bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studies on 1‐β‐D‐Arabinofuranosyl‐cytosine‐Resistant Mutants of Chinese‐Hamster Fibroblasts
Authors: Robert de Saint‐Vincent Bruno , Buttin Gérard 
Published: March 2005

The biochemical basis of resistance to 1‐β‐D‐arabinofuranosyl‐cytosine (Ara‐Cyt), an analog of cytidine, was explored in two lines of Chinese‐hamster fibroblasts, one of which exhibits a temperature‐dependent resistance to the drug. In both cases, an absolute defect in the capacity to phosphorylate Ara‐Cyt was demonstrated under growth conditions which confer complete resistance.
Both lines are unable to utilize appreciably exogenous deoxycytidine (dCyd) for nDNA synthesis, thus suggesting that in hamster fibroblasts, as in other previously studied mammalian cells Ara‐Cyt and dCyd are phosphorylated by the same pathway.
However, the level of dCyd kinase activity in crude extracts of the Ara‐Cyt‐kinase‐deficient cells is not greatly reduced. This is explained by the existence in wild‐type cells of two dCyt kinases which can be separated by DEAE‐cellulose chromatography: dCyd kinase 1, which is devoid of activity on Ara‐Cyt, is present in both wild‐type and mutant cells, while dCyd kinase 2, which phosphorylates Ara‐Cyt, is detectable in wild‐type Ara‐Cyt‐sensitive cells only.
The inefficiency of dCyd kinase 1 alone to ensure conversion of exogenous dCyd for its incorporation into nDNA is best accounted for by the mitochondrial location of this enzyme: this is established both by subcellular fractionation experiments and by the demonstration that incorporation of exogenous dCyd into mtDNA is unchanged in the Ara‐Cyt‐resistant mutants.
Membrane Adenosine Triphosphatase of Micrococcus lysodeikticus
Authors: Andreu Jose Manuel , Muñoz Emilio , Albendea Jose Antonio 
Published: March 2005

The adenosine triphosphatase of Micrococcus lysodeikticus has been purified by Sephadex G‐200 filtration in 1 mM EDTA‐50 mM NH4HCO3 buffer pH 9.0, after its selective release (“shock wash”) from the membranes. The use of the EDTA‐ammonium bicarbonate buffer, instead of the Tris‐CI buffers previously used (Muñoz et al., 1969), shortened the process of purification and improved its yield. The purified ATPase did not show trypsin‐stimulated activity, unlike the crude soluble enzyme which presented an approximately 50% ATPase stimulation by trypsin. The purified protein gave a single band in 7% or 10% polyacrylamide‐gel electrophoresis. Sedimentation‐velocity experiments provided new criteria of protein purity, although they indicated a possible dissociation of the protein at low concentrations. We calculated a so20, w value of 13.6 S by linear extrapolation. Sedimentation‐equilibrium experiments confirmed both the homogeneity and partial dissociation of the native enzyme. Hence, we determined molecular weights of 330000–350000. The amino acid composition of M. lysodeikticus ATPase resembles those of other ATPases, but contains lower percentages of proline and lysine. It reveals a medium hydrophobicity, which agrees with its properties as membrane‐associated enzyme. A partial specific volume of 0.74 ml/mg was calculated from the amino acid composition. Sodium dodecylsulphate—7% polyacrylamide electrophoresis revealed five components (relative mobilities were: 0.71, 0.75, 0.79, 0.92 and 1.0). The componente(s) of faster mobility appeared to be contaminant(s). The other four components were considered as the ATPase subunits (α, β, γ and δ). Similar subunit patterns were obtained by dissociation with phenol‐acetic acid and urea‐acetic acid. The α and β‐subunits were present in greater amounts than the γ and δ‐subunits. The γ‐subunits showed variations depending on the enzyme preparation. The other three subunits (“intrinsic” subunits) were found in all preparations with approximately constant molar ratios. We therefore conclude that the 33000—350000 molecular weight must correspond to a complex of the “intrinsic” subunits. The molecular weight of each subunit calculated from its relative mobility in dodecylsulphate‐polyacrylamide electrophoresis were: α= 52500 ± 5000, β= 47000 ± 4500, γ= 41500 ± 4000 and δ= 28500 ± 2000. We propose a formula α3β3δ(+γ) for the native ATPase non‐stimulated by trypsin. M. lysodeikticus APTase shows marked structural similarities with beef‐heart mitochondrial ATPase. The presence of minor subunits (γ, δ) does not seem to account for the trypsin stimulation of the enzyme.
Brown‐Adipose‐Tissue Mitochondria
Authors: Nicholls David G. , Lindberg Olov 
Published: March 2005

