bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multi‐Enzyme Complex of Glutamine‐Dependent Carbamoyl‐Phosphate Synthetase with Aspartate Carbamoyltransferase and Dihydroorotase from Rat Ascites‐Hepatoma Cells
Authors: MORI Masataka , TATIBANA Masamiti 
Published: March 2005

Glutamine‐dependent carbamoyl‐phosphate synthetase, the first enzyme of de novo pyrimidine nucleotide biosynthesis, was purified 1300‐fold from Yoshida ascites hepatoma cells (AH 13) as a multi‐enzyme complex with aspartate carbamoyltransferase and dihydroorotase, using dimethyl sulfoxide, glycerol and dithiothreitol as enzyme stabilizers. The ratios of the three enzyme activities were initially 1:65:9.2 and were essentially constant throughout the purification, and the final fraction had ratios of 1:37:11 (dihydroorotase assayed in the forward direction). The purified complex had a specific activity of the synthetase of 0.51 μmol × min−1× (mg protein)−1. It was essentially homogeneous on agarose/acrylamide composite gel electrophoresis, analytical ultra‐centrifugation, and electrofocusing; it had an apparent pI of 5.5. The complex had a sedimentation coefficient (s20, w) of 24.6 S in the presence of 30% (v/v) dimethyl sulfoxide and 5% (w/v) glycerol, and its molecular weight was estimated to be 870000 by sedimentation equilibrium studies.On polyacrylamide gel electrophoresis in sodium dodecyl sulfate the complex migrated as a single protein band with a molecular weight of 210000, indicating that the complex is composed of four to five subunits of similar size. Sucrose gradient centrifugation in the presence of 1.5 M sodium chloride partially dissociated the three enzyme activities. Limited proteolysis of the complex with pancreatic elastase caused its dissociation and made separable the three activities. The synthetase was recovered as a 197000‐molecular‐weight fragment and the carbamoyltransferase as a 145000‐molecular‐weight fragment. Dihydroorotase was initially recovered as a 197000‐molecular‐weight fragment and was further converted to a 43000‐molecular‐weight fragment. It appears difficult to reconcile these results with the view that the three enzyme activities are located on the same polypeptide chain.
Cytochrome c from Schizosaccharomyces pombe
Authors: SIMON‐BECAM Anne‐Marie , CLAISSE Maurice , LEDERER Florence 
Published: March 2005

The amino acid sequence of Schizosaccharomyces pombe cytochrome c has been established by automatic degradation of the protein and by manual degradation of fragments obtained by cyanogen bromide cleavage and chymotryptic digestion. The chymotryptic peptides were aligned by homology with other known cytochrome c sequences. The protein is 108 residues long, with a four‐residue amino‐terminal tail. It has only one methionine residue and differs from other fungal cytochromes c in lacking the one‐residue deletion at the C‐terminal end.After a cyanogen bromide step, an unexpected cleavage of the peptide chain before a cysteine residue was observed. This is ascribed to formation of a dehydroalanyl residue during an incomplete S‐carboxymethylation of the apoprotein, and subsequent cleavage under acidic conditions. Experimental evidence is presented in favour of the proposed mechanisms.
Biosynthesis of Thromboxane B2 and 12‐l‐Hydroxy‐5,8,10‐heptadecatrienoic Acid in Human Platelets
Authors: RAZ Amiram , AHARONY David , KENIG‐WAKSHAL Rina 
Published: March 2005

