bluuebunny/crossref_metadata_2025 huggingface dataset:
The Primary Structure of the Porcine Luteinizing‐Hormone α‐Subunit
Authors: Maghuin‐Rogister Guy , Combarnous Yves , Hennen Georges
Published: March 2005

The primary structure of porcine luteinizing hormone α‐subunit was established by determination of the amino acid sequence of its tryptic peptides, which were aligned by homology with the known sequence of ovine luteinizing hormone α‐subunit. Its polypeptide chain is shorter by six amino acid residues at its amino‐terminus, when compared to the ovine α‐subunit. Furthermore four amino acid replacements are observed in the porcine as compared with the ovine chain.

Heterogeneity of both carbohydrate prosthetic groups of porcine luteinizing hormone α‐subunit is indicated by the different sugar compositions of the glycopeptides isolated in the course of this work.
The Protective Effect of Thiosulfate upon the Inactivation of Aspartate
Authors: Cavallini Doriano , Federici Giorgio , Bossa Francesco , Granata Filippo
Published: March 2005

The inactivation of aspartate aminotransferase in the course of the incubation with l‐serine O‐sulfate is protected by thiosulfate. It has been found that also l‐cysteine sulfinic acid is an inactivator when it is incubated with the enzyme, while l‐serine‐O‐phosphate and l‐cysteine‐S‐sulfonate are inactive. The inactivation by l‐cysteine sulfinic acid is increased by the simultaneous addition of 2‐oxoglutarate and is protected by thiosulfate. Cysteine‐S‐sulfonate has been detected in the incubates of the enzyme with l‐serine‐O‐sulfate protected with thiosulfate. It has been concluded that the protection by thiosulfate is due to the β‐addition of thiosulfate to the inactivator aminoacrylic acid. The resulting cysteine‐S‐sulfonate thus represents an inactive dead‐end product formed in the course of the protection by thiosulfate. It is suggested that this reaction could play the role of protecting enzymes sensitive to aminoacrylic acid and of inserting inorganic sulfur into aminoacrylic acid, producing a cysteine derivative.
Further Evidence for the Pool Function of Ubiquinone as Derived from the Inhibition of the Electron Transport by Antimycin
Authors: Kröger Achim , Klingenberg Martin , Schweidler Sabine
Published: March 2005

The role of ubiquinone (Q) in the respiratory chain is studied by analyzing the inhibition by antimycin of the electron transport in uncoupled submitochondrial particles. As a result, the antimycin inhibition curve of substrate oxidation is quantitatively explained.

The participation of Q in the electron transport is required for the occurrence of nonlinear antimycin inhibition curves as demonstrated with external hydroquinones of different redox potentials as hydrogen donors. The respiration is non‐linearly inhibited with a hydroquinone of sufficiently low redox potential to reduce the mitochondrial Q (menadiol) whereas a linear inhibition curve is found with hydroquinones of higher redox potentials (durohydroquinone, dimethoxy‐dimethylbenzohydroquinone). If Q is extracted, the inhibition curve also with menadiol becomes linear. This demonstrates the relation of the nonlinearity of the inhibition curve to the function of Q as a distributor of hydrogen between parallel cytochrome chains.

The electron transport through the cytochrome segment is inhibited linearly with increasing amounts of antimycin as demonstrated by the linear titration of three different functions: (a) the oxidation rate of Qred on addition of oxygen to anaerobic submitochondrial particles, (b) the repiratory activity with added hydroquinones, (c) the acceptor activity as defined by the ratio of the respiration rate to the redox state of Q (Kröger, Klingenberg and Schweidler, 1973).

The ratio of donor to acceptor activity to Q (Vred/Vox) is the main parameter in controlling the shape of the antimycin titration curve. This curve becomes more nonlinear with decreasing this ratio. Also the redox state of Q is nonlinearly dependent on the amount of antimycin and controlled by the donor/acceptor activity ratio.

A theoretical hyperbolic relation of the rate of substrate oxidation to the amount of antimycin is derived, involving the ratio donor to acceptor activity and the linear inhibition by antimycin of the electron transport through the cytochrome segment. With independently measured donor/acceptor activity ratios, antimycin titration curves are calculated which give a good fit to the experimental titrations over the range from highly nonlinear to almost linear curves. The observed nonlinearity is thus recognized to result from a hyperbolic relationship. Also the calculated titration curves for the redox state of Q are in agreement with the experiments.

