bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of dexamethasone and cAMP on tyrosine aminotransferase expression in cultured fetal rat hepatocytes
Authors: SHELLY Leslie L. , YEOH George C. T.
Published: March 2005

Fetal hepatocyte cultures were used to investigate tyrosine aminotransferase (TyrAT) expression during development. Previous studies showed that TyrAT is synthesized by hepatocytes isolated from 15‐day‐gestation fetuses maintained in culture for two or more days, then exposed to dexamethasone. TyrAT expression was essentially undetectable on the first day of culture of hepatocytes derived from 15‐day‐gestation, or less mature, fetuses. Dexamethasone and cAMP are potent inducers of TyrAT and they synergistically induce TyrAT to extremely high levels when added simultaneously to cultured fetal hepatocytes.

The effects of dibutyryl‐cAMP (Bt2cAMP) alone and in combination with dexamethasone on TyrAT expression are investigated. Hepatocytes isolated from 15‐day‐gestation fetuses exposed to both inducers possessed detectable levels of TyrAT activity and mRNA on day 1 of culture, and this increased by day 3. In contrast, hepatocytes exposed to either inducing agent alone were essentially negative on day 1, but positive on day 3. This was shown to be a consequence of transcription. When 13‐day‐gestation hepatocytes were maintained in culture under identical conditions detectable levels of TyrAT mRNA were evident on day 1, and this increased by day 3.

Immunocytochemical studies revealed that the appearance and subsequent increase in TyrAT production elicited by dexamethasone and Bt2cAMP were due to changes in the proportion of hepatocytes expressing the enzyme. Therefore, in the presence of both dexamethasone and Bt2cAMP, TyrAT expression can be detected in some cells at an earlier stage of liver development than reported previously.
Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII
Authors: ROSE Matthias , ALBIG Werner , ENTIAN Karl‐Dieter
Published: March 2005

Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102–246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C‐terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression.

To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose‐phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double‐null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose‐phosphorylating activity, which is threefold increased compared to wild‐type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.
Properties of the elongation factor 1α in the thermoacidophilic archaebacterium Sulfolobus solfataricus
Authors: MASULLO Mariorosario , RAIMO Gennaro , PARENTE Augusto , GAMBACORTA Agata , DE ROSA Mario , BOCCHINI Vincenzo
Published: March 2005

The elongation factor 1α (aEF‐1α) was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus by chromatographic procedures utilising DEAE‐Sepharose, hydroxyapatite and FPLC on Mono S. The purified protein binds [3H]GDP at a 1:1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP. These findings indicate that aEF‐1α is the counterpart of the eubacterial elongation factor Tu (EF‐Tu).

Purified aEF‐1α is a monomeric protein with a relative molecular mass of 49 000 as determined by SDS/PAGE and by gel filtration on Sephadex G‐100; its isoelectric point is 9.1. The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF‐Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF‐1α from Artemia salina or of archaebacterial EF‐1α from Methanococcus vannielii. The close similarities between the average hydrophobicity and the numbers of hydrogen‐bond‐forming or non‐helix‐forming residues suggest that common structural features exist among the factors compared.

aEF‐1α shows remarkable thermophilic properties, as demonstrated by the rate of [3H]GDP binding which increases with temperature, reaching a maximum at 95°C; it is also quite heat‐resistant, since after a 6‐h exposure at 60°C and 87°C the residual [3H]GDP‐binding ability was still 90% and 54% of the control, respectively.

The affinity of aEF‐1α for GDP and GTP was also evaluated. At 80°C Ka′ for GDP was about 30‐fold higher than Ka′ for GTP; at the same temperature Kd′ for GDP was 1.7 μM and Kd′ for GTP was 50 μM; these values were 300‐fold and 100‐fold higher, respectively, than those reported for E. coli EF‐Tu at 30°C; compared to the values at 0°C of EF‐Tu from E. coli and T. thermophilus or EF‐1α from A. salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors. At 80°C k′−1 GDP and k′+1 GDP of aEF‐1α were 7.8 min−1 and 4.6 min−1·μM−1, respectively; comparison with the values attributed to E. coli EF‐Tu at 30°C indicates that the dissociation rate constant increases and the association rate constant decreases, thus indicating that the higher Kd′ for GDP of aEF‐1α is due to a synchronous alteration of the rates of both the dissociation and the association processes between GDP and aEF‐1α.

Besides aEF‐1α, the other two elongation factors, aEF‐2 and aEF‐1β were also present in S. solfataricus, as suggested by the occurrence of reactions typical of each of them. This allows the hypothesis that in this extremophilic archaebacterium the protein synthesis process follows the same general pattern found in all other living organisms.
The location of the polyphosphate‐binding sites on cytochrome c measured by NMR paramagnetic difference spectroscopy
Authors: CONCAR David W. , WHITFORD David , WILLIAMS R. J. P.
Published: March 2005

