bluuebunny/crossref_metadata_2025 huggingface dataset:
The folding and solution conformation of penicillin G acylase
Authors: LINDSAY Christopher D. , PAIN Roger H.
Published: March 2005

The solution conformation properties of penicillin G acylase (EC 3.5.1.11) have been characterised by nearand far‐ultraviolet circular dichroism, steady‐state and time‐resolved fluorescence spectroscopy and differential sedimentation velocity. The enzyme (86 kDa) was found to be spherical and stable, unfolding over a narrow range of urea concentrations in an apparently cooperative fashion with a mid‐point of 4.5 M urea. Separation of its constituent α and β peptides (23.8 kDa and 62.2 kDa, respectively) was accompanied by loss of enzyme activity and unfolding, the kinetics of unfolding being highly dependent upon urea concentration. Urea gradient gel electrophoresis showed that the separated β peptide aggregates over a wide range of urea concentrations but that the α peptide refolds reversibly to a compact state. Physical studies showed that the refolded α peptide has a compact but asymmetric structure with more α helix than the native enzyme, but is more sensitive to denaturant. The latter is suggested to be due to a hydrophobic patch detected by 8‐anilino‐1‐naphthalene sulfonic acid binding and which is normally covered by the β peptide in the native enzyme. The results of these investigations indicate that the α peptide constitutes a folding domain and suggest that it plays a key role in folding of the precursor for penicillin acylase.
A critical crosslink region in human‐bone‐derived collagen type I
Authors: BÄTGE Boris , NOTBOHM Holger , DIEBOLD Joachim , LEHMANN Hartwig , BODO Michael , DEUTZMANN Rainer , MÜLLER Peter K.
Published: March 2005

Collagen was extracted from human adult bone by limited pepsin digestion and collagen types were purified by consecutive salt precipitation first under neutral and then under acid conditions. In SDS/PAGE, all collagen type I preparations showed a protein band [α1s(I)] migrating between α(I) and α2(I) as well as a band [α2s(I)] migrating in front of α2(I). The collagenous nature of the pepsin‐stable α1s(I) protein was clearly demonstrated by digestion with human‐leucocyte‐derived collagenase, immunoblotting with antibodies against collagen type I and amino acid analysis. Partial amino acid sequencing of α1(I) and α1s(I) identified α1s(I) as a shortened α1(I) chain due to a specific cleavage site between residues Leu95 and Asp96 which is in close vicinity to the hydroxylysine‐derived crosslink at position 87. In circular dichroism, the proportion of thermally labile collagen molecules was proportional to the amount of shortened α1(I) and α2(I) chains, respectively. The melting temperature was found to be 36 ± 0.5°C as judged from circular dichroism and susceptibility to proteolysis.

Our data provide clear evidence that a shortened α1‐derived collagen chain can be extracted from human adult bone whereas it is hardly found in human skin. The unique cleavage site might provide important information about the collagen I molecule embedded in the calcified matrix of human bone.
Pyridoxal phosphate inhibits the DNA‐binding activity of the ecdysteroid receptor
Authors: OŻYHAR Andrzej , KILTZ Hans‐Hermann , PONGS Olaf
Published: March 2005

The effect of pyridoxal 5′‐phosphate on the binding of the ecdysteroid receptor from a nuclear extract of Drosophila melanogaster to DNA‐cellulose was studied. The binding of hormone‐receptor complexes to DNA‐cellulose was completely blocked after a 30‐min incubation with 3 mM pyridoxal 5′‐phosphate at 0–4°C. The effect was specific for pyridoxal 5′‐phosphate since related compounds (pyridoxal, pyridoxamine 5′‐phosphate and pyridoxamine) were not effective or gave only 17% inhibition (pyridoxal). Under standard conditions, none of the compounds tested exerted a significant effect on the stability of [3H](20R,22R)‐2β,3β,14α,20,22‐pentahydroxy‐5β‐cholest‐7‐en‐6‐one ([3H]ponasterone A)‐receptor complexes. The loss of DNA‐binding activity caused by pyridoxal 5′‐phosphate is accompanied by changes in the molecular properties of [3H]ponasterone‐A–receptor complexes. A shift of [3H]ponasterone‐A binding was observed from the 8.0–8.5 S to the 4.5–5.0 S region, when [3H]ponasterone‐A–receptor complexes were exposed to pyridoxal 5′‐phosphate during sucrosegradient centrifugation.

The inhibition of DNA‐cellulose binding by pyridoxal 5′‐phosphate can be reversed. Probably, pyridoxal 5′‐phosphate forms a Schiff base with a critical lysine group of the ecdysteroid receptor, presumably at its DNA‐binding site. The hormone‐receptor complexes obtained after removal of pyridoxal 5′‐phosphate had the same affinity for DNA‐cellulose as ‘native’ complexes.

DNA–cellulose‐bound [3H]ponasterone‐A complexes were efficiently eluted from DNA‐cellulose with pyridoxal 5′‐phosphate in 0.1 M K Cl resulting in a 104‐fold purification of the ecdysteroid receptor.

