bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification et caractérisation partielle des deux malate synthases d'Escherichia coli
Authors: Falmagne Paul , Wiame Jean‐Marie 
Published: March 2005

A simple procedure is reported for the purification of the two malate synthases of Escherichia coli involved in the use of acetate (A) and glycolate (G), respectively, as carbon source. Both isoenzymes have been isolated simultaneously and in equally high amounts from a strain constitutive for the glyoxylate cycle, after growth on glycolate. After separation from each other by column chromatography on DEAE‐cellulose, the malate synthases have been purified by heating, ammonium sulfate precipitation and molecular sieve chromatography on a Sephadex G‐200 column, in the constant presence of their specific protectors, glycolate for malate synthase A, and oxalate for malate synthase G.The purified preparations so obtained appeared to be homogeneous on the basis of analytical ultracentrifugation and moving‐boundary electrophoresis.The sedimentation coefficient at infinite dilution was 4.6 S for malate synthase A and 5.2 S for malate synthase G; their electrophoretic mobilities at pH 8 were 4.3 X 10−5 cm2 V−1 s−1 and 4.1 × 10−5 cm2 V−1 s−1, respectively.The apparent molecular weight of malate synthase A, estimated by Archibald's method, was 54000; that of malate synthase G, estimated by light scattering, was 52000.Threonine and serine have been found, by Sanger's method, to be N‐terminal residues of malate synthases A and G, respectively.
Detection of α‐Glycosidic Bonds in the Ganglioside GM1 by Stereospecific Enzymatic Degradation
Authors: Werries Eckhard , Buddecke Eckhart 
Published: March 2005

The monosialosyl‐N‐tetraglycosyl‐ceramide (GM1) isolated from ox brain and submitted to a stepwise degradation by specific α‐ and β‐glycosidases isolated and purified from ox spleen.
By the sequential action of β‐galactosidase and β‐N‐acetylhexosaminidase, 100% of the terminal galactose and the N‐acetylgalactosamine residues were removed, while the corresponding α‐glucosidases were completely ineffective. When the sialic acid was removed from the remaining sialated dihexosyl ceramide, the internal galactose residue becomes suspectible again to β‐galactosidase which removed up to 89%, but α‐galactosidase also hydrolyzed 2% of the internal galactose residues. A subsequent action of α‐glucosidase hydrolyzed 3% whereas β‐galactosidase released 47% of the remaining glucose moiety.
The results reported prove a β‐configuration of the glycosidic‐linked terminal galactose and of the penultimate N‐acetylgalactosamine moiety of ganglioside GM1 throughout. A β‐configurated glycosidic linkage is also predominant for the internal galactose and the glucose residues, but the presence of a small percentage of α‐glycosidic bonds must be concluded from their susceptibility to α‐galactosidase and α‐glucosidase, respectively.
Acyl‐Donor Specificities of Partially Purified 1‐Acylglycerophosphate Acyltransferase, 2‐Acylglycerophosphate Acyltransferase and 1‐Acylglycerophosphorylcholine Acyltransferase from Rat‐Liver Microsomes
Authors: Yamashita Satoshi , Hosaka Kohei , Numa Shosaku 
Published: March 2005

1. A partially purified preparation containing 1‐acylglycerophosphate acyltransferase, 2‐acylglycerophosphate acyltransferase and 1‐acylglycerophosphorylcholine acyltransferase was obtained from rat liver microsomes which were resolved with a nonionic detergent, Triton X‐100, in glycine buffer pH 8.6. The purification procedure involves molecular sieve chromatography and sucrose density gradient centrifugation.2. The partially purified 1‐acylglycerophosphate acyltransferase exhibits a significant specificity for mono‐ and dienoic fatty acyl‐CoA thioesters. The effectiveness of the various acyl‐donors examined is as follows: oleyl‐CoA > linoleyl‐CoA ∼ palmitoleyl‐CoA > palmityl‐CoA ∼ myristyl‐CoA; arachidonyl‐CoA, stearyl‐CoA and lauryl‐CoA are almost ineffective. The newly formed ester bond is sensitive to the action of phospholipase A2.3. The partially purified 2‐acylglycerophosphate acyltransferase shows a predominant selectivity for saturated acyl‐CoA thioesters. Stearyl‐CoA is the most effective acyl‐donor, and palmityl‐CoA is utilized fairly efficiently.4. The partially purified 1‐acylglycerophosphorylcholine acyltransferase is specific for arachidonyl‐CoA.5. These findings, together with our previous studies on the acyl‐donor specificity of glycerophosphate acyltransferase, indicate that the asymmetric fatty acid distribution found in naturally occurring glycerolipids results mostly from the specific at the step of phosphatidic acid synthesis and partly from the deacylation‐reacylation cycle operating after the synthesis de novo of phospholipids.
Synthesis of Lens Protein in vitro
Authors: Strous Ger J. A. M. , van Westreenen Hanske , Bloemendal Hans 
Published: March 2005