Analyses of unimolecular electron self‐exchange reactions provide a comparatively simple and direct approach to understanding biological electron transfer. Such studies are currently limited by a lack of well characterised aggregating systems. In the presence of sodium hexametaphosphate, cytochrome c forms stable protein aggregates as a result of binding hexametaphosphate at a single site on its surface (preceding paper in this issue of the journal). Here we report the location of the principal polyphosphate binding site on the surface of cytochrome c for both hexametaphosphate and a second polyphosphate, tripolyphosphate determined using 1H‐NMR spectroscopy in conjunction with the relaxation probe potassium hexacyanochromium(III). Addition of either hexametaphosphate or tripolyphosphate to ferricytochrome c in the presence of the relaxation probe causes a decrease in intensity of several resonances in the paramagnetic difference spectrum, including Phe82 ortho/meta, Ile85 δ methyl and Ile9 γ methyl. Together these effects put the site of polyphosphate binding close to lysines 13, 86, and 87. Additionally the effect of sodium tripolyphosphate and sodium trimetaphosphate on cytochrome c aggregation is described. The potential role of this site in anion‐induced cytochrome c aggregation is discussed.
Two‐dimensional 1H‐NMR studies of phospholipase‐A2‐inhibitor complexes bound to a micellar lipid‐water interface
Authors: DEKKER Niek , PETERS Anton R. , SLOTBOOM Arend J. , BOELENS Rolf , KAPTEIN Robert , DIJKMAN Ruud , DE HAAS Gerard
Published: March 2005

One‐ and two‐dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2 with substrate analogs bound to a micellar lipid‐water interface of fully deuterated dodecylphosphocholine. The interactions between the inhibitor and the enzyme were localized by comparison of the two‐dimensional NOE spectra recorded for the enzyme‐inhibitor complex using both protonated and selectively deuterated inhibitors. These experiments led us to the following conclusions for the phospholipase‐A2‐micelle complex: (i) the 38‐kDa phospholipase A2 complex gives NMR spectra with relatively narrow lines, which is indicative of high mobility of the enzyme; (ii) the residues Ala1, Trp3, Phe63 and Tyr69 located in the interface recognition site, as well as Phe22, Tyr75, Phe106 and Tyr111 are involved in the micelle‐binding process; (iii) when present on the micelle, phospholipase A2 is stereospecific for the inhibitor binding; (iv) the inhibitor, (R)‐dodecyl‐2‐aminohexanol‐1‐phosphoglycol, binds stoichiometrically to phospholipase A2 with high affinity (Kd≤ 10 μM); (v) the inhibitor binds in the active site of the enzyme, which is evidenced by large chemical‐shift differences for Phe5, Ile9, Phe22, His48, Tyr52 and Phe106; (vi) the acyl chain of the inhibitor makes hydrophobic contacts (< 0.4 nm) near Phe5, Ile9, Phe22 and Phe106.

Comparison of our results on the enzyme‐inhibitor‐micelle ternary complex with the crystal structure of the enzyme‐inhibitor complex [Thunnissen, M. M. G. M., AB, E., Kalk, K. H., Drenth, J., Dijkstra, B. W., Kuipers, O. P., Dijkman, R., de Haas, G. H. & Verheij, H. M. (1990) Nature 347, 689–691] shows that the mode of inhibitor binding is similar.
Isoforms of human cytochrome‐c oxidase
Authors: VAN KUILENBURG André B. P. , DEKKER Henk L. , VAN DEN BOGERT Coby , NIEBOER Popko , VAN GELDER Bob F. , MUIJSERS Anton O.
Published: March 2005

The subunit pattern and the steady‐state kinetics of cytochrome‐c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome‐c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in staining intensity and electrophoretic mobility. No differences in subunit pattern were observed between the other nucleus‐encoded subunits of the various cytochrome‐c oxidase preparations.

Tissue homogenates, in which cytochrome‐c oxidase was solubilised with laurylmaltoside, were directly used in the assays to study the cytochrome‐c oxidase steady‐state kinetics. Cytochrome‐c oxidase concentrations were determined by immunopurification followed by separation and densitometric analysis of subunit IV. When studied in a medium of low ionic strength, the biphasic kinetics of the steady‐state reaction between human ferrocytochrome c and the four human cytochrome‐c oxidase preparations revealed large differences for the low‐affinity TNmax (maximal turnover number) value, ranging from 77 s−1 for kidney to 273 s−1 for liver cytochrome‐c oxidase at pH 7.4, I= 18 mM. It is proposed that the low‐affinity kinetic phase reflects an internal electron‐transfer step. For the steady‐state reaction of human heart cytochrome‐c oxidase with human cytochrome c, Km and TNmax values of 9 μM and 114 s−1 were found, respectively, at high ionic strength (I= 200 mM, pH 7.4). Only minor differences were observed in the steady‐state activity of the various human cytochrome‐c oxidases.

The interaction between human cytochrome‐c oxidase and human cytochrome‐c proved to be highly specific. At high ionic strength, a large decrease in steady‐state activity was observed when reduced horse, rat or bovine cytochrome c was used as substrate. Both the steady‐state TNmax and Km parameters were strongly affected by the type of cytochrome c used. Our findings emphasize the importance of using human cytochrome c in kinetic assays performed with tissues from patients with a suspected cytochrome‐c oxidase deficiency.
The specificity of chymotrypsin
Authors: SCHELLENBERGER Volker , BRAUNE Katrin , HOFMANN Hans‐Jörg , JAKUBKE Hans‐Dieter
Published: March 2005

From the literature we collected all available quantitative data on the chymotrypsin‐catalyzed hydrolysis of series of amino acid and peptide substrates. Utilizing this data base, we performed calculations on their quantitative structure/activity relationship (QSAR). The substrates were considered to be composed of fragments; log(kcat/Km) values for the substrates resulted from additive contributions of their fragments. Despite the fact that the kinetic constants in the data base were determined by different authors under various reaction conditions, the data are well described by the simple additivity model. Obviously, the intrinsic specificity of chymotrypsin dominates the influence of varying reaction conditions.
Comparative study of the structure/function relationship of wild‐type and structurally modified maltopentaose‐producing amylase
Authors: CANDUSSIO Anton , SCHMID Gerhard , BÖCK August
Published: March 2005