The results reflect possible structural similarities between ecdysteroid and vertebrate steroid receptors.
Metabolic control analysis
Authors: FELL David A. , SAURO Herbert M.
Published: March 2005

Differing views have been given in the literature as to whether the presence in a pathway of an enzyme at a concentration comparable to that of its substrate affects the values of control coefficients and the theorems of metabolic control analysis. Here we argue in favour of one of those views: that there is no effect unless the enzyme sequesters a substrate that contains a conserved moiety. In this particular case, we derive both a general criterion for estimating whether such an effect will be of a significant magnitude, and equations for determining the changes in the flux control coefficients. The nature of the phenomenom and the application of the equations are illustrated with a numerical simulation.
Large‐scale purification of choline acetyltransferase and production of highly specific antisera
Authors: OSTERMANN Christel , DICKMANN Ulrich , MULEY Thomas , MÄDER Michael
Published: March 2005

Choline acetyltransferase (ChAT) was purified by immunoaffinity chromatography using a covalently immobilized monoclonal antibody. In a two‐step procedure, 10 kg porcine brain yielded 750 μg active enzyme of apparent homogeneity. This amount of ChAT was purified routinely. The purification factor was 18000 and the yield of activity 4.3%. The affinity resin was stable under the experimental conditions applied and was used many times. The highly purified enzyme was subsequently employed to obtain a specific anti‐ChAT antiserum of high titer.
Structural features of the lipopeptidophosphoglycan from Trypanosoma cruzi common with the glycophosphatidylinositol anchors
Authors: de LEDERKREMER Rosa M. , LIMA Carlos , RAMIREZ María I. , CASAL Olga L.
Published: March 2005

The lipopeptidophosphoglycan (LPPG) from Trypanosoma cruzi, a major constituent of the plasma membrane of epimastigote forms, has been now extracted with butanol/water from delipidated cells and purified by hydrophobic chromatography. We have found that the LPPG undergoes two reactions, characteristic of the glycosylphosphatidylinositol anchors: (a) cleavage of the ceramide by phosphatidylinositol‐specific phospholipase C(PtdIns‐specific phospholipase C) from Bacillus thuringiensis, (b) nitrous acid deamination of the non‐N‐acylated glucosamine. Palmitoylsphinganine, palmitoylsphingosine, lignoceroylsphinganine and, as minor components, the stearoylceramides were identified by gas liquid chromatography/mass spectrometry.

The presence of cross reacting determinant (CRD) epitopes in the glycophosphoinositol released by PtdIns‐specific phospholipase C was investigated by direct and inhibition ELISA. A sample of glycophosphoinositol containing 5 μg carbohydrate caused 60% inhibition of the binding of anti‐CRD antibodies raised against the soluble form of variant surface glycoprotein.
Characterization of the steady‐state calcium fluxes in skeletal sarcoplasmic reticulum vesicles
Authors: SOLER Fernando , TERUEL Jose A. , FERNANDEZ‐BELDA Francisco , GOMEZ‐FERNANDEZ Juan C.
Published: March 2005

Unidirectional Ca2+ fluxes (influx and efflux), supported by ATP as a phosphate‐donor substrate, were measured without alteration of the lumenal Ca2+ content in longitudinal sarcoplasmic reticulum vesicles. The referred fluxes are dependent on extravesicular Ca2+, ATP and ADP. They are unaffected by ruthenium red but inhibited by quercetin. The Ca2+ fluxes at steady state are drastically diminished when ATP is substituted by acetylphosphate although the addition of 10 μM ADP increases the apparent rate constants more than eight fold. The observed fluxes appear to be dependent on Ca2+‐ATPase phosphoenzyme transitions. The results indicate that: (a) the slow Ca2+ release, due to the passive permeability of the membrane, is a minor component of the total Ca2+ efflux, and (b) the ATPase protein is basically operating as a Ca2+/Ca2+ exchanger at steady state. Kinetic resolution of the Ca2+ fluxes, measured by isotopic tracer and rapid filtration techniques can be recreated by computer simulation of the ATPase reaction cycle featuring some modifications to account for the fast Ca2+/Ca2+ exchange and the uncoupling effect observed at steady state.
Control of respiration and oxidative phosphorylation in isolated rat liver cells
Authors: BROWN Guy C. , LAKIN‐THOMAS Patricia L. , BRAND Martin D.
Published: March 2005

NAD(P)H fluorescence, mitochondrial membrane potential and respiration rate were measured and manipulated in isolated liver cells from fed and starved rats in order to characterize control of mitochondrial respiration and phosphorylation. Increased mitochondrial NADH supply stimulated respiration and this accounted for most of the stimulation of respiration by vasopressin and extracellular ATP. From the response of respiration to NADH it was estimated that the control coefficient over respiration of the processes that supply mitochondrial NADH was about 0.15–0.3 in cells from fed rats.

Inhibition of the ATP synthase with oligomycin increased the mitochondrial membrane potential and decreased respiration in cells from fed rats, while the uncoupler carbonyl cyanide p‐trifluoromethoxyphenylhydrazone had the opposite effect. There was a unique relationship between respiration and membrane potential irrespective of the ATP content of the cells indicating that phosphorylation potential controls respiration solely via phosphorylation (rather than by controlling NADH supply).

From the response of respiration to the mitochondrial membrane potential (ΔΨM) it was estimated that the control coefficients over respiration rate in cells from fed rats were: 0.29 by the processes that generate ΔΨM, 0.49 by the process of ATP synthesis, transport and consumption, and 0.22 by the processes that cycle protons across the inner mitochondrial membrane other than via ATP synthesis (e.g. the passive proton leak). Control coefficients over the rate of mitochondrial ATP synthesis were 0.23, 0.84 and −0.07, respectively, by the same processes. The control distribution in cells from starved rats was similar.
Synchronous differentiation of Trypanosoma brucei from bloodstream to procyclic forms in vitro
Authors: ZIEGELBAUER Karl , QUINTEN Margret , SCHWARZ Heinz , PEARSON Terry W. , OVERATH Peter
Published: March 2005

The differentiation of mammalian stage Trypanosoma brucei bloodstream forms comprising predominantly parasites of intermediate and stumpy morphology to the procyclic forms characteristic for the insect midgut stage was studied in vitro. Differentiation of the cell population occurred synchronously as judged by the synthesis of the surface glycoprotein, procyclin, characteristic of the arising procyclic forms and the loss of the membrane‐form variant surface glycoprotein, the coat protein of bloodstream forms. The change in surface antigens took place within 12 h in the absence of cell growth; subsequently, the procyclic cells divided exponentially. As defined in this study, T. brucei may be a useful model to follow other changes in gene expression, metabolism or ultrastructure during differentiation of a unicellular eucaryote.
On the roles of magnesium and spermidine in the isoleucyl‐tRNA synthetase reaction
Authors: AIRAS R. Kalervo
Published: March 2005

