bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of ovariectomy and resistance training on lipid content in skeletal muscle, liver, and heart; fat depots; and lipid profile
Authors: Leite Richard Diego , Prestes Jonato , Bernardes Celene Fernandes , Shiguemoto Gilberto Eiji , Pereira Guilherme Borges , Duarte Josiane Oliveira , Domingos Mateus Moraes , Baldissera Vilmar , de Andrade Perez Sérgio Eduardo
Published: December 2009

The aim of the present study was to investigate the effects of resistance training on skeletal muscle lipid content, liver lipid content, heart lipid content, fat depots, and lipid profile in ovariectomized rats. Wistar adult female rats were divided into 4 groups (n = 10 per group): sedentary (Sed-Intact), sedentary ovariectomized (Sed-Ovx), strength trained (ChronicEx-intact), and strength trained ovariectomized (ChronicEx-Ovx). A 12-week strength-training period was used, during which the animals climbed a 1.1-m vertical ladder with weights attached to their tails. The sessions were performed once every 3 days, with 4–9 climbs and 8–12 dynamic movements per climb. Ovariectomy increased liver lipid content and fat depots, and heart and muscle lipid content. There was an increase in the atherogenic index and a negative change in lipid profile because of the ovariectomy. Resistance training decreased lipid content in the liver, soleus, and tibialis anterior, decreased fat depots (mesenteric and retroperitoneal), and changed the lipid profile, independently of ovarian hormone status. These results indicate the potential benefits of resistance training as an alternative strategy to control the effects of ovariectomy on fat depot, lipid profile, and tissue lipid content.
Mathematical modeling and simulation of hexane degradation in fungal and bacterial biofilters: effective diffusivity and partition aspectsThis article is one of a selection of papers published in this Special Issue on Biological Air Treatment.
Authors: Arriaga Sonia , Revah Sergio
Published: December 2009

Mathematical modeling in the biofiltration of volatile organic compounds is a valuable tool for performance prediction and in scaling up. Majority of the published models include parameters obtained from fitting experimental data, thus masking their real influence as they are lumped generally. The present work aims to evaluate experimentally some of the most relevant parameters including kinetic constant, partition coefficient in the biofilm, biofilm thickness, superficial area, and effective diffusivity. For the fungal biofilm, all the parameters mentioned above were obtained experimentally; and for the bacterial biofilm, the biofilm thickness and some intrinsic parameters used to obtain the first-order kinetic constant were taken from the literature. These parameters were then incorporated in a mathematical model to describe the steady-state degradation of hexane in bacterial and fungal biofilters operating under continuous mode. Experimentally, the dimensionless partition coefficients (mG) indicated that hexane was 4 and 35 times more soluble in the bacterial (mG = 9.14) and fungal (mG = 0.88) biofilters, respectively, than in water (mG = 30.4). Comparison of model estimates with experimental concentration profiles of the pollutant along the height of the biofilters proves that the first-order limited by reaction model was appropriate to interpret the experimental results with a small error of ∼1%.
Waste gas treatment in bioreactors: environmental engineering aspectsThis article is one of a selection of papers published in this Special Issue on Biological Air Treatment.
Authors: Kennes Christian , Montes María , López M. Estefanía , Veiga María C.
Published: December 2009

This paper gives an overview of the most important bioreactor configurations used in waste gas treatment, i.e., conventional and trickling biofilters, the bioscrubber, suspended-growth bioreactors, as well as two-liquid phase systems (stirred tank bioreactor and biotrickling filter). The historical evolution, main characteristics, and recent developments are described in each case.
Catalytic removal of propene and toluene in air over noble metal catalystThis article is one of a selection of papers published in this Special Issue on Biological Air Treatment.
Authors: Benard S. , Ousmane M. , Retailleau L. , Boreave A. , Vernoux P. , Giroir-Fendler A.
Published: December 2009

In this paper we present the study of catalytic oxidation of volatile organic compound (VOC) traces in air over Pt/γ-Al2O3 catalysts. The study was carried out using a laboratory fixed-bed catalytic reactor (FBCR) with catalytic oxidation of propene and toluene as model reactions. For better understanding of catalytic activity in a FBCR, the influence of operating parameters such as of platinum (Pt) loading, total gas flow rate, VOC concentration, and oxygen content were studied. The results showed that catalytic activity increases with increase in VOC concentration and a decrease in total gas flow rate. This displayed behaviour is instrumental to working at a low temperature region for complete oxidation of volatile organic compounds.
Traitement des lisiers de porc par un procédé de biofiltration : Étude de formation du méthaneLe présent ouvrage fait partie d'une série d'articles publiés dans ce numéro spécial sur le traitement biologique de l'air.
Authors: Lessard P. , Bourgault C. , LeBihan Y. , Turgeon N. , Aubry G. , Buelna G.
Published: December 2009

Il est admis que la production de méthane (CH4) à partir de la décomposition de la matière organique est effectuée par des microorganismes méthanogènes en condition anaérobie. C’est pour cette raison que le CH4, un gaz à effet de serre (GES) possédant un pouvoir de réchauffement global (PRG) équivalent à 25 fois celui du CO2, n’est généralement pas considéré dans les protocoles de quantification de GES concernant les procédés de traitement d’eaux usées par voie aérobie. Or, selon le type de procédé aérobie et les conditions d’opération, du méthane peut effectivement être produit. Cette note technique montre des résultats obtenus lors d’essais pilotes en laboratoire avec un biofiltre traitant du lisier de porc. Il en ressort clairement que la formation de méthane est possible avec ce type de procédé, et que les concentrations générées peuvent être non négligeables. Par conséquent, cet élément doit donc être pris en compte pour l’optimisation des procédés de traitement aérobie en rapport aux aspects liés à la réduction des GES et la lutte aux changements climatiques.
Characterization techniques of packing material colonization in gas biofiltration processesThis article is one of a selection of papers published in this Special Issue on Biological Air Treatment.
Authors: Andres Yves , Dumont Eric , Gerente Claire
Published: December 2009

