bluuebunny/crossref_metadata_2025_split huggingface dataset:
Control of the Activity of Phosphoertolpyruvate Carboxykinase and the Level of ItsmRNA in Livers of Newborn Rats
Authors: BENVENISTY Nissim , SIMCHON Edit Ben , COHEN Hannah , MENCHER David , MEYUHAS Oded , RESHEF Lea 
Published: March 2005

Streptozotocin treatment produces a typical experimental diabetes in neonates exhibiting hyperglycemia, glucosuria, ketonemia and increased level of free fatty acids in the blood. The liver is affected as well, with reduced activity of glycogen synthase and a corresponding decrease in the content of liver glycogen.In contrast, the activity of liver cytosolic phosphoenolpyruvate carboxykinase and the level of its mRNA are not affected. Using a cDNA containing P‐pyruvate carboxykinase sequence, the relative abundance of the enzyme mRNA was estimated. The level of the mRNA was readily observed increasing by glucocorticoid treatment or decreasing in response to administered load of glucose. These parallel the changes observed in the activity of the enzyme under these treatments, indicating that the level of P‐pyruvate carboxykinase mRNA actually determines that of the cnzyme. The failure of diabetes to incrcase the level of enzyme mRNA and the limited response to glucose loading strongly suggest that the mechanisms controlling the level of P‐pyruvate carboxykinase mRNA in neonates are relatively resistant to insulin. This is unique to nconates, since in both the adult and the fetal liver, P‐pyruvate carboxykinase readily responds to insulin. The minimal levels of glucocorticoids characteristic of neonates may be associated with this phenomenon.
Purification and Characterization of a Melanoma Cell Plasminogen Activator
Authors: WALLÉN Per , POHL Gunnar , BERGSDORF Nils , RÅNBY Mats , NY Tor , JÖRNVALL Hans 
Published: March 2005

The plasminogen activator from a human melanoma cell line was purified with immunoadsorption as a major step. the cells were cultured in the presence of aprotinin in order to avoid proteolysis. A three‐step purification involved adsorption on antibodies to porcine tissue plasminogen activator before chromatographies on arginine‐Sepharose and Sephadex G‐150. All solvents contained Tween‐80 (0.01%) and, except for the last step, aprotinin. The final product had a specific acitivity of about 220000 IU/mg measured against the WHO urokinase standard. The activator obtained has an apparent Mr of 72000 and consists of single‐chain molecules.Evidence was obtained that four different types of activator variants occur. First and known previously, the one‐chain form can be proteolytically cleaved into a two‐chain form. Secondly, both the one‐chain and two‐chain molecules exhibit two forms with molecular weight differences of about 3000 (possibly due to carbohydrate differences). Thirdly, the one‐chain preparations contain two variants, each constituting about 50% of the material and differing in length by three N‐terminal amino acids. Finally, a possible positional microheterogeneity was detected.Digestion with plasmin yields the two‐chain form with disulfide‐bonded polypeptide chains, ‘A′ and ‘B′ (from the N‐terminal and C‐terminal parts, respectively). At the same time, the variability of the original N terminus is removed. The A chain keeps the two Mr variants (now about 40000 and 37000, respectively). The B chain (Mr about 33000) contains thee active site of the molecule, as demonstrated by labelling with [3H]diisopropyl phosphofluoridate, and is homologous to the enzymatically active chains of thrombin, plasmin and other serine proteases. In contrast to these enzymes, the plasminogen activator is enzymatically active in the one‐chain form. A speculative explanation for this activity may possibly be the presence of an ɛ‐amino group of a lysine residue at a position close to the bond cleaved in the two‐chain form.
Interaction of a Coenzyme Analog with Aspartate Aminotransferase Isoenzymes in the Crystal
Authors: OTTONELLO Simone , MOZZARELLI Andrea , ROSSI Gian Luigi , CAROTTI Daniela , RIVA Francesca 
Published: March 2005