The reaction of isoleucyl‐tRNA synthetase from Escherichia coli B was analysed by deriving total steady‐state rate equations for the ATP/PPi exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2+ inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme‐bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine · ATP complex. Spermidine · ATP is a weaker substrate. The role of spermidine · ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines.
Hepatic processing and biliary secretion of the cholesteryl esters from β very‐low‐density lipoproteins in the rat
Authors: De WATER Ruud , HESSELS Esther M. A. J. , BAKKEREN Hille F. , VAN BERKEL Theo J. C.
Published: March 2005

β‐Migrating very‐low‐density lipoproteins (β‐VLDL) are cholesteryl‐ester‐enriched lipoproteins which accumulate in the serum of cholesterol‐fed animals or patients with type III hyperlipoproteinemia. In the rat, β‐VLDL are rapidly cleared by the liver and parenchymal liver cells form the major site for uptake. In this investigation, β‐VLDL were labeled with [3H]cholesteryl esters and the hepatic intracellular transport of these esters was followed. 2 min after injection, the major part of the [3H]cholesteryl esters is already associated with the liver and a significant proportion is recovered in endosomes. Up to 25 min after injection, an increase in radioactivity in the lysosomal compartment is noticed. This radioactivity initially represents cholesteryl esters, while from 25 min onward, radioactivity is mainly present in unesterified cholesterol. Between 45 min and 90 min after β‐VLDL injection, specific transfer of unesterified [3H]cholesterol to the endoplasmic reticulum is observed, while by 3 h the majority is located in this fraction. The appearance of radioactivity in the bile was rather slow as compared to the rapid initial uptake and processing, and up to 5 h after injection only 10% of the injected dose had reached the bile (mainly as bile acids). 72 h after injection, the amount of the injected radioactivity recovered in the bile had increased to 50%. Chloroquine treatment of the rats inhibited the hydrolysis of the cholesteryl esters and the appearance of radioactivity in the bile was retarded.

It is concluded that β‐VLDL are rapidly processed by parenchymal liver cells and that the cholesteryl esters from β‐VLDL are hydrolyzed in the lysosomal compartment. Unesterified cholesterol remains associated with the endoplasmic reticulum for a prolonged time, although ultimately the majority will be secreted into the bile as bile acids. The effective operation of this pathway will prevent extrahepatic accumulation of cholesteryl esters from β‐VLDL, while the prolonged residence time of unesterified cholesterol in the endoplasmic reticulum might be important for regulation of low‐density lipoprotein (LDL) receptors in liver and thus for LDL levels in the blood.
The bovine plasma retinol‐binding protein
Authors: BERNI Rodolfo , STOPPINI Monica , ZAPPONI Maria Carla , MELONI Maria Laura , MONACO Hugo L. , ZANOTTI Giuseppe
Published: March 2005

The primary structure of bovine plasma retinol‐binding protein (RBP) has been determined and found to be more than 90% identical to human and rabbit RBPs, and more than 80% identical to rat RBP. Main changes in amino acid sequence are observed in two regions on the surface of the protein molecule (residues 138–148 and 169–183).

The interactions of bovine RBP with bovine and human transthyretins were investigated using the technique of fluorescence polarization. Bovine RBP was able to form high affinity complexes (K′d= 0.34 ± 0.02 μM) with both bovine and human transthyretins, displaying a stoichiometry of approximately 2 molecules RBP/molecule transthyretin in both cases. The sites that participate in protein‐protein interactions are thus very similar, and this tends to exclude the involvement of the superficial regions more significantly substituted in mammalian RBPs (residues 138–151 and 167–183) in the protein–protein recognition.

Bovine RBP has been crystallized (space group P212121, with a= 4.61 nm, b= 4.91 nm, c= 7.61 nm) and the crystals are suitable for high‐resolution X‐ray diffraction studies.
Changes in the phosphorylation state of the inhibitory guanine‐nucleotide‐binding protein Gi‐2 in hepatocytes from lean (Fa/Fa) and obese (fa/fa) Zucker rats
Authors: BUSHFIELD Mark , PYNE Nigel J. , HOUSLAY Miles D.
Published: March 2005

Treatment of intact, 32Pi‐labelled hepatocytes from lean Zucker rats with a range of agents including 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA), vasopressin, and angiotensin II elicited substantial increases in the phosphorylation of the α‐subunit of the inhibitory G protein of adenylate cyclase (αGi‐2). These agonist‐induced phosphorylations of αGi‐2 were associated with loss of Gi function as assessed by the ability of low concentrations of guanylyl 5′‐[β,γ imido]triphosphate (p[NH]ppG) to inhibit forskolin‐stimulated adenylate cyclase activity. Hepatocytes from obese Zucker rats displayed a resistance to both agonist‐induced phosphorylation of αGi‐2 and to p[NH]ppG‐mediated inhibition of adenylate cyclase. The basal level of αGi‐2 phosphorylation in hepatocytes from obese Zucker rats was considerably greater at 1.06 ± 0.09 mol phosphate/mol αGi‐2 than in hepatocytes from lean animals which gave 0.54 ± 0.09 mol phosphate/mol αGi‐2. Incubation with TPA (10 ng/ml, 15 min) approximately doubled the level of phosphorylation of αGi‐2 in the hepatocytes from lean animals but had little effect on the phosphorylation of αGi‐2 in hepatocytes from obese animals. Incubation of hepatocytes from lean animals with ligands which lead to the phosphorylation of αGi‐2 abolished the ability of low concentrations of p[NH]ppG to inhibit adenylate cyclase expressed in isolated membranes. Treatment of hepatocyte plasma membranes from lean but not obese Zucker rats with pure protein kinase C led to the phosphorylation of αGi‐2. The resistance to protein‐kinase‐C‐mediated phosphorylation in hepatocyte membranes from obese animals could be overcome by treatment of the membranes with alkaline phosphatase. These results indicate that the defect in guanine‐nucleotide‐mediated ‘Gi function’ seen in obese Zucker rats may be due to an inactivating phosphorylation of αGi‐2.
Nitrous oxide reductase from denitrifying Pseudomonas stutzeri
Authors: ZUMFT Walter G. , VIEBROCK‐SAMBALE Adelheid , BRAUN Cornelia
Published: March 2005