Human platelet microsomes convert prostaglandin H2 to thromboxane B2 and 12‐l‐hydroxy‐5,8,10‐heptadecatrienoic acid (12OH‐17:3) in approximately equimolar amounts. The synthesizing activities of both products appear to go in parallel, both activities gradually decline upon storage and are equally destroyed by heat inactivation. Furthermore imidazole, a potent inhibitor of thromboxane synthetase activity, is an equally effective inhibitor of 12OH‐17:3 formation in platelets. These results suggest that both thromboxane B2 and 12OH‐17:3 are derived from a common intermediate.We propose two alternative pathways for the conversion of prostaglandin H2 to thromboxane A2 and 12OH‐17:3 in human platelets. The first pathway depicts thromboxane A2 as the common intermediate for the formation of both thromboxane B2 and 12OH‐17:3. In the second pathway, prostaglanding H2 is converted to an activated intermediate which is converted to either thromboxane A2 or 12OH‐17:3.
The Acceptor for Polar Head Groups of the Lipid A Component of Salmonella Lipopolysaccharides
Authors: LEHMANN Volker , REDMOND John , EGAN Aubrey , MINNER Inge 
Published: March 2005

We describe here experiments which determine at which stage in the lipid A biosynthesis the polar head groups 4‐aminoarabinose, phosphorylethanolamine and 3‐deoxy‐d‐manno‐octulosonic acid are transferred to the diphosphorylated glucosamine backbone of the lipid A structure. Use was made of a conditional lethal mutant of Salmonella typhimurium (Ts1) which is defective in the synthesis of 3‐deoxy‐d‐manno‐octulosonic acid 8‐phosphate and accumulates under nonpermissive conditions an underacylated lipid A intermediate [Lehmann, Rupprecht and Osborn (1977) Eur. J. Biochem. 76, 41–49]. Pulse‐chase experiments, including a detailed analysis of radioactive pulse and chase products, demonstrated that this underacylated compound is a key intermediate in the lipid A synthesis. It can serve as direct acceptor for the incorporation of the polar head groups 4‐aminoarabinose, phosphorylethanolamine and 3‐deoxy‐d‐manno‐octulosonic acid.On the basis of these findings some steps in the sequence of reactions involved in the lipid A biosynthesis are proposed.
Hormonal Regulation of Phosphoenolpyruvate Carboxykinase in Primary Cultures of Adult‐Rat Liver Parenchymal Cells
Authors: OLIVER Ivan T. , EDWARDS Anthony M. , PITOT Henry C. 
Published: March 2005

Adult rat liver parenchymal cells, maintained in primary monolayer culture on floating collagen gels, were used for studies on the hormonal regulation of phosphoenolyruvate carboxykinse. In cells maintained in a defined, serum‐free medium containing insulin but no other hormones, phosphoenolpyruvate carboxykinse activity expressed on the basis of cell DNA content decreased over 3 days in culture with a half‐life of 9–10 h. Treatment of cells with glucagon or N6, O2′‐dibutyryladenosine 3′:5′ monophosphate (Bt2‐cAMP) caused an increase in enxyme activity up to 4‐fold higher than in untreated cells. There was little or no response during the first 24 h of culture but addition of either glucagon or Bt2‐cAMP after this period resulted in significant enzyme induction within 3 h. When cells were maintained for 2–3 days after attachment directly to palstic culture dishes rather than to collagen gels, similar inductive responses to glucagon or Bt2‐cAMP were observed. Induction by glucagon was shown to be dose‐dependent; a significant response was evident at 0.1 nM gucagon.Addition of epinephrine to cultured cells at concentration between 10 nM to 0.1 mM was without effect on phosphoenolpyruvate carboxykinase. Induction by Bt2‐cAMP was completely prevented by the simultaneous addition of actinomycin D (0.2 μg/ml) or cordycepin (10 μg/ml) suggesting that the nucletide acts or transcription to increase phosphoenolpyruvate carboxykinase synthesis.
Identification of Some Metabolic Products of 5′‐Deoxy‐5′‐S‐Isobutylthioadenosine, an Inhibitor of Virus‐Induced Cell Transformation
Authors: LAWRENCE Franqoise , RICHOU Mireille , VEDEL Michde , FARRUGIA Giselle , BLANCHARD Pierre , ROBERT‐GERO Malka 
Published: March 2005