The interaction between the coenzyme derivative 4′‐N‐(2,4‐dinitro‐5‐fluorophenyl)‐pyridoxamine 5′‐phosphate with cytoplasmic and mitochondrial apo‐aspartate aminotransferase in the crystalline state was investigated to establish whether the structural differences, known to exist between the active sites of the two isoenzymes in solution, are maintained in the crystal although they are not apparent from the available crystallographic data.In the crystal, as in solution, both apo‐isoenzymes reversibly bind the coenzyme derivative and catalyze a slow cleavage reaction, by which pyridoxal 5′‐phosphate is produced and bound to the active‐site lysine. In the case of the cytoplasmic isoenzyme, however, in the crystal as in solution, the initial complex can follow an alternative reaction path that leads to the formation of a covalent bond between the active‐site lysine and the C‐5 of the 2,4‐dinitrophenyl moiety of the reagent.Therefore, crystal‐packing forces neither abolish the active site properties that are needed to cleave the specifically bound reagent and are common to the two isoenzymes nor mask the subtle differences that allow for the selective irreversible labeling of the cytoplasmic isoenzyme.
The Refined Structure of the Selenoenzyme Glutathione Peroxidase at 0.2‐nm Resolution
Authors: EPP Otto , LADENSTEIN Rudolf , WENDEL Albrecht 
Published: March 2005

The crystal structure of bovine erythrocyte glutathione peroxidase has been refined by a combined procedure of restrained crystallographic refinement and energy minimization at 0.20 nm resolution. The final R value at this resolution is 0.178. The r.m.s. deviation of main‐chain atoms of the two independently refined monomers is 0.019 nm. The structure at 0.28 nm resolution, which has been determined by multiple isomorphous replacement, served as a starting model.The refined model allowed a detailed survey of the hydrogen‐bonding pattern and of the subunit contact areas in the molecule. The model contains 165 solvent molecules per dimer, all taken as water molecules. The mobility of the structure was derived from the individual atomic temperature factors. The complete tetramer, including the active sites, seems to be rather rigid, except for narrow loops near to the N‐terminal ends and some β turns exposed to solvent.The active centres of glutathione peroxidase are found in flat depressions on the molecular surface. The catalytically active selenocysteine residues could be located at the N‐terminal ends of α helices forming βαβ substructures together with two adjacent parallel β strands. In the vicinity of the reactive group some aromatic amino acid side‐chains could be localized. Especially Trp‐148, which could be hydrogen bonded to SeCys‐35, may play a functional role during catalysis.The results of substrate and inhibitor binding studies in solution and in the crystalline state could be interpreted by an apparent half‐site reactivity of glutathione peroxidase. The enzyme seems to react in the sense of negative cooperativity with dimers being the functional units.Based on difference Fourier analyses of appropriate derivatives a reasonable model of glutathione binding is presented. Among the residues which could be of functional importance are Arg‐40, Gln‐130 and Arg‐167, presumably forming salt bridges and a hydrogen bond to the glutathione molecule.In conclusion, a general picture of a minimal reaction mechanism, which is in good agreement with functional and structural data, is proposed. The main reaction of the catalytic cycle presumably shuttles between the selenolate and the selenenic acid state of SeCys‐35.
p‐Hydroxybenzoate Hydroxylase from Pseudomonas fluorescens
Authors: WEIJER Wicher J. , HOFSTEENGE Jan , BEINTEMA Jaap J. , WIERENGA Rik K. , DRENTH Jan 
Published: March 2005

The complete primary and tertiary structure of p‐hydroxybenzoate hydroxylase is now known. The amino acid sequences of the two largest CNBr peptides have been fitted to the electron‐density map at 0.25‐nm resolution. The parts of the polypeptide chain contributing the residues to the FAD‐binding site and the residues of the substrate‐binding site have been identified. The active site is located in a large hydrophobic area enclosed by all domains of the enzyme structure. Here the substrate, p‐hydroxybenzoate, is bound near, but not in direct contact with, the isoalloxazine ring system of FAD.Many side chains from the C‐terminal part of the polypeptide chain are involved in subunit‐subunit interactions. In the center of one of the largely hydrophobic contact areas between the subunits, a cluster of six aromatic amino acids was found.
Purification and Properties of 2‐Aminoethylphosphonate: Pyruvate Aminotransferase from Pseudomonas aeruginusa
Authors: DUMORA Catherine , LACOSTE Anne‐Marie , CASSAIGNE André 
Published: March 2005