Nitrous oxide (N2O) respiration by the denitrifying bacterium Pseudomonas stutzeri requires the synthesis of the multi‐copper enzyme N2O reductase. nosZ, the structural gene for this enzyme, is part of a DNA region of approximately 8 kbp that carries several essential genes. Insertional mutagenesis of the region downstream of nosZ generates apoenzyme‐synthesizing strains, which argues for the existence of functions for copper acquisition or copper processing, or both, for N2O reductase. The relevant DNA region of approximately 3.2 kbp was sequenced and found to consist of three genes, nosDFY, presumably within a single transcriptional unit. Cellular concentration, copper content, and location of the N2O reductase protein were studied with mutants which were affected in the three genes.

Interactions of the deduced gene products among each other and with the cytoplasmic membrane appear to be analogous to those of the components of osmotic‐shock‐sensitive bacterial transport systems. The 33.8‐kDa product of the nosF gene belongs to the family of nucleotide‐binding proteins [C. Higgins et al. (1986) Nature 323, 448–450]. Its amino acid sequence shows two canonical nucleotide‐binding motifs, and the positional identity of amino acids to members of this family is around 30%. The 29.4‐kDa product of the nos Y gene is a hydrophobic protein with six predicted transmembrane helices and an export signal. The 48.2‐kDa product of the nosD gene is a periplasmic component; it carries an export signal and is a hydrophilic protein. N2O reductase itself is a periplasmic enzyme. Our results provide evidence for an auxiliary system of Cu processing and suggest its involvement in the periplasmic biosynthesis of the Cu centers of N2O reductase.
Isolation and characterization of cytochrome c550 from the methylamine‐oxidizing electron‐transport chain of Thiobacillus versutus
Authors: LOMMEN Arjen , RATSMA Age , BIJLSMA Niels , CANTERS Gerard W. , VAN WIELINK John E. , FRANK Johannes , VAN BEEUMEN Jozef
Published: March 2005

The isolation and purification of cytochrome c550 from the methylamine‐oxidizing electron‐transport chain in Thiobacillus versutus is reported. The cytochrome is a single‐heme‐containing type I cytochrome c with a relative molecular mass of 16 ± 1 kDa, an isoelectric point of 4.6 ± 0.1, a midpoint potential of 272 ± 3 mV at pH < 4 and 255 ± 5 mV at pH = 7.0, and an axial coordination of the Fe by a methionine and a histidine. The midpoint potential decreases with increasing pH due to the deprotonation of a group tentatively identified as a propionate (pKa= 6.5 ± 0.1 and 6.7 ± 0.1 in the oxidized and reduced protein, respectively) and a change in the Fe coordination at pH > 10. The electron‐self‐exchange rate appears to depend strongly on the ionic strength of the solution and is relatively insensitive to changes in pH. At 313 K and pH 5.2 the electron‐exchange rate amounts to 0.7 × 102 M−1 s−1 and 5.3 × 102 M−1 s−1 at I= 40 mM and I= 200 mM, respectively. Amino acid composition and molar absorption coefficients at various wavelengths are reported. Resonances of heme protons and the ±H3 group of the ligand methionine of the Fe have been identified in the 1H‐NMR spectrum of the reduced as well as the oxidized cytochrome.
Expression of argininosuccinate lyase mRNA in foetal hepatocytes
Authors: HUSSON Annie , RENOUF Sylvie , FAIRAND Alain , BUQUET Catherine , BENAMAR Malika , VAILLANT René
Published: March 2005

Argininosuccinate lyase (ASL), the fourth enzyme of the urea cycle, belongs to a group of liver enzymes appearing in the late foetal period in the rat. Several hormones, including glucocorticosteroids and insulin have been implicated in the control of the development of this enzyme activity. In this study, the cloned cDNA was used to measure the relative abundance of ASL mRNA in the livers of rats at various stages of perinatal development and in cultured foetal hepatocytes during hormonal manipulations.

The ASL mRNA was first detectable on day 15.5 of gestation and increased in amount concomitantly with the rise in the enzyme activity, suggesting that the appearance of enzyme activity reflects the turning on of specific gene transcription. When foetal hepatocytes were exposed to dexamethasone, an increase in ASL mRNA was detected, which was completely abolished by addition of actinomycin D, suggesting a transcriptional effect of the steroid. In contrast, administration of cortisol to foetuses in utero had no effect on the mRNA level, suggesting that the steroid action is inhibited in the intra‐uterine environment. Insulin might be the inhibiting factor since it completely repressed the dexamethasone‐induced accumulation of ASL mRNA in foetal hepatocytes. These data were confirmed in vivo by experiments using streptozotocin, which produces insulin‐depleted foetuses and causes the accumulation of ASL mRNA. This regulation of ASL mRNA by glucocorticoids and insulin could account for the modulation of the enzyme activity observed in vivo and in vitro.
Purification and characterization of DNA ligase II from Drosophila melanogaster
Authors: TAKAHASHI Miho , TOMIZAWA Kayoko
Published: March 2005

