bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction of Biguanides with Mitochondrial and Synthetic Membranes
Authors: SCHÄFER Günter , RIEGER Ekkehard 
Published: March 2005

Primary reactions of the energy‐conserving pathway in oxidative phosphorylation are inhibited by biguanides and alkylguanidines. The inhibitors bind to phospholipids of mitochondria and synthetic membranes with identical affinities and are assumed to modify the physical properties of membranes, as for example cation conductance.The study compares effects of biguanides on respiratory‐linked ion fluxes across mitochondrial membranes with those on the conductance and the electrical capacitance of phospholipid bilayers. The following observations have been made:1. The rate of energy‐linked mitochondrial swelling and the rate of proton uptake during phosphorylation of ADP is inhibited in presence of biguanides. The activity of different compounds is proportional to their binding affinity to phospholipids.2. Oscillatory volume changes of mitochondria are largely abolished by biguanides.3. Biguanides cause a small increase of electrical conductance of phospholipid bilayers. This is only in part due to charge transfer by the lypophilic biguanide cations. It is suggested that also structural changes are induced by biguanides altering the surface area per molecule of phospholipid. At high biguanide concentrations structural reorganization is also expressed by changes in electrical capacitance. Both effects are saturable and proportional to binding affinity.4. Biguanides affect the electrical conductance of bilayers in the opposite way but to the same extent with positively charged species (K+/ionophore) and with negatively charged species (uncouplers of oxidative phosphorylation). This is explained by generation of a positive surface potential at phospholipid containing membranes.5. The data fit excellently the application of diffuse double‐layer theory to lipid bilayers. The relative effectiveness of different biguanides on model membranes is identical to that on ion fluxes with mitochondrial membranes. From this it is concluded that a similar mechanism of action applies to synthetic and natural membranes as well, suggesting that screening of functional negative sites interferes with the translocation of cations linked to electron transport and the primary reactions in conservation of respiratory energy.
Deficiency of Subunit Two of Cytochrome Oxidase in the mi‐3 Cytoplasmic Mutant of Neurospora crassa
Authors: BERTRAND Helmut , WERNER Sigurd 
Published: March 2005

The formation of cytochrome oxidase was examined in the cytochrome‐aa3‐deficient mi‐3 cyto‐plasmic mutant ofNeurospora crassa. The treatment of mitochondria of mutant mi‐3 with antibody to cytochrome oxidase yields a precipitate of cross‐reacting material consisting predominantly of cytochrome oxidase subunits 5 (mol. wt 14000) and 6 (mol. wt 12000) and small amounts of subunits 4 (mol. wt 16000) and 7 (mol. wt 10000), but devoid of the mitochondrially synthesized, high molecular weight polypeptides, namely subunits 1 (mol. wt 41000), 2 (mol. wt 28000) and 3 (mol. wt 21000).On the other hand, a qualitative examination of the products of mitochondrial translation in mutant mi‐3 revealed that large molecular weight polypeptides are synthesized. However, a comparison of the dodecyl sulfate acrylamide gel electrophoresis patterns of the mitochondrial translation products of mutant mi‐3 and wild‐type indicated that the cytoplasmic mutant is deficient in polypeptides in the molecular weight ranges of 28000 and 21000, respectively.By means of subunit specific antibodies it could be shown that the mitochondria of mutant mi‐3 contain virtually undetectable amounts of subunit 2 of cytochrome oxidase, but synthesize easily detectable quantities of subunits 1 and 3. Those subunits that are present in the mitochondria of mutant mi‐3 do not appear to form a definitive complex with each other.The above results are discussed in view of earlier observations indicating that mutant mi‐3 may be defective in a regulatory circuit that controls the formation of cytochrome aa3. The synthesis of subunit 2 of cytochrome oxidase could be the affected process, and the relative deficiency of this polypeptide may limit the assembly of cytochrome aa3 in the mutant.
Synthesis and Transport of Myosin in Physarum polycephalum
Authors: SCHWÄRZLER Markus , JOCKUSCH Brigitte M. , HALL Len , BRAUN Richard 
Published: March 2005