N‐terminal acetylation of the lens protein α‐crystallin has been studied in a lens cell‐free system. This system is capable of synthesizing de novoα‐crystallin, a protein with acetylmethionine as N‐terminus. When [35S]Met‐tRNAfMet is used as radioactive precursor only the α‐crystallin chains are labeled in the N‐terminal position. This phenomenon enables the study of the process of acetylation. Newly synthesized polypeptides could be separated according to chain length. Electrophoretic analysis of thermolytic peptides revealed that acetylation of the N‐terminus occurs while the chain is still on the ribosome.It is concluded that the N‐terminal acetylation takes place after chain initiation and before completion of the polypeptide chain.
Inhibition of Enzymes which Interact with Citrate by (—)Hydroxycitrate and 1,2,3,‐Tricarboxybenzene
Authors: Cheema‐Dhadli Surinder , Halperin Mitchell L. , Leznoff Clifford C. 
Published: March 2005

The effects of two citrate analogues, (—)hydroxycitrate and 1,2,3‐tricarboxybenzene, were studied using rat liver enzymes which interact with citrate.The most pronounced effect of 1,2,3‐tricarboxybenzene was inhibition of acetyl‐CoA carboxylase (Ki 20 μM). It also inhibited the mitochondrial citrate transporter (50% inhibition at 3 mM), but was not a substrate for this transporter.ATP‐citrate lyase was markedly inhibited by both free (—)hydroxycitrate (Ki 8 μM) and (—)hydroxycitrate lactone (Ki 50 to 100 μM). Acetyl‐CoA carboxylase was activated by both the forms of (—)hydroxycitrate (Ka 0.7 mM and 1.6 mM, respectively). (—)Hydroxycitrate is a substrate for the mitochondrial citrate transporter, but its rate of transport is less than 10% of that of citrate.Other citrate metabolizing enzymes also were inhibited by 1,2,3‐tricarboxybenzene and (—)hydroxycitrate but much higher concentrations were required.
Poly(Adenosine Diphosphoribose) Polymerase in Mammalian Nuclei
Authors: Stone Peter R. , Shall Sydney 
Published: March 2005

1. The ability of isolated LS‐cell nuclei to incorporate [adenine‐3H]‐NAD into acid‐insoluble products is demonstrated.2. The incorporation has a pH optimum at 8.5 and an apparent temperature optimum at 25 °C.3. The optimum Mg2+ concentration is dependent upon the NAD concentration; at 1.5 μM NAD it is 2 mM; at 1.5 mM NAD it is 3 mM; and at 9 mM NAD it is above 20 mM Mg2+ Mg2+ can be replaced by Ca2+.4. Dithiothreitol or mercaptoethanol, up to 10 mM, enhances the incorporation of NAD.5. With the optimal conditions the incorporation of NAD (V) is 128 nmol NAD×5 min−1×mg DNA−1 and the Km is 1.47 ± 0.18 mM. Nicotinamide inhibits the incorporation competitively with a Ki of 14.3 ± 1 μM and thymidine inhibits competitively with a Ki of 32.5 μM.6. The incorporation is not affected by added DNA or poly(U). It is not affected by ribonuclease but is inhibited by about 50% on treatment with deoxyribonuclease.7. The enzyme system is unstable and the decay is temperature‐dependent; there is little decay at 0 °C but above this temperature the rate of decay follows an Arrhenius relationship. The initial rates of enzyme activity also show a temperature dependence with a linear Arrhenius plot. The temperature optimum at 25 °C is the arithmetic result of these two opposing processes.
Adenylate‐Cyclase Activity of Rat‐Liver Plasma Membranes
Authors: Leray Françoise , Chambaut Anne‐Marie , Perrenoud Marie‐Lise , Hanoune Jacques 
Published: March 2005

