated in the presence of 20 mM or 50 mM N‐(phosphonomethyl)glycine.
The cytosolic and glycosomal glyceraldehyde‐3‐phosphate dehydrogenase from Trypanosoma brucei
Authors: LAMBEIR Anne‐Marie , LOISEAU Ann M. , KUNTZ Douglas A. , VELLIEUX Frederic M. , MICHELS Paul A. M. , OPPERDOES F. R. 
Published: March 2005

The protozoan haemoflagellate Trypanosoma brucei has two NAD‐dependent glyceraldehyde‐3‐phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody‐like organelle that fulfils an essential role in glycolysis. The kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms. Both trypanosome enzymes had pH/activity profiles similar to that of other glyceraldehyde‐3‐phosphate dehydrogenases, with optimal activity around pH 8.5–9. Only the yeast enzyme showed its maximal activity at a lower pH. The glycosomal enzyme was more sensitive to changes in ionic strength below 0.1 M, while the cytosolic enzyme resembled more the enzymes from rabbit muscle, human erythrocytes and yeast. The affinity for NAD of the glycosomal enzyme was 5–10‐fold lower than that of the cytosolic, as well as the other enzymes. A similar, but less pronounced, difference was found for its affinity for NADH. These differences are explained by a number of amino acid substitutions in the NAD‐binding domain of the glycosomal isoenzyme.In addition, the effects of suramin, gossypol, agaricic acid and pentalenolactone on the trypanosome enzymes were studied. The trypanocidal drug suramin inhibited both enzymes, but in a different manner. Inhibition of the cytosolic enzyme was competitive with NAD, while in the case of the glycosomal isoenzyme, with NAD as substrate, the drug had an effect both on Km and Vmax. The most potent inhibitor was pentalenolactone, which at micromolar concentrations inhibited the glycosomal enzyme and the enzymes from yeast and Bacillus stearothermophilus in a reversible manner, while the rabbit muscle enzyme was irreversibly inhibited.
α‐Factor‐leader‐directed secretion of recombinant human‐insulin‐like growth factor I from Saccharomyces cerevisiae
Authors: STEUBE Klaus , CHAUDHURI Bhabatosh , MÄRKI Walter , MERRYWEATHER James P. , HEIM Jutta 
Published: March 2005

A synthetic gene coding for human‐insulin‐like growth factor I (IGFI) was fused to the leader sequence of yeast prepro‐α‐factor and expressed in Saccharomyces cerevisiae under the control of a glyceraldehyde‐3‐phosphate dehydrogenase promoter fragment. Recombinant IGFI was found inside yeast cells and secreted into the medium. The secreted IGFI migrated on SDS gels with the same electrophoretic mobility as authentic IGFI, i.e. at about 7.5 kDa. HPLC analysis of secreted IGFI revealed the presence of the correctly folded, genuine molecule as well as an isomeric byproduct of equal molecular mass but with two of the three disulfide bonds interchanged.Inside exponentially growing cells the 7.5‐kDa IGFI was also found, along with up to four additional IGFI‐related polypeptides of higher molecular mass. By endoglycosidase F treatment the three polypeptides between 19–26 kDa were converted to a single peptide of 17 kDa. Since this peptide also reacted with an anti‐α‐factor antibody, it represents most likely the unglycosylated α‐factor–IGFI fusion precursor.Pulse‐chase experiments established the precursor nature of the intracellular higher‐molecular‐mass IGFI species. Conversion of the primary translation product to the differently glycosylated IGFI precursor proteins and into the mature form occurred very rapidly, within 2 min. Rapid maturation was, however, not followed by an equally rapid secretion of the mature form into the medium: only after 30–40 min did IGFI appear outside the cells.We therefore postulate the presence of an as yet undefined Golgi or post‐Golgi bottleneck representing a major obstacle in secretion of recombinant IGFI from S. cerevisiae cells.
The complex between a tissue inhibitor of metalloproteinases (TIMP‐2) and 72‐kDa progelatinase is a metalloproteinase inhibitor
Authors: KOLKENBROCK Hansjörg , ORGEL Dagmar , HECKER‐KIA Adelheid , NOACK Wolfgang , ULBRICH Norbert 
Published: March 2005