Biological air treatment is largely used for the decontamination of large air streams with low pollutant concentrations. As a result of microbial metabolism, the pollutants are degraded into carbon dioxide and water or converted into cell biomass. Three main research domains have emerged within the field of biofiltration: filter bed development, process optimization, and microbiological studies (community, nutrients, …). The objective of this paper is to focus on the microbial activities in air biofiltration processes. The nutrient contribution and the packing material effects are described. Special attention is paid to the description of techniques for the characterization and identification of the microbial population.
Cytoplasmic compartmentation of glucose 6-phosphate
Authors: Marcus O. , Kalant N.
Published: December 2009

When rat diaphragm, intact or homogenized, was incubated with [14C]glucose, the initial specific activity of the fructose 6-phosphate was higher than that of glucose 6-phosphate (glc-6-P). This relationship was also found when the cytosol was incubated with the labeled glucose. The early time course of production of glc-6-P from glucose and its removal provide evidence that newly synthesized glc-6-P does not equilibrate rapidly with the total pool of this metabolite and is preferentially removed by isomerization. It is concluded that glc-6-P is functionally compartmented within the cytoplasm.
Morphine-like activity of sheep β-lipotropin and of its tryptic fragments
Authors: Seidah Nabil G. , Lis Martin , Gianoulakis Christina , Routhier Richard , Benjannet Suzanne , Schiller Peter W. , Chretien Michel
Published: December 2009

Sheep β-lipotropin (β-LPH) (sequence 1–91) was selectively cleaved with trypsin after blocking the ε-amino groups of lysine with citraconic anhydride. The resulting peptides were purified by a combination of cation-exchange chromatography and high-voltage electrophoresis. The purified fragments were then tested for their morphine-like activity in the mouse vas deferens bioassay. The active peptides were 61–91 and 61–80. All other regions of the molecule investigated were not active. Moreover, the peptides 61–91 and 61–80 were about as active as the synthetic methionine-enkephalin, and in turn these were about 100 times more active than β-LPH itself. The inhibition of electrically stimulated mouse vas deferens by these peptides is reversed by naloxone, and suggests a competitive character of interaction. It is thus concluded that the active core for the morphine-like activity in the mouse vas deferens bioassay is the fragment 61–65 of β-LPH.
Glycosylation of apoproteins of rat very low density lipoproteins during transit through the hepatic Golgi apparatus
Authors: Dolphin Peter J. , Rubinstein David
Published: December 2009

The glycosylation of apo very low density lipoproteins (apo-VLDL) in vivo was studied by following the incorporation of [14C]glucosamine into several groups of apoproteins of VLDL isolated from hepatic Golgi fractions and from serum of sucrose-fed, colchicine-treated rats. Simultaneous incorporation of [3H]leucine was used to quantitate the apoproteins following separation by polyacrylamide gel electrophoresis. Experimental conditions were selected so that the 14C:3H ratio in the apoproteins permitted estimations of the extent of glycosylation by glucosamine and its metabolites. A rapidly decreasing 14C:3H ratio was noted in serum apo-VLDL for the first 30 min after administration of the isotopically labelled precursors, followed by stabilization of the ratio. These data are consistent with the glycosylation of a preformed pool of apo-VLDL, probably apo-B. Glucosamine was progressively incorporated into apo-VLDL during transition from the forming face of the Golgi apparatus to the secretory vesicles, as indicated by an increasing 14C:3H ratio. On the other hand, the ratio of the rapidly migrating apoproteins of VLDL, corresponding to the apo-C-II and apo-C-III, showed the opposite trend, as did total apo high density lipoprotein (apo-HDL) and the rapidly migrating bands of apo-HDL. Division of the rapidly migrating apoproteins of VLDL into upper bands (probably apo-C-II and apo-C-III-0) and lower bands (probably apo-C-III-3) resulted in a 14C:3H ratio near zero in the upper band apoproteins, consistent with the absence of carbohydrates. The lower band showed a rising 14C:3H ratio during transition through the Golgi apparatus, suggesting increased glycosylation. The decreasing 14C:3H ratio in the rapidly migrating proteins is therefore due to the acquisition of apo-C-II and apo-C-III-0 by VLDL during passage from the forming face to the secretory vesicles of the Golgi apparatus.
Wheat embryo ribonucleates. VII. Rapid, efficient and selective formation of 5S–18S and 5.8S–26S hybrids in an aqueous solution of the four ribosomal polynucleotides, and the results of a search for the corresponding hybrids in wheat embryo ribosomes
Authors: Oakden K. M. , Azad A. A. , Lane B. G.
Published: December 2009