The adenylate‐cyclase activity from a rat‐liver plasma‐membrane preparation purified according to the Neville's procedure was increased 10 to 12‐fold by glucagon, but the addition of epinephrine, at any concentration, failed to activate the enzyme. When the membranes were isolated from adrenalectomized animals, the responses of the enzyme to glucagon and NaF were enhanced and mainly the adenylate‐cyclase activity became sensitive to epinephrine with an optimal effect at 0.05 mM epinephrine. Intraperitoneal administration of cortisol for 2 days to the adrenalectomized animals reversed the effect of the hormonal deprivation. But addition of cortisol in vitro was without effect.The use of liver plasma membranes from adrenalectomized rats is therefore proposed as a tool to study the adenylate‐cyclase activation by both glucagon and epinephrine. The epinephrine‐sensitive adenylate‐cyclase activity was completely inhibited by propranolol but not by phentolamine, thus confirming that the metabolic effect of epinephrine is linked to a β‐like receptor. Epinephrine and glucagon effects upon adenylate‐cyclase activity were both inhibited by concentration of calcium above 0.01 mM but differed with respect to the Mg‐ATP concentration. The two hormonally stimulated adenylate‐cyclase activities were found to be additive.The effect of epinephrine upon adenylate‐cyclase activity was more sensitive to stimulation by low concentration of GTP and other nucleotides than the effect of glucagon. Addition of insulin in vitro at any concentration failed to alter the adenylate‐cyclase activity regardless of the concentration of magnesium, the presence of glucose or GTP.
Sex‐Dependent Differences in Derangements of Biliary‐Steroid‐Excretion Patterns Produced by Cyanoketone in Rats
Authors: Begue Rene‐Jean , Goldman Allen S. , Gustafsson Jan‐Åke , Gustafsson Sven A. 
Published: March 2005

Intramuscular injections of 25 mg/kg body weight of 2α‐cyano‐4,4,17α‐trimethyl‐5‐androsten‐17β‐ol‐3‐one (cyanoketone) produces and immediate cessation of excretion of practically all corticosteroid metabolites (3α, 11β,15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one and 3α‐ and 3β,11β, 21‐trihydroxy‐5α‐pregnan‐20‐one) in bile from female rats. Instead, large amounts of 3β‐hydroxy‐5‐pregnen‐20‐one, 3β, 16α‐dihydroxy‐5‐pregnen‐20‐one and 5‐pregnene‐3β,20α‐diol are excreted. The total daily excretion of steroids in bile from treated female animals is 2580 ± 580 μg compared to 850 μg in a female rat injected with solvent only. When ovariectomized rats are treated with cyanoketone, the principal biliary metabolites are 3β‐hydroxy‐5‐pregnen‐20‐one and 3β,16α‐dihydroxy‐5‐pregnen‐20‐one and the total daily excretion of steroids measured is 1186 ± 197 μg indicating that ovaries contribute about 50% of the total output of 3β‐hydroxy‐Δ5‐steroids in treated, intact female rats. As late as 28 days after a single injection of cyanoketone 3β‐hydroxy‐Δ5‐steroids (44 μg/24 h) are excreted in bile and the total excretion of corticosteroid metabolites is still low (72 μg/24 h) at this time. 130 days after a single dose, no 3β‐hydroxy‐Δ5‐steroids can be identified in bile but the total corticosteroid excretion is still less than 2 standard deviations below values of untreated intact animals (315 μg/24 h). Thus, in females cyanoketone has both an immediate enzyme‐inhibiting effect reflected by rapid onset of action after administration and, in addition, a long‐term depressing action on enzyme biosynthesis manifested by low total steroid excretion values measured at least as long as 130 days after injection of inhibitor.Administration of cyanoketone to male rats gives rise to a small biliary excretion of 3β‐hydroxy‐Δ5‐steroids (3β,16α‐dihydroxy‐5‐pregnen‐20‐one, 5‐pregnene‐3β,17α,20α‐triol and 5‐pregnene‐3β,20α‐diol) and a decrease in corticosteroid (mainly 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one) excretion. Corticosteroid output reaches normal values about 24 h after injection of inhibitor. Testectomy eliminates biliary 3β‐hydroxy‐Δ5‐steroids. These findings indicate that cyanoketone produces biliary 3β‐hydroxy‐Δ5‐steroid mainly of testicular origin. Moreover, it appears that the 3β‐hydroxy‐Δ5‐steroid oxidoreductase system in the adrenals and testes of the male rat is inhibited by cyanoketone considerably less efficiently and for a shorter duration than that in the adrenals and ovaries of the female.
Sequence of Events in Initiation of Protein Synthesis
Authors: Benne Rob , Ebes Frieda , Voorma Harry O. 
Published: March 2005