2‐Aminoethylphosphonate aminotransferase has been purified to homogeneity with a yield of 15% from cell extracts of Pseudomonas aeruginosa. The molecular weight of the enzyme was estimated by gel filtration to be 65000 ± 2000. Sodium dedecyl sulfate/polyacrylamide gel electrophoresis yielded a molecular weight of 16500 ± 1000, suggesting a tetrameric model for this protein. The absorption spectrum exhibits maxima at 280 nm, 335 nm and 415 nm which are characteristic of a pyridoxal‐phosphate‐dependent enzyme: 4 mol of pyridoxal 5′‐phosphate/mol of enzyme have been found.This aminotransferase catalyzes the transfer of the amino group of 2‐aminoethylphosphonate (ciliatine) to pyruvate to give 2‐phosphonoacetaldehyde and alanine. A pH optimum between 8.5–9 and an activity increasing from 30°C to 50°C have been observe. The reaction follows Michaelis‐Menten kinetics with Km values of 3.85 mM and 3.5 mM for ciliatine and pyruvate respectively.This enzyme shows a very high specificity since ciliatine and pyruvate are pyruvate are the only amino donor and acceptor respectively. Methyl, ethyl and propylphosphonic acids are better competitors towards ciliatine than their α‐amino derivatives. 3‐Aminopropylphosphonate, the higher homologue of ciliatine, is recognized neither as a substrate nor as an inhibitor. The enzyme activity is significantly affected by carbonyl reagents and by HgCl2.Transamination of 2‐aminoethylphosphonate is the first step of a double‐step pathway which leads to the cleavage of its C‐P bond.
Study of the Positive Control of the General Amino‐Acid Permease and Other Ammonia‐Sensitive Uptake Systems by the Product of the NPR1 Gene in the Yeast Saccharomyces cerevisiae
Authors: GRENSON Marcelle 
Published: March 2005

Mutations at the NPR1 genetic locus are known to inactivate (totally or partially) at least five distinct ammonia‐sensitive permeases. Mutants with thermosensitive NPR1 gene product (nprts) have been used to discriminate between three possible roles of this protein, namely (a) a common constituent of a set of ammoni‐sensitive permeases; (b) a common activator of these permeases; (c) a common positive factor necessary for their synthesis.Inactivation of the general amino‐acid permease was observed upon transfer of nprts mutant cells to a non‐permissive temperature. Under the same conditions, the general amino‐acid permease of the wild‐type cells remained active for several hours even when protein synthesis was inhibited by nitrogen starvation or by cycloheximide.Mutations at three unlinked loci, namely the PGR site (located in the GAP1 structural gene of the permease), and the unlinked MUT2 and MUT4 loci restore the general amino‐acid permease activity in npr1 mutants.The results are interpreted as indicating that the NPR1 product is necessary for the reactivation of the general amino‐acid permease which seems to be continuously inactivated by a regulatory process mediated by the MUT2 and the MUT4 gene products acting at the level of the PGR site of the general amino‐acid permease molecule.The proline permease and the ureidosuccinic‐acid permease seem to be subject to the same double regulation by inactivation‐reactivation of the permeases and by repression of their synthesis.A tentative scheme of the regulation of the general amino‐acid permease is presented.
The Relationship between Extramitochondrial Ca2+ Concentration, Respiratory Rate, and Membrane Potential in Mitochondria from Brown Adipose Tissue of the Rate
Authors: NEDERGAARD Jan 
Published: March 2005

The ability of isolated mitochondria from rat brown‐adipose tissue to regulate extramitochondrial Ca2+ (measured by arsenazo) was studied in relation to their ability to produce heat (measured polarographically). The energetic state of the mitochondria was expressed as a membrane potential, Δψ (estimated with safranine), and was varied semi‐physiologically by the use of different GDP concentrations. In these mitochondria GDP binds to the 32‐kDa polypeptide, thermogenin, which regulates coupling.Ca2+ uptake (at 5 μM extramitochondrial Ca2+) was maximal at Δψ > 150mV. Basal Ca2+ release increased from 1 to 2 nmol × min−1× mg −1 below 150 mV. Na+‐stimulated rate of Ca2+ release was stable within the investigated Δψ span (100–160 mV).Initial Ca2+ levels were maintained below 0.2 μM for 100mV < Δψ < 160 mV. Ca2+ levels maintained after Ca2+ challenge (20 nmol Ca2+× mg−1 were below 0.4μM for Δψ > 135 mM.Respiration was unstimulated for Δψ > 150 mV and was maximal at Δψ≤ 135mV. In the presence of well‐oxidised substrates, the respiration at maximally activated thermogenin was markedly below fully uncoupled respiration and was probably limited by thermogenin activity–i.e. by a limited H+ reentry (OH− exit) and therefore by a membrane potential maintained at about 135 mV.It is concluded that at membrane potentials of 135 mV and above the mitochondria exhibit full Ca2+ control and are able to regulate thermogenic output up to maximum without interfering with this Ca2+ control. Membrane potential probably does decrease below 135 mV in vivo.Therefore, Ca2+ homeostasis and thermogenesis are non‐interfering and can be hormonally independently regulated, e.g. by α‐adrenergic and β‐adrenergic stimuli, respectively.
Isolation and Characterization of Multiforms of Aldehyde Reductase in Chicken Kidney
Authors: HARA Akira , DEYASHIKI Yoshihiro , NAKAYAMA Toshihiro , SAWADA Hideo 
Published: March 2005

