bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mitochondrial Adenylate Kinase from Chicken Liver
Authors: WATANABE Kiyotaka , KUBO Shuichiro 
Published: July 2010

Mitochondrial adenylate kinase has been purified 5400‐fold chicken liver extract in an overall yield of 36%. The purified enzyme has a specific activity of 810 U/mg, a molecular weight of 28000, and the following amino acid composition:21 aspartic acid or asparagines, 14 threonine, 17 serine, 27 glutamic acid or glutamine, 16 proline, 22 glycine, 22 alanine, 15 valine, 6 methionine, 11 isoleucine, 29 leucine, 5 tryosine, 7 phenylalanine, 16 lysine, disulfide bond and one sulfhydryl group. The disulfide bond is related to the active conformation of the enzyme, whereas the sulfhydryl group does not contribute to the enzyme activity. The sulfhydryl group is easily oxidized in the presence of Cu2+ resulting in the formation of dimmer with about one half of the specific activity of the monomer. The enzyme is similar to porcine heart mitochondrial adenylate kinase in antigenicity but different from chicken cytosolic adenylate kinase.mitochondrial adenylate kinase was synthesized in the mRNA‐dependent rabbit reticulocyte lysate system programmed with total chicken liver RNA. The mobility in sodium dodecylsulfate gel electrophoresis of the product obtained in vitro was the same as that of the purified mitochondrial aadenylate kinase. This evidence indicates that the mitochondrial adenylate kinase is synthesized as a polupeptide with a molecular weight indistinguishable from that of the mature protein.
New Mutants Resistant to Glucose Repression Affected in the Regulation of the NADH Reoxidation
Authors: GUERIN Martine , CAMOUGRAND Nadine , VELOURS Giséle , GUÉRIN Bernard 
Published: July 2010

Spontaneous mutants resistant to vanadate, arsenate or thiophosphate were isolated from a haploid strain of Saccharomyces cerevisiae. These three anions have an inhibitory effect on some mitochondrial functions and at the level of glyceraldehyde 3‐phosphate dehydrogenase, a glycolysis enzyme. All the selected mutants had the same phenotype: they were deficient in alcohol dehydrogenase I, the terminal enzyme of the glycolysis, and possessed a high content of cytochrome c oxidase, the terminal enzyme of the respiratory chain. Moreover, cytochrome c oxidase biosynthesis had become insensitive to the catabolite repression. while the biosynthesis of the other enzymes sensitive to this phenomenon were always inhibited by glucose.Metabolic effects of this pleiotropic mutation manifested themselves in the following ways.1. Growth rate and final cell mass were enhanced, compared to the wild type, when cells were grown on glucose or on glycerol, but not on lactate or ethanol.2. Growth under anaerobiosis was nil and mutants did not ferment.3. Mitochondria1 respiration of the mutant strains was identical to the wild type with succinate or 2‐oxoglutarate as substrate, and weak with ethanol. But with added NADH, respiration rate of the mutants was higher than that of the wild type and partially insensitive to antimycin, even when cells were grown in repression conditions.It is postulated that in mutants strains, NADH produced at the level of glyceialdehyde 3‐phosphate dehydrogenase, failing to be reoxidized via alcohol dehydrogenase, could be reoxidized with a high turnover owing to the enhancement of the amount of cytochrome c oxidase. Since NADH reoxidation is partially insensitive to antimycin, a secondary pathway going from external NADH dehydrogenase to cytochrome c oxidase is suggested.
Temperature Dependence of ADP/ATP Translocation in Mitochondria
Authors: KLINGENBERG Martin , GREBE Karin , APPEL Maria 
Published: July 2010