Immunological techniques have been used to study the rate of synthesis and intracellular transport of myosin in the slime mould Physarum polycephalum. Quantitative precipitation of myosin in homogenates of Physarum was achieved using an antimyosin antibody produced in rabbit in response to purified Physarum myosin. Dodecylsulphate‐gel electrophoresis revealed that about 50% of the precipitated material is myosin.The rates of synthesis of total cellular protein and myosin were measured over the mitotic cycle. Both were found to increase exponentially or linearly between two successive nuclear divisions. Similarly, no difference in the proportion of myosin‐synthesising polysomes, assayed by precipitation with antimyosin serum, could be detected between the S phase and G2 phase of the mitotic cycle.Myosin makes up nearly 2% of total plasmodial proteins. Its transport into the nucleus occurs predominantly during the G2 phase.
Synthesis and Kinetic Properties of a New NAD+ Derivative Carrying a Vinyl Group
Authors: MURAMATSU Minoru , URABE Itaru , YAMADA Yasuhiro , OKADA Hirosuke 
Published: March 2005

A method for the synthesis of NAD+‐N6‐[N‐(N‐acryloyl‐1‐methoxycarbonyl‐5‐aminopentyl)‐propioamide] (monomeric NAD+ derivative), a new NAD+ derivative carrying a vinyl group, was described. By copolymerization with acrylamide 90% of the NAD+ derivative was converted to a water‐soluble macromolecular NAD+ derivative (polymeric NAD+ derivative). Both NAD+ derivatives were reduced completely with yeast alcohol dehydrogenase.High cofactor activities relative to free NAD+ and NADH were obtained for the monomeric derivatives of NAD+ (71–86%) and NADH (66–87%) with yeast alcohol, horse liver alcohol, lactate, and malate dehydrogenases. Lower but substantial relative cofactor activities were obtained for the polymeric derivatives of NAD+ (18–33%) and NADH (28–60%) with yeast alcohol, horse liver alcohol, and malate dehydrogenases. But lactate dehydrogenase had negligible activity both for the polymeric NAD+ and NADH derivatives.Kinetic studies were carried out with yeast alcohol dehydrogenase and lactate dehydrogenase, and the following kinetic constants were determined: the maximum velocity (V), the limiting Michaelis constants for coenzyme (Ka) and for substrate (Kb), and the dissociation constant of the enzyme‐coenzyme complex (Kia). In the reaction system with the monomeric NAD+ derivative and yeast alcohol dehydrogenase, the Kia value was similar to that for NAD+ and the values of V, Ka, and Kb were 38, 30, 21% of those for NAD+. In the reaction system with the polymeric NAD+ derivative and yeast alcohol dehydrogenase, the values of V, Ka, Kb, and Kia were 0.5, 4, 3, 3 times larger than those for the monomer. In the reaction system with the monomeric NAD+ derivative and lactate dehydrogenase, the values of V, Ka, Kb and Kia were 39, 16, 36, 57% of those for NAD+. From these results the following conclusions were obtained: (a) the modification of NAD+ by alkylating at position 6 in the adenine moiety of NAD+ caused a decrease in the V value, possibly due to configurational alterations of the binary or ternary coenzyme complexes of the enzymes resulting in decreased cofactor activity of NAD+, but the decrease in the activity was somewhat compensated by the decrease in the values of Ka, Kb, and Kia also caused by the modification; (b) the incorporation of the NAD+ derivative into the side chain of polyacrylamide caused decrease in the affinity for the enzyme and further decrease in the V value, and thus decreased the cofactor activity to below that of the monomeric NAD+ derivative.
Analysis of Calf‐Thymus Satellite DNA: Evidence for Specific Methylation of Cytosine in C‐G Sequences
Authors: GAUTIER Fritz , BÜNEMANN Hans , GROTJAHN Lutz 
Published: March 2005