Human rheumatoid synovial cells in culture secrete both 72‐kDa progelatinase and a complex consisting of 72‐kDa progelatinase and a 24‐kDa inhibitor of metalloproteinases, TIMP‐2. In addition, the culture medium contains TIMP‐1, the classical inhibitor of metalloproteinases, with a molecular mass of 30 kDa. TIMP‐1 does not form a complex with free 72‐kDa progelatinase.Free progelatinase and progelatinase complexed with TIMP‐2 can be activated with the organomercury compound p‐aminophenylmercury acetate. The activated complex shows less than 10% the enzyme activity of activated free gelatinase.The progelatinase – TIMP‐2 complex could be shown to be an inhibitor for other metalloproteinases, such as gelatinase and collagenase secreted by human rheumatoid synovia fibroblasts, as well as for the corresponding enzymes from human neutrophils.
Synergistic effects of Ca2+ and wheat germ agglutinin on the lamellar‐hexagonal (HII) phase transition of glycophorin‐containing egg‐phosphatidylethanolamine membranes
Authors: TAMPÉ Robert , GALLA Hans‐Joachim 
Published: March 2005

Glycophorin has been reconstituted into egg phosphatidylethanolamine (egg‐PtdEtn) membranes. Stable vesicles were obtained at a molar ratio of 8 × 10−4 of inserted protein/lipid. This macroscopic change from lipid aggregates to lipid vesicles was followed by density gradient centrifugation. Vesicles formed in the presence of protein enclose the dye calcein and are stable with time and temperature. Membrane aggregation does not occur, as was demonstrated by energy‐transfer experiments. The phase transition from the fluid lamellar Lα phase to the inverted hexagonal HII phase observed at 29°C in pure egg‐PtdEtn membranes is suppressed and finally disappears in the presence of glycophorin. The transition enthalpy decreases linearly from ΔH= 4 kJ/mol in pure lipids to zero at a protein/lipid molar ratio of 1:1000.Ca2+ ions and wheat germ lectin act synergistically on the phase behavior of vesicles containing glycophorin and phosphatidylethanolamine. Differential scanning calorimetry scans show that the lamellar‐to‐hexagonal phase transition is reinduced. The membranes aggregate and exchange lipid as could be demonstrated by energy transfer experiments. The dye calcein is released but only if the temperature exceeds the lamellar‐to‐hexagonal phase transition temperature of the pure egg‐PtdEtn.
Inhibition of hepatic proteolysis by insulin
Authors: HALLBRUCKER Christian , vom DAHL Stephan , LANG Florian , GEROK Wolfgang , HÄUSSINGER Dieter 
Published: March 2005

Proteolysis was measured as [3H]leucine release from isolated perfused livers from rats, which had been labeled in vivo by an intraperitoneal injection of [3H]leucine about 16 h prior to the perfusion experiment. In livers from fed rats, insulin (35 nM) inhibited [3H]leucine release by 24.5 ± 1.3% (n= 15) and led to an amiloride‐sensitive, bumetanide‐sensitive and furosemide‐sensitive net K+ uptake of 5.53 ± 0.31 μmol · g−1 (n= 15). Both the insulin effects on net K+ uptake and on [3H]leucine release were diminished by about 65% or 55% in presence of furosemide (0.1 mM) or bumetanide (5 μM), respectively. The insulin‐induced net K+ uptake was virtually abolished in the presence of amiloride (1 mM) plus furosemide (0.1 mM).
In perfused livers from 24‐h‐starved rats, both the insulin‐stimulated net K+ uptake and the insulin‐induced inhibition of [3H]leucine release were about 80% lower than observed in experiments with livers from fed rats. The insulin effects on K+ balance and [3H]leucine release were not significantly influenced in the presence of glycine (2 mM), although glycine itself inhibited [3H]leucine release by 30.3 ± 0.3% (n= 4) and 13.8 ± 1.2% (n= 5) in livers from starved and fed rats, respectively. When livers from fed rats were preswollen by hypoosmotic perfusion (225 mOsmol · l−1), both the insulin‐induced net K+ uptake and the inhibition of [3H]leucine release were diminished by 50–60%.
During inhibition of [3H]leucine release by insulin, further addition of glucagon (100 nM) led to a marked net K+ release from the liver (3.82 ± 0.24 μmol · g−1), which was accompanied by stimulation of [3H]leucine release by 16.4 ± 4.6% (n= 4).
Ba2+ (1 mM) infusion led to a net K+ uptake by the liver of 3.2 ± 0.2 μmol · g−1 (n= 4) and simultaneously inhibited [3H]leucine release by 12.4 ± 1.7% (n= 4).
There was a close relationship between the Ba2+ or insulin‐induced net K+ uptake and the degree of inhibition of [3H]leucine release, even when the K+ response to insulin was modulated by bumetanide, furosemide, glucagon, hypotonic or glycine‐induced cell swelling or the nutritional state.
The data suggest that the insulin‐induced net K+ uptake involves activation of both NaCl/KCl cotransport and Na+/H+ exchange. The findings further suggest that modification of insulin‐induced intracellular net K+ accumulation parallels the antiproteolytic potency of the hormone, consistent with a role of hormone‐induced ion movements across the plasma membrane in mediating the antiproteolytic effect of insulin in liver.
Localization on the mitochondrial F1 ATPase α subunit of an epitope masked in the membrane‐bound enzyme using a monoclonal antibody and synthetic peptides
Authors: MORADI‐AMÉLI Mahnaz , CLERC François Frédéric , CIEUR Franck , SEIBERRAS Ghislaine , GODINOT Catherine 
Published: March 2005