(1) If wheat embryo 5S and 5.8S rRNA are differentially labelled, it can be shown that there is highly selective association of 5S [14C]RNA with 18S rRNA, and of 5.8S [3H]RNA with 26S rRNA when a solution (0.3 M NaCl) that contains approximately equimolar amounts of the four ribosomal polynucleotides is heated briefly (3 min) at 60 °C.(2) Comparison of Tm values and melting profiles for laboratory-prepared and natural 5.8S–26S rRNA hybrids suggests that restoration of the natural union between 5.8S and 26S rRNA can be achieved with facility and fidelity in the laboratory.(3) Union between 5.8S and 26S rRNA remains intact when wheat embryo ribosomes are disintegrated either by digestion with pronase or by treatment with sodium dodecyl sulphate, but the same treatments release 5S and 18S rRNA as freely migrating electrophoretic components.(4) Intact 18S and 26S rRNA can be prepared from small and large subunits, respectively, when wheat embryo ribosomes are dissociated by treatment with 0.5 M KCl.(5) Incidental to the principal investigation, it has been shown that, even after storage for more than 6 years at − 70 °C, commercial supplies of roller-milled wheat germ yield S23 extracts that are very active in the cell-free translation of globin mRNA.(6) The physicochemical and possible biochemical significance of various types of intermolecular complexing between pairs of ribosomal polynucleotides is a subject of discussion.
The effect of diabetes and insulin on adipose lactate dehydrogenase isozymes
Authors: Chang Albert Y. , Rothrock Donna
Published: December 2009

Adipose lactate dehydrogenase (LDH) (EC 1.1.1.27) isozyme distribution was altered in streptozotocin-diabetic and fasting rats resulting from a relative reduction of subunit A. Treatment with insulin for 2 days partially restored the relative content of isozyme 5 to control values in the diabetic rats, and the effect of insulin was not inhibited by simultaneous injection of actinomycin D or puromycin. When the epididymal adipose tissues isolated from control animals were incubated in vitro with dibutyryl adenosine 3′,5′-cyclic monophosphate or epinephrine, a relative decrease in subunit A was observed; whereas, either compound caused an increase in subunit A in diabetic tissues. The findings suggest that the redistribution of LDH isozyme under these conditions is to prevent excessive accumulation of lactate in the tissue.
Preferential uptake of D-glucose by plasma membranes isolated from human adipose tissue
Authors: Brenner Bluma G. , Kahlenberg Arthur
Published: December 2009

A sensitive method for the measurement of the stereospecific uptake of D-glucose by plasma membranes isolated from human adipose tissue has been developed. The method is based on the difference in uptake of L-[l4C]glucose and D-[3H]glucose as measured by the retention of radioactivity by the membrane preparation collected on Millipore filters. This D-glucose-uptake activity was reversible and did not involve any chemical alteration of the sugar. All uptake activity was lost upon boiling the membrane preparation for 5–10 min. All of the hydroxyl groups of D-glucose appear to be involved in a concerted fashion in the uptake reaction.The D-glucose-uptake activity was shown to be closely associated with glucose transport in adipose cells, since it exhibited the following properties characteristic of this carrier-mediated transport system, (a) The uptake was specific for the D-isomer of glucose, (b) Saturation of D-glucose uptake occurred with increasing concentrations of D-glucose. (c) The uptake activity was inhibited by N-ethylmaleimide and phloretin, two reagents previously reported to inhibit D-glucose transport.We conclude that plasma membranes isolated from human adipose tissue retain the glucose transport activity of the intact cells and can be used in subsequent attempts at the isolation and characterization of this transport system.
Translation of protamine mRNA in a rabbit reticulocyte cell-free system
Authors: Gedamu Lashitew , Dixon Gordon H.
Published: December 2009

Protamine mRNA isolated from the microsomal and postribosomal supernatant fractions of trout testis in poly A(+) (polyadenylated RNA) and poly A(−) (RNA devoid of poly A(+)) forms (Gedamu, L. &Dixon, G.H. (1976)7. Biol. Chem. 251, 1446–1454 and 1455–1463) was translated in the heterologous rabbit reticulocyte cell-free system; the products were shown to be identical in mobility with authentic protamine by polyacrylamide and starch gel electrophoresis. Chromatography, on carboxymethyl cellulose (Whatman CM-52), of the labelled polypeptide products synthesized in this cell-free system in the presence of poly A(+) and poly A(−) mRNA fractions also showed that [14C]arginine was incorporated into all three protamine components resolved in this system, but there was an unequal and variable incorporation of label into the three components with different preparations of mRNA. These results were interpreted as showing that the population of subcomponents of the protamine mRNA coding for the three different protamine polypeptides varied in batches of trout testis at differing stages of development. In addition, the proportion of mRNA components varied between the poly A(+) and poly A(−) editions of the mRNA, and it appeared that the poly A(−) mRNA fraction might represent the product of deadenylation of an earlier population of poly A(+) mRNA.
Selective enzymatic radioactive and spin labelling of phospholipids in biological membranes: application to study of temperature-induced changes of microsomal phosphatidylinositols and mitochondrial polyglycerophosphatides
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

A new method for the covalent radioactive and spin labelling of lipids within isolated biological membranes has been described in detail and applied to studies of temperature-induced changes of microsomal and mitochondrial membranes. The method is based on the enzymatic use of radioactive substrates carrying covalently bound doxyl derivatives of stearic acid in the biosynthesis of phospholipids in isolated membranes. Radioactive- and spin-labelled lipids bound to the microsomal and mitochondrial membranes were then used as internal probes in monitoring temperature-induced changes of these membranes. Since the analysis of isolated radioactive- and spin-labelled lipids revealed the exact composition of membrane-bound labelled lipids, specific temperature-induced changes were correlated with specific lipids of examined membranes. Phosphatidylinositol of microsomal membranes and polyglycerophosphatides (phosphatidyl-glycerol and cardiolipin) of mitochondrial and inner mitochondrial membranes were found to be involved in the apparent formation of lipid clusters at around 20–30 °C. Cardiolipin was found to be involved in the fluidization of inner mitochondrial membranes. These findings are discussed in view of the present state of knowledge of the organization of lipids in biological membranes.
Enzymic preparation of adenosine 5′-[β-32P]triphosphate
Authors: Leung Kwok-Luen , Yamazaki Hiroshi
Published: December 2009