Three multiforms of NADPH‐dependent aldehyde reductase have been purified to homogeneity from chicken kidney. The enzymes were monomeric proteins with similar molecular weights around 39000 but with different pI values. Two of them exhibited, almost the same heat stability, a broad optimal pH of 6–6.5 and preference for NADPH as a cofactor. They were high‐Km aldehyde reductases which reduced various aldehydes, hexonates, α‐diketones, xylose, glucuronolactone and ethyl acetoacetate, and which were inhibited to similar degrees by heavy metal ions, organic acids, p‐chloromercuribenzoate, indomethacin and phenobarbital. The third aldehyde reductase was distinct from the other two enzymes in its heat lability, sharp acidic pH optimum of 6.0, and dual cofactor specificity. The enzyme showed low Km values for the above‐mentioned substrates except hexonates and 4‐carboxybenzaldehyde, and it specifically reduced lauryl aldehyde, acetoin and ketosteroids. This enzyme was less sensitive to the above‐mentioned inhibitors than the high‐Km aldehyde reductases, but the enzyme was moderately inhibited by chlorpromazine and was activated about 1.5 times by the addition of sulfate ions.It was demonstrated using antibodies against one of the high‐Km forms and the low‐Km form of aldehyde reductase that the two high‐Km forms were immunologically identical with each other but not with the low‐Km form, and that the high‐Km and low‐Km forms were widely distributed in various tissues of chicken.
Hepatocyte Heterogeneity in Glutamine and Ammonia Metabolism and the Role of an Intercellular Glutamine Cycle during Ureogenesis in Perfused Rat Liver
Authors: HÄUSSINGER Dieter 
Published: March 2005

The metabolism of glutamine and ammonia was studied in isolated perfused rat liver in relation to its dependence on the direction of perfusion by comparing the physiological antegrade (portal to caval vein) to the retrograde direction (caval to portal vein).
Added ammonium ions are mainly converted to urea in antegrade and to glutamine in retrograde perfusions. In the absence of added ammonia, endogenously arising ammonium ions are converted to glutamine in antegrade, but are washed out in retrograde perfusions. When glutamine synthetase is inhibited by methionine sulfoximine, direction of perfusion has no effect on urea synthesis from added or endogenous ammonia.
14CO2 production from [1‐14C]glutamine is higher in antegrade than in retrograde perfusions as a consequence of label dilution during retrograde perfusions.
The results are explained by substrate and enzyme activity gradients along the liver lobule under conditions of limiting ammonia supply for glutamine and urea synthesis, and they are consistent with a perivenous localization of glutamine synthetase and a predominantly periportal localization of glutaminase and urea synthesis. Further, the data indicate a predominantly periportal localization of endogenous ammonia production. The results provide a basis for an intercellular (as opposed to intracellular) glutamine cycling and its role under different metabolic conditions.
Acyl‐Coenzyme A: Cholesterol Acyltransferase
Authors: SYNOURI‐VRETTAKOU Stella , MITROPOULOS Konstantinos A. 
Published: March 2005