Digestion of purified calf tymus satellite I (θ= 1.714 g/cm3) with a series of restriction enzymes shows that modification in this satellite occurs preferentially in the sequence C‐G. This was also shown to be the case in the other satellites and in bulk chromosomal calf thymus DNA.Cloning of purified satellite I DNA in Escherichia coli makes sites, previously modified, available for cutting with certain restriction enzymes. All these ‘new’ sites contain the sequence C‐G.High‐resolution mass spectroscopy establishes that the satellites contain a low concentration of 5‐methylcytosine. This iners that methylation which inhibits restriction enzyme cutting must occur preferentially in the sequence C‐G. Hybridization of cRNA of cloned satellite I DNA with the satellites III (θ= 1.706 g/cm3) and IV (θ= 1.710 g/cm3) shows that there is no or little sequence homology between these satellites. Digestion of calf thymus satellite I DNA with endoR ·EcoRI and subsequent hybridization studies with the fragments shows two EcoRI fragments in addition to the usual 1400‐base‐pair EcoRI repeat unit.
Accumulation of Hypothiocyanite Ion during Peroxidase‐Catalyzed Oxidation of Thiocyanate Ion
Authors: AUNE Thomas M. , THOMAS Edwin L. 
Published: March 2005

An oxidized form of thiocyanate accumulated during incubation of lactoperoxidase or myeloperoxidase with thiocyanate ion and low concentrations of peroxide. Addition of one mole of peroxide resulted in loss of one mole of thiocyanate and formation of one mole of the oxidation product. The oxidation product reacted with two moles of sulfhydryl to yield one mole of disulfide and one mole of thiocyanate. The same product was obtained by alkaline hydrolysis of thiocyanogen. The product is proposed to be hypothiocyanite ion. The peroxidases may catalyze the oxidation of thiocyanate to hypothiocyanite, or to thiocyanogen. Hypothiocyanite may be the antimicrobial agent formed during peroxidase‐catalyzed oxidation of thiocyanate.Hypothiocyanite decomposed slowly with a second‐order rate constant of 0.1 M−1 s−1 at 25°C. When the amount of peroxide added approached the amount of thiocyanate, the yield of hypothiocyanite reached a maximum and then declined. This result suggested that hypothiocyanite was oxidized by excess peroxide. Efforts to produce high concentrations of hypothiocyanite were unsuccessful, even when thiocyanate was well in excess of peroxide. As the hypothiocyanite concentration approached 0.5 mM, decomposition was accelerated and small amounts of cyanide were produced. Hypothiocyanite reacted stoichiometrically with cyanide to give products that did not oxidize sulfhydryls. Hydrolysis of thiocyanogen at neutral pH also resulted in rapid decomposition and formation of cyanide.
Fast Kinetic Studies on the Interaction of Cholinergic Agonists with the Membrane‐Bound Acetylcholine Receptor from Torpedo marmorata as Revealed by Quinacrine Fluorescence
Authors: GRÜNHAGEN Hans‐Heinrich , IWATSUBO Motohiro , CHANGEUX Jean‐Pierre 
Published: March 2005