Endogenously uncoupled mitochondria from the brown adipose tissue of cold‐adapted hamsters, rats and guinea‐pigs swell in KC1, KBr, KNO3 and KCNS in the presence of valinomycin. Identical rates of swelling are obtained employing the ammonium salts or the potassium salts in the presence of nigericin.
Albumin has no effect on the swelling rates in these potassium salts plus valinomycin, but is strongly inhibitory with both potassium salts plus nigericin and with ammonium salts. Albumin inhibits swelling in potassium phosphate plus valinomycin but is without effect in potassium phosphate plus nigericin or in ammonium phosphate. All these inhibitions may be reversed by uncoupler.
These results are interpreted in terms of electrogenic entry of Cl‐, Br‐, NO3‐ and CNS‐, and of a high native proton permeability which is abolished by albumin.
GDP inhibits swelling in KCl and KBr in the presence of either valinomycin or nigericin. The inhibition is not reversed by uncoupler. Exchange of 36Cl‐ both into and out of the sucroseimpermeable space is strongly inhibited by GDP. The effectiveness of different nucleotides in inhibiting 36Cl‐ efflux is identical to their effectiveness previously observed for uncoupler‐releasable respiratory inhibition in the presence of albumin.
GDP only partially inhibits NO3‐ permeation and is without demonstrable effect on thiocyanate, phosphate or acetate entry, but only if no proton movements are involved. Otherwise, in these systems, some limitation of proton permeation is observed which may be reversed by carbonyl cyanide p‐trifluoromethoxyphenylhydrazone.
The entry of sodium appears to be by electroneutral exchange and not to be affected by albumin or nucleotide.
The use of the ion permeability properties as a tool to investigate the relationship between membrane structure and energy conservation is discussed.
2‐Amino‐2,6‐Dideoxy‐D‐Glucose (D‐Quinovosamine): a Constituent of the Lipopolysaccharides of Vibrio cholerae
Authors: Jann Barbara , Jann Klaus , Beyaert Guido O. 
Published: March 2005

From the lipopolysaccharides of Vibrio cholerae strains Inaba and Ogawa, an unusual amino sugar was isolated.By sequential oxidation with sodium metaperiodate and hypoiodide and by oxidation with periodate followed by detection of the acetaldehyde formed, the amino sugar was characterized as a 2‐amino‐dideoxyaldose.The hydrochloride of the amino sugar was compared with those of 2‐amino‐2,6‐dideoxy‐d‐glucose, 2‐amino‐2,6‐dideoxy‐l‐galactose, 2‐amino‐2,6‐dideoxy‐d‐mannose, 2‐amino‐2,6‐dideoxy‐d‐gulose, and 2‐amino‐2,6‐dideoxy‐d‐allose in an amino acid analyzer. The amino sugar alcohols were studied by paper electrophoresis in molybdate buffer.From the results obtained the amino sugar was identified as 2‐amino‐2,6‐dideoxyglucose (quinovosamine). Its optical rotation indicated it to be of the d‐configuration.
Membranes of Glyoxysomes from Castor‐Bean Endosperm
Authors: Bieglmayer Christian , Ruis Helmut , Graf Jürg 
Published: March 2005

A procedure was developed for the isolation of membranes of glyoxysomes from the endosperm of germinating castor beans (Ricinus communis L. var. zanzibariensis). These membranes were prepared by osmotic‐shock treatment of isolated glyoxysomes. In some cases this was followed by mild sonication. They were isolated by centrifugation on to a 57% (w/w) sucrose layer and purified by isopycnic sucrose gradient centrifugation.The membrane preparations were characterized by electronmicroscopy and assayed for enzyme activities. They consisted mainly of ghost‐type vesicles and were found to contain a major part of fatty acid β‐oxidation, malate synthase, citrate synthase and antimycin‐A‐insensitive NADH‐cytochrome c reductase activities of the whole glyoxysomes. Malate dehydrogenase may also be bound to the membrane to a significant extent. A tentative scheme is postulated describing the structural organization of fatty acid β‐oxidation and of the glyoxylate cycle in glyoxysomes.
The Reaction of Active‐Site Inhibitors with Elastase Using a New Assay Substrate
Authors: Thomson Alan , Denniss Iain S. 
Published: March 2005