Amylase A‐180, which is secreted by a new alkaliphilic organism, isolate 163‐26, consists of a single type of polypeptide chain of 186.5 kDa and hydrolyses starch by exo‐attack releasing malto‐pentaose as preferential product. The structure/function relationship of this unusual starch‐degrading enzyme was analysed by introducing 3′ deletions into the structural gene. It was found that removal of up to a 110‐kDa portion from the C‐terminus leaving 563 N‐terminal amino acids still led to the formation of a fully active enzyme. The part of the structural gene coding for these 563 N‐terminal amino acids was fused with the signal peptide‐encoding segment of the cyclodextrin glucanotransferase gene from Klebsiella oxytoca and was cloned into an expression vector. The resulting truncated A‐180 derivative, A‐180/21, was efficiently transported through the cytoplasmic membrane and released into the medium by an Escherichia coli strain which ‘leaks’ periplasmatic components. A‐180/21 was purified and its catalytic properties, i.e. specific activity and product specificity, proved to be identical to those of the wild‐type enzyme; however, in contrast to the wild‐type enzyme, it was unable to bind to raw starch and it displayed an altered temperature and pH dependence of activity.
Selective 13C‐labelling of cyclosporin A
Authors: SENN Hans , WEBER Christoph , KOBEL Hans , TRABER René
Published: March 2005

Cyclosporin A is biosynthetically labelled with 13C by growing an overproducing strain of Tolypocladium inflatum on minimal media containing either [1‐13C]‐, [2‐13C]‐, [3‐13C]‐ or [6‐13C]glucose as the only carbon source. NMR analysis of the 13C‐labelled peptide showed a labelling pattern in which 13C occurs at specific sites. These can be predicted by consideration of the relevant biosynthetic pathways. Quantitation of the site‐specific enrichments revealed that the 13C‐label incorporation is efficient and selective. Metabolic fluxes through alternative pathways can also be estimated from these results. Isotopically labelled peptides will be a very useful tool for the study of molecular interactions with their receptors.
Structure/function relationships in mitochondrial cytochrome b revealed by the kinetic and circular dichroic properties of two yeast inhibitor‐resistant mutants
Authors: TRON Thierry , CRIMI Massimo , COLSON Anne‐Marie , DEGLI ESPOSTI Mauro
Published: March 2005

The kinetic and circular dichroic properties of two yeast mutants that are resistant towards specific inhibitors of the mitochondrial cytochrome bc1 complex have been characterized. Both of these mutants have an altered cytochrome b gene in which aromatic residues are exchanged with non‐polar residues in a highly conserved region of the protein.

The mutant resistant to myxothiazol and mucidin that contains the substitution Phe129 → Leu is not greatly affected either in its ubiquinol: cytochrome c reductase or in the spectral properties of cytochrome b. On the other hand, the mutant resistant to stigmatellin that contains the substitution Ile147 → Phe shows a large decrease of the catalytic efficiency for ubiquinol and of the maximal turnover of its reductase activity. This stigmatellin mutant also shows an altered circular‐dichroic spectrum of the low‐potential haem of cytochrome b. This study provides biochemical and biophysical information for identifying a region in mitochondrial cytochrome b that may fulfill a crucial role in the binding of ubiquinol to the bc1 complex. The results are discussed also in terms of the structural model of cytochrome b having a core of four transmembrane helices.
Characterization of a specific ecdysteroid receptor‐DNA complex reveals common properties for invertebrate and vertebrate hormone‐receptor/DNA interactions
Authors: OżYHAR Andrzej , STRANGMANN‐DIEKMANN Magdalene , KILTZ Hans‐Hermann , PONGS Olaf
Published: March 2005

A gel‐filtration assay has been developed with which the specific interaction between Drosophila melanogaster ecdysteroid receptor and the 20‐hydroxy‐ecdysone responsive element of the hsp27 gene promoter region was characterized in terms of complex formation, saturation of DNA binding and the apparent molecular mass of the complex. The hsp27 DNA‐binding sequence for ecdysteroid receptor in vitro was delimited by footprinting and mutational analysis. The combined results show that ecdysteroid receptor binds as a dimer to an imperfect palindromic sequence (GGTTCAATGCACT) closely resembling the structures of the different vertebrate steroidhormone‐responsive elements reported so far.
Tendamistat (12–26) fragment
Authors: BLANCO Francisco J. , JIMÉNEZ M. Angeles , RICO Manuel , SANTORO Jorge , HERRANZ José , NIETO José L.
Published: March 2005

In order to determine whether regions of a protein that are turns in the native structure are able to maintain such a structure when isolated, we have studied the conformational properties of various peptide fragments corresponding to the 12–26‐peptide region of the α‐amylase inhibitor tendamistat, by NMR. Amide solvent accessibility, NOE spectroscopy (NOESY) and rotating‐frame NOE spectroscopy (ROESY) data strongly support the conclusion that the 12–26 and 15–23 peptides adopt in aqueous solution, a set of turn‐like structures located around the central region of their corresponding polypeptidic chains, the same region where a β turn exists in the native protein. Such a set of structures are destabilized when one residue located within the native β turn of the 15–23 peptide is modified Trp 18→Ser. Our results indicate that the tendency to bend in a predetermined region of a protein chain seems to exist from the very beginning of the folding process and therefore it could drive the folding instead of being a consequence of the tertiary assembly of the protein.
Purification and properties of a novel type of exo‐1,4‐β‐glucanase (Avicelase II) from the cellulolytic thermophile Clostridium stercorarium
Authors: BRONNENMEIER Karin , RÜCKNAGEL Karl P. , STAUDENBAUER Walter L.
Published: March 2005