A simple method for the preparation of adenosine 5′-[β-32P]triphosphate is described. When [32P]orthophosphate was incubated with polyadenylate and phosphoenolpyruvate in the presence of polynucleotide phosphorylase (EC 2.7.7.8) and pyruvate kinase (EC 2.7.1.40), up to 75% of 32P radioactivity was recovered in ATP. [32P]ATP was purified to 99.5% radiochemical purity by chromatography on polyethyleneimine-cellulose thin-layer plates. Analysis of hydrolysis products of [32P]ATP with apyrase (EC 3.6.1.5) indicates that 32P in the β-phosphate position accounts for all 32P label in ATP.
Further evidence for the presence of androgen receptors in the lungs and in the head appendages of the cock
Authors: Dubé Jean Y. , Chapdelaine Pierre , Tremblay Roland R.
Published: December 2009

The presence of cytoplasmic dihydrotestosterone receptors in the lungs, the comb, the wattle, and the ear lobes of the cock was demonstrated by sucrose density-gradient centrifugation. All these tissues exhibited saturable 'in vitro' binding of dihydrotestosterone in the 8–11S region of the gradient. When 0.5 M KCl was added to the lung, wattle, and comb cytosols and to the gradients, the radioactive dihydrotestosterone migrated in the 4–5S region. These studies suggest that the mechanism of action of androgens in the head appendages of the cock and in other target tissues is similar.
Human pituitary thyrotropin and gonadotropins: a comparative study of their in vitro lipolytic activity
Authors: Sairam M. R.
Published: December 2009

Highly purified preparations of human thyrotropin, follitropin, lutropin, and choriogonadotropin were evaluated for their ability to stimulate lipolysis in isolated rat adipocytes. Thyrotropin was the most active of all while follitropin showed some activity. Lutropin and choriogonadotropin were essentially inactive. The isolated subunits of thyrotropin were inactive and 60–100% activity was regenerated upon recombination. The results provide direct evidence that lipolytic activity is intrinsic to human thyrotropin. The hybrid of human lutropin α + thyrotropin β was considerably less active than the natural recombinant (thyrotropin α + β), possibly indicating the presence of some differences in the α subunits.
Purification and characterization of human muscle pyruvate kinase
Authors: Harkins Richard N. , Black John A. , Rittenberg Marvin B.
Published: December 2009

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240 700 and has a sedimentation coefficient (s20,w) of 10.04 S. It contains four subunits with identical molecular weights of 61 000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.
Phosphatidic acid synthesis in the heart. 1. Effect of age and species difference in the mitochondrial and microsomal synthesis
Authors: Kako K. J. , Zaror-Behrens G. , Peckett S. D.
Published: December 2009

Rates of syntheses of monoacyl- and diacyl-glycerol 3-P (phosphate) were determined in the mitochondrial and microsomal fractions prepared from hearts of rats and rabbits, to compare characteristics of the acylation reactions by the two subcellular fractions. The assays were carried out with the subcellular fractions prepared from (i) hearts of hyperthyroid animals, and (ii) hearts of newborn and weanling rats. In addition, the effect of an addition of bovine serum albumin in the assay system was examined. (1) Administration of thyroid hormones increased the acyltransferase activity in rabbit hearts but not that in rat hearts. (2) Mitochondrial and microsomal fractions of hearts of newborn rats acylated glycerol 3-P at a rate 1.3–4 times greater than those of adult rats. The rate of acylation by the mitochondrial fraction of weanling rats was also high, but the rate of microsomal acylation was slightly lower than that of adult rats. By contrast, in newborn rats, diacylglycerol 3-P formation by the liver microsomes was not greater than that of the adults, although its formation by the newborn liver mitochondria was greater. (3) The accumulation of monoacylglycerol 3-P during the assay was accelerated by the addition of increasing amounts of bovine serum albumin. Therefore, the monoacylglycerol 3-P formation was less than 10% of the diacylglycerol 3-P formation in the assay containing no albumin and with the rat subcellular fractions, whereas nearly four times more monoacyl- than diacyl-glycerol 3-P was synthesized in the presence of 20 mg albumin. (4) The ratio of monoacyl- to diacyl-glycerol 3-P formation by the mitochondrial fraction was greater than that of microsomal fraction at any concentration of albumin in both rats and rabbits. At an equal albumin concentration in the assay, relatively more diacyl- than monoacyl-glycerol 3-P was formed in the mitochondrial fraction of newborn rat hearts as compared with adult hearts. (5) In conclusion, our data concerning the age and species differences in acyltransferase activities support a view that the mitochondrial fraction of both rat and rabbit hearts, in addition to the microsomal enzymes, is capable of catalyzing the de novo synthesis of phosphatidic acid.
Characterization of an activating factor required for hydrolysis of Gm2 ganglioside catalyzed by hexosaminidase A
Authors: Hechtman Peter
Published: December 2009