1. Some active‐site directed chloroketone derivatives of elastase have been synthesised and characterised. Their inhibition constants and rate constants for inhibition of elastase have been determined.2. A new spectrophotometric substrate of elastase has been developed.3. Inhibition of elastase increases in efficiency with length of polypeptide chain except that in N‐acetylated derivatives the dipeptide is significantly less reactive when bound to the enzyme than is a simple alanine derivative.
Immunochemical Studies on Rat‐Liver Microsomal NAD Glycohydrolase
Authors: Bock Karl Walter , Matern Siegfried 
Published: March 2005

Rabbit antiserum was prepared against rat liver microsomal NAD glycohydrolase solubilized by lipase treatment and subsequently purified by successive DEAE‐cellulose, Sephadex G‐100 chromatography and polyacrylamide gel electrophoresis. The antibody inhibited the purified enzyme 60–80%, but the membrane‐bound and Triton X‐100 solubilized enzyme up to 20–30% only. A single precipitation line was produced when the antibody reacted with the purified enzyme, and with crude lipase and Triton X‐100 solubilized enzyme preparations. NAD glycohydrolase from liver rough‐microsomal and plasma‐membrane fractions reacted identically.The antibody to rat liver NAD glycohydrolase similarly inhibited the lipase or Triton X‐100 solubilized and membrane‐bound NAD glycohydrolase from rat spleen and brain as well as from mouse liver but not from bovine liver.
Further Studies on the Mechanism of Action of UDP‐Apiose/UDP‐Xylose Synthase from Cell Cultures of Parsley
Authors: Baron Diethard , Grisebach Hans 
Published: March 2005

1. When uridine diphopho‐d‐[U‐14C]xylose was incubated with the UDP‐apiose/UDP‐xylose synthase from cell suspension cultures of parsley no radioactivity was detected in UDP‐apiose (UDP‐Api).2. UDP‐d‐glucuronic acid (UDP‐GlcUA) labeled with tritum at either C‐4 or C‐5 was converted to UDP‐Api/UDP‐Xyl synthase. Free apiose was reduced to apiitol which was oxidized with periodic acid to glycolic acid. In both cases oxidation of glycolic acid with glycollate oxidase from spinach to glyoxalic acid released all the tritium into the water. This result proves that during the hydride shift from UDP‐[4‐3H]GlcUA tritium is transfered only to the pro‐R position at C‐3′ of apiose and that inversion of configuration occurs at C‐4 of apiose in the synthase reaction.3. When the UDP‐Api/UDP‐Xyl synthase reaction was carried out in a 3H1HO/2H2O mixture, a total of 1.5 mol 3H per mol apiose was incorporated with correction made for the product isotope effect. By degradation of apiose it was proved that 1 mol 3H was located at C‐4 and 0.5 3H per mol sugar at C‐3′ of apiose.4. After addition of NaB3H4 to the enzyme incubation with UDP‐d‐[U‐14C]GlcUA and hydrolysis of the nucleiotide sugars, arabinose, xylose and glucuronic acid with a constant 3H/14C ratio were isolated after extensive purification. it was shown by degradation of xylose that over 90% of the tritium was located at C‐4 of this sugar. These results prove the existence of a 4‐keto intermediate and the formation of UDP‐4‐keto‐glucuronic acid as an intermediate in the decarboxylation step.5. The results presented can best be explained by an aldolase type mechanism for the UDP‐Api/UDP‐Xyl synthase reaction.
Bacteriophage T7‐DNA Replication in vitro: an Experimental System
Authors: Ferdinand Franz Josef , Krause Elke , Knippers Rolf , Strätling Wolf 
Published: March 2005

We describe an experimental system which consists of intact double‐stranded T7 DNA and an unfractionated protein extract from bacteriophage‐infected cells. Under proper conditions, DNA replication is initiate. Evidence will be presented that, at least the viral DNA polymerase and the function of the viral gene‐4‐protein are required for initiation. Circumstantial evidence suggests that the viral RNA polymerasee is also necessary for initiation.After successful initiation a part of the viral genome is semiconservatively replicated.
Studies on the Metabolic Effect Induced in the Rat by a High‐Fat Diet
Authors: Zaragoza‐Hermans Nelly M. 
Published: March 2005