Avicelase II was purified to homogeneity from culture supernatants of Clostridium stercorarium. A complete separation from the major cellulolytic enzyme activity (avicelase I) was achieved by FPLC gel filtration on Superose 12 due to selective retardation of avicelase II. The enzyme has an apparent molecular mass of 87 kDa and a pI of 3.9. Determination of the N‐terminal amino acid indicates that avicelase II is not a proteolytically processed product of avicelase I. Maximal activity of avicelase II is observed between pH 5 and 6. In the presence of Ca2+, the enzyme is highly thermostable, exhibiting a temperature optimum around 75°C. Hydrolysis of avicel occurs at a linear rate for three days at 70°C. Avicelase II is active towards unsubstituted celluloses, cellotetraose and larger cellodextrins. It lacks activity towards carboxymethylcellulose and barley β‐glucan. Unlike other bacterial exoglucanases, avicelase II does not hydrolyze aryl‐β‐d‐cellobiosides. Avicel is degraded to cellobiose and cellotriose at a molar ratio of approximately 4:1. With acid‐swollen avicel as substrate, cellotetraose is also formed as an intermediary product, which is further cleaved to cellobiose. The degradation patterns of reduced cellodextrins differ from that expected for a cellobiohydrolase attacking the non‐reducing ends of chains; cellopentaitol is degraded to cellobiitol and cellotriose, while cellohexaitol is initially cleaved into cellobiitol and cellotetraose. These findings, taken together, indicate that avicelase II represents a novel type of exoglucanase (cellodextrinohydrolase), which, depending on the accessibility of the substrate, releases cellotetraose, cellotriose, or cellobiose from the non‐reducing end of the cellulose chains.
O‐specific polysaccharide of Hafnia alvei lipopolysaccharide isolated from strain 1211
Authors: KATZENELLENBOGEN Ewa , ROMANOWSKA Elzbieta , DABROWSKI Ursula , DABROWSKI Janusz
Published: March 2005

The Hafnia alvei strain 1211 O‐specific polysaccharide is composed of 3‐amino‐N‐(d‐3′‐hydroxybutyryl)‐3,6‐dideoxy‐d‐galactose, N‐acetyl‐d‐galactosamine, N‐acetyl‐d‐glucosamine and d‐glucose (1:1:2:2). On the basis of sugar and methylation analyses, Smith degradation, and one‐ and two‐dimensional 1H‐ and 13C‐NMR spectroscopy, the polysaccharide was shown to be an O‐acetylated polymer of the repeating hexasaccharide unit, → 2d(4‐OAc)Fucp3NAcylβ1 → 6dGlcpNAcα1 → (dGlcpβ1 → 3)4dGalpNAcα1 → 3dGlcpNAcβ1 → 2dGlcpβ1 →, where dFucp3NAcyl = 3‐amino‐N‐(d‐3′‐hydroxybutyryl)‐3,6‐dideoxy‐d‐galactopyranose. The O‐specific polysaccharide showed some microheterogeneity due to incomplete substitution by terminal glucose.
Glycosphingolipids in insects
Authors: HELLING Friedhelm , DENNIS Roger D. , WESKE Bianka , NORES Gustavo , PETER‐KATALINIC Jasna , DABROWSKI Ursula , EGGE Heinz , WIEGANDT Herbert
Published: March 2005

A group of Calliphora vicina pupal glycolipids could be segregated from the neutral glycosphingolipids, according to their two‐dimensional TLC migration properties and positive reactions toward ninhydrin and fluorescamine spray reagents. These classified zwitterionic glycolipids were isolated by silica‐gel column chromatography and characterized by the presence of a N‐acetyl‐glucosamine‐bound phosphoethanolamine residue. The structural elucidation of the oligosaccharide moieties was performed by the determination of constituent carbohydrates as alditol acetates, linkage analysis by permethylation, exoglycosidase cleavage, fast‐atombombardment mass spectrometry and NMR spectroscopy. The dominant fatty acid and sphingoid base species of the ceramide moieties were C20:0 (arachidic acid) and C14:1 (tetradecasphing‐4‐enine), respectively. The chemical structures of the zwitterionic, biogenetic glycosphingolipid series were determined as:

(PEtn‐6′)GlcNAc(β1–3)Man(β1–4)GlcβCer;

GalNAc(β1–4) (PEtn‐6′)GlcNAc(β1–3)Man(β1–4)Glcβ Cer;

GalNAc(α1–4)GalNAc(β1–4) (PEtn‐6′)GlcNAc (β1–3)Man(β1–4)GlcβCer;

Gal(β1–3)GalNAc(β1–4) (PEtn‐6′)GlcNAc(β1–3)Man(β1–4)Glcβ Cer;

Gal(β1–3)GalNAc(α1–4)GalNAc(β1–4) (PEtn‐6′)GlcNAc(β1–3)Man(β1–4)Glcβ Cer;

GlcNAc(β1–3)Gal(β1–3)GalNAc(α1–4)GalNAc(β1–4) (PEtn‐6′)GlcNAc(β1–3)Man(β1–4)Glcβ Cer.
Conformational dynamics and solvent viscosity effects in carboxypeptidase‐A‐catalyzed benzoylglycylphenyllactate hydrolysis
Authors: GOGUADZE Nina G. , HAMMERSTAD‐PEDERSEN Jan M. , KHOSHTARIYA Dimitrij E. , ULSTRUP Jens
Published: March 2005

We have used a new approach to the dynamics of hydrolytic metalloenzyme catalysis based on investigations of both external solvent viscosity effects and kinetic 2H isotope effects. The former reflects solvent and protein dynamics, and the nuclear reorganization distribution among damped protein motion and intramolecular friction‐free nuclear motion. The isotope effect represents proton tunnelling and reorganization in the hydrogen bond network around the active site.