Drosophila melanogaster contains DNA ligases I and II. The activity of DNA ligase I is especially high during early embryonic periods, but decreases rapidly afterwards. Although the activity of DNA ligase II is low, it persists throughout all developmental stages. The specific activity of DNA ligase II is high in embryos, but the total activity per body mass was highest in pupae. To characterize the properties of DNA ligase II further and to clarify its differences from DNA ligase I, DNA ligase II was prepared from pupae of D. melanogaster. The enzyme was purified about 3200‐fold by ammonium sulfate fractionation (40–70% saturation), phosphocellulose (P11) and Ultrogel column chromatography. Some of the properties have been reported previously. The isoelectric point of DNA ligase II was 6.4 while those of DNA ligase I were 4.9 and 5.8. The optimum pH of DNA ligase II was 7.8–8.1 but 8.0–8.5 for DNA ligase I.

The molecular masses of DNA ligase II adducts with AMP were determined as 90 and 70 kDa. These adducts were degraded to 42 and 14.4 kDa by trypsin digestion. For preparation of monoclonal antibodies, a mouse was immunized with the purified enzyme. Two clones, 10‐6 and 3‐3 IgM, were obtained and purified from mouse ascites. These antibodies showed both binding and neutralizing activities toward DNA ligase II from D. melanogaster, but did not react with DNA ligase I from the same origin.

These results showed clearly that DNA ligases I and II have different properties and suggest they have different roles during the developmental stages of D. melanogaster.
Biotransformation of intracellular minerals by zinc ions in vivo and in vitro
Authors: TAYLOR Marina G. , GREAVES G. Neville , SIMKISS Kenneth
Published: March 2005

Inorganic granules of the general type CaMgP2O7 are found within cells of a wide range of species. The granules are amorphous to X‐rays and occur in membrane‐bound compartments. They accumulate a variety of cations from the body fluids of these animals.

The interaction of zinc ions with pyrophosphate granules from cells of the snail Helix aspersa have been studied in vivo and in vitro by X‐ray absorption spectroscopy and X‐ray diffraction. The in vivo deposits incorporate zinc and remain amorphous although the pyrophosphate is hydrolysed to orthophosphate. In the in vitro reaction the mineral becomes crystalline but there is little change in the form of the anion. The results clearly indicate a role for the cellular system in maintaining the amorphous state and in the hydrolysis of pyrophosphate to orthophosphate. As such they provide a model system for some of the changes observed in precursors of bone mineral and in pathological changes in the calcification of cartilage.
myo‐Inositol metabolites as cellular signals
Authors: DOWNES C. Peter , MACPHEE Colin H.
Published: March 2005

The discovery of the second‐messenger functions of inositol 1,4,5‐trisphosphate and diacylglycerol, the products of hormone‐stimulated inositol phospholipid hydrolysis, marked a turning point in studies of hormone function. This review focusses on the myo‐inositol moiety which is involved in an increasingly complex network of metabolic interconversions. myo‐Inositol metabolites identified in eukaryotic cells include at least six glycerophospholipid isomers and some 25 distinct inositol phosphates which differ in the number and distribution of phosphate groups around the inositol ring. This apparent complexity can be simplified by assigning groups of myo‐inositol metabolites to distinct functional compartments. For example, the phosphatidylinositol 4‐kinase pathway functions to generate inositol phospholipids that are substrates for hormone‐sensitive forms of inositol‐phospholipid phospholipase C, whilst the newly discovered phosphatidylinositol 3‐kinase pathway generates lipids that are resistant to such enzymes and may function directly as novel mitogenic signals. Inositol phosphate metabolism functions to terminate the second‐messenger activity of inositol 1,4,5‐trisphosphate, to recycle the latter's myo‐inositol moiety and, perhaps, to generate additional signal molecules such as inositol 1,3,4,5‐tetrakisphosphate, inositol pentakisphosphate and inositol hexakisphosphate. In addition to providing a more complete picture of the pathways of myo‐inositol metabolism, recent studies have made rapid progress in understanding the molecular basis underlying hormonal stimulation of inositol‐phospholipid‐specific phospholipase C and inositol 1,4,5‐trisphosphate‐mediated Ca2+ mobilisation.
Coamplification of μ class glutathione S‐transferase genes and an adenylate deaminase gene in coformycin‐resistant Chinese hamster fibroblasts
Authors: ROBERT DE SAINT VINCENT Bruno , HYRIEN Olivier , DEBATISSE Michelle , BUTTIN Gérard
Published: March 2005

In Chinese hamster fibroblasts, we previously detected an expressed gene located near the AMP deaminase gene. This gene was named Y1. Upon selection for resistance to coformycin, an inhibitor of AMP deaminase activity, both genes were amplified in several mutants. We have determined the complete nucleotide sequence of Y1 cDNA and identified the Y1 gene as a μ class glutathione S‐transferase gene by comparison with sequences present in a data bank. Accordingly, Y1‐amplified mutants express an increased glutathione S‐transferase activity toward 1‐chloro‐2,4‐dinitrobenzene; this activity, as well as the abundance of the corresponding RNA, appears, however, to reach a limit despite further increase in the Y1 gene copy number during successive amplification steps. Southern blot experiments showed that Y1 belongs to a multigene family, all or part of which has been amplified in mutant lines. These data provide a method to amplify and to overexpress the μ class of the glutathione S‐transferase gene family on the basis of its linkage with the AMP deaminase gene.
Purification and characterization of an exo‐1,4‐β‐galactanase from a strain of Bacillus subtilis
Authors: NAKANO Hirofumi , TAKENISHI Shigeyuki , KITAHATA Sumio , KINUGASA Haruko , WATANABE Yasuto
Published: March 2005

