bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulatory Properties of the Pyruvate‐Dehydrogenase Complex from Escherichia coli
Authors: Bisswanger Hans , Henning Ulf 
Published: March 2005

It is shown that the pyruvate‐dehydrogenase complex from Escherichia coli K12 produces, under certain conditions, positive cooperativity for the substrate pyruvate and negative cooperativity for the inhibitor acetyl‐CoA. The target of the two effectors is the pyruvate dehydrogenase component of the complex. Suggestive evidence indicates that pyruvate cooperativity may mostly be of intramolecular nature (i.e., occurring within individual pyruvate dehydrogenase molecules) although some interactions between such molecules become apparent in the presence of acetyl‐CoA inhibition.Negative cooperativity exerted by acetyl‐CoA may not be a direct consequence of ligand‐induced conformational changes of or non‐exclusive ligand binding. It was found that a partially inactivated pyruvate dehydrogenase component is activated by acetyl‐CoA rather than inhibited. It has not been possible to demonstrate the occurrence of this activation with the fully active enzyme component; nonetheless, it does not seem unlikely that the negative cooperativity observed is the consequence of acetyl‐CoA both activating and inhibiting (presumably by acting at different sites) pyruvate dehydrogenase catalysis.
Nuclear Dependency of Chloroplast Proteins in Acetabularia
Authors: Apel Klaus , Schweiger Hans‐Georg 
Published: March 2005

A membrane protein fraction, insoluble in water, has been prepared from Acetabularia chloroplasts. The greater part of these proteins could be dissolved in a phenol—formic acid—water mixture (2:1:1, v/v/v). The dissolved proteins were subjected to gel electrophoresis. The electrophoretic pattern was reproducible for one species.The protein patterns of A. calyculus and A. mediterranea were composed of 8 peaks common for both species and of at least 3 peaks which were species‐specific.By means of nuclear transplantation and implantation techniques it was shown that the species‐specific chloroplast‐proteins were nucleus‐dependent and therefore were coded by the nuclear DNA. Six weeks after the operation the patterns of the transplants and implants had changed into that of the nucleus donor species. It was concluded that the mRNA of the nucleus acceptor cell lost its stability under the influence of the implanted or transplanted nucleus.Both cycloheximide and chloramphenicol inhibited the incorporation of radioactive amino acids into the insoluble chloroplast‐protein fraction.The electrophoretic pattern of the labelled chloroplast‐proteins consisted of at least 3 peaks. Cycloheximide inhibited the incorporation into peak 3, whereas chloramphenicol affected the incorporation into the two other peaks. Isolated chloroplasts incorporated amino acids only into peaks 1 and 2. Peak 3 was absent in the pattern in vitro.The protein groups which were involved in the formation of chloroplasts were synthesized either by cytoplasmic 80 S or by 70 S chloroplast ribosomes. It was concluded that the autonomy of the chloroplasts was limited by the influence of the nucleus.
Structural Studies of Alcohol Dehydrogenase from Human Liver
Authors: Jörnvall Hans , Pietruszko Regina 
Published: March 2005

Tryptic peptide maps of human liver alcohol dehydrogenase show this protein to be homologous to the corresponding protein of the horse. A subunit molecular weight very close to 40000 is established for the human enzyme.Sequence analysis of about one quarter of the tryptic peptides of human alcohol dehydrogenase show identical residues to the horse enzyme at about 90% of the positions, which is considered fairly representative of the general resemblance between the two proteins. All amino acid exchanges found are compatible with one‐base mutations in the genetic code.Two different types of subunits of human liver alcohol dehydrogenase are identified. They are essentially similar but differ at some positions, one of which is No. 43 (valine in one subunit and alanine in the other). Still other subunit types may exist. The known occurrence of isoenzymes of human liver alcohol dehydrogenase may therefore be explained at least in part by subunits of different primary structures.The amino acid differences between the subunits of the human enzyme are not found at the same positions as those between the two types of subunit of the horse enzyme. The structural differences between subunits from the two species seem greater than between the subunits within either species. Isoenzyme differences may, therefore, have evolved independently in the two species. The suggestion that some of the amino acid exchanges between the horse subunits may be directly involved in the substrate binding is supported.The isoenzyme and species differences at position 43 is only three residues away from the reactive “active site” cysteine residue. The region around this important residue is thus not kept constant in liver alcohol dehydrogenase during evolution.
The Effect of Magnesium‐Ion Concentration on the Translation of Phage‐f2 RNA in a Cell‐Free System of Escherichia coli
Authors: Zagórski Włodzimierz , Filipowicz Witold , Wodnar Aleksandra , Leonowicz Andrzej , Zagórska Ludwika , Szafrański Przemysław 
Published: March 2005