The epitope of the monoclonal antibody 20D6 was localized by N‐terminal sequencing of the smallest immunoreactive peptides obtained after CNBr and trypsin cleavage of the F1α subunit of the mitochondrial ATPase/ATP synthase. Immunochemical analysis of overlapping synthetic octapeptides, covering the immunoreactive peptide sequence, has defined the seven‐amino‐acid sequence recognized by 20D6 as 84EGDIVKR90. The binding of 20D6 was lost after substituting either I87 by K or S, or R90 by C or A as it occurs in the α subunit sequence of Escherichia coli or chloroplast ATPase, respectively. This explained the lack of immunoreactivity of 20D6 to these species and indicated the importance of charged as well as hydrophobic residues in the epitope. Immunochemical analysis of synthetic peptides by polyclonal anti‐F1 antisera showed that this region is highly immunodominant.In a competitive ELISA, the monoclonal antibody bound with similar affinity to F1 in the presence and absence of substrate as well as to cold dissociated F1, indicating that the epitope was located on the surface of the α subunit and not buried between F1 subunits. The lack of binding of 20D6 when F1 is bound to the membrane showed that the epitope exposed at the surface of purified soluble F1 became masked after binding to the membrane. This suggests that it is located at the interface between F1 and the membrane.
On the role of polysialoglycosphingolipids as tetanus toxin receptors
Authors: SCHIAVO Giampietro , DEMEL Rudy , MONTECUCCO Cesare 
Published: March 2005

Lipid monolayers of different compositions were used to study the interaction of tetanus toxin with membrane lipids and to evaluate the role of polysialoglycosphingolipids as membrane receptors.At neutral pH, the toxin binds to dioleoylglycerophosphocholine monolayers and inserts into the phospholipid layer. This effect is potentiated by acidic phospholipids without an apparent preference for a single class of phospholipids. Polysialoglycosphingolipids further increase the fixation and penetration of tetanus toxin in lipid monolayers, but no specific requirement for a particular ganglioside was identified. The ganglioside effect is abolished in the presence of other nervous tissue lipids: cerebrosides and glycosphingolipid sulfates are partially responsible for this effect.The penetration of tetanus toxin in the lipid monolayer is pH dependent. It increases with lowering pH, it is facilitated by acidic phospholipids and by glycosphingolipid sulfates and it is mediated both by hydrophobic and electrostatic interactions as deduced from an analysis of the effect of ionic strength. Fragment B of tetanus toxin (light chain plus the 50‐kDa N‐terminal part of the heavy chain) is involved in the low‐pH‐driven lipid interaction of the toxin.On the basis of the present findings, the possible role of polysialoglycosphingolipids in the neurospecific binding of tetanus toxin is discussed.
Degradation of heme by a soluble peptide of heme oxygenase obtained from rat liver microsomes by mild trypsinization
Authors: YOSHIDA Tadashi , ISHIKAWA Kazunobu , SATO Michihiko 
Published: March 2005

A tryptic peptide of heme oxygenase obtained after solubilization of rat liver microsomes by mild trypsin treatment was purified. The purified peptide gave only a single protein band with a molecular mass of 28 kDa on SDS/PAGE.The tryptic peptide, like the native heme oxygenase, readily bound with substrate heme forming a hemeprotein transiently. The absorption spectra of the ferric, ferrous, ferrous‐CO and ferrous‐O2 forms of the resulting complex resembled those of the corresponding forms of the complex of heme and the native enzyme. Ferric heme bound to the tryptic peptide was quantitatively decomposed to biliverdin on incubation with a mixture of ascorbic acid and desferrioxamine, indicating that the tryptic peptide still retained catalytic activity. These observations suggest that heme oxygenase has two domains, a hydrophilic and a hydrophobic domain, and that the two domains are folded almost independently of each other.An NADPH‐cytochrome‐P‐450 reductase system composed of NADPH and detergent‐solubilized NADPH‐cytochrome‐P‐450 reductase readily reduced the ferric heme bound to the tryptic peptide, but failed to transfer the second electron required for rapid heme degradation, suggesting that the hydrophobic domain of heme oxygenase is important for receiving the second electron from the reductase.