The deviation of the experiment from the calculated curves near equimolarity between the amount of antimycin and inhibition sites can be accounted for by the dissociation of antimycin from the site of inhibition.
Structural Properties of the Creatine‐Kinase Active Site Studied by Chromophoric‐Reagent Labelling
Authors: Roustan Claude , Brevet Annie , Pradel Louise‐Anne
Published: March 2005

The environment of the active site of creatine kinase has been investigated by means of three chromophoric reagents: 2‐hydroxy‐5‐nitrobenzyl bromide, 2‐methoxy‐5‐nitrobenzyl bromide and 2‐(dansylamino)ethyl monophosphate.

The first two reagents are incorporated at the essential thiol groups. The effect of pH on the spectra of the 2‐hydroxy‐5‐nitrobenzyl‐labelled creatine kinase suggests that this reagent lies inside the active site near an ionized residue, while the 2‐methoxy‐5‐nitrobenzyl group is apparently exposed to the medium. Conformational changes induced by the binding of different ligands, including nucleotide substrates or anions, produce only perturbations of the 2‐hydroxy‐5‐nitro‐benzyl chromophore.

The 2‐(dansylamino)ethyl monophosphate (which is really an analogue of the nucleotide substrate) interacts with the nucleotide site of creatine and arginine kinases in a relatively hydrophobic environment.
Homology between Enterotoxin Protein and Spore Structural Protein in Clostridium perfringens Type A
Authors: Frieben William R. , Duncan Charles L.
Published: March 2005

Clean, intact spores from three enterotoxin positive (ent+) and three enterotoxin‐negative (ent−) strains of Clostridium perfringens type A were treated with urea/mercaptoethanol, alkaline mercaptoethanol, or alkaline dithiothreitol, reagents known to solubilize spore coat proteins. A protein fraction comprising approximately 15 to 30% of the total spore protein was extracted. Protein solubilized by each method contained a fraction that was precipitated by anti‐enterotoxin serum and that had enterotoxin‐like biological activity neutralizable with anti‐enterotoxin serum but not commercial C. perfringens type A antitoxin. Highly purified C. perfringens type A enterotoxin was precipitated by antiserum prepared against the solubilized spore protein from an ent+ and an ent− strain. These antisera also neutralized the biological activity of C. perfringens type A enterotoxin. Examination of “core” and “coat” fractions prepared from intact spores revealed that the extractable enterotoxin‐like protein was associated with the spore coat fraction. It is concluded that the enterotoxin of C. perfringens type A is a structural component of the spore coat.
β‐Chain Allostery in the Frozen Quaternary T‐Structure of Haemoglobin M Iwate
Authors: Gersonde Klaus , Overkamp Matthias , Sick Hinrich , Trittelvitz Eberhard , Junge Wolfgang
Published: March 2005

Two hybrid species of haemoglobin M Iwate exist: α2Mmetβmetβdeoxy and α2Mmetβ2deoxy These species differ in their ligand and effector binding properties.

The α2Mmetβmetβdeoxy hybrid is characterized by a Bohr effect, while the Hill coefficient is n= 1.00. The energy of the interaction between the Bohr protons and the ligand‐binding site corresponds to the interaction energy found in monomeric haemoglobins and amounts to 0.55 kcal/mol. One molecule of 2, 3‐bisphosphoglycerate per mole is bound at neutral pH, while at alkaline pH two co‐operative molecules are bound. Binding of 2,3‐bisphosphogly‐cerate reduces the ligand affinity and shifts the pK value of the Bohr proton binding site to the alkaline region.

The α2Mmetβ2deoxy hybrid shows a pH‐dependent co‐operativity of the ligand‐binding sites (n= 1.8 at pH 9). The co‐operativity is weakened by binding of protons or 2,3‐bisphospho‐glycerate. The latter lowers the ligand affinity, but in the alkaline region no influence is observed. Furthermore the Bohr effect curve is shifted to the alkaline region by this compound but the amplitude remains unchanged.

The results of the present paper are interpreted in terms of intra‐chain and β‐β inter‐chain interactions, the latter being responsible for a consecutive change of the ligand affinity in a “frozen” T‐state molecule.
On the Reactivity of the Thiol Group of Thiolsubtilisin
Authors: Polgár László , Halász Piroska , Moravcsik Ernó
Published: March 2005

The reaction of thiolsubtilisin with iodoacetamide shows that in the alkaline pH‐range the –SH group of the enzyme reacts like a simple thiol compound such as mercaptoacetate. On the other hand, around neutral pH the enzyme displays enhanced reactivity compared with that of mercaptoacetate. On the basis of measurements in 2H2O it is concluded that a mercaptide‐imidazolium ion‐pair is formed in thiolsubtilisin and this accounts for the enhanced reactivity around neutral pH.