The temperature dependence of the adenine nucleotide exchange in mitochondria has been determined by employing a rapid mixing, quenching and sampling apparatus and the inhibitor quench‐back exchange method. Thus the exchange is resolved down to 0.1 s. Rates are evaluated from accumulating the time‐dependent progress at about 10 points.The exchange rate in liver mitochondria was determined from –10°C to +10°C in the presence of 20% glycol, from 0°C to 25°C, and from 20°C to 40°C under partial inhibition by carboxyatractylate. The total range between –10°C to + 40°C has only one temperature break at 13°C. From the Arrhenius plot between –10°C to +13°C, EA∼ 140 kJ and above 13°C, EA∼ 56 kJ is evaluated, corresponding to a Q10 of 8 and 2 respectively.In beef heart mitochondria the exchange rate was measured between 0°C and 20°C, and between 15°C and 30°C under partial inhibition with carboxyatractylate. There is a temperature break around 14°C with EA∼ 143 kJ between 0°C and 14°C and EA∼ 60 kJ from 15°C to 30°C.The extrapolated translocation rates at 37°C are 500 and 1800 μmol min−1 (g protein)−1 for rat liver and for beef heart mitochondria respectively.The temperature break is suggested to reflect a conformation change since there is no reversed break at low temperature, the temperature break changes in sonic particles and no lipid phase transition at 14°C in mitochondria has been reported.
Induction in Primary Culture of ‘Gluconeogenic’ and ‘Glycolytic’ Hepatocytes Resembling Periportal and Perivenous Cells
Authors: PROBST Irmelin , SCHWARTZ Peter , JUNGERMANN Kurt 
Published: July 2010

Adult rat hepatocytes were kept in primary culture for 48 h under different hormonal conditions to induce an enzyme pattern which with respect to carbohydrate metabolism approximated that of periportal and perivenous hepatocytes in vivo.

Glucagon‐treated cells compared with control cells possessed a lower activity of glucokinase, a 4.5‐fold higher activity of phosphoenolpyruvate carboxykinase and unchanged levels of glucose‐6‐phosphatase, phosphofructokinase, fructose‐bisphosphatase and pyruvate kinase; they resembled in a first approximation the periportal cell type and are called for simplicity ‘periportal’. Inversely, insulin‐treated cells compared with control cells contained a 2.2‐fold higher activity of glucokinase, a slightly decreased activity of phosphoenolpyruvate carboxykinase, increased activities of phosphofructokinase and pyruvate kinase and unaltered levels of glucose‐6‐phosphatase and fructose‐bisphosphatase; they resembled perivenous cells and are called simply ‘perivenous’. Gluconeogenesis and glycolysis were studied under various substrate and hormone concentrations.
Physiological concentrations of glucose (5 mM) and lactate (2 mM) gave about 80% saturation of gluconeogenesis from lactate and less than 15% saturation of glycolysis at a simultaneous 40% inhibition of the glycolytic rate by lactate.
Comparison of the two cell types showed that under identical assay conditions (5 mM glucose, 2 mM lactate, 0.5 nM insulin, 0.1 μM dexamethasone) gluconeogenesis was 1.5‐fold faster in the ‘periportal’ cells and glycolysis was 2.4‐fold faster in the ‘perivenous’ cells.
Metabolic rates were under short‐term hormonal control. Insulin increased glycolysis three fold in both cell types with a half‐maximal effect at about 0.4 nM, but did not influence the gluconeogenic rate. Glucagon inhibited glycolysis by 70% with a half‐maximal effect at about 0.1 nM. Gluconeogenesis was stimulated by glucagon (half‐maximal dose: 0.5 nM) 1.8‐fold only in ‘periportal’ cells containing high phosphoenolpyruvate carboxykinase activity, not in the ‘perivenous’ cells with a low level of this enzyme.
A comparison of the two cell types showed that with maximally stimulating hormone concentrations gluconeogenesis was threefold faster in ‘periportal’ cells and glycolysis was eightfold faster in ‘perivenous’ cells.

The results support the view that periportal and perivenous hepatocytes in vivo catalyse gluconeogenesis and glycolysis at inverse rates.
Optical Characteristics of All Individual Proteins from the Small Subunit of Escherichia coli Ribosomes
Authors: VENYAMINOV Sergei Yu. , GOGIA Zurab V. 
Published: July 2010

The procedure of isolation and renaturation of all ribosomal proteins from the 30‐S subunit of Escherichia coli ribosomes is described. Absorption spectra of these proteins in the near‐ultraviolet region have been measured and molar absorption coefficients have been determined on the basis of nitrogen content. Molar absorption coefficients have been calculated for 20 proteins with a known amino acid sequence and the calculated values have been compared with the experimentally determined ones. The absorption spectra obtained allow an easy, precise and highly reproducible spectrophotometric determination of the concentration of individual ribosomal proteins.Circular dichroic spectra of 21 individual proteins from the 30‐S subunit of E. coli ribosomes were measured in the range 184–310 nm. The secondary structure of the proteins studied was calculated from the spectra in the range 190–240 nm. Almost all proteins (except proteins S12, S17, S18 and S19) are characterized by a high content of secondary structure. Circular dichroic spectra in the near‐ultraviolet region (240–310 nm) indicate that the side groups of aromatic amino acids are fixed in the tertiary structure of the proteins studied.Some internal characteristics (independent of the measurement conditions) of the circular dichroic spectrum in the far‐ultraviolet region were proposed as a measure of the resemblance to the native state of ribosomal proteins; these characteristics may be useful for comparison of protein preparations obtained by different methods in different laboratories.
Inhibition of Free and Bound Trypsin‐Like Enzymes
Authors: STEVEN Frank S. , GRIFFIN Margaret M. , ITZHAKI Shaul 
Published: July 2010