Separation of the hexosaminidase A (EC 3.2.1.52) and B isozymes of human liver by ion-exchange chromatography results in recovery of greater than 80% of the activity in crude extracts when synthetic substrates are used to monitor enzyme activity. Only 15% of hexosaminidase activity toward the N-acetylgalactosaminyl (N-acetylneuraminyl) galactosyl glucosylceramide (Gm2 ganglioside) substrate is recovered and all of this activity is associated with the hexosaminidase A fraction.The low level of Gm2 ganglioside hydrolase activity in the hexosaminidase A fraction could be enhanced by coincubation with column fractions which contain hexosaminidase B. The activating factor, which has been partially purified by gel filtration, is a heat-stable protein with a molecular weight of 36 000 and is without enzyme activity toward hexosaminidase substrates.Highly purified hexosaminidase A or crude hexosaminidase A recovered after gel filtration on Sephadex G-100 has no Gm2 ganglioside hydrolase activity. The Gm2 ganglioside hydrolase activity of these hexosaminidase A preparations can be completely restored by addition of activating factor. The activating factor does not affect the rate of hydrolysis of synthetic substrate or asialo Gm2 ganglioside catalyzed by hexosaminidase A.
Glutamine synthetase IX. Purification and characterization of the enzyme from sheep spleen
Authors: Wu Chung
Published: December 2009

Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) has been purified about 550-fold from sheep spleen. The subunit weight of the enzyme is estimated to be 48 000. Sedimentation coefficient determination by density gradient centrifugation gives a value of 15.0 S. The approximate molecular weight calculated from the S value is 378 500. In addition, electron micrographs of the enzyme show an 'H' shape. Hence, the protein appears to have eight subunits. In sheep spleen, the enzyme resides chiefly in the soluble fraction of the cell. The amino acid composition of the enzyme from spleen shows similarity to that from other sources. The enzyme activity is nearly five times as high in Mg2+ as in Mn2+. ATP inhibits the enzyme; the inhibition is competitive with respect to Mg2+ ATP. A number of compounds, such as D-alanine, AMP, creatine phosphate, arsenite in combination with 2,3-dimercaptopropanol, and 2-amino-4-phosphonobutyrate, also inhibit the enzyme. The inhibition by the last compound is competitive with respect to glutamate. D-Glutamate and α-methyl-DL-glutamate can serve as substrates in the synthesis reaction, but N-methyl-DL-glutamate cannot. On the other hand, neither D-glutamine nor N-acetyl-L-glutamine can replace L-glutamine as a substrate in the γ-glutamyl transfer reaction of the enzyme. Inhibition by Mn2+ and ATP and its reversal by Mg2+ have been discussed as a means of regulating the enzyme activity in mammalian tissues.
An effect of enzyme and ligand concentration on the state of aggregation of aspartate transcarbamylase of E. coli: I. The binding of CTP and ATP to the enzyme
Authors: Cook Robert A. , Milne Judi A.
Published: December 2009

Detailed binding studies of the inhibitor, cytidine triphosphate (CTP), to native Escherichia coli aspartate transcarbamylase (EC 2.1.3.2) reveal significant changes in subunit interaction when enzyme concentration is altered. In contrast, similar binding studies of the activator, adenosine triphosphate (ATP), do not reveal such changes, but do indicate more complex subunit interactions than previously reported. Equilibrium dialysis studies at 4 °C are consistent with six binding sites for CTP and ATP per enzyme molecule of molecular weight 310 000, at all enzyme concentrations. CTP binding studies reveal a progressive change from apparent positive to negative cooperativity as the enzyme concentration is decreased. ATP binding studies reveal complex subunit interactions involving a mixture of apparent negative and positive cooperativity. Sucrose gradient studies indicate the presence of at least three enzymatically active polymeric forms of the enzyme. The preliminary sedimentation studies indicate possible ligand and enzyme concentration perturbations of a preexisting association equilibrium in the aspartate transcarbamylase system. The binding data are therefore interpreted in terms of an association model.
Factors affecting inosinate synthesis and inosine triphosphate accumulation in human erythrocytes
Authors: Henderson J. Frank , Zombor George , Fraser Joyce H. , McCoy Ernest E. , Verhoef Vernon , Morris Allan J.
Published: December 2009

Measurements of rates of inosinate synthesis from radioactive hypoxanthine by human erythrocytes show a large degree of individual variation. Rates of inosinate synthesis also vary with the pH and phosphate concentration of the incubation medium. This may be due to changes in the rate of phosphoribosyl pyrophosphate synthesis, and the stimulatory effect of phosphate on this process seems to be more important than the inhibitory effect of 2,3-diphosphoglycerate. The rate of inosinate synthesis, and especially the extent of accumulation of inosine triphosphate, increase disproportionately with time of incubation up to at least 24 h. Storage of erythrocytes also tends to increase inosinate synthesis and inosine triphosphate accumulation.
The metabolism of 3-deoxy-3-fluoro-D-glucose by Locusta migratoria and Schistocerca gregaria
Authors: Romaschin A. , Taylor N. F. , Smith D. A. , Lopes D.
Published: December 2009

3-Deoxy-3-fluoro-D-glucose (3FG) administered by injection is toxic to adult Locusta migratoria or Schistocerca gregaria (LD50, 4.8 mg/g). Temperature-programmed and isothermal gas chromatographic analysis of poisoned locust haemolymph reveals the presence of a fluorinated metabolite identified as 3-deoxy-3-fluoro-D-glucitol (3FGL). The enzymes responsible for the accumulation of this metabolite are located in the fat body of the insect and partially purified as aldose reductase (alditol: NADP+ 1-oxidoreductase, EC 1.1.1.21) and sorbitol dehydrogenase (L-iditol: NAD+ 5-oxidoreductase, EC 1.1.1.14). 3FGL is shown to be both a competitive inhibitor of the NAD-linked sorbitol dehydrogenase with Ki 8.2 × 10−2 M as well as a substrate with Km 0.5 M. A kinetic rate equation is derived and verified to account for the kinetic duality of 3FGL. These results partially explain the toxic effects of 3FG and are consistent with, the presence of a hitherto undetected sorbitol metabolism in locusts.
In vitro and in vivo studies on the metabolism of estrogens and their sulfates in guinea pigs
Authors: Hobkirk R. , Freeman D. J. , Harvey P. R. C. , Nilsen Mona , Jennings Barbara
Published: December 2009