The phage‐f2 specific protein synthesis was studied in the cell‐free system of Escherichia coli at 10.6–18.6 mM Mg2+. The products formed under the direction of f2 RNA between 12.6 mM and 18.6 mM Mg2+ were separated by polyacrylamide gel electrophoresis and found to correspond to phage‐specific proteins, i. e. RNA synthetase and the coat protein. The rate of f2‐RNA translation was optimal at 12.6 mM Mg2+. Lowering of Mg2+ concentration from the optimal value to 10.6 mM caused the lack of formation of complete RNA synthetase while the coat protein was still synthesised.This was due to a sharp decrease in rate of polypeptide‐chain elongation, which led to incomplete translation of the longer gene. Our results indicate that, within the studied range of Mg2+ concentration, specific initiation of f2‐RNA translation occurs even at 18.6 mM Mg2+.
Studies on the Mode of Action of Nalidixic Acid
Authors: Pedrini Antonia M. , Geroldi Diego , Siccardi Antonio , Falaschi Arturo 
Published: March 2005

Nalidixic acid, a very specific inhibitor of bacterial DNA synthesis, has been studied for its action on purified enzymes acting on DNA and in subcellular DNA‐synthesizing systems. The drug does not inhibit the following enzymes: DNA polymerase I, endonuclease I, exonuclease I, II and III from Escherichia coli, polynucleotide‐ligase and DNA methyl‐transferase from T4‐ infected E. coli, DNA polymerase from Bacillus subtilis. A significant inhibition of ATP‐dependent DNA synthesis is observed in cells of a strain of E. coli lacking DNA polymerase I after short treatments with toluene (2 min); longer treatments reduce the rate of synthesis and abolish its sensitivity to nalidixic acid. Equally insensitive are the ATP‐dependent DNA synthesizing systems bound to membrane fractions of E. coli and B. subtilis.The results suggest a mode of action through a still unidentified physiological component of the growing point apparatus, and supply a new criterion of physiological integrity of subcellular DNA‐synthesizing systems, namely their sensitivity to nalidixic acid.
RNA Polymerase from Eukaryotic Cells
Authors: Mondal Hrishikes , Mandal Radha K. , Biswas Birendra B. 
Published: March 2005

This paper describes the isolation and purification of two RNA polymerases (CI and CII) and several protein fractions (A, B and C) from the chromosomal acidic proteins of coconut endosperm nuclei. The method involves disruption of isolated nuclei in dilute salt medium, centrifugation to sediment the crude chromatin, solubilization of chromatin in concentrated salt, dialysis of soluble chromatin to dilute salt to precipitate DNA‐histone complex and centrifugation to get the acidic proteins in the supernatant. Further purification involves ammonium sulphate fractionation and chromatography on DEAE‐cellulose and QAE‐Sephadex.RNA polymerase CI purified through the QAE‐Sephadex step gives a single band on polyacrylamide gel electrophoresis. In gels containing dodecylsulfate, this enzyme shows multiple bands indicating its subunit nature. The pH optima for both RNA polymerases is 8.0. RNA polymerase CI is maximally activated by Mn2+; while CII, by Mg2+. The activities of both the enzymes are stimulated by fraction B. All the fractions except A are substantially free from nucleases. Both RNA polymerases require the addition of DNA for activity. Fraction B is ineffective either with denatured coconut DNA or native λ DNA.
Porcine Carboxypeptidase B
Authors: Sokolovsky Mordechai , Eisenbach Lea 
Published: March 2005

Coupling of porcine carboxypeptidase B with diazotized arsanilic acid labels an active‐site tyrosine residue of the enzyme. Tyrosine modification is accompanied by a change in the activities of carboxypeptidase B towards both basic and non‐basic substrates. The major arsanilazotyrosine‐containing peptide, Thr‐Ile‐Tyr‐Pro‐Ala, isolated by affinity chromatography is homologous with the peptide containing tyrosine‐248 from the active center of bovine carboxypeptidase A.Formation of the arsanilazotyrosine residue generates extrinsic Cotton effects above 300 nm. The dichroic spectrum of the azochromophore constitutes a sensitive probe of conformational changes accompanying the interaction of arsanilazocarboxypeptidase B with inhibitors, as monitored by changes in the circular dichroic spectrum. The changes are consistent with the hypothesis that the tyrosine residue is a component of the active‐site region.
Chemical Modification and Kinetic Studies on Glucarate Hydro‐Lyase from a Species of Pseudomonas A
Authors: Jeffcoat Roger 
Published: March 2005