Adipose tissue of rats fed from weaning a fat diet or a control carbohydrate diet, was incubated with [U‐14C]glucose or [6‐14C]glucose (20 mM) or with unlabelled glucose (20 mM) and trace amount of [U‐14C]pyruvate, in the presence of insulin (1000 μU/ml). Fluxes of carbon through various metabolic pathways were calculated.In adipose tissue of fat‐fed rats, the rate of glucose carbon flow at the level of pyruvate decarboxylation and in fatty acid synthesis from acetyl‐CoA, was reduced to 5% or less of the control. The rate of glycolysis was only decreased to 20% while the greatest part of intermediary pyruvate was diverted into lactate.Recycling of pyruvate through a partial reversal of glycolysis was also demonstrated and was concomitant with an exchange of 14C with CO2, accounting for up to 35% of the total 14CO2 production from [U‐14C]glucose.A balance of production and utilization of reduced nucleotide coenzymes in the cytoplasm during glucose metabolism was established. In contrast with the control tissue, a net excess was obtained in adipose tissue of fat‐fed rats. Alteration of the cytoplasmic redox state was also indicated by an abnormally high lactate/pyruvate ratio.Several aspects of the glucose metabolism show the existence of a high energetic level or ATP:ADP ratio.The significance of the findings is discussed with regard to the control of lipogenesis in adipose tissue.
The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K 12
Authors: Véron Michel , Saari John C. , Villar‐Palasi Carlos , Cohen Georges N. 
Published: March 2005

The interactions between the catalytic regions of the subunit of the bifunctional enzyme aspartokinase I‐homoserine dehydrogenase I, although not necessary for the catalytic functions, are shown to be of utmost importance for the regulation of the activities of the enzyme.The study of the effect of a number of –SH reagents on the protein shows that the inhibition of the homoserine dehydrogenase activity by the allosteric effector l‐threonine is closely correlated with the existence of an active aspartokinase region.The kinetic study of the proteolytic reaction leading to the previously described homoserine dehydrogenase fragment indicates that a dissociation is a necessary requirement for the digestion of the aspartokinase region.Cross‐linking with dimethylsuberimidate and proteolytic degradation of the fully cross‐linked tetramer have been used to show that the oligomeric interactions, necessary to build the tetrameric structure, involve the kinase region only.A tentative model is proposed showing aspartokinase I‐homoserine dehydrogenase I as an isologous tetramer in which the aspartokinase regions, organized as a “core”, are surrounded by the homoserine dehydrogenase regions.
On the Mechanism of Bongkrekate Effect on the Mitochondrial Adenine‐Nucleotide Carrier as Studied through the Binding of ADP
Authors: Klingenberg Martin , Buchholz Marlies 
Published: March 2005

Further unique effects of bongkrekate on the binding of ADP to the inner mitochondrial membrane are described in addition to a preceding report by us and explained so as to demonstrate the reorientation of a mobile carrier across the membrane.1. The dependence of the bongkrekate‐induced increase of ADP binding is nonlinear with respect to the bongkrekate concentration. Conscquently the apparent Kd for ADP decreases nonlinearly with bongkrekate concentration. The nonlinearity is kinetic in nature and is not explained by regulatory (allosteric) effects. The nonlinearity is diminished at lower pH.2. The binding of bongkrekate appears to be dependent on ADP indicating a reciprocal enhancement effect between ADP and bongkrekate binding. As a consequence the binding of high‐affinity nucleotide (such as ADP) is relatively more increased by bongkrekate the binding of nucleotides with less affinity such as dADP. This specificity preference for ADP is also increased by bongkrekate as compared to other analogues such as formycin diphosphate, and the imidodiphosphate and methylene diphosphate derivations of adenylate. The counteraction of bongkrekate and atractylate on ADP binding also depends on the cooperation between ADP and bongkrekate.3. An apparent irreversibility of the bongkrekate‐induced ADP binding is demonstrated by several effects. In the course of time bongkrekate + ADP supersede the counteraction of atractylate, even at higher atractylate concentration. Also carboxy‐atractylate, with still higher affinity than atractylate, cannot abolish the bongkrekate. [14C]ADP once fixed with bongkrekate at low pH cannot be removed by exchange with excess ADP or atractylate even at pH 8, although at this pH bongkrekate is unable to induce a binding increase.4. The results can be explained by the “reorientation mechanism” of the bongkrekate effect by which the ADP · carrier complex is translocated inside and all carriers become trapped inside by bongkrekate as the immobile carrier · bongkrekate complex. The reciprocal enhancement of bongkrekate and ADP binding is explained by the fact, that ADP is required to move the carrier inside where bongkrekate can bind to the carrier. The irreversibility of the bongkrekate effect results from the shift of the carrier inside and the resulting removal of the carrier out of the equilibrium with external ligands such as atractylate or ADP. The results thus produce the most direct evidence so far available on the molecular level, for the existence of a mobile metabolite carrier.
Isolation and Characterization of Ribosome‐Free Cytoplasmic Messenger‐Ribonucleoprotein Complexes from Avian Erythroblasts
Authors: Gander Eugen S. , Stewart Andrew G. , Morel Carlos M. , Scherrer Klaus 
Published: March 2005