An arabinogalactan 4‐β‐d‐galactanohydrolase was purified to a homogeneous state from the culture filtrate of a strain of Bacillus subtilis. The enzyme have a molecular mass of 36 kDa and an isoelectric point of pH 7.9. The enzyme is most active at around pH 6.5–7 and at 60°C, and is stable between pH 6–10 and below 55°C. Hg2+ and Cu2+ inhibit the activity. The enzyme hydrolyze soybean arabinogalactan which contains β‐1,4‐galactosidic linkages in its main chain structure, but not other polysaccharides with β‐1,3‐galactosidic linkages. The hydrolysis products from soybean arabinogalactan are predominantly galactobiose with a small amount of galactotetraose. The enzyme is an exo‐enzyme and the ability to transfer galactobiose to other galactobiose molecules is indicated by the formation of galactotetraose.
Evidence for a super‐reduced cobamide as the major corrinoid fraction in vivo and a histidine residue as a cobalt ligand of the p‐cresolyl cobamide in the acetogenic bacterium Sporomusa ovata
Authors: STUPPERICH Erhard , EISINGER Hans Jürgen , ALBRACHT Simon P. J.
Published: March 2005

The redox state of cobalt in p‐cresolyl cobamide and one of its axial ligands were determined by EPR spectroscopy of Sporomusa ovata as harvested. The analyses revealed that < 2% (< 30 nmol/g dry cells) of the total corrinoids (> 2400 nmol/g dry cells) were in a low‐spin Co(II) complex. The amount increased to about 15% (190–450 nmol/g dry cells) upon partial oxidation by air, indicating that the original valence state of cobalt was a Co(I) prior to this treatment. The cob(I)amide was quantified as Co(III)‐CH3 after methylation by iodomethane. More than 45% (1100 nmol/g dry cells) of the extractable corrinoids were in the methylated form, whereas non‐treated cells revealed less then 1% (< 15 nmol/g dry cells) of light‐sensitive corrinoids. EPR spectra of the Co(II) complex exhibited a threefold N‐hyperfine splitting in the gz region, which was similar to vitamin B12. Cells grown with [1,3‐15N2]histidine showed a twofold N‐hyperfine splitting, demonstrating that the axial N ligand of the corrinoid was derived from the imidazole group of histidine. It is concluded that the super‐nucleophilic p‐cresolyl cob(I)amide is the major corrinoid complex in vivo and that it is stabilized by its protein(s). The Co(II) ion of the prosthetic group was coordinated by one histidine residue of the apoprotein(s).
A multinuclear magnetic resonance study of a cls11 mutant showing the Pet− phenotype of Saccharomyces cerevisiae
Authors: GALONS Jean‐Philippe , TANIDA Isei , OHYA Yoshikazu , ANRAKU Yasuhiro , ARATA Yoji
Published: March 2005

Energetic and intermediary metabolism was studied in a Pet− mutant of Saccharomyces cerevisiae with a calcium‐sensitive phenotype that shows an inability to grow when cultured in a medium containing non‐fermentable substrates. The perchloric acid extracts were prepared from suspensions of cls11 mutant and wild‐type cells incubated with [1,3‐13C]glycerol or [2‐13C]acetate, and analyzed by 31P, 13C and 1H NMR. 31P‐ and 1H‐NMR spectra showed significant differences between cls11 and wild‐type cells at the level of amino acids, the storage carbohydrate trehalose (higher in mutant cells), and sugar phosphates (higher in wild‐type cells). 13C‐NMR spectra revealed major differences in the steady‐state labelling of glutamate carbons. For incubations with [1,3‐13C]glycerol, we estimated from the relative 13C enrichment of glutamate carbons that acetyl‐CoA C2 is 43% C13 labelled in wild‐type and 10%13C labelled in mutant cells, respectively. For incubations with [2‐13C]acetate, we calculated that the ratio of the relative flux through the glyoxylate shunt versus oxidative reactions is 58% in wild‐type cells and 44% in the cls11 mutant cells. Again, a dilution of the relative enrichment of C2 of acetyl‐CoA was observed in the mutant cells (89%) compared to the wild‐type cells (97%). These results are discussed in terms of pleiotropic defects in non‐fermentable carbon metabolism in mutant cells.
Two glyceraldehyde‐3‐phosphate dehydrogenase isozymes from the koningic acid (heptelidic acid) producer Trichoderma koningii
Authors: SAKAI Kaoru , HASUMI Keiji , ENDO Akira
Published: March 2005

The sesquiterpene lactone koningic acid (heptelidic acid) irreversibly inactivated glyceraldehyde‐3‐phosphate dehydrogenase [d‐glyceraldehyde 3‐phosphate:NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) (GAPDH) and thus inhibits glycolysis. The koningic‐acid‐producing strain of Trichoderma koningii M3947 was shown to contain the koningic‐acid‐resistant GAPDH isozyme (GAPDH I) under conditions of koningic acid production. In peptone‐rich medium, however, no koningic‐acid production was observed, and the koningic‐acid‐sensitive GAPDH isozyme (GAPDH II), in addition to the resistant enzyme, was produced. Both enzymes were tetramer with a molecular mass of 152 kDa (4 × 38 kDa) and lost enzyme activity when two of the four cysteine residues reacted with koningic acid. The apparent Km values of GAPDH I and II for glyceraldehyde 3‐phosphate were 0.54 mM and 0.33 mM, respectively. The former isozyme was inhibited 50% by 1 mM koningic acid but not affected at 0.1 mM, while the latter isozyme was inhibited 50% at 0.01 mM. The immunochemical properties and partial amino acid sequences suggested that the two isozymes have different molecular structures. These results suggest that GAPDH I is responsible for the glycolysis in T. koningii when koningic acid is produced.
Purification and characterization of a cysteine proteinase from silkworm eggs
Authors: KAGEYAMA Takashi , TAKAHASHI Susumu Y.
Published: March 2005