We illustrate the approach by new spectrophotometric and pH‐titration data for carboxypeptidase‐A‐catalyzed benzoylglycyl‐l‐phenyllactate hydrolysis. This substrate exhibits both a significant inverse fractional power law viscosity dependence over wide ranges controlled by glycerol and sucrose, and a kinetic 2H isotope effect of 1.65. The analogous benzoylglycylphenylalanine hydrolysis has a smaller isotope effect (1.3) and no viscosity dependence. Viscosity variation has no effect on the CD spectra in the 180–240‐nm range. In terms of stochastic chemical rate theory, the data correspond to an enzyme‐peptide substrate complex with a ‘tight’ structure protected from the solvent. In comparison, the enzyme‐ester substrate complex is ‘softer’, strongly coupled to the solvent, and the rate‐determining step is accompanied by proton transfer or by substantial reorganization in the hydrogen bonds near the active site.
Inactivation of chloroplast H+‐ATPase by modification of Lysβ359, Lysα176 and Lysα266
Authors: HORBACH Martina , MEYER Helmut E. , BICKEL‐SANDKÖTTER Susannę
Published: March 2005

Treatment of isolated, latent chloroplast ATPase with pyridoxal‐5‐phosphate (pyridoxal‐P) in presence of Mg2+ causes inhibition of dithiothreitol‐activated plus heat‐activated ATP hydrolysis. The amount of [3H]pyridoxal‐P bound to chloroplast coupling factor 1 (CF1) was estimated to run up to 6 ± 1 pyridoxal‐P/ enzyme, almost equally distributed between the α‐ and β‐subunits. Inactivation, however, is complete after binding of 1.5–2 pyridoxal‐P/CF1, suggesting that two covalently modified lysines prevent the activation of the enzyme. ADP as well as ATP in presence of Mg2+ protects the enzyme against inactivation and concomittantly prevents incorporation of a part of the 3H‐labeled pyridoxal‐P into β‐ and α‐subunits. Phosphate prevents labeling of the α‐subunit, but has only a minor effect on protection against inactivation. The data indicate a binding site at the interface between the α‐ and β‐subunits. Cleavage of the pyridoxal‐P‐labeled subunits with cyanogen bromide followed by sequence analysis of the labeled peptides led to the detection of Lysβ359, Lysα176 and Lysα266, which are closely related to proposed nucleotide‐binding regions of the α‐ and β‐subunits.
Identification and characterization of a Saccharomyces cerevisiae gene (PAR1) conferring resistance to iron chelators
Authors: SCHNELL Norbert , ENTIAN Karl‐Dieter
Published: March 2005

o‐Phenanthroline (1,10‐phenanthroline) is a chemical known to chelate iron and other transition metal ions. This compound was added to solid yeast media to reduce the concentration of biologically available iron. Other essential divalent cations, like Zn2+ or Cu2+, which could also be bound, were supplemented. Growth of wild‐type yeast strains was totally inhibited at specific concentrations of the chelator. However, several cells containing plasmids of a multicopy vector genomic library of S. cerevisiae could be selected by growth on these media. All of the resistant clones carried a single additional gene, PAR1 on their multicopy plasmids.

Plasmid‐directed overexpression of PAR1 increased the resistance of transformants to o‐phenanthroline and additionally conferred resistance to 1‐nitroso‐2‐naphthol, an iron(III)‐binding molecule with different coordinating ligands. By supplementing the o‐phenanthroline‐containing media with several different metal ions, it could be proved that the selection plates really caused a specific iron limitation. These observations clearly demonstrated that the overexpressed PAR1 gene enables the cell to compete with iron‐chelating organic molecules.

PAR1 null mutants, constructed by insertion of the LEU2 gene into the open reading frame, showed a remarkable phenotype: they did not grow on slightly alkaline buffered media (pH > 7) and became hypersensitive to oxidative stress by hydrogen peroxide. Of several heavy metal ions, such as Fe3+, Fe2+, Co2+, Ni2+, Cu2+ and Zn2+, tested for supplementation of the alkaline growth deficiency, only iron, either added in the ferrous or ferric form, was able to restore cellular growth.