Two RNA · protein complexes were isolated from duck erythroblast postribosomal supernatants. Their nominal sedimentation values on sucrose gradients are 12 S and 20 S, respectively. The 12‐S particle contains a 4–6 S RNA, in the 20‐S particle a 9‐S RNA is found. This 9‐S RNA is shown to direct the synthesis of all duck globin chains in a cell‐free, messenger RNA‐dependent protein‐synthesizing system.The protein moiety of these ribosome‐free particles is described and compared with the proteins found in the mRNA · protein complex liberated by EDTA from polyribosomes. We show that in the free‐cytoplasmic particles no protein can be found which is identical to any of the polypeptides associated with polyribosomal mRNA. Some of these protein are phosphorylated and contain phosphoserine. The electrophoretic patterns of phosphorylated proteins from the two globin mRNA‐containing complexes differ, as do those of the unlabelled polypeptides. We conclude that the mRNA‐associated protein population is exchanged when the mRNA enters the translation machinery.The possible role of the RNA‐associated proteins in the post‐transcriptional and translational control of eukaryotic protein synthesis is discussed.
A New Purification Procedure for Yeast Phosphofructokinase Minimizing Proteolytic Degradation
Authors: Diezel Wolfgang , Böhme Hans‐Joachim , Nissler Karl , Freyer Renate , Heilmann Werner , Kopperschläger Gerhard , Hofmann Eberhard 
Published: March 2005

A new and efficient method for preparation of pure yeast phosphofructokinase is described, leading rapidly to an enzyme with minimum proteolytic alterations. Essential is an affinity chromatography step on the reactive dye Cibacron blue F3G‐A® which has been covalently bound to Sephadex G‐100. The enzyme prepared by this method has a significantly higher sedimentation constant (20.5 S) than those forms of yeast phosphofructokinase purified by procedures previously described (17.8 S) and is composed of two different kinds of subunits having molecular weights of 130000 and 96000, respectively. By gel filtration various enzyme fractions have been obtained, containing either mainly subunits of the 130000 or of the 96000 type, respectively. Samples of phosphofructokinase which have been prepared by the original and by the new method were compared by means of analytical gel chromatography and dodecyl‐sulphate electrophoresis.In the course of the preparation procedure described previously, the enzyme is evidently proteolytically converted to the 570000 molecule by degradation of the subunits having 130000 molecular weight to those of 96000.
Enzymatic Iodination of Protein
Authors: Pommier Jacques , Sokoloff Louis , Nunez Jacques 
Published: March 2005

1. The initial rate of I2 formation catalyzed by horse‐radish peroxidase exhibited a clear sigmoid relationship with respect to the concentration of I−. These data were found to fit an equation based on a model which predicts a second‐order dependence on iodide concentration and are consistent with a bimolecular reaction between two I−ions on the surface of the enzyme. Such a model presumes two sites for the substrate on the enzyme.2. The initial rate of lysozyme iodination also exhibited a clear sigmoid relationship with respect to concentration of I−. The sigmoidicity increased with the lysozyme concentration. These experimental data fit a random‐ordered sequence of substrate fixation but with one of the two possible sequences kinetically preferred. These results also suggest that lysozyme interacts with the enzyme and that there are two sites for substrate addition on the surface of the enzyme.3. When the ratio of the concentrations of both substrates, iodide and lysozyme, is varied, the rates of formation of each product, I2 or iodinated protein, also vary; the lower the iodide/protein ratio, the lower the I2 yield and the higher the rate of protein iodination. Studies on the influence of pH on the nature of the product showed that I2 formation is favored at acidic pH and protein iodination at more alkaline pH. These results are also consistent with a two‐site model for the enzyme, both sites being able to fix either two iodide ions or one iodide ion and one lysozyme molecule. The affinity of the sites for each one of the two substrates differs according to the pH. This conclusion was confirmed by the observation that iodination of free tyrosine could be obtained with very good yields provided that the pH of the reaction was adjusted to avoid either complete dimerization of free tyrosine, which is favored at alkaline pH, or I2 formation which is favored at low pH.4. These results and other quantitative data on the stoichiometry of H2O2 consumption do not establish unequivocally which is the oxidized iodide‐reacting species, I+ or I°. However, they indicate that I2 is not the iodinating species in the protein iodination reaction catalyzed by horse‐radish peroxidase.