Glucarate hydro‐lyase, obtained from cells of Pseudomonas A grown on glucarate as the sole source of carbon, was purified 40‐fold in 70% yield.
The effects of a variety of chemical modifiers and competitive inhibitors on the enzyme activity were studied.
Enzyme activity was irreversibly lost by incubating the enzyme with p‐chloromercuribenzene sulphonate.
(+)‐Tartrate, a competitive inhibitor of the enzyme, protected the enzyme from inhibition by this reagent but was not effective when the enzyme was inhibited by coupling it to a diazonium salt. Glucarate, unlike (+)‐tartrate, was an effective protecting group against this type of inhibition.
The roles of the modified amino acid residues are discussed.
Light‐Absorption Studies on Neutral Flavin Radicals
Authors: Müller Franz , Brüstlein Martin , Hemmerich Peter , Massey Vincent , Walker Wolfram H. 
Published: March 2005

Quantitative light absorption data of the neutral free flavosemiquinone are reported. It is shown that the visible range of the spectrum of the radical is drastically dependent on the solvent. The spectra of the neutral blue flavosemiquinone are compared with those of the blue flavoprotein radicals and conclusions are drawn concerning the environment of the protein‐bound flavosemiquinone.The stabilization of the neutral blue flavosemiquinone through N(5)‐alkylation and/or π‐complex formation with aromatic compounds is described.The light absorption characteristics of a tautomeric neutral, but red‐coloured, flavosemiquinone are described. The results are compared with those obtained from the free anionic and protein‐bound red flavosemiquinones. The two types of radicals cannot be distinguished from each other by electron spin resonance spectrometry but may be recognized by means of light absorption spectrophotometry. The possible biological significance of the neutral red flavosemiquinone is discussed. The optical properties of this radical are compared with those of free and protein‐bound anionic flavosemiquinones.
La D‐altronate: NAD‐oxydoréductase d'Escherichia coli K12 Purification, proprié0tés et individualité
Authors: Portalier Raymond C. , Stoeber François R. 
Published: March 2005

D‐Altronate: NAD‐oxidoreductase of Escherichia coli K12 has been purified. The purification consists of four steps: nucleic acids precipitation with protamine sulfate; ammonium sulfate precipitation; ion exchange chromatography on DEAE‐cellulose and Sephadex G‐150 gel filtration. The overall procedure results in a 53‐fold purification with a yield of 60%.Altronate oxidoreductase is an inducible enzyme: galacturonate, tagaturonate, glucuronate and fructuronate are good inducers. The activity remains stable at 4 °C or at —20 °C for several months.Optimal activity is obtained at pH 6.3 for tagaturonate reduction, and at pH 8.9 for altronate oxidation. Altronate oxidoreductase does not require any cofactor, except NADH.The apparent Michaelis constants have been determined: for tagaturonate, the Km is 350 μM; for NADH, 60 μM; for altronate, 90 μM, and for NAD+, 110 μM. Moreover, altronate oxidoreductase is active with NADPH as coenzyme and the apparent Km value for NADPH is 400 μM. Other carbohydrates tested are unsuitable as substrates. Many other carbohydrates tested do not prove to be inhibitors.The value for the energy of activation is 38 kJ/mol (9 kcal/mol); the Q10 is 1.5. The results of heat inactivation of altronate oxidoreductase (58.5 °C) indicate homogeneous enzyme activity in crude extract. Thermal inactivations also indicate that the same enzyme, altronate oxidoreductase, is induced by galacturonate and glucuronate, and catalyzes the direct reactions with NADH or NADPH. Finally, it has been confirmed by heat inactivation that there is no interference between specific assays of altronate and mannonate oxidoreductases.Compounds such as NADH, NADPH, altronate and mannonate protect enzymatic activity from degradation by heat, whereas NAD+, tagaturonate and fructuronate do not. NADH, in particular, is effective as a stabilizer; this ability is proportional to its concentration.Enzyme inhibition with para‐chloromercuribenzoate is non‐competitive and the apparent K1 is 1 mM. para‐Chloromercuribenzoate also inactivates altronate oxidoreductase indicating homogeneous activity and specificity of the assay method in crude extracts.Chromatographic properties of the enzyme are identical whether the inducer is galacturonate or glucuronate.Using paper chromatography, the reaction product of enzyme action on D‐tagaturonate is identified as D‐altronate.
Purification and Characterization of Pyruvate‐Dehydrogenase Phosphatase from Pig‐Heart Muscle
Authors: Siess Elmar A. , Wieland Otto H. 
Published: March 2005