Labelled estradiol-17β(E2) or estrone (E1), when incubated with guinea pig liver slices, is metabolized by two main pathways. Part of each substrate is converted to estrone-3-glucuronide and estradiol-3-glucuronide. A further part of each is metabolized to estradiol-3-sulfate (E23S) and estrone-3-sulfate (E13S), which are interconverted. The latter conjugate appears to be the substrate for a 16α-hydroxylase forming 16α-hydroxyestrone-3-sulfate (16αOHE13S). This, in turn, is further sulfurylated to yield 16α-hydroxyestrone-3,16-disulfate, accompanied by estriol-3,16-disulfate. A relatively small amount of tentatively identified '6-hydroxyestrone disulfate' accompanies these other two diconjugates. The guinea pig liver system suggests itself as a useful and relatively simple model for further study of 16α-hydroxylation of E13S. The use of the latter as a natural substrate in the system in vitro is supported by our observation that E13S and E23S are present in liver, kidney, blood, gallbladder bile, intestine, uterus, and placenta after injection of labelled E2 into mature male and female guinea pigs. Some evidence has been obtained for the disulfate fraction (above) in liver and bile after injection of labelled E1.
Metabolism of tryptophan and serotonin by the chick pineal gland in organ culture
Authors: Wainwright S. D.
Published: December 2009

Marked differences were seen between the metabolism of L-[3-14C]tryptophan and of [2-14C]serotonin by the intact chick pineal gland in organ culture. The major metabolite of tryptophan recovered by our procedures was melatonin, which accounted for about half the radioactivity recovered as metabolic products. In contrast, the principal product of serotonin metabolism recovered was hydroxyindoleacetic acid, and the yield of products derived through monoamine oxidase (EC 1.4.3.4) activity vastly exceeded that of melatonin. Metabolism of tryptophan yielded a much larger proportion of methylated metabolites among the products recovered than did metabolism of serotonin. However, the yield of methoxyindoleacetic acid from serotonin was greater than that from tryptophan. Serotonin formed endogenously and serotonin supplied exogenously appear to enter two or more largely distinct metabolic pools.
The chemical and kinetic consequences of the modification of papain by N-bromosuccinimide
Authors: Glick Bernard R. , Brubacher Lewis J.
Published: December 2009

Nonactivated papain was treated with N-bromosuccinimide at pH 4.75. The N-bromosuccinimide-modified enzyme was characterized by (1) the change in absorbance at 280 nm, (2) amino acid analysis, (3) separate chemical determinations of tryptophan and tyrosine, (4) difference spectroscopy, and (5) an N-terminal residue determination. It is concluded that N-bromosuccinimide in sevenfold molar excess oxidizes one tryptophan and two to three tyrosine residues per molecule of nonactivated papain, without causing peptide chain cleavage. Kinetic studies with several substrates and competitive peptide inhibitors were performed at pH 6 using the N-bromosuccinimide-modified papain. In addition, the kinetics of the modified enzyme with the substrate α-N-benzoyl-L-arginine ethyl ester were studied in the region of pH 3.5–9.0. All substrates (and inhibitors) tested, with the exception of α-N-benzoyl-L-arginine p-nitroanilide, displayed approximately a twofold decrease in both kcat and Km (or Ki), relative to the native enzyme. It is concluded that the key tryptophan residue which is modified is probably Trp-177.
Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells
Authors: Gupta Radhey S. , Flintoff Wayne F. , Siminovitch Louis
Published: December 2009

We have previously described methotrexate-resistant Chinese hamster ovary cells which appear to contain normal levels of a structurally altered dihydrofolate reductase (EC 1.5.1.3) (Flintoff, W. F., Davidson, S. V., and Siminovitch, L. (1976) Somatic Cell Genet. 2, 245–261). By selecting for increased resistance from these class I cells, class III resistant cells were isolated which appeared to possess an increased activity of the altered enzyme. In this report, we describe the purification and several properties of the reductase from wild-type cells, two independently selected class I cells, and a class III resistant cell. The reductases from wild-type and resistant cells had similar specific activities using folate and dihydrofolate as substrates, and similar molecular weights as determined by sodium dodecyl sulfate gel electrophoresis. The mutant enzymes, however, were about six- to eight-fold more resistant to inhibition by methotrexate than the wild-type enzyme, suggesting a decreased affinity of the mutant reductases to methotrexate-binding. Small differences between various enzymes were also seen in other physicochemical properties such as pH optima and Km values for folate, and in their heat stabilities, which suggest that different structural alterations may lead to the same mutant phenotype. As expected from earlier studies with crude extracts, class III cells did produce a higher (about 10-fold) yield of the reductase than the class I or wild-type cells.
Reversibility of the ampicillin- and nitrite-induced inactivation of β-lactamase I
Authors: Durkin J. P. , Dmitrienko G. I. , Viswanatha T.
Published: December 2009