Eggs of the silkworm, Bombyx mori, contain a high level of a proteinase which is most active in acidic pH region. The proteinase was purified from an extract of eggs by a six‐step procedure which included conventional chromatographic fractionations. The molecular mass of the proteinase was estimated to be 350 kDa by gel filtration and 47 kDa by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels, suggesting an octameric structure. The amino acid composition was found to resemble that of mammalian lysosomal cysteine proteinases, in particular cathepsin L. The NH2‐terminal 10‐residue sequence is Val‐Gln‐Phe‐Phe‐Asp‐Leu‐Val‐Lys‐Glu‐Glu‐. The enzyme appears to be a member of the class of cysteine proteinases since it was strongly inhibited by sulfhydryl‐reactive compounds and N‐[N‐(1,3‐trans‐carboxyoxiran‐2‐carbonyl)‐l‐leucyl]‐agmatine (E‐64). The enzyme hydrolyzed various protein substrates, such as hemoglobin, vitellogenin, vitellin, and lipophorin, with maximal activity around pH 3–3.5. The specificity of the cleavage sites in the oxidized B chain of insulin was rather well defined and there was high affinity for hydrophobic residues at the P2 and P3 positions. The cysteine proteinase is thought to be involved in protein degradation during embryonic development of silkworm eggs.
Cooperative homotropic interaction of l‐noradrenaline with the catalytic site of phenylalanine 4‐monooxygenase
Authors: MARTÍNEZ Aurora , HAAVIK Jan , FLATMARK Torgeir
Published: March 2005

Catecholamines (adrenaline, noradrenaline and dopamine) are potent inhibitors of phenylalanine 4‐monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1). The amines bind to the enzyme by a direct coordination to the high‐spin (S= 5/2) Fe(III) at the active site (charge transfer interaction), as seen by resonance Raman and EPR spectroscopy. Experimental evidence is presented that a group with an apparent pKa value of about 5.1 (20°C) is involved in the interaction between the catecholamine and the enzyme. The high‐affinity binding of l‐noradrenaline to phenylalanine hydroxylase, as studied by equilibrium microdialysis (anaerobically) and ultrafiltration (aerobically), shows positive cooperativity (h= 1.9); at pH 7.2 and 20°C the rat enzyme binds about 0.5 mol l‐noradrenaline/mol subunit with a half‐maximal binding (S50) at 0.25 μM l‐noradrenaline. No binding to the ferrous form of the enzyme was observed. The affinity decreases with decreasing pH, by phosphorylation and by preincubation of the enzyme with the substrate l‐phenylalanine, while it increases after alkylation of the enzyme with the activator N‐ethylmaleimide. Preincubation of the enzyme with l‐phenylalanine also leads to a complete loss of the cooperativity of l‐noradrenaline binding (h= 1.0). The many similarities in binding properties of the inhibitor l‐noradrenaline and the activator/substrate l‐phenylalanine makes it likely that the cooperative interactions of these effectors are due to their binding to the same site. The high‐affinity of catecholamines to phenylalanine hydroxylase is a valuable probe to study the active site of this enzyme and is also relevant for the homologous enzyme tyrosine hydroxylase, which is purified as a stable catecholamine‐Fe(III) complex.
Ornithine/phosphate antiport in rat kidney mitochondria
Authors: PASSARELLA Salvatore , ATLANTE Anna , QUAGLIARIELLO Ernesto
Published: March 2005

[14C]Ornithine uptake by rat kidney mitochondria has been investigated according to the stop inhibitor method by using praseodimium chloride as an inhibitor. The existence of an ornithine/Pi exchange was found occurring with 1:1 stoichiometry. Both uptake and efflux follow first‐order kinetics with a k of 2.4 min−1. Uptake increases with increasing pH. The activation energy for the process is 58.6 kJ/mol and Q10 is 2.6. Ornithine/Pi exchange is electrical and energy‐dependent, as suggested by the sensitivity of the process to the ionophores valinomycin and nigericin. Measurements both of proton movement across the mitochondrial membrane and of membrane potential strongly suggest that ornithine uptake into mitochondria is driven by the electrochemical proton gradient via the dependent ornithine/Pi translocator and ΔpH‐dependent Pi carrier.
Linear dichroism studies of tryptophanase and its quasisubstrate complexes oriented in polyacrylamide gel
Authors: ZAKOMIRDINA Lyudmila N. , SAKHAROVA Irene S. , TORCHINSKY Yuri M.
Published: March 2005

Tryptophanase from Escherichia coli was oriented in a compressed slab of polyacrylamide gel and its linear dichroism (LD) and absorption spectra have been measured. The free enzyme displays four LD bands at 305, 340, 425 and 490 nm. Two bands at 340 and 425 nm belong to the internal coenzyme‐lysine aldimine. The 305‐nm band apparently belongs to an aromatic amino acid residue. The 490‐nm band disappears after treatment with NaBH4 or after incubation with l‐alanine and subsequent dialysis. It is suggested that the 490‐nm band belongs to a quinonoid enzyme subform. The reaction of tryptophanase with threo‐3‐phenyl‐dl‐serine, l‐threonine and d‐alanine leads to formation of an external aldimine with an intense absorption band at 420–425 nm. The values of reduced LD (ΔA/A) in this band strongly differ from that in the 420‐nm band of the free enzyme. The LD value of the complex with d‐alanine is intermediate between those of the free enzyme and the complex with 3‐phenylserine. In the presence of indole the complex with d‐alanine displays the same LD as that observed with 3‐phenylserine. The reaction of tryptophanase with l‐alanine or oxindolyl‐l‐alanine leads to formation of a quinonoid intermediate with an absorption band near 500 nm. The LD value in this band is close to that of an external aldimine with l‐threonine. It is concluded that reorientations of the coenzyme occur in the course of the tryptophanase reaction.
Lipáses catalyse hydrolysis of fatty acid anhydrides
Authors: PIERONI Gérard , FOURNERON Jean‐Dominique
Published: March 2005