Iodoacetamide reacts with the –SH group of the enzyme, as compared to mercaptoacetate, at a relatively higher rate than bromo‐, chloro‐, and fluoroacetamide. This might be due to the less polar environment around the –SH group on the surface of the enzyme than in water, as indicated by alkylation of mercaptoacetate with iodo‐ and chloroacetamide in the presence of dioxane.

By alkylating thiolsubtilisin with enantiomeric reactants, a slight conformational change at the active site, not measurable with ordinary physical methods, could be established above pH · 9, whereas the overall protein structure is stable up to pH · 11. D‐2‐Bromo‐n‐butyramide reacts with thiolsubtilisin about 60 times as fast as D‐2‐bromopropionamide. This indicates that hydrophobic interaction between the enzyme and a methyl group of the substrate can enhance the reaction rate by more than one order of magnitude. Model building of the active site suggests that Ala‐152 and Thr‐220 of thiolsubtilisin may form van der Waals interactions with D‐2‐bromo‐n‐butyramide if the alkylating agent is properly positioned for reaction. No such interaction is possible between D‐2‐bromopropionamide and thiolsubtilisin.
Studies on the Metabolism of Steroids in the Foetus
Authors: Plasse Jean‐Claude , Lisboa Belisário Penna
Published: March 2005

The metabolism of testosterone in human foetal adrenal glands at mid‐gestation was investigated in vitro. After incubations with microsome preparations in a medium supplemented with NADPH‐regenerating system, the metabolites were isolated by thin‐layer and gas‐liquid chromatography, and identified by gas chromatography‐mass spectrometry. 4‐Androstenedione, 3β‐hydroxy‐4‐androsten‐17‐one and 4‐androstene‐3α (and 3β),17β‐diols were identified in both incubation series.

The presence of 1β‐, 2α‐, 2β‐, 6α‐ and 6β‐hydroxylase activities in foetal adrenal glands was demonstrated by the isolation of the corresponding testosterone derivatives. The biosynthesis of these steroids in human adrenal gland has not been demonstrated before.

Because 3β‐hydroxy‐4‐androsten‐17‐one and epimeric 4‐androstene‐3α (and 3β), 17β‐diols could be isolated in these incubation studies the presence of 3α, 3β‐ and 17β‐hydroxysteroid oxidoreductase in the adrenal gland preparation is very likely. Neither 5α(H)‐ nor 5β(H)‐ring reductase activities have been detected.
Studies on the Conformational Changes of Plasminogen Induced during Activation to Plasmin and by 6‐Aminohexanoic Acid
Authors: Sjöholm Ingvar , Wiman Björn , Walléan Per
Published: March 2005

The hydrodynamic properties of plasminogen are highly affected during the activation process as found by studies on the sedimentation‐velocity, sedimentation‐equilibrium behaviour, gelfiltration behaviour of plasminogen A, plasminogen B and of plasmin inactivated by diisopropylphosphorofluoridate (Dip‐plasmin). The molecular weight was estimated to 92 000, 84000 and 81 000 for plasminogen A, B and Dip‐plasmin, respectively and the frictional ratio, f/f0, was found to be 1.50, 1.56 and 1.55.

The circular dichroism (CD) spectra of human plasminogen A, plasminogen B and Dip‐plasmin have been studied at pH 7.4 in 0.1 M KCI. The far‐ultraviolet spectra are almost identical for all the samples and indicate that they contain a large proportion of random structure (about 80%) and about 20%β‐structure, while no significant amounts of α‐helix could be detected. In the near‐ultraviolet region, large significant differences were seen, which are consistent with a stepwise activation of plasminogen A via plasminogen B to plasmin.

The effects of 6‐aminohexanoic acid and l‐lysine on the CD spectra of plasminogen A and of the activation peptide on that of plasminogen B indicate that the amino‐terminal peptidic material has great affinity for the rest of the protein at pH 7.4 and greatly affects the CD spectrum between 250–280 nm. In this area, the spectra of Dip‐plasmin and plasminogen B are almost identical and the same spectrum is also obtained with plasminogen A when treated 0.01 M 6‐aminohexanoic acid or 0.1 M l‐lysine. The conformation around the amino‐terminal part of Dip‐plasmin and around the internal peptide bond cleaved during the activation to plasmin seems to be of great importance for the CD spectra at 280–305 nm.
On Steroid Conjugates in Plasma
Authors: Kurtenbach Peter , Benes Peter , Oertel Georg W.
Published: March 2005

By incubation with washed mitochondria from guinea pig liver [7α‐3H]dehydroepiandroster‐one sulfate can be converted into a 3H‐labelled lipophile sulfoconjugate. Concerning the overall reaction a Km‐value of approximately 6 μM was derived from the substrate concentration curve. Optimal yields of lipophile steroid sulfoconjugates were obtained at a pH of 8.0. Of the various coenzymes tested CTP appeared to be essential for the formation of lipophile sulfoconjugates. Whereas magnesium ions stimulated the conversion of [7α‐3H]dehydroepiandrosterone sulfate, the biosynthesis of lipophile steroid sulfoconjugates was inhibited by sodium fluoride or p‐chloromercuribenzoate.
Studies on the Properties of Fish Hemoglobins
Authors: Giardina Bruno , Brunori Maurizio , Binotti Ines , Giovenco Silvia , Antonini Eraldo
Published: March 2005

The ligand‐binding kinetics of the isolated component from trout (Salmo irideus) blood has been extensively investigated; it appears to be fully consistent with their equilibrium behaviour, described previously. At alkaline pH (∼ 7.5), in a region where the Root effect is not operative, comparison of the kinetics of the various components (Hb trout I, II and IV) shows considerable similarities between them. For Hb trout I (and II) the time course of the reaction with both O2 and CO is practically pH independent. On the other hand Hb trout IV, characterized by a Root effect, displays a strong dependence on pH of the kinetics of ligand binding. Thus in the pH region where the Root effect becomes operative there is a large change of the character, as well as of the speed, of the kinetics with both O2 and CO.

The Root effect characteristic of Hb trout IV appears to originate, from a kinetic point of view, in a pH‐dependent change of both the “on” and “off” constants, although at least for O2 the effect of pH on the “off” kinetic constants is much more marked.
Purification and Molecular Properties of Yeast Hemoglobin
Authors: Oshino Reiko , Asakura Toshio , Takio Koji , Oshino Nozomu , Chance Britton , Hagihara Bunji
Published: March 2005

Yeast hemoglobin from Candida mycoderma has been purified by ammonium sulfate fractionation and subsequent DEAE‐cellulose column chromatography. A minimum molecular weight estimated from the result of amino acid analysis, as well as dodecylsulfate gel electrophoresis and gel filtration on Sephadex G‐100, is 50000. The absorption spectra of ferrous yeast hemoglobin in the oxy, deoxy and carboxy forms were almost identical to those of other hemoglobins, with the exception of a small contribution from flavin. The molecule has two prosthetic groups, FAD and protoheme. The results obtained by dodecylsulfate‐gel electrophoresis and 8 M urea treatment suggest that both prosthetic groups are attached on a single peptide chain, which appears to exist as a monomer. Yeast hemoglobin, in the ferrous form, is capable of binding oxygen reversibly. The oxygen affinity of yeast hemoglobin is 20 nM (0.01 mm Hg) and a rate constant of oxygen dissociation is 17 s−1 at pH 7.0, 23°C. Half‐reduction potentials of heme and flavin moieties were ‐60 mV and about ‐150 mV, respectively, in 0.1 M phosphate buffer pH 7.0 at 25°C.
Mode of Action of the Macrolide‐Type Antibiotic, Chlorothricin
Authors: Schindler Peter W. , Zäuhner Hans
Published: March 2005

The antibiotic, chlorothricin, inhibits the reaction catalyzed by pyruvate carboxylase from Bacillus stearothermophilus. With steady‐state kinetic measurements the following effects can be observed. (a) Inhibition of the overall reaction is competitive with respect to CoASAc, the allosteric activator of this enzyme, and non‐competitive with respect to both MgATP and pyruvate (substrates). (b) Chlorothricin raises the Hill coefficient, n, of CoASAc from 2 to nearly 3. On the other hand, interaction of the antibiotic with enzyme is characterized by n= 3. (c) The Michaelis‐Menten‐type kinetic of MgATP is changed to a kinetic showing substrate inhibition, while the substrate activation of pyruvate carboxylase by pyruvate is suppressed upon the addition of chlorothricin. The data presented in this paper strongly suggest that the conformation of site 1 of pyruvate carboxylase responsible for the regulation of the overall enzyme activity is influenced by chlorothricin and CoASAc in an antagonistic manner. A model is proposed which assumes the existence of hybrid enzyme forms.
Biosynthesis of Fatty Acids and Cholesterol in Isolated Rat‐Liver Parenchymal Cells
Authors: Nilsson Åke , Sundler Roger , ÅKesson Björn
Published: March 2005

The fatty acid and cholesterol synthesis in isolated rat‐liver parenchymal cells was studied with tritiated water as the radioactive precursor. In cells from rats fed a diet containing 3% fat the rate of fatty acid biosynthesis was 1.25 and the cholesterol biosynthesis 0.68 nmol/mg protein per h. This means that the rate of cholesterol biosynthesis was in the same range as in earlier studies with perfused rat livers, whereas the fatty acid synthesis was about ten times lower than in the intact organ. Addition of palmitic or oleic acid to the medium caused up to 81% inhibition of the fatty acid synthesis, whereas the cholesterogenesis was either unaffected or inhibited to a lesser extent. The data therefore provide direct evidence for a feedback inhibition of hepatic lipogenesis, caused by the uptake of long‐chain fatty acids. Octanoic and dodecanoic acid were not as efficient inhibitors of the fatty acid biosynthesis. Instead the addition of these fatty acids to the medium in increasing concentrations caused an increasing inhibition of the cholesterol biosynthesis.
5‐Aminolevulinic‐Acid Synthetase of Rhodopseudomonas spheroides Y
Authors: Fanica‐Gaignier Michèle , Clement‐Metral Jenny
Published: March 2005

The two species of 5‐aminolevulinic acid synthetase were purified about 4200‐fold for fraction I and about 2000‐fold for fraction II. They were obtained homogenous in disc electrophoresis. These two enzymes have the same molecular weight (about 100 000) and contain one molar equivalent of pyridoxal phosphate. This cofactor is linked as a substituted aldamine derivative (330 nm) at neutral pH values and as an easily dissociable Schiff base in the acid or very basic ranges. An –SH group may be involved in the formation of the aldamine derivative. They differ by their isoelectric point (pI= 5.1 for fraction I and pI= 6.0 for fraction II) and by their number of –SH groups (1 for fraction I and 7 for fraction II). The results suggest that the two synthetases are two isofunctional enzymes.
5‐Aminolevulinic‐Acid Synthetase of Rhodopseudomonas spheroides Y
Authors: Fanica‐Gaignier Michèle , Clement‐Metral Jenny
Published: March 2005

The results of initial velocity studies on the 5‐aminolevulinic acid synthetase reaction and product inhibition studies are consistent with an ordered bi‐bi mechanism in which glycine binds to the enzyme first and 5‐aminolevulinic acid dissociates last. The results are confirmed by the spectral changes of holoenzyme caused by the substrates.

5‐Aminolevulinic acid synthetase activity is inhibited only by adenosine triphosphate, guanosine triphosphate and pyrophosphate. This inhibition is competitive with glycine and non‐competitive with succinyl‐CoA. Experiments with [α‐32P]ATP, [γ‐32P]ATP and [32P2]pyrophosphate have shown that adenosine triphosphate and pyrophosphate are bound on each of both forms of 5‐aminolevulinic acid synthetase. The ratio is one mole per mole of enzyme. The estimation of –SH groups with 5,5′‐dithiobis‐(2‐nitrobenzoic acid) after binding of inhibitor shows that one –SH is masked. After p‐hydroxymercuribenzoate action on the enzyme, 5‐aminolevulinic acid synthetase does not bind anymore [32P]ATP and is completely inactive. We suggest that ATP and pyrophosphate inhibit 5‐aminolevulinic acid synthetase by acting on an –SH group of the active center of 5‐aminolevulinic acid synthetase.
The Use of Affinity Chromatography in Determining the Sites of Protein‐Protein Interaction Relative to the Binding Sites of Substrates in Gramicidin S Synthetase
Authors: Pass Lidia , Zimmer Trine‐Lise , Laland Søren G.
Published: March 2005

Columns containing one of the amino acids in gramicidin S bound through its carboxyl group to the amino group of amino‐alkyl‐Sepharose (CNBr‐activated Sepharose + 3,3′‐diaminodipropylamine) have been prepared and were found to exhibit affinity for the corresponding enzymes (light or heavy) of gramicidin S synthetase. The d