β-Lactamase I was isolated from Bacillus cereus 569/H. Treatment with ampicillin in the presence of sodium nitrite at pH 4 or 5 resulted in the inactivation of the enzyme presumably by modification of a carboxyl group in the active site. However, this inactivation was rapidly reversible at neutral pH and the available evidence points to the participation of a second carboxyl group which is involved in the reactivation process.
An essential arginine residue in the active-site pocket of glycogen phosphorylase
Authors: Li E. C. Y. , Fletterick R. J. , Sygusch J. , Madsen N. B.
Published: December 2009

Phosphorylases a and b (EC 2.4.1.1) were inactivated by selective modification of arginyl residues on reaction with 2,3-butanedione in sodium borate buffer. The rate of inactivation was slightly greater for phosphorylase a than b. The course of inactivation followed pseudo-first-order kinetics with some deviations at low rates or at more than 60% inactivation. The rate of inactivation was first order with respect to butanedione concentration. The inactivation was partially reversible, and ultracentrifugal studies showed no change in subunit association or dissociation. Amino acid analyses indicated that several arginines were modified during inactivation and that no other amino acid was affected. Protection from inactivation was provided by the substrate glucose 1-phosphate (G1P), alone or together with the allosteric activator AMP, as well as by the competitive inhibitor UDP-glucose. The rate of inactivation of phosphorylase b was also retarded by the presence of AMP alone. Glycogen did not have any appreciable effect on inactivation. The Km of G1P for phosphorylase a remained constant over the course of inactivation, while the Km values of G1P and AMP for phosphorylase b increased. The modification of cross-linked tetragonal microcrystals of phosphorylase a followed the same trend as the enzyme in solution, although the rate of inactivation was slower. The X-ray crystallography studies at 6 Å (1 Å = 0.1 nm) resolution, of butanedione-treated cross-linked tetragonal crystals of phosphorylase a showed a large new peak of electron density at the end of a long side chain in the active-site pocket. The substrates G1P and arsenate, as well as UDP-glucose, had previously been shown to bind in that location. Other, small peaks of electron density were found in locations on the outside of the protein molecule. UDP-glucose failed to bind to the active site of crystals which had been treated with butanedione, while AMP, which also binds in the active-site pocket, showed a lower occupancy. This work indicates the presence of a functional arginine residue at the binding site for G1P in glycogen phosphorylases a and b.
The inhibition by substrate analogues of γ-aminobutyrate aminotransferase from mitochondria of different subcellular fractions of rat brain
Authors: Tunnicliff G. , Ngo T. T. , Rojo-Ortega J. M. , Barbeau A.
Published: December 2009

Cytoplasmic-mitochondria-enriched and synaptosome-enriched subcellular fractions were prepared from rat brain to study certain kinetic properties of γ-aminobutyrate:α-oxoglutarate aminotransferase (GABA-T) (EC 2.6.1.19) from each of the sources. From this study two differences emerged. Firstly, the cytoplasmic enzyme exhibited an eightfold greater affinity for γ-aminobutyric acid (GABA) than did the synaptosomal GABA-T; the Km being 6.5 mM and 53 mM, respectively. Secondly, synaptosomal GABA-T is comparatively more susceptible to inhibition by the substrate analogues 2,4-diaminobutyric acid (DABA) and aminooxyacetic acid (AOAA) than is the enzyme from the cytoplasmic mitochondrial fraction. In each case the inhibition was of a competitive nature with respect to GABA. The Ki for the DABA was 13 mM for the cytoplasmic-derived enzyme and 8 mM for the synaptosomal enzyme. With AOAA the Ki was 0.1 μM and 0.06 μM for the synaptosomal and cytoplasmic mitochondrial enzyme, respectively. These results provide further evidence that GABA-T from cytoplasmic mitochondria is different in several respects from the enzyme found in synaptosomes.
Effect of ribonucleotides on substrate availability for DNA polymerase assays in cytoplasmic fractions of rat intestinal mucosa
Authors: Zbarsky S. H. , Chevalier S.
Published: December 2009

The deoxyribonucleoside triphosphate substrates for DNA synthesis were hydrolysed during the DNA polymerase (EC 2.7.7.7) assay with cytoplasmic subcellular fractions of rat intestinal mucosa. Presumably because of phosphatase (EC 3.1.3.2) activity in these fractions, inorganic phosphate was liberated from the nucleotides, and radioactive thymidine triphosphate was shown to be degraded to thymidine di- and mono-phosphate, thymidine, and thymine. Addition of ATP to the postmicrosomal supernatant increased its DNA polymerase activity by sparing the deoxyribonucleotide precursors from enzymatic degradation.
A simple method for the determination of the O-acetyl substitution pattern of the sialic acids of colonic epithelial glycoprotein
Authors: Reid P. E. , Culling C. F. A. , Ramey C. W. , Dunn W. L. , Clay M. G.
Published: December 2009

A simple, rapid scheme is described for the quantitative division of the sialic acids of a glycoprotein into four classes: (a) those bearing no O-acyl substituents, (b) those with an O-acyl substituent at position C7, (c) those substituted at C9, and (d) those substituted at C8 or which are either di-(C7C8, C7C9, C8C9) or tri-(C7C8C9) substituted. This scheme can be applied to samples containing as little as 230 μg of sialic acid and requires neither acid hydrolysis nor expensive instrumentation. In combination with previously published techniques it is possible to estimate: (a) the side-chain substitution pattern of those sialic acids which are Vibrio Cholera neuraminidase (EC 3.2.1.18) insensitive, (b) the percentage of sialic acids which are Vibrio Cholera neuraminidase insensitive because of an ester substituent at either position C4 or C1, and (c) the percentage of those sialic acids which bear more than one side-chain substituent.The application of this procedure to epithelial glycoproteins isolated from the colons of man and rat and to a commercial sample of bovine submaxillary mucin is described. Evidence is presented which suggests that either these glycoproteins contain little or no C9-O-acetyl sialic acids and (or) that, under the conditions used, such acids are oxidized to completion.
Uridine phosphorylase activity of isolated plasma membranes of rat liver
Authors: Bose Ratna , Yamada Esther W.
Published: December 2009