Rapid changes of fluorescence intensity take place after fast mixing of quinacrine‐labelled receptorrich membrane fragments from Torpedo marmorata with cholinergic effectors and are recorded in the time range of 1 – 1000 ms, accessible to the stopped‐flow technique. Agonists such as acetylcholine, phenyltrimethylammonium, carbamylcholine, suberyldicholine or choline cause the change, but not antagonists such as d‐tubocurarine, flaxedil or Naja nigricollisα‐toxin. In addition, preincubation of the membrane fragments with N. nigricollisα‐toxin blocks the response to agonists. Ceruleotoxin, a toxin from Bungarus ceruleus venom which blocks the postsynaptic response to acetylcholine at a site distinct from the α‐toxin site, causes a decrease of the apparent amplitude of the fast signal. In the presence of 0.05 mM tetram, an acetylcholinesterase inhibitor, the kinetics of the fast response to acetylcholine do not change; in the second or minute range and in the case of acetylcholine, however, a decrease of fluorescence intensity occurs in the absence of acetylcholinesterase inhibitor showing that the fast fluorescence signal is reversible.Under the present experimental conditions the observed fast kinetics do not vary with the concentration of receptor sites or quinacrine but do vary with the concentration of agonist. The data can be analyzed in terms of the phenomenological reaction scheme , when k2»k3, if the fluorescence signal monitors the isomerization from A to A*. The following values of the constants are found: for acetylcholine: k4= (1.5 ± 0.5) s−1, k3= (60 ± 40) s−1, k2≥ 5 × 102 s−1 and k1≥ 107l·mol−1 s−1, K1=k2/k1= (7 ± 3) × 10−5 M, K=K1·k4/k3= (2 ± 1) × 10−6M; for carbamylcholine: k4= (0.2 ± 0.1) s−1, k3= (10 ± 5) s−1, k2× 102 s−1 and k1≥ 3 × 105 s−1, K1= (3 ± 1) × 10−4 M and K= (4 ± 3) × 10−6 M. The values of the apparent dissociation constants determined from the variation of the amplitudes of the fast effect for acetylcholine and carbamylcholine are respectively (1.0 ± 0.5) × 10−6 M and (5 ± 2) × 10−6 M. Some variability in the absolute values of the constants is noticed from one preparation to another and, for a given preparation, changes with age. Sets of kinetic constants have also been determined in the case of choline and suberyldicholine. At 20 °C, the Q10 is about 2.5 in the presence of 0.03 mM carbamylcholine and 1.3 in the presence of 0.01 M carbamylcholine; it is 1.3 with 0.05 M acetylcholine.The data are interpreted in terms of a three‐state model and their physiological significance is discussed.
Adenosine Triphosphatase Activity Associated with Purified Cholinergic Synaptic Vesicles of Torpedo marmorata
Authors: BREER Heinz , MORRIS Stephen J. , WHITTAKER Victor P. 
Published: March 2005

A rapid method for purifying Torpedo electric organ vesicles is described, which employs an iso‐osmotic continuous sucrose‐glycine gradient followed by chromatography on CPG‐10‐3000 porous glass beads.The synaptic vesicles have a buoyant density of 1.057 g/ml. The purified vesicles are free of cholinesterase, lactate dehydrogenase and Na+,K+‐stimulated ATPase activity. They contain a ouabaininsensitive, Na+,K+‐inhibited, Mg2+,Ca2+‐stimulated ATPase activity. This is further stimulated by acetylcholine but not by choline.
Mechanism of the Prolyl Hydroxylase Reaction
Authors: MYLLYLÄ Raili , TUDERMAN Leena , KIVIRIKKO Kari I. 
Published: March 2005