5′‐Deoxy‐5′‐S‐isobutylthiodenosine (iBuS)5′ Ado has been shown to be rapidly degraded to 5‐deoxy‐5‐S‐isobutylthioribose and adenine in procaryotes. In chick embryo fibroblasts there are two metabolic pathways for (iBuS)5′ Ado degradation: (a) oxidative deamination into 5′‐deoxy‐5′‐S‐isobutylthioinosine (the main product) and (b) hydrolysis into 5‐deoxy‐5‐S‐isobutylthioribose plus adenine. The latter reaction is not due to bacterial contamination, since the same results were obtained under sterile conditions and in chick embryo fibroblasts in culture.The inhibition of the virus‐induced cell transformation reported by us previously was due to (iBuS)5′ Ado rather than to the main metabolic product of this molecule in chick embryo fibroblasts.
Distribution of AMP‐Deaminase Isozymes in Rat Tissues
Authors: OGASAWARA Nobuaki , GOTO Haruko , YAMADA Yasukazu , WATANABE Tomomasa 
Published: March 2005

The distribution of AMP deaminase isozymes in rat tissues was analyzed by electrophoresis on cellulose acetate membrane, by chromatography on phosphocellulose column, and by the application of immunological technique empolying specific antisera against three parental AMP deaminases (isozymes A, B and C). Skeletal muscle extracts and diaphragm extracts contain a single identical isozyme, isozyme A. The major isozyme species of liver, kindney and testes are also identical and they are isozyme B. Heart extracts contains isozyme C exclusively. Extracts of brain, lung and spleen contain five isozymes, presumably a complete set of five B–C hybrids.
Developmental patterns of AMP deaminase isozyme were studied. In early postnatal life, extracts of heart, liver, kidney ad lung contain five isozymes similar to those observed in adult brain. During postnatal development, a shift to isozyme C occurs in heart, whereas a shift to isozyme B occurs in liver and kidney. Five isozymes in lung remain throught development. In brain a shift ofB to five isozymes is observed during de3velopment. Isozyme A is the predominant form in muscle throught postnatal development.
AMP deaminase in the regenerating liver was analyzed, but the data indicated that thre was no change of isozyme distribution during hepatic regeneration.
The Synthesis of a Photoreactive Puromycin Analogue and Its Application for Labeling Proteins in the 50‐S Subunit of Escherichia coli Ribosomes
Authors: KRASSNIGG Franz , ERDMANN Volker A. , FASOLD Hugo 
Published: March 2005

A photoreactive puromycin analogue, 6‐dimethylamino‐9‐[3‐(p‐azido‐l‐β‐phenlalanylamino)‐3‐deoxy‐β‐ribofuranosyl] purine, was synthesized. Biological activity was demonstrated by inhibition of the poly(U)‐directed phenylalanine‐incorporation system and by decomposition of isolated polysoomes from Escherichia coli.The 3H‐labeled puromycin analogue was covalently attache to the 50‐S subunit of isolate 70‐S ribosomes from Escherichia coli after irradiation. More than 90% of the radioactivity was bounmd to the protein fraction. The 70‐S proteins were separted by two‐dimensional gel electrophoresis. The proteins labeled primarily were those of the 50‐S subunit, identified as L6, L13, L18, L22 and L25. On the basis of the affnity label used and supportive data from the literature, it is concluded that these proteins are at the active center of the 50‐S particle and probably bellong to the region of the ribosomal A site.
Limited Tryptic Digestion of Human Serum Low‐Density Lipoprotein: Islation and Characterisation of the‐Deficient Particle and of Its Apoprotien
Authors: CHAPMAN M. John , GOLDSTEIN Sonia , MILLS Gervase L. , LAGRANGE D. , TAYLAUR C. E. 
Published: March 2005