The inhibition of β‐naphthylamidase activity of free trypsin, trypsin‐Sepharose and a trypsin‐like neutral protease on the surface of tumour cells have been studied in independent systems and with mixtures of free trypsin plus surface‐bound trypsin.
Kinetic data have demonstrated that high‐molecular‐weight (protein) inhibitors of free trypsin are less effective inhibitors of trypsin‐Sepharose and fail to inhibit the cell surface neutral protease.
Inhibition of mixtures of free trypsin plus trypsin‐Sepharose follows independent kinetic plots for each component. The free trypsin is reacted before any Sepharose‐bound trypsin reacts with high‐molecular‐weight inhibitors.
Low‐molecular‐weight inhibitors of trypsin also inhibit bound trypsin equally well.
Papain‐derived peptides from high‐molecular‐weight inhibitors of trypsin inhibit free trypsin, trypsin‐Sepharose and the cell‐surface neutral protease almost equally well.
Fluorescence microscopy has shown that a high‐molecular‐weight inhibitor of trypsin does not bind to the tumour cell‐surface neutral protease, but it does bind to trypsin‐Sepharose.
The cell‐surface neutral protease has been shown to be capable of activation of latent β‐naphthylamidase activity in the presence of excess extracellular inhibitors of free trypsin.
The mechanism by which trypsin‐Sepharose remains partially active in the presence of excess inhibitor necessary to inhibit an equivalent quantity of free trypsin has been discussed.
These studies indicate that a search for inhibitors which are selectively active against the cell‐surface neutral protease and have no action on trypsin‐like enzymes in free solution must take into account the modifying effects of the cell surface on neutral protease activity.
Solubilization and Characterization of the Initial Enzymes of the Dolichol Pathway from Yeast
Authors: SHARMA Chandra B. , LEHLE Ludwig , TANNER Widmar 
Published: July 2010

Preparation and purification of substrate amounts of radioactive as well as non‐radioactive dolichyl diphosphate N‐acetylglucosamine and dolichyl diphosphate chitobiose made it possible to test and characterize tentatively the first three reactions of the dolichol pathway (enzyme I–III). The test conditions are described in detail.All three enzymes were solubilized from yeast membranes with detergents. Enzyme II and III were purified to give a purification factor of 35‐fold and 70‐fold, respectively. The reactions required divalent metal ions with an optimum concentration of 10 mM Mg2+. Enzyme II was stimulated almost to the same extent also by Ca2+.The Km values for UDP‐N‐acetylglucosamine for enzyme I and II were 15 and 10 μM, respectively, and for GDP‐mannose (enzyme III) 7 μM. The apparent Km values for the lipophilic acceptor was 180 μM for enzyme I (dolichyl phosphate), 40 μM for enzyme II (dolichyl diphosphate N‐acetylglucosamine) and 17 μM for enzyme III (dolichyl diphosphate chitobiose). The corresponding V values were approximately 1, 10, and 50 nmol × h−1× mg protein−1. All reactions were inhibited by nucleoside diphosphates.
Self‐Inactivation by 13‐Hydroperoxylinoleic Acid and Lipohydroperoxidase Activity of the Reticulocyte Lipoxygenase
Authors: HÄRTEL Brigitte , LUDWIG Peter , SCHEWE Tankred , RAPOPORT Samuel M. 
Published: July 2010