Pyruvate‐dehydrogenase phosphatase was extensively purified from pig heart muscle.
The molecular weight was determined to be in the range between 92000 and 95000. By dodecylsulfate‐gel electrophoresis there was no indication for a subunit structure of the protein.
The phosphatase is dependent on Mg2+ or Mn2+. Half‐maximal activity was obtained, in histidine buffer, at 2.5 mM MgCl2 and 1.8 mM MnCl2, respectively.
Fluoride at a concentration of 0.6 mM inhibited phosphatase activity by 50% in the presence of Mg2+, but not of Mn2+. The enzyme was not affected by alkylating thiol‐reagents.
Studies with sequestering agents suggest that pyruvate‐dehydrogenase phosphatase contains a metal which, besides Mg2+ or Mn2+, is essential for its function.
Interaction of Rose Bengal with Apo‐Hemoproteins
Authors: Coulson Andrew F. W. , Yonetani Takashi 
Published: March 2005

The interaction of “ose bengal with apo‐hemoproteins was found to give rise to a large absorption change near 550 nm. This was used to study the binding of the dye to apo‐cytochrome c peroxidase, apo‐myoglobin, apo‐horseradish peroxidase and apo‐hemoglobin. In all cases there was about one tight and a number of weak binding sites per heme‐binding site, and the binding was impaired in the presence of stoichiometric quantities of heme.Rose bengal was found to be an effective photo‐oxidation sensitizer for apo‐cytochrome c peroxidase. Kinetic evidence was obtained consistent with the possibility that binding of the dye to the protein is an important step in the photo‐oxidation process. Heme was found to protect the enzyme from loss of enzymic activity caused by photo‐oxidation. Photo‐oxidation does not simply destroy the binding of heme to apo‐cytochrome c peroxidase, but produces changes in the spectrum of the bound heme. There is a loss of ability of recombined enzyme to form an enzyme substrate complex in the presence of stoichiometric quantities of hydrogen peroxide, which apparently causes the overall loss of enzymic activity. Photo‐oxidation was accompanied by loss of one tryptophan and two histidine residues per mole of protein, but apparently only one of the latter is essential to the enzymic activity.It was concluded that rose bengal can act as a specific modifying agent for the heme site in apo‐hemo‐proteins, and that this specificity arises from specificity of binding to the heme site. It is suggested that specificity of binding may provide a general basis for the observation of specific destruction of functionally interesting residues in photo‐oxidation.
Studies on the Metabolic Effects Induced in the Rat by a High‐Fat Diet
Authors: Bringolf Michel , Zaragoza Nelly , Rivier Denis , Felber Jean‐Pierre 
Published: March 2005

Rats were fed from weaning during 3–4 weeks a high fat carbohydrate‐poor diet. Control groups received a diet in which fat was completely replaced by starch in the same caloric proportion.
The effect of this high‐fat diet on the metabolism of pyruvate in diaphragm in vitro was investigated. Glycogen breakdown and glyceride content of the tissue were also measured.
Fat diet was shown to induce a reduction in 14CO2 production from labelled pyruvate and in the pyruvate uptake. Acetate oxidation was found to be unchanged after fat feeding. This gives evidence for an inhibition of the pyruvate dehydrogenase reaction in diaphragm from fat‐fed rats.
The breakdown of glycogen during either an incubation in vitro or a 24‐h fast was significantly reduced in diaphragm from fat‐fed rats.
Diaphragm glycerides were found to be increased four‐fold by fat diet. Examination by light‐microscopy of muscle sections have shown that glycerides were present in the muscle fibre and not in adipose tissue cells in the preparation.
Inhibition of the oxidation of fatty acids by 2‐bromostearate was shown to reverse completely the impairment of pyruvate metabolism and to stimulate glycolysis. In diaphragm from carbohydrate‐fed rats 2‐bromostearate induced no significant effect on carbohydrate metabolism. Similar results were obtained with glucose as substrate.
Rats were fed the high‐fat diet and then the high‐carbohydrate one for a few days. This change of diet was shown to induce a rapid reversal of the inhibition of glucose oxidation in diaphragm in vitro.
These results indicate that the impairment of carbohydrate metabolism in diaphragm from fat‐fed rats is probably due to an increased oxidation of fatty acids as observed in muscle from alloxan‐diabetic or starved rats rather than to enzymatic adaptations.
The Respiratory‐Chain NADPH Dehydrogenase ofAzotobacter vinelandii
Authors: Ackrell Brian A. C. , Erickson Sandra K. , Jones Colin W. 
Published: March 2005