Limited tryptic digestion of human serum low‐density (LD) lipoprotein (ϱ 1.024–1.045 g/m) under defined conditons permitted isolation by gel filtration chromatography of a stable, protein‐deficient lipoprotein; the liberated protein was separated as a mixture of peptedes of low molecular weight (< 5000). Comparison of the chemical, physical and immunological characterstics of the trypsin‐trated LD‐lipoprotein with those of the native preparation revealed several differences, including (a) a diminished protein content (loss of some 20–25% of the total protien of LD‐lipoprotein) and increased proportions of the various components, except for triglyceride (probably resulting from a loss of bound free fatty acids with the liberated peptides); (b) a greater heterogeneity in particle size and slightly larger mean diameter; (c) a lower hydrated density and greater peak sf rate than the native LD‐lipoprotein (d) an increased net negative charge; and (e) a partial immunological idensity between LD‐lipoprotein and the corresponding trysin‐treated fraction.While the amino acid compositions of the protein moieties of LD‐lipoprotein and of trypsin‐treated LD‐lipoprotein were essentially identical, trypsin‐treated apo‐LD‐lipoprotein was distinct in its complete solubility in urea‐containing buffers at high concentrations, and also in its partial solubility in buffers lacking denaturing agents.Comparison of the apoproteins of the native and trypsin‐treated LD‐lipoproteins by electrophoretic techniques based on molecular weight revealed a transformation of the high‐molecualar weight material (> 250000) characterstic of apo‐LD lipoprotein into several polypeptide species (10 major forms) ranging in size form 161500 to about 10000. The largest of these (band b1: 161500) could be completely dissociated into smaller components (b2: 93500 and b3: 77000) upon extensive heat treatement at 90°C. Elecstrophoresis of the soluble fraction of apo‐LD‐lipoprotien and of that from its trysin‐treated counterpart in polyacylamide gels containing urea at basic pH showed the didapperance of the small amounts (< 5%) of C apoproteins of apo‐LD‐lipoprotein upon tryptic treatment. These results, which were hightly reproducible in LD‐lipoprotein prepartions from different individuals, suggest that trypsin‐treated LD‐lipoprotein may provide a model for investigation of the organisation and structural role of the principal apoprotein (apoprotein‐B) in the LD‐llipoprotein molecule.
Influence of Magnesium on the Steady‐State‐Derived Order of Substrate Addition and Product Release in tRNA Trp Aminoacylation by Beef Pancreas Tryptophan: tRNA Ligase
Authors: MÉRAULT Guy , GRAVES Pierre‐Vincent , LABOUESSE Bernard , LABOUESSE Julie 
Published: March 2005

The order of the partial reactions in the aminoacylation of tRNATrpbeef by tryptophan : tRNA ligase from beef pancreas has been explored by measurements of intial velocity and product inhibition according to the analytical framework described by Cleland [Biochim. Biophys. Acta. 67, 104– 137, 173– 187, 188– 196 (1963)]. The kinetic patterns are suggestive of a ping‐pong mechanism (bi‐uni uni‐bi). The oreder of addition of tryptophan and ATP that one can derive is crucially dependent on the conditions of magnesium concentration used. Random fixation of tryptophan and ATP is observed in studies performed at catalytically optimal magnesium concentration. Linear‐ordered fixation of the two substrates, ATP binding first, is observed under conditions where magnesium concentration is in excess, which are not catalytically optimal. The analysis of the inhibition patterns taking into account the calculated concentrations of teh main active species Mg · ATP2− and Mg · P2O2−7 favors the random substrate‐binding mechanism derived under optimal magnesium conditions. Under both conditions, adjusted and fixed magnesium, the release of products, AMP and tryptophanyl‐tRNATrpbeef is, ordered, AMP released first.The change in the product nhibition patterns of aminoacylation of tRNATrpbeef by beef pancreas tryptophan: tRNA ligase under different magnesium concentration conditions casts some doubt on the significance of the comparison of mechanisms derived for amino‐acid: tRNA ligases of different sources.
Adenosine 3′:5′‐Monophosphate and Guanosine 3′:5′‐Monophosphate: Levels and Cyclase Activities in Liver and Adipose Tissue from Diabetic Mice (db/db)
Authors: LEVILLIERS Jacqueline , PAIRAULT Jacques , LECOT Françoise , TOURNEMOLLE Annie , LAUDAT Marie‐Hélène 
Published: March 2005