Initiation factors recycle during the assembly of a 70‐S initiation complex. At early stages the factors are present, but they are absent in the mature 70‐S initiation complex.Experiments described in this paper deal with the sequence of events and the contribution of each factor in the assembly process. Initiation factors IF‐1 and IF‐2 bind to 30‐S subunits independent of the presence of the other components involved in initiation. The fate of IF‐2 during 70‐S initiation complex formation has been studied in a system devoid of IF‐1 and in a system in which GTP is replaced by 5′‐guanylylmethylenediphosphonate (Guo‐5′‐P2‐CH2‐P). In both cases it is found that IF‐2 is not released from the 70‐S initiation complex. This leads to the inhibition of the elongation factor EF‐Tu dependent binding of the second aminoacyl‐tRNA, coded for by the messenger. The data are interpreted by assuming partly overlapping binding sites for EF‐Tu and IF‐2.
Products of Mitochondrial Protein Synthesis in Neurospora crassa
Authors: Sebald Walter , Machleidt Werner , Otto Joachim 
Published: March 2005

Cytochrome oxidase isolated from Neurospora crassa was resolved into seven protein components by electrophoresis in polyacrylamide gels containing sodium dodecylsulfate. The apparent molecular weights were determined to be 41000, 28500, 21000, 16000, 14000, 11500 and 10000 for the components 1,2,3,4,5,6, and 7, respectively. The components 1,2 and 3 are synthesized on mitochondrial ribosomes as shown by the incorporation of radioactive amino acids in the presence of cycloheximide.Amino‐acid analysis of the isolated components 1, 2 and 3 revealed a high content of apolar amino acids and a low content of basic amino acids compared to an average amino‐acid composition of components 4–7.Components 1, 2 and 3 contribute 27.9%, 18% and 14.2% to the whole cytochrome oxidase protein. This was calculated from the contributions of the single components to the total leucine content of the enzyme and the leucine contents (nmol leucine per mg protein) of the single components as determined by amino‐acid analysis. Equimolar relations of the components 1, 2 and 3 are found by dividing the amounts of protein by their apparent molecular weights. A stoichiometry of 1:1:1 results assuming a minimal molecular weight of 150000 for the whole cytochrome oxidase protein.On the basis of the heme a content a molecular weight of about 70000 per heme group was determined, using an absorption coefficient Δɛ605 (reduced minus oxidized) of 12 mM−1 cm−1. It is concluded that the smallest structural unit of cytochrome oxidase contains two heme groups.
Interaction of Haemoglobin with Ions
Authors: Berger Hartmut , Jänig Gerd‐Rüdiger , Gerber Gerhard , Ruckpaul Klaus , Rapoport Samuel M. 
Published: March 2005

Under simulated intracellular conditions (pH 7.2, 37 °C, 130 mM KCl, 20 mM NaCl and haemoglobin concentrations > 1.5 mM) the conditional association constants for the binding of ATP and 2,3‐bisphosphoglycerate (P2‐glycerate) to deoxygenated and oxygenated haemoglobin (Hb and HbO2, respectively) and the influence of Mg2+ on the binding of phosphocompounds were determined by means of a cation‐sensitive electrode and ultrafiltration.The K′ass for the binding of ATP to Hb is 2.6 mM−1 and 0.36 mM−1 for HbO2. P2‐glycerate is bound to Hb with K′ass of 5 mM−1 and to HbO2 with a K′ass of 0.25 mM−1. The anions compete with each other in binding. Mg2+ is not bound to haemoglobin but it reduces the binding of the phosphocompounds by complexing with them. It decreases the shift of the oxygen binding curve produced by ATP. MgATP is weakly bound to Hb with a K′ass of 0.14 mM−′ and to HbO2 with a K′ass of 0.039 mM−1. Binding of the Mg ·P2‐glycerate complex to haemoglobin was not established.The calculated values for the dependence of p50 (the partial pressure of oxygen at half‐saturation of haemoglobin) on the free concentration of P2‐glycerate, based on the K′ass of Hb and HbO2 complexes, agree well with the experimental data on erythrolysates and intact human red blood cells. The K′ass are furthermore an appropriate basis for the estimation of the intraerythrocatic state of Mg2+ and the metabolites.
Interaction of Haemoglobin with Ions
Authors: Gerber Gerhard , Berger Hartmut , Jänig Gerd‐Rüdiger , Rapoport Samuel M. 
Published: March 2005