It can be concluded from the DNA sequence that PAR1 encodes a highly acidic protein of 650 residues with mostly hydrophilic character. Some interesting repetitive amino acid motifs, such as (Asp‐Asn)4 or Cys‐Ser‐Glu, may act as metal‐binding sites. The possible role of PAR1 is discussed.
Characteristics of short‐chain alcohol dehydrogenases and related enzymes
Authors: PERSSON Bengt , KROOK Maria , JÖRNVALL Hans
Published: March 2005

Different short‐chain dehydrogenases are distantly related, constituting a protein family now known from at least 20 separate enzymes characterized, but with extensive differences, especially in the C‐terminal third of their sequences. Many of the first known members were prokaryotic, but recent additions include mammalian enzymes from placenta, liver and other tissues, including 15‐hydroxyprostaglandin, 17β‐hydroxysteroid and 11β‐hydroxy‐steroid dehydrogenases. In addition, species variants, isozyme‐like multiplicities and mutants have been reported for several of the structures. Alignments of the different enzymes reveal large homologous parts, with clustered similarities indicating regions of special functional/structural importance. Several of these derive from relationships within a common type of coenzyme‐binding domain, but central‐chain patterns of similarity go beyond this domain. Total residue identities between enzyme pairs are typically around 25%, but single forms deviate more or less (14–58%). Only six of the 250‐odd residues are strictly conserved and seven more are conserved in all but single cases. Over one third of the conserved residues are glycine, showing the importance of conformational and spatial restrictions. Secondary structure predictions, residue distributions and hydrophilicity profiles outline a common, N‐terminal coenzyme‐binding domain similar to that of other dehydrogenases, and a C‐terminal domain with unique segments and presumably individual functions in each case. Strictly conserved residues of possible functional interest are limited, essentially only three polar residues, Asp64, Tyr152 and Lys156 (in the numbering of Drosophila alcohol dehydrogenase), but no histidine or cysteine residue like in the completely different, classical medium‐chain alcohol dehydrogenase family. Asp64 is in the suggested coenzyme‐binding domain, whereas Tyr152 and Lys156 are close to the center of the protein chain, at a putative inter‐domain, active‐site segment. Consequently, the overall comparisons suggest the possibility of related mechanisms and domain properties for different members of the short‐chain family.
Competitive inhibition of liver glucokinase by its regulatory protein
Authors: VANDERCAMMEN Annick , VAN SCHAFTINGEN Emile
Published: March 2005

The regulatory protein of rat liver glucokinase (hexokinase IV or D) behaved as a fully competitive inhibitor of this enzyme when glucose was the variable substrate, i.e. it increased the half‐saturating concentration of glucose as a linear function of its concentration without affecting V (velocity at infinite concentration of substrate). The inhibition by the regulatory protein and that by palmitoyl‐CoA were synergistic with that by N‐acetyl‐glucosamine, indicating that the two former inhibitors bind to a site distinct from the catalytic site. In contrast, the effects of the regulatory protein and palmitoyl‐CoA were competitive with each other, indicating that these two inhibitors bind to the same site.

The regulatory protein exerted a non‐competitive inhibition with respect to Mg‐ATP at concentrations of this nucleotide <0.5 mM. At higher concentrations, the latter antagonized the inhibition by the regulatory protein partly by decreasing the apparent affinity for fructose 6‐phosphate.

The following anions inhibited glucokinase non‐competitively with respect to glucose: Pi, sulfate, I−, Br−, NO−3, Cl−, F− and acetate. Pi and sulfate, at concentrations in the millimolar range, decreased the inhibition by the regulatory protein by competing with fructose 6‐phosphate. Monovalent anions also antagonized the inhibition by the regulatory protein with the following order of potency: I− >Br− > NO3− > Cl − > F− acetate and their effect was non‐competitive with respect to fructose 6‐phosphate.

Glucokinase from Buffo marinus and pig liver were, like the rat liver enzyme, inhibited by the regulatory protein, as well as by palmitoyl‐CoA at micromolar concentrations. In contrast, neither compound inhibited hexokinases from rat brain, beef heart or yeast, or the low‐Km specific glucokinase from Bacillus stearothermophilus.
Effectors of the regulatory protein acting on liver glucokinase: a kinetic investigation
Authors: DETHEUX Michel , VANDERCAMMEN Annick , VAN SCHAFTINGEN Emile
Published: March 2005

In the absence of fructose 6‐phosphate, the regulatory protein of rat liver glucokinase (hexokinase IV or D) inhibited this enzyme, though with a much (15‐fold) lower potency than in the presence of a saturating concentration of fructose 6‐phosphate. Evidence is provided that this inhibition is not due to contaminating fructose 6‐phosphate.

In the presence of regulatory protein, sorbitol 6‐phosphate, a potent analog of fructose 6‐phosphate, exerted a hyperbolic, partial inhibition on glucokinase, the degree of which increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between the rates in the absence and in the presence of sorbitol 6‐phosphate versus 1/[sorbitol 6‐phosphate] at various concentrations of regulatory protein were linear, and demonstrated that the apparent affinity for sorbitol 6‐phosphate increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between 1/v in the presence and in the absence of sorbitol 6‐phosphate versus 1/[sorbitol 6‐phosphate] were also linear and crossed the axis at a value independent of the concentration of regulatory protein. Fructose 1‐phosphate released the inhibition exerted by the regulatory protein in a hyperbolic fashion. The concentration of this effector required for a half‐maximal effect increased linearly with the concentrations of sorbitol 6‐phosphate and of regulatory protein. These results are consistent with a model in which the regulatory protein exists under two conformations, one form which binds inhibitors and glucokinase, and the other which binds activators, although not glucokinase.