The adenylate and guanylate cyclase activities and the adenosine and guanosine 3′:5′‐monophosphate (cyclic AMP and cyclic GMP) contents have been investigated in the liver and adipose tissue of control homozygote (+m/+m), heterozygote (db+/+m) and diabetic homozygote (db+/db+) mice (C57BL/Ks‐J‐db m strain).The response to (−)‐isoproterenol of the adenylate cyclase system in isolated fat cells, as well as in adipocyte plasma membranes, from diabetic mice (db+/db+), is impaired as compared to their thin littermates (+m/+m) and also to heterozygote (db+/+m) mice. The increase in cyclic AMP formation elicited by (−)‐isoproterenol was 10‐fold and 4‐fold higher in isolated adipocytes and in adipocyte plasma membranes respectively from lean control as compared to diabetic mice. The cyclic AMP content of adipose tissue from diabetic mice (44.0 ± 7.6 fmol per mg of wet weight) was markedly lower than that from control and heterozygote mice (128.3 ± 41.5 and 128.4 ± 34.5 fmol per mg of wet weight, respectively). In contrast the basal adenylate cyclase activities of liver homogenates and the activties stimulated by glucagon, epinephrine and fluoride were similar in the three groups of animals. Similarly, the hepatic cyclic AMP content did not differ in the three genotypes of mice. These findings indicate that disturbances in cyclic AMP metabolism in adipose tissue from diabetic mice might result from a phenomenon secondary to the diabetic syndrome.The specific activity of guanylate cyclase in liver and adipocytes progressively decreased from lean control (+m/+m) to heterozygote (db+/+m) to homozygote diabetic (db+/db+) mice; e.g. the guanylate cyclase activity in adipocyte plasma membranes was markedly diminished in diabetic mice (18.9 ± 1.9, 12.7 ± 0.9 and 4.7 ± 1.4 pmol cyclic GMP · min−1· (mg protein)−1 for homozygote control, heterozygote and diabetic mice, respectively). A similar situation was noted in respect of the cyclic GMP levels in the liver and adipose tissue of these animals. Thus, the cyclic GMP content in tissues from diabetic mice represented only 13% and 43% of that in the liver and adipose tissue respectively of control mice. These data are consistent with the existence of a dosage effect of the db gene on cyclic GMP metabolism. On the other hand identical maximal guanylate cyclase activities were obtained after preincubation with Triton X‐100 of adipocyte plasma membranes from the three kinds of mice. At least, there was a 19‐fold and 2.5‐fold stimulation of guanylate cyclase activity by sodium azide in liver homogenates from homozygote diabetic mice and homozygote control, respectively; the hypersensitivity to NaN3 of guanylate cyclase activity in liver and adipose tissue could be related to a disturbance in peroxide metabolism in the tissues of the mutant.
Isolation and Characterization of the Hemoglobin from the Lanceolate Fluke Dicrocoelium dendriticum
Authors: TUCHSCHMID Peter E. , KUNZ Peter A. , WILSON Kenneth J. 
Published: March 2005