The preincubation at 37°C of rat liver microsomal fraction, followed by re‐isolation of the treated vesicles, results in a time‐dependent increase in the activity of acyl‐CoA: cholesterol acyltransferase. The presence of cholesterol‐phospholipid (1:1, mol/mol) liposomes results in higher rate of increase in activity and under these conditions the rate of increase is liposomal cholesterol concentration‐dependent.The preincubation of the microsomal fraction in the presence of [3H]cholesterol‐phospholipid liposomes results in transfer of [3H]cholesterol to the re‐isolated microsomal vesicles and this transfer follows first‐order kinetics in respect to the donor concentration. These preincubations result also in a time‐dependent and liposomal cholesterol concentration‐dependent increase in the incorporation of [3H]cholesterol into the cholesteryl oleate produced on assay of cholesterol acyltransferase activity. From specific radioactivity data of the cholesteryl esters synthesised on assay of cholesterol acyltransferase in treated microsomal preparations, the rate of liposomal [3H]cholesterol equilibration with the cholesterol acyltransferase substrate pool can be calculated. The half‐time of this transfer decreased with the concentration of liposomal cholesterol present during the preincubation. The activation energy for the transfer of liposomal cholesterol to the cholesterol acyltranferase substrate pool was 87.9 kJ/mol and was independent of the concentration of liposomal cholesterol. The activation energy for the rate of increase of total cholesteryl oleate was similar to this value for low concentrations of liposomal cholesterol and progressively decreased with increasing concentrations of liposomal cholesterol. The liposomal cholesterol and progressively decreased with increasing concentrations of liposomal cholesterol. The data suggest that under the present conditions, the time‐dependent and temperature‐dependent increase in cholesterol acyltransferase activity is due to the transfer of non‐esterified cholesterol from other microsomal and/or liposomal vesicles to the vesicles that contain the enzyme and therefore to increased availability of substrate.
The Phytochrome‐Controlled Accumulation of mRNA Sequences Encoding the Light‐Harvesting Chlorophyll a/b Protein of Barley (Hordeum vulgare L.)
Authors: GOLLMER Ingrid , APEL Klaus 
Published: March 2005

Double‐stranded cDNA was synthesized from polysomal poly(A)‐containing RNA of illuminated barley plants and was inserted into the PstI site of the bacterial plasmid pBR322. Two different strategies were used for the screening of the bacterial colonies. Light‐regulated sequences were detected by differential hybridization with cDNA of polyadenylated RNA from dark‐grown and illuminated barley plants. For a second screening step partially purified mRNAs encoding the light‐harvesting chlorophyll a/b protein were used for the synthesis of another cDNA probe. By using these procedures a cDNA clone was isolated which encodes a constitutive polypeptide of the light‐harvesting chlorophyll a/b protein.This cloned cDNA has been used to assess the effect of phytochrome on the steady‐state level of mRNA sequences encoding the light‐harvesting chlorophyll a/b protein. The mRNA is almost undetectable in dark‐grown plants. Following treatment with red light, the concentration of this mRNA sequence increases rapidly during the subsequent dark period. This red light effect can be reversed substantially by irradiation with far‐red light. These results indicate that not only the amount of mRNA activity as shown previously, but also the steady‐state level of mRNA sequences encoding chrome. the light‐harvesting chlbrophyll a/b is contrdlled by phytochrome.
Neutron Diffraction of Chromatin in Interphase Nuclei and Metaphase Chromosomes
Authors: IBEL Konrad , KLINGHOLZ Reiner , STRÄTLING Wolf H. , BOGENBERGER Jakob , FITTLER Friedrich 
Published: March 2005

We have used neutron diffraction to study chromatin structure in interphase nuclei and metaphase chromosomes as a function of decreasing ion concentration.Aliquots of a suspension of rat liver nuclei prepared in a polyamine‐free buffer were washed in buffers of 1/3, 1/6 and 1/12 if the original concentration of monovalent and divalent cations (40 mM KC1; 20 mM NaCl; 1.2 mM MgCl2). After the first dilution step (l/1 to 1/3), only small changes occurred in the diffraction pattern. They can be interpreted by a loosening of the original structure, i.e. by the formation of isolated buffer‐filled spaces with an overall size of the order of 35 ‐ 45 nm. Drastic changes in the diffraction pattern were observed, however, when the nuclei were washed in the more diluted buffers (1/6 and 1/12). The profiles of the distances distribution functions indicate the formation of supranucleosonial particles with an overall diameter of 40–50 nm. The compact chromatin structure disassembled directly into these fundamental structural units.Structural transformations in the Chinese hamster ovary metaphase chromosomes were induced by diminishing the Ca2+ ion concentration of the buffer from originally 3.0 mM to 0.3 mM and/or by increasing the pH value of the buffer from originally 7.0 up to 8.0. The neutron diffraction patterns remained essentially unchanged during these treatments, i.e. the decondensation of the chromosomes as observed in the light microscope is not accompanied by disassembly at the ultrastructural level between 2 nm and 150 nm.