The kinetics of the prolyl hydroxylase reaction were studied with pure enzyme from chick embryos by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other substrates were held constant. Intersecting lines were obtained in double‐reciprocal plots for all possible pairs of Fe2+, 2‐oxoglutarate, O2 and the polypeptide substrate, whereas parallel lines were obtained for pairs involving ascorbate with each substrate. In addition, parallel lines were obtained when the polypeptide substrate concentration was varied at different fixed 2‐oxoglutarate concentrations in the presence of saturating O2 concentration. Poly(L‐proline) was a competitive inhibitor with respect to the polypeptide substrate, but uncompetitive with respect to Fe2+ and 2‐oxoglutarate. High concentrations of the polypeptide substrate inhibited the reaction, this substrate inhibition being competitive with respect to Fe2‐ and 2‐oxoglutarate. Succinate, CO2 and collagen were product inhibitors, succinate inhibiting the reaction competitively with respect to 2‐oxoglutarate, but noncompetitively with respect to the other substrates, and collagen noncompetitively with respect to all substrates. The apparent Km and Ks values for the substrates and Ki values for the inhibitors are given.These and additional data would be consistent with a tentative reaction scheme involving an ordered binding of Fe2+, 2‐oxoglutarate, O2 and the polypeptide substrate to the enzyme in this order, the binding of Fe2+ being at thermodynamic equilibrium. The enzyme can also react directly with the polypeptide substrate or its analogue poly(L‐proline) under certain conditions, forming dead‐end complexes. The products are released only after the hydroxylation, possibly in the order: the hydroxylated polypeptide, CO2 and succinate. Ascorbate may react either with enzyme·Fe before the release of Fe2+ or with free enzyme before the binding of Fe2+, but a reaction with ascorbate at any stage after the release of the first product is not excluded. The mechanism proposed is not entirely identical with either of the main two previous suggestions for the mechanism of 2‐oxoglutarate dioxygenases.
Structure and Function of Ornithine Carbamoyltransferases
Authors: LEGRAIN Christiane , STALON Victor , NOULLEZ Jean‐Pierre , MERCENIER Annick , SIMON Jean‐Paul , BROMAN Kathleen , WIAME Jean‐Marie 
Published: March 2005

The reaction catalyzed by ornithine carbamoyltransferase can participate in either the anabolism or the catabolism of arginine. The carbamoylation of ornithine, yielding citrulline, is involved in the biosynthetic sequences; the reverse reaction, the phosphorolysis of citrulline, is the second step of the arginine deiminase pathway.The ornithine carbamoyltransferases of a number of microorganisms which can fulfil both of these functions have been studied in this work. This group of organisms was found to possess two distinct ornithine carbamoyltransferases. The functions of these enzymes were surmised by determining the type of genetic regulation to which they were subjected.The kinetic properties of these various enzymes have been determined. All of them, regardless of the role they play in the cell, catalyze both the synthesis and arsenolysis of citrulline. The anabolic transferase of Pseudomonas is the only enzyme which displays functional irreversibility.A comparison of the quaternary structure of these transferases was performed and reveals interesting features in relation to the metabolic function of these enzymes. All well‐characterized anabolic enzymes have low molecular weights (from 150000–105000) and are likely to be trimers. Catabolic enzymes, with the exception of those of Bacillus licheniformis and halobacterium salinarium, display much higher molecular weights and more elaborate quaternary structure.The properties of these two groups of transferases are discussed in relation to their metabolic role in the cells.
A Cytoplasmic High Affinity Estrogen‐Binding Protein in the Embryonic Chicken Liver
Authors: GSCHWENDT Michael , KITTSTEIN Walter 
Published: March 2005

1. Embryonic chicken liver contains cytoplasmic estrogen‐binding sites with high affinity for estradiol (Kd= 1 × 10−9 M) and with low capacity (200–1100 binding sites/cell, depending on the age of the embryo).2. Only estrogenic hormones are bound with high affinity.3. At least the estrogen‐binding moiety of these binding sites is a protein.4. By agarose gel chromatography an apparent molecular weight of 50 000–60 000 daltons was determined, very similar to that of the nuclear estrogen‐binding protein.5. In low salt the cytoplasmic binding sites exhibit a stong tendency to aggregate, which is abolished by mild trypsination.6. A single injection of estradiol into the egg seems to cause a translocation of the cytoplasmic binding protein to the nucleus.7. During the embryonic development the number of binding sites increases up to the age of 19 days. Thereafter it decreases again reaching a constant low level soon after hatching.8. Data are presented to show that this low level of binding sites can not be due solely to a degradation or inhibition in vitro by a component eventually present in the liver homogenate of hatched animals.
Adenosylhomocysteinase from Yellow Lupin Seeds
Authors: GURANOWSKI Andrzej , PAWELKIEWICZ Jerzy 
Published: March 2005