Sorbitol 6‐phosphate, 2‐deoxysorbitol 6‐phosphate and mannitol 1‐phosphate, all analogs of the open‐chain configuration of fructose 6‐phosphate, inhibited glucokinase in the presence of regulatory protein at lower concentrations than fructose 6‐phosphate, whereas fixed analogs of the furanose form of fructose 6‐phosphate were inactive or behaved as activators. This indicated that fructose 6‐phosphate in its open‐chain configuration is recognized by the regulatory protein. A series of compounds exerted an activating effect. These included, in order of decreasing potency: fructose 1‐phosphate, psicose 1‐phosphate, ribitol 5‐phosphate, analogs of fructose 1‐phosphate and of ribitol 5‐phosphate and, at much higher concentrations, inorganic phosphate.
The discovery of glycogenin and the priming mechanism for glycogen biogenesis
Authors: SMYTHE Carl , COHEN Philip
Published: March 2005

The biogenesis of glycogen in skeletal muscle requires a priming mechanism that has recently been elucidated. The first step is catalysed by a protein tyrosine glucosyltransferase and involves the formation of a novel glycosidic linkage, namely the covalent attachment of glucose to a single tyrosine residue (Tyr194) on a priming protein, termed glycogenin. The next stage is the extension of the glucan chain from Tyr194 and involves the sequential addition of up to seven further glucosyl residues. This reaction is brought about autocatalytically by glycogenin itself, which is a Mn2+/Mg2+‐dependent UDP‐Glc‐requiring glucosyltransferase. The glucan primer is elongated by glycogen synthase, but only when glycogenin and glycogen synthase are complexed together. Glycogen synthase dissociates from glycogenin during the synthesis of a glycogen molecule, enabling glycogen molecules to reach their maximum theoretical size. Each mature glycogen β particle in muscle contains one molecule of glycogenin attached covalently, and an average one glycogen synthase catalytic subunit bound non‐covalently. As evidence accumulates that a priming protein may be a fundamental property of polysaccharide synthesis in general, the molecular details of mammalian glycogen biogenesis may serve as a useful model for other systems.
Primary structure of nuclease P1 from Penicillium citrinum
Authors: MAEKAWA Kazuhiko , TSUNASAWA Susumu , DIBÓ Gábor , SAKIYAMA Fumio
Published: March 2005

The primary structure of nuclease P1, which cleaves both RNA and single‐stranded DNA, from Penicillium citrinum was elucidated. The complete amino acid sequence consisting of 270 residues was determined by analysis of peptides obtained by digestion with Achromobacter Protease I of the reduced and S‐aminoethylated protein and by digestion with Staphylococcus aureus V8 protease of the reduced and S‐carboxymethylated protein. Four half‐cystine residues were assigned to Cys72–Cys217 and Cys80–Cys85. N‐Glycosylated asparagine residues were identified at positions 92, 138, 184 and 197. Fast‐atom‐bombardment and laser‐ionization MS were successfully used to confirm the determined amino acid sequences of peptides and to estimate the molecular mass of this glycoprotein having heterogenous sugar moieties, respectively. Comparison of the amino acid sequence of nuclease P1 with other nucleases revealed that the protein has a high degree of sequence identity (50%) with nuclease S1 from Aspergillus oryzae. The His‐Phe‐Xaa‐Asp‐Ala sequence (positions 60–64) is similar to the sequence (His‐Phe‐Asp‐Ala) involving the active‐site His119 of bovine pancreatic RNase A, and the Pro‐Leu‐His sequence (positions 124–126) is identical with the sequence involving the active‐site His134 of porcine pancreatic D Nase I.
Chemical structure of the carbohydrate backbone of Vibrio parahaemolyticus serotype 012 lipopolysaccharide
Authors: KONDO Seiichi , ZÄHRINGER Ulrich , SEYDEL Ulrich , SINNWELL Volker , HISATSUNE Kazuhito , RIETSCHEL Ernst Th.
Published: March 2005

The chemical structure of the saccharide portion of Vibrio parahaemolyticus serotype 012 lipopolysaccharide was studied. Using chemical degradation and modification, as well as methylation analysis in combination with GLC‐MS, laser‐desorption mass spectrometry and 1H‐NMR and 13C‐NMR spectroscopy, the carbohydrate backbone of the lipopolysaccharide was characterized as a branched decasaccharide with the following structure:

image

In the native lipopolysaccharide two additional phosphate groups are present and 3‐deoxy‐d‐threo‐hexulosonic acid and d‐galacturonic acid are bound via acid‐labile linkages.
A fluorescence spectroscopic study of glutaminyl‐tRNA synthetase from Escherichia coli and its implications for the enzyme mechanism
Authors: BHATTACHARYYA Tista , BHATTACHARYYA Anusree , ROY Siddhartha
Published: March 2005

Interaction between Escherichia coli glutaminyl‐tRNA synthetase (GlnRS) and its substrates have been studied by fluorescence quenching. In the absence of other substrates, glutamine, tRNAGln and ATP bind with dissociation constants of 460, 0.22 and 180 μM, respectively. The presence of other substrates has either no effect or, at best a weak effect, on binding of ligands. Attempts to isolate enzyme‐bound aminoacyl adenylate did not succeed. Binding of the phosphodiester, 5′‐(methyl)adenosine monophosphate (MeAMP), to GlnRS was studied by fluorescence quenching and radioactive‐ligand binding. tRNA also only has a weak effect on phosphodiester binding.

Selectively pyrene‐labeled GlnRS was used to obtain shape and size information for free GlnRS. A comparison with the GlnRS shape in the GlnRS/tRNAGln crystal structure indicates that no major change in shape and size occurs upon tRNAGln binding to GlnRS. 5,5′‐Bis(8‐anilino‐1‐naphthalene sulfonate) (bis‐ANS), a non‐covalent fluorescent probe, was also used to probe for conformational changes in GlnRS. This probe also indicated that no major conformational change occurs upon tRNAGln binding.