The intracellular distribution of ATP, 2,3‐bisphosphoglycerate(P2‐glycerate) and Mg2+ was calculated for the oxygenated and deoxygenated human erythrocyte for the normal range and that of pathophysiological variations based on the association constants of the relevant complexes.The data indicate that about 20% of ATP is bound both in the oxygenated and deoxygenated cells, while 39 and 73% of P2‐glycerate is bound under these conditions. An increase of the free Mg2+ concentration from 0.7 to 1.1 mM is produced by complete deoxygenation of haemoglobin.The calculations would indicate that during deoxygenation of haemoglobin the hexokinase reacts to the increased concentration of the activator Mg2+ and the decline of the inhibitor P2‐glycerate with an elevation of its activity which corresponds to experimental data on intact erythrocytes.The P2‐glycerate formation rate in deoxygenated cells is stimulated about 2.5 times in comparison to oxygenated cells as estimated from the free concentration of P2‐glycerate and the kinetic constants of bisphosphoglycerate mutase.An assessment of the possible influence of other anions including bicarbonate shows that the distribution of the species of ATP, P2‐glycerate and Mg2+ are changed by less than 20%.Together with the data presented in the accompanying paper, the results given here indicate that the constants and estimates are approximately valid for intracellular conditions.
Molecular Properties of Lactic Dehydrogenase under the Conditions of the Enzymatic Test
Authors: Bartholmes Peter , Durchschlag Helmut , Jaenicke Rainer 
Published: March 2005

In order to investigate the correlation of enzymatic activity and subunit structure of lactic dehydrogenase from pig heart, the molecular weight, activity and conformation of the enzyme were determined under conditions of the enzymatic test, chosing optimum conditions of the medium regarding subunit dissociation or hybridization and biological function.At concentrations of 10mg/ml to 0.1 μg/ml no dissociation is to be observed. A population analysis on the basis of high‐speed equilibrium ultracentrifugation does not show significant fractions of other species besides the tetramer. At c < 0.1 μg/ml both activity transport and gelfiltration prove dissociation to the dimer to occur which is accompanied by deactivation. The first‐order rate constant of the dissociation reaction is of the order of 0.002 min−1.In the presence of the coenzyme, reassociation and reactivation take place, the binding of NADH leading to a tightening of the quaternary structure (Δscorr= 0.17 S). Nicotinamide which does not influence the activity of the native enzyme inhibits reassociation, while ATP does not show any appreciable effect on the dissociation reaction.The lack of enzymatic activity of the dimer in the presence of nicotinamide and the parallel occurrence of subunit dissociation and deactivation at c < 0.1 μg/ml confirm previous observations implying enzymatic activity to require the intact quaternary structure of the enzyme.
The Size of the Substrate‐Binding Site of an Aspergillus niger Extracellular Endopolygalacturonase
Authors: Rexová‐Benková Ľubomíra 
Published: March 2005

The action pattern and kinetics of an Aspergillus niger extracellular endopolygalacturonase were studied with oligogalacturoic acids (digalacturonic acid through hexagalacturonic acid) and their derivatives in which the terminal aldehyde group was reduced. The rate of hydrolysis catalyzed by the enzyme decreases with the shortening of oligogalacturonide chain length; digalacturonic acid is not hydrolyzed. With tetragalacturonic acid one productive complex is formed resulting in (1 + 3) cleavage. Two and three modes of cleavage occur with pentagalacturonic acid and hexagalacturonic acid, respectively. The enzyme is competitively inhibited by trigalacturonic acid whereas digalacturonic acid does not affect the activity. Reduced pentagalacturonic acid forms one productive complex. Reducing trigalacturonic acid and nonreducing digalacturonic acid are the products of the reaction. Lower reduced oligogalacturonic acids are not degraded by the enzyme.The action pattern and k2 values obtained with the oligouronides suggest that the binding site of the endopolygalacturonase contains four subsites and that the catalytic groups of the enzyme are located in the proximity of the first bond, counting from the reducing end, of the substrate segment bound in the complex.
Structural Aspects of Heterologous Blood‐Group‐P1 Substance
Authors: Anstee David J. , Pardoe Grace I. 
Published: March 2005

Blood‐group‐P1 substance from sheep hydatid‐cyst fluid was purified by a combination of gel filtration through Sepharose 6B and preparative polyacrylamide‐gel electrophoresis. The resultant glycoprotein contained D‐galactose; D‐glucose; N‐acetyl‐D‐glucosamine and N‐acetyl‐D‐galactosamine in molar ratios 5.2:1:2:2; unidentified carbohydrate was also present. The amino‐acid composition resembled that of vertebrate A, B, H and Lewis active glycoproteins.Prolonged incubation of P1 substance with coffee bean α‐galactosidase resulted in the liberation of 17–24% of the total D‐galactose present and a reduction in blood‐group‐P1 inhibitory activity. The serological activity of the glycoprotein was unaffected by treatment with pronase or papain.P1 substance has receptors for the agglutinins from Salmo trutta, Clupea harengus, Soja hispida, Maclura aurantiaca and Triticum vulgaris. Some loss of receptor for Soja hispida and Maclura aurantiaca occurred after α‐D‐galactosidase treatment.