Adenosylhomocysteinase from yellow lupin seeds (Lupinus luteus) has been purified to homogeneity.Active enzyme, Mr= 110 000, consists of two probably identical subunits with Mr= 55 000 as judged by gel filtration and dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of 2‐mercaptoethanol. The isoelectric point of the enzyme was shown to be 4.9 ± 0.1. It was demonstrated by disc and pore gradient electrophoresis that the most purified fraction formed multimers. The enzyme shows optimum activity at pH 8.5–9.0. Km values are 2.3 μM, 4.6 mM and 12 μM for adenosine, DL‐homocysteine and S‐adenosyl‐L‐homocysteine, respectively. The energy of activation for S‐adenosylhomocysteine synthesis was estimated as 14.4 kcal/mol (60.2 kJ/mol) and temperature coefficient as 2.4. The equilibrium constant for the hydrolysis of S‐adenosylhomocysteine amounts to 5 × 10−7 M. Anti‐sulfhydryl reagents such as p‐hydroxymercuribenzoate and N‐ethylmaleimide acted as irreversible inhibitors. The enzyme exhibits high specificity for homocysteine whereas some of the rare nucleosides tested could substitute for adenosine.
The Preparation of Ligandin with Glutathione‐S‐Transferase Activity from Porcine Liver Cytosol by Affinity Chromatography on Bromosulphophthalein‐Sepharose
Authors: GRAHNÉN Anders , SJÖHOLM Ingvar 
Published: March 2005

A simple and rapid method for the purification of porcine ligandin with glutathione‐S‐transferase activity is presented. After ion‐exchange chromatography on DEAE‐Sephadex, ligandin is isolated from porcine liver cytosol by affinity chromatography on bromosulphophthalein‐Sepharose and gel filtration on Sephadex G‐100. Evidence is presented that the purified ligandin is homogeneous with respect to polyacrylamide‐gel electrophoresis (7.5%) and sodium dodecylsulphate‐gel electrophoresis.Physico‐chemical investigations show that the purified ligandin has properties similar to those of ligandin isolated from other species with respect to molecular weight, amino‐acid composition, secondary structure and catalytic activity. As is the case for human and rat ligandin, porcine ligandin binds bilirubin. Evidence is also presented that porcine liver cytosol contains several bromosulphophthalein‐binding proteins with basic isoelectric points lacking catalytic activity.
Properties of the Complex between Histone H1 and Poly(ADP‐ribose) Synthesised in HeLa Cell Nuclei
Authors: STONE Peter R. , LORIMER Wishard S. , KIDWELL William R. 
Published: March 2005

Preparations of H1 histone from HeLa cell nuclei incubated with [3H]NAD to permit poly(ADP‐ribose) synthesis were electrophoresed on polyacrylamide gels. The incorporated radioactivity migrated as a sharply defined peak in association with a protein band which moved more slowly than H1, the major protein component. The following observations indicate that this complex is composed of two molecules of H1 and a single chain of poly(ADP‐ribose) with one detectable covalent linkage of polymer to protein.

The [14C]arginine/[3H]lysine ratio is identical in H1 histone and in the protein moiety of the complex.
Protein is displaced from H1 histone to the complex during poly(ADP‐ribose) synthesis. At least 90% of the protein in the complex (stainable protein and labelled protein) is derived from H1.
Sedimentation rate studies indicate a molecular weight of the complex about twice that of H1 histone.
The average chain length of the polymer is 15 ADP‐ribose units and there are 7–8 ADP‐ribose units for each molecule of H1 histone in the ‘complex’.
Poly(ADP‐ribose) glycohydrolase, which hydrolyses the polymer exoglycosidically from the AMP terminus, degrades the complex producing ADP‐ribose and mono‐ADP‐ribosylated H1 histone which co‐electrophoreses with unmodified H1.

Although only one covalent linkage between protein and polymer has been detected, the ‘complex’ does not dissociate when electrophoresed on dodecylsulfate gels. Nor can the noncovalently linked H1 histone of the complex readily exchange with free H1. Complex formation does not occur when purified poly(ADP‐ribose) and H1 are mixed.