Regio‐specific and non‐regio‐specific lipases from mammals and microorganisms catalyse the hydrolysis of short, medium and long‐chain fatty acid anhydrides. All the lipases tested in the present study can catalyse the hydrolysis of pure fatty acid anhydrides more efficiently than that of glycerol tributyrate. Molecular turnovers more than four times higher than that measured using glycerol tributyrate were calculated. The presence of 0.5% (by mass) anhydride in a triacylglyceride can double the initial rate of proton release during enzymatic hydrolysis. This should be taken into account when testing the chain specificity of a lipase for various synthetic substrates. Lipase inhibition was found to be associated very often with anhydride hydrolysis. The inhibition rates depended on the anhydride and the origin of the lipase. Inhibition of lipase activity is probably due to the formation of a poorly reversible acyl‐lipase complex which differs from the classical fully reversible acyl‐lipase complex at the catalytic centre.
Ontogeny and perinatal modulation of gene expression in rat brown adipose tissue
Authors: GIRALT Marta , MARTIN Immaculada , IGLESIAS Roser , VIÑAS Octavi , VILLARROYA Francesc , MAMPEL Teresa
Published: March 2005

We have performed a sequential study on the abundance of the mRNA for uncoupling protein (UCP), subunit II of cytochrome‐c oxidase (COII) and lipoprotein lipase in brown adipose tissue during the fetal and postnatal periods. Moreover, we have determined whether these parameters can be modulated by ambient temperature in the early hours after birth, and at which point in development this sensitivity first appears.

UCP gene expression in the fetal and neonatal period has particular features when compared with overall mitochondriogenesis (COII mRNA expression) or with the expression of lipoprotein lipase mRNA. There is a specific induction of UCP gene expression between days 18 and 19 of pregnancy followed by a specific increase of UCP gene expression in utero and a further increase after birth. The acquisition of the physiological apparatus capable of the response to UCP and lipoprotein lipase gene expression to the environmental temperature is not achieved until the last day of fetal development. This result suggests that mechanisms of β‐adrenergic modulation of gene expression in brown fat are already established at birth.

From an experiment on iopanoic acid treatment of pregnant mothers, it was concluded that iodothyronine 5′‐deiodinase activity is not necessary for the expression of the mRNAs for UCP, COII and lipoprotein lipase in the fetus whereas it is necessary for the acquisition of temperature sensitivity to these parameters at birth.
The tissue‐specific expression of the thyroglobulin gene requires interaction between thyroid‐specific and ubiquitous factors
Authors: SINCLAIR Alison J. , LONIGRO Renata , CIVITAREALE Donato , GHIBELLI Lina , DI LAURO Roberto
Published: March 2005

Thyroid‐specific expression of the rat thyroglobulin gene is mediated by transcriptional control. Sufficient DNA sequence information to confer thyroid‐specific expression to a heterologous gene is contained between positions −168 and +39. DNA‐binding studies have demonstrated that this region interacts with two thyroid‐specific factors (TTF‐1 and TTF‐2), and a ubiquitous factor (UFA). Here we have characterized three elements within the promoter, A, K, and C, which are important for promoter activity in thyroid cells. We have shown by mutational analysis that the interaction of TTF‐1 with the A and C regions, UFA with the A region, and TTF‐2 with the K region are required for full promoter activity. The complex interactions in the A region can be replaced by the substitution of the UFA/TTF‐1‐binding site with a high‐affinity TTF‐1 binding site. There is a correlation between the presence of TTF‐1 and TTF‐2 DNA‐binding activities and the expression of thyroglobulin, which implies that the mechanism restricting thyroglobulin expression to thyroid cells is mediated through the control of the expression, or the activity, of TTF‐1 and TTF‐2.
Bovine hemoglobin pseudo‐crosslinked with mono(3,5‐dibromosalicyl)‐fumarate
Authors: FRONTICELLI Clara , BUCCI Enrico , RAZYNSKA Anna , SZNAJDER Joanna , URBAITIS Barbara , GRYCZYNSKI Zygmunt
Published: March 2005

Under oxygenated conditions bovine hemoglobin reacts with mono(3,5‐dibromosalicyl)‐fumarate which specifically acylates the EF5 lysines in the β‐cleft of the protein. The chemical modification introduces in the molecule a pseudo‐crosslink which hinders the dissociation of the hemoglobin molecule into dimers. Retention time in circulation of the chemically modified bovine hemoglobin, measured in the rat, is increased fivefold with respect to untreated bovine hemoglobin. The oxygen affinity at 37°C and at pH 7.4, has a P50= 5.4 kPa and a value of n= 1.9. Under the same experimental conditions the oxygen affinity is not sensitive to anions and polyanions whereas it is sensitive to CO2. The Bohr effect is shifted toward the alkaline pH range by 0.5–1: the maximum number of protons released is 1.5/tetramer, similar to normal bovine hemoglobin (1.8 protons/tetramer). Analysis of the binding isotherms, using the two‐state Monod‐Wyman‐Changeux model and fixing the value of the allosteric constant L= 105, shows that the oxygen affinity of the T structure is not modified, and that the low oxygen affinity of the system is due to a decrease of the oxygen affinity of the R structure. Analysis using the sequential Adair model shows a modification of the overall binding constants and suggests a redistribution of the intermediate species of oxygenation.