We conclude that lack of tRNA‐independent pyrophosphate‐exchange activity in this enzyme is not a result of either lack of glutamine or ATP binding in the absence of tRNA, or formation of aminoacyl adenylate and slow release of pyrophosphate. A conformational change is implied upon tRNA binding, which promotes pyrophosphate exchange. Fluorescence studies indicate that this conformational change must be limited and local in nature.
Reversible high hydrostatic pressure inactivation of phosphofructokinase from Escherichia coli
Authors: DEVILLE‐BONNE Dominique , ELSE Anthony J.
Published: March 2005

Tetrameric Escherichia coli phosphofructokinase dissociates reversibly on incubation under hydrostatic pressures of 80 MPa and above, yielding inactive dimers and monomers. The transition is dependent upon enzyme concentration and presence of ligands. The substrate, d‐fructose 6‐phosphate, which bridges the intersubunit interface at the active site, produces a massive stabilization to pressure, whereas ATP, which binds to only one subunit, induces only a mild stabilization. Both the positive allosteric regulator, GDP, and the negative allosteric regulator, phosphoenolpyruvate, whose binding sites lie at the other subunit interface, produce an intermediate effect. Of these ligands, only ATP increases the rate of reactivation after depressurization.
Isolation and characterisation of porcine sorbin
Authors: VAGNE‐DESCROIX Monique , PANSU Danielle , JÖRNVALL Hans , CARLQUIST Mats , GUIGNARD Henri , JOURDAN Gerard , DESVIGNE Annie , COLLINET Martine , CAILLET Catherine , MUTT Viktor
Published: March 2005

Sorbin has been isolated from extracts of porcine upper intestine, and the biological activity in absorbing water and electrolytes utilized to monitor the purification procedure. Pure sorbin was obtained in a yield of about 1 mg/Mg boiled intestine. The protein chain has 153 amino acid residues and the primary structure was determined by analyses of CNBr‐cleaved fragments and four enzymatic digests. The protein has a free N‐terminal Met and an amidated C‐terminal Ala. No structural similarity was observed with other known proteins in data bases, but several segments have special properties and the C‐terminal half is rich in Pro and Arg.
Collagen‐binding domain of human plasma fibronectin contains a latent type‐IV collagenase
Authors: LAMBERT VIDMAR Smilja , LOTTSPEICH Friedrich , EMOD Istvan , IMHOFF Jean‐Marie , KEIL‐DLOUHA Vera
Published: March 2005

Cleavage of the 45‐kDa gelatin‐binding fragment of human plasma fibronectin with fibronectinase resulted in the activation of two forms of metalloproteinase with different substrate specificities. The 40‐kDa FN‐type‐IV collagenase A degrades heat‐denatured type‐I collagen, laminin and also native collagen type IV. The 27‐kDa FN‐type‐IV collagenase B degrades native collagen type IV, but it does not cleave laminin and only poorly degrades gelatin. Both enzymes begin with the same N‐terminal sequence VYQPQPH‐ (residues 262–268 of fibronectin) but, contrary to the FN‐type‐IV collagenase A, the FN‐type‐IV collagenase B has lost the C‐terminal region of type I repeats, where the major gelatin‐binding determinants of fibronectin are located. The FN‐type‐IV collagenases A and B are sequentially similar to the middle domain (domain II) of collagenase type IV, secreted by H‐ras‐transformed human bronchial epithelial cells.

Substrate and inhibition specificity of FN‐type‐IV collagenase A and B are different from those of FN‐gelatinase and FN‐laminase, isolated previously from the central and C‐terminal fibronectin domains, respectively. The substrate specificity of both enzymes, characterized in this study, is also different from that of already known matrix‐degrading metalloproteinases.
Low membrane transport activity for cystine in resting and mitogenically stimulated human lymphocyte preparations and human T cell clones
Authors: GMÜNDER Helmut , ECK Hans‐Peter , DRÖGE Wulf
Published: March 2005

In order to determine whether the cysteine requirement of human T lineage cells is met primarily by extracellular cysteine or by cystine, amino‐acid‐transport activities were measured in resting and mitogenically stimulated human peripheral blood lymphocytes (PBL) and several human T cell clones and T cell tumors. The transport activity of the small neutral amino acids cysteine and alanine (ASC system) and the transport of the cationic amino acid arginine (y+ system) were found to be markedly increased after stimulation of PBL by the T cell mitogen phytohemagglutinin from Phaseolus vulgaris. The anionic transport activity for cystine and glutamate (xc− system), in contrast, was extremely weak in both resting and activated human PBL and also in all human T cell lines under test. The weak system xc− activity of human T lineage cells was further confirmed by an independent line of experiments showing that an increase of the extracellular concentration of glutamate, i.e. a competitive inhibitor of cystine transport, causes a decrease in the intracellular cystine levels in cells of the promonocytic line U937, but not in T lineage cells (Molt‐4). A third set of experiments showed that the rate of DNA synthesis in mitogenically stimulated human PBL is strongly influenced by variations of the extracellular cysteine level, even in cultures with relatively high and approximately physiological concentrations of cysteine. Cysteine cannot be replaced in this case by the addition of corresponding amounts of cystine or methionine. This demonstrates an important functional consequence of the weak cystine transport activity of human lymphocytes. The results may be relevant for the pathogenetic mechanism of the acquired immunodeficiency syndrome, since the mean plasma cysteine concentration of human‐immunodeficiency‐virus‐1‐seropositive persons was found to be strongly decreased in comparison with that of healthy blood donors, and since the cysteine level even of healthy persons is extremely low in comparison with all other protein‐forming amino acids.