The self‐inactivation of lipoxygenase from rabbit reticulocytes with linoleic acid at 37°C is caused by the product 13‐hydroperoxylinoleic acid. This inactivation is promoted by either oxygen or linoleic acid.
Lipohydroperoxidase activity was demonstrated with 13‐hydroperoxylinoleic acid plus linoleic acid as hydrogen donor under anaerobic conditions at 2°C. The products were 13‐hydroxylinoleic acid, oxodienes and compounds of non‐diene structure similar to those produced by soybean lipoxygenase‐1.
13‐Hydroperoxylinoleic acid also changed the absorbance and fluorescence properties of reticulocyte lipoxygenase. The results indicate that one equivalent of 13‐hydroperoxylinoleic acid converts the enzyme from the ferrous state into the ferric state as described for soybean lipoxygenase‐1. The spectral changes were reversed by sodium borohydride at 2°C, but not at 37°C; it is assumed that the ferric form of reticulocyte lipoxygenase suffers inactivation.
An Alteration in Phosphofructokinase 2 of Escherichia coli which Impairs Gluconeogenic Growth and Improves Growth on Sugars
Authors: DALDAL Fevzi , BABUL Jorge , GUIXÉ Victoria , FRAENKEL Dan G. 
Published: July 2010

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkBl mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., ‘gluconeogenic growth’). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkBl*. We show here that strains with pfkBl* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6‐bisphosphate.The Km for fructose 6‐phosphate is low (about 5 × 10−5 M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6‐bisphosphate and very low levels of hexose monophosphates.These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a ‘non‐allosteric’ protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569–574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6‐bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.
The Subcellular Localization of DNA Components from Cyanophora paradoxa, a Flagellate Containing Endosymbiotic Cyanelles
Authors: BOHNERT Hans J. , CROUSE Edwin J. , POUYET Jean , MUCKE Herrmann , LÖFFELHARDT Wolfgang 
Published: July 2010

Cyanophora paradoxa, a unicellular flagellate, contains cyanelles which are supposed to be cyanobacterial origin. DNA was isolated from subcellular fractions and separated according to density components in CsCl density gradients.The main DNA component, comprising more than 90% of the total DNA, has a buoyant density of 1.724 g · cm−3. Several subsfractions in the range from 1.718 g · cm−3 to 1.735 g · cm−3 are contained in this component. This DNA of high complexity was considered to be host nuclear DNA.The DNA from the endosymbiotic cyanelles, which were isolated, treated with DNase, and purified by sucrose density gradient centrifugation exhibited a buoyant density of 1.692 g · cm−3 in one strain and 1.695 g · cm−3 in a second strain. Both cyanelle DNAs (cyDNA) have a complexity of approximately 126 · 103 base pairs and comprise about 5% of the total cellular DNA content.Two additional DNA components of low complexity were isolated from crude cyanelle pellets obtained without DNase treatment. The larger of these, approximately 48 × 103 base pairs in size, had a density of approximately 1.688 g · cm−3. The second component, about 15 × 103 base pairs in size, banded in the density range between 1.710 g · cm−3 and 1.720 g · cm−3. The latter is associated with nuclear DNA. The 48 × 10−3 base‐pair component was located in the cytosol and could be obtained after CsCl/ethidium bromide density gradient centrifugation at the position of covalently closed circular DNA. Both these components amounted to approximately 0.5–1% of total DNA.A further DNA component with a complexity of more than 150 × 103 base pairs, enriched in fractions where mitochondria are expected, was not characterized further. The density was intermediate between cyDNA and nuclear DNA (1.710–1.720 g · cm−3) and it amounted to 1–2% of the total DNA.Our results indicate that the DNA from cyanelles, believed to be endosymbiotic cyanobacteria, is not more complex than higher plant chloroplast DNAs.
Nuclear Magnetic Resonance of Protamines
Authors: FERRARA Luciano , NAPOLITANO Roberto , PAOLILLO Livio , WURZBURGER Silvana , ANDINI Salvatore , TONIOLO Claudio , BONORA Gian M. 
Published: July 2010

The three main fractions of clupeine, the protamine extracted from herring sperm, have been investigated by 13C nuclear magnetic resonance techniques.The dynamic behaviour, examined through the evaluation of the spin lattic relaxation times (T1) of individual carbon resonances assigned to both backbone and side chains, reveals interesting features. The relaxation times of backbone α‐carbons, interpreted on the basis of an axially symmetric ellipsoid, point to the clupeine fractions as being essentially extended in aqueous solution. These times remain constant along the polypeptide chain and are of the order of 0.16 ± 0.02 s. Conversely, the side chains show different flexibilities in the presence of monophosphate counterions, thus demonstrating a diverging behaviour which may be biologically relevant. In particular, the side‐chain flexibilities of fraction YI decrease, while those of fractions Z and YII are either constant or increase.Comparison of these data with the viscosity measurements helps in explaining the viscosity changes observed in the presence of phosphate.
Blood Group A and H Determinants in Polyglycosyl Peptides of A1 and A2 Erythrocytes
Authors: VIITALA Juha , FINNE Jukka , KRUSIUS Tom 
Published: July 2010