The hemoglobin of the flatworm Dicrocoelium dendriticum, a lanceolate fluke which infests the hepatic ducts of certain mammals, has been isolated by gel filtration and ion‐exchange chromatography. The molecular weight of the denatured protein was found to be 15500, a value in the same range as hemoglobin subunits. The fact that the native hemoglobin has an apparent molecular weight of 22000 in 0.01 M phosphate buffer, pH 7.4, suggests limited aggregation. The protein contains, as all other myoglobins and hemoglobins, one molecule of non‐covalently associated ferroprotoporphyrin IX per polypeptide chain. It forms the same ligand derivatives with very similar spectral properties as vertebrate hemoglobins. The high oxygen affinity (p50 is 0.07–0.1 mmHg or 9.3–13.3 Pa at 20 °C and pH 7.0) and the absence of heme‐heme interaction (Hill coefficient nH= 1.0) are properties which this heme protein shares with other monomeric hemoglobins from invertebrate and lower vertebrate organisms.The native hemoglobin exists in two forms, having isoelectric points of 4.51 and 4.53, which do not differ in their amino‐acid compositions. Dansylation indicated that the amino‐terminal amino‐acid residue is alanine. The carboxy‐terminal sequence, determined by carboxypeptidase A digestion of the globin, is ‐His‐Ala‐Leu.
Alanine Metabolism in Rat Liver Mitochondria
Authors: DIETERLE Peter , BRAWAND Fritz , MOSER Ulrich K. , WALTER Paul 
Published: March 2005

In the presence of 2‐oxoglutarate, bicarbonate, Pi and ATP, rat liver mitochondria were found to metabolize alanine. The main products were glutamate, aspartate, malate and citrate. Pyruvate did not accumulate but reached a low steady‐state concentration. Addition of octanoate caused a strong stimulation of alanine metabolism and a concomitant decrease in pyruvate concentration. NO inhibition of alanine metabolism was obtained in the presence of inhibitors of mitochondrial pyruvate transport, α‐cyano‐4‐hydroxy‐cinnamate and α‐cyano‐cinnamate, showing that alanine was not transaminated by a contamination of the mitochondria by cytosolic alanine aminotransferase. In the presence of l‐cycloserine, alanine metabolism in mitochondria was strongly inhibited indicating that in contrast to earlier reports l‐cycloserine can enter the mitochondria.In isolated liver cells, addition of α‐cyano‐cinnamate caused a strong pyruvate accumulation and a nearly complete inhibition of gluconeogenesis with serine as substrate, whereas with alanine no pyruvate increase and a weak inhibition of gluconeogenesis was observed.From a comparison of the employed substrate concentrations and of the observed rates of alanine transamination in intact mitochondria with those under physiological conditions in cellular systems, it is concluded that cellular conversion of alanine to pyruvate most likely occurs to a great extent within the mitochondria as has been proposed earlier by DeRosa and Swick (1975) J. Biol. Chem. 250, 7961‐7967.
Incorporation Rates of [1‐14C]Glycerol into the Molecular Species of Alkyl Ether Phospholipids of Ehrlich Ascites Tumor Cells in vivo
Authors: WAKU Keizo , NAKAZAWA Yasuo 
Published: March 2005

[1‐14C]Glycerol was injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor cells and the lipids were extracted from the cells after selected periods. 1‐O‐Alkyl‐2‐acyl‐3‐acetyl‐glycerols and 1,2‐diacyl‐3‐acetylglycerols were prepared from the ethanolamine and choline phosphoglycerides and fractionated by AgNO3‐impregnated thin‐layer chromatography into seven molecular species. Bands 1‐7 obtained in this way can be designated as saturated, monoene, diene (11), diene (02), triene, tetraene and pentaene + hexaene species, respectively. Band 7, composed mainly of hexaene species, was predominant among those derived from ether phospholipids. The predominant species was band 4 (saturated/linoleate) in both diacyl ethanolamine and choline phospholipids.The specific radioactivities of the fractionated species were determined. In diacyl or alkyl ether compounds of ethanolamine and choline phosphoglycerides, disaturated species turned over very rapidly, while in alkyl ether ethanolamine phosphoglyceride, hexaene + pentaene molecular species turned over rapidly. In diacyl compounds of the ethanolamine phosphoglyceride, hexaene molecular species also turned over at a high rate. In diacyl ethanolamine or choline phosphoglycerides, dienoic species turned over rather slowly.