NADPH dehydrogenase activity ofAzotobacter vinelandiirespiratory membranes is inhibited by adenine nucleotides (pure competitive inhibition; AMP>ADP>ATP) and by NAD+(partially competitive, allosteric inhibition). No allosteric activators, other than NADPH, were found for this enzyme.The dehydrogenase exhibits kinetic properties compatible with the presence of a catalytic site, which binds one molecule of NADPH (or one molecule of adenine nucleotide), and an apparently acid‐sensitive, site (or sites) which binds one molecule of either NADPH (positive homotropic effector) or NAD+(negative heterotropic effector).These results suggest that in the presence of NAD+or low energy charge, NADPH may be converted to NADHviathe NADPHNAD+transhydrogenase, and thence oxidisedviathe more efficiently phosphorylating NADH dehydrogenase.The prime function of the NADPH dehydrogenase is probably to oxidise the excess NADPH which might be expected to accumulate when nitrogen‐fixation is switched off (e.g.under highly aerobic conditions) and thus to contribute to the phenomenon of nitrogenase protection by enhanced respiration and also to the maintenance of the desired intracellular NADPH/NADP+ratio.
Ribonuclease T1 Spectrophotometric Investigations of the Interaction of the Enzyme with Substrate Analogues
Authors: Epinatjeff Christa , Pongs Olaf 
Published: March 2005

Spectral changes on interaction of ribonuclease T1 with a number of substrate analogues have been measured. For guanosine and guanosine monophosphates these changes are the same as those produced by protonation of the guanine chromophore. It is concluded that the guanine ring of the substrate analogues is protonated by ribonuclease T1 in position N‐7. Evidence is presented for a similar interaction of the enzyme with the purine ring of adenosine monophosphate.From the magnitude of the spectral changes binding constants have been determined. They decrease in the order guanosine 2′‐phosphate > guanosine 3′‐phosphate > guanosine 5′‐phosphate > guanosine. The temperature dependence of the binding constants has been measured and the changes in enthalpy, free energy and entropy, which occur upon the RNAase T1‐ligand interaction, have been calculated.The pH dependencies of the different ligand · enzyme complex formations revealed several pKa values (2.5,3.7,6.5,8.5). The pKa values of 2.5 and 6.5 have been assigned to protonations of the ligand. The pKa values of 3.7 and 8.5 have been assigned to specific groups in the active site of ribonuclease T1, which are involved in the recognition of the guanine base. From the pH dependencies of the guanosine monophosphate/enzyme interaction it is concluded that only the monoanionic form of guanosine monophosphate can interact with the enzyme.The nature of binding of guanosine monophosphate to ribonuclease T1 is discussed. It is proposed that the positions N‐1 and N‐7 of the guanine base are recognized by a basic and an acid group in the active site of ribonuclease T1 and that the monoanionic phosphate moiety interacts with a basic group of the enzyme.
Effect of Degradation of Rat‐Liver Ribosomal RNA on the Structure of the Ribosomal Subunits
Authors: Hüvös Piroska , Gaál Ödön , Vereczkey László 
Published: March 2005

The effect of pancreatic ribonuclease on the sedimentation properties of rat liver ribosomal RNA and subunits was studied. When undissociated ribosomes were treated with ribonuclease both the smaller and larger subunits were found to retain original sedimentation properties in spite of the fact, that they contained no more intact 18‐S or 28‐S RNA, respectively.Treatment of ribosomes with EDTA caused different changes in the ribonuclease sensitivity of the two subunits: the smaller subunit became more sensitive towards nucleolytic degradation than the larger one. On the other hand, addition of EDTA to the ribosomes enhanced the ribonuclease sensitivity of 28‐S RNA to a greater extent than that of 18‐S RNA.