Plasma membranes were isolated from rat liver homogenates either by differential centrifugation or by fractionation in discontinuous sucrose density gradients. Both membrane preparations contained about 17% of the total uridine phosphorylase (EC 2.4.2.3) activity and 44% of the total 5′-nucleotidase (EC 3.1.3.5). The enrichment factor for uridine phosphorylase in the fractions prepared by differential centrifugation was about 2.8 and by the gradient method, as much as 11.0; the respective enrichment factors for 5′-nucleotidase were 1.8 and 9.5. Uridine phosphorylase activity of isolated plasma membrane fractions was stimulated 2.5-fold by 0.1% Triton X-100. Unlike the cytosol enzyme, uridine phosphorylase of plasma membranes showed little or no deoxyuridine-cleaving activity. Contamination of the membrane fractions by thymidine phosphorylase (EC 2.4.2.4) of the cytosol was negligible.The other subcellular organelles obtained by either procedure and characterized by marker enzyme activities were found not to contain significant uridine phosphorylase activity; the cytosol fractions contained just over 70% of the total uridine phosphorylase activity with an enrichment of only about 2.8-fold. The activity of the cytosol enzyme was not stimulated by Triton X-100.
Correlation between organophosphate poisoning, acetylcholinesterase inhibition, and increased cyclic GMP levels in malathion-treated insects
Authors: Bodnaryk Robert P.
Published: December 2009

Organophosphate poisoning with malathion caused large increases (up to 125 and 440%, respectively) in the level of cyclic GMP in larvae of Mamestra configurata Wlk. and in the fly Sarcophaga bullata Parker. Cyclic AMP was little affected. The malathion-induced increase in cyclic GMP was time and dose dependent. Time-course studies with the head and thorax of S. bullata demonstrated that the increase in cyclic GMP level occurred precipitously after a lag period of about 1 h, during which time the activity of acetylcholinesterase (EC 3.1.1.7) was progressively inhibited. The abrupt increase in cyclic GMP began when acetylcholinesterase activity had been inhibited to a sufficient extent to permit accumulation of acetylcholine. It is suggested that the accumulation of acetylcholine in the malathion-poisoned insects caused cyclic GMP levels to rise.Cyclic GMP may have a role in cholinergic transmission in normally functioning insect neural tissue. Increased levels of cyclic GMP induced by organophosphate and organochlorine (Bodnaryk, R. P. (1976) Can. J. Biochem. 54, 957–962) insecticides appear to be a vital and previously unrecognized biochemical lesion in insects poisoned by these compounds.
The syntheses of α-D-glucopyranose-1-phosphates labelled with 18O in the phosphoryl or ester oxygen positions
Authors: Kokesh Fritz C. , Kakuda Yukio
Published: December 2009

Phosphoryl-oxygen-labelled α-D-glucopyranose-1-phosphate (Glc1P) has been prepared by the hydrolysis in 18O-enriched water of α-D-glucopyranose-1,2-cyclic phosphate catalyzed by extracts of Saccharomyces fragilis. Ester-oxygen-labelled α-D-galactopyranose-1-phosphate has been prepared by galactokinase-(EC 2.7.1.6)-catalyzed phosphorylation of D-[1-18O]-galactose by ATP, and it has been isomerized to ester-oxygen-labelled Glc1P using galactose-1-phosphate uridyltransferase (EC 2.7.7.12) and UDPgalactose 4-epimerase (EC 5.1.3.2). These labelled glucose phosphates are for use in oxygen-exchange experiments.
A convenient preparation of β-N-acetylhexosaminidase from commercial bovine serum albumin
Authors: Kanfer Julian N. , Spielvogel Christine
Published: December 2009

A simple procedure for the preparation of β-N-acetylhexosaminidase (EC 3.2.1.52), free from other glycosidases, from commercial bovine serum albumin is described. It has been fractionated into three peaks on a DEAE-cellulose column. The Km and V values for 4-methylumbel-liferyl-β-D-N-acetylglucosaminide (4MU-gluNAc), 4-methylumbelliferyl-β-D-N-acetylgalactosaminide (4MU-galNAc), p-nitrophenyl-β-D-N-acetylglucosaminide, and p-nitrophenyl-β-D-N-acetylgalactosaminide for each was established. The inhibition by several structural analogues on 4MU-gluNAc and 4MU-galNAc hydrolysis by the enzyme present in peak 1 was investigated.
Determination of the preferred tautomeric form of histamine by 13C nmr spectroscopy
Authors: Reynolds William F. , Tzeng Chau W.
Published: December 2009

The pH-dependent 13C chemical shifts for histamine indicate an approximate 4:1 preference for the Nτ-H tautomer of the imidazole ring, similar to that previously deduced for L-histidine. It is concluded that the 13C chemical shift method is a complimentary technique to the method of determining tautomer preference from pK values. Factors determining the tautomer preference in histamine and L-histidine are discussed.