Polyglycosyl peptides were isolated from delipidated erythrocyte membranes of human blood‐group A1 and A2 erythrocytes by extensive pronase digestion and gel filtration. As estimated by the amounts of N‐acetyl‐galactosamine and 2‐O‐substituted galactose residues about 85% of the possible acceptor sites (H determinants) were saturated with A determinants in A1 polyglycosyl peptides whereas only 25% of H sites were filled in A2 glycopeptides. The distribution of A and H determinants in the glycopeptides was studied by affinity chromatography with Sepharose‐bound Bandeiraea simplicifolia I‐lectin (binds blood‐group A and B determinants) and Ulex europaeus I‐lectin (binds blood‐group H determinants). About 55% of the polyglycosyl peptides contained A, H, or A and H determinants in both A1 and A2 blood subgroups. 48% of the polyglycosyl peptides of blood group A1 and 10% of A2 bound to Bs I‐lectin. 25% of the polyglycosyl peptides in A1 and 53% in A2 carried H determinants.The molecular size, monosaccharide composition and the substitution pattern of the monosaccharides in the Bs‐I‐bound polyglycosyl peptides were very similar in both A1 and A2 blood groups. The only difference was the amount of N‐acetylgalactosamine which was on the average 3.7 mol/mol in A1 and 2.5 mol/mol in A2. The active fraction was found to be heterogeneous with respect to the amount of A determinants, which varied from 1 to 6 per glycopeptide in A1 and A2 polyglycosyl peptides. The findings do not indicate a structural difference between blood‐group A1 and A2 polyglycosyl peptides and state chemically that A1 glycopeptides contain more A determinants than A2 glycopeptides.
Phosphorylation of the Type‐II Regulatory Subunit of Cyclic‐AMP‐Dependent Protein Kinase by Glycogen Synthase Kinase 3 and Glycogen Synthase Kinase 5
Authors: HEMMINGS Brian A. , AITKEN Alastair , COHEN Philip , RYMOND Michael , HOFMANN Franz 
Published: July 2010

The regulatory (RII) subunit of type‐II cyclic‐AMP‐dependent protein kinase from bovine heart is phosphorylated at a significant rate in vitro by glycogen synthase kinase 3 and glycogen synthase 5, but not by glycogen synthase kinase 4 or phosphorylase kinase. The regulatory (RI) subunit of type‐I cyclic‐AMP‐dependent protein kinase from rabbit skeletal muscle is not phosphorylated by any of these four protein kinases.Glycogen synthase kinase 3 phosphorylates two serine residues on the RII subunit located 44 and 47 amino acids from the N terminus of the polypeptide chain. Glycogen synthase kinase 5 phosphorylates serine‐74 and serine‐76. These sites are distinct from the residue phosphorylated by the catalytic subunit of cyclic‐AMP‐dependent protein kinase (serine‐95).The RII subunit, as normally isolated, contains 1.5–1.8 mol alkali‐labile phosphate per subunit. At least 80% of this phosphate (∼ 1.3 mol/subunit) is located in the thermolytic peptide containing serine‐74 and serine‐76, demonstrating that phosphorylation of the RII subunit by glycogen synthase kinase 5 occurs in vivo. Only small amounts of phosphate (∼ 0.1 mol/subunit) are associated with the thermolytic peptides containing serine‐44/serine‐47 and serine‐95.The phosphorylation sites on the RII subunit are organised in a strikingly similar manner to those of glycogen synthase, the amino acid sequences in the immediate vicinity of the phosphorylation sites showing a particular resemblance. These include the presence of a number of proline residues near the sites phosphorylated by glycogen synthase kinase 3, five consecutive acidic residues C‐terminal to the sites phosphorylated by glycogen synthase kinase 5, and two adjacent arginine residues just N‐terminal to the sites phosphorylated by the catalytic subunit of cyclic‐AMP‐dependent protein kinase.Glycogen synthase kinase 5 is very similar or identical to the enzyme that has been variously termed casein kinase TS, casein kinase 2, casein kinase G, casein kinase N‐II or troponin‐T kinase. The biological role of this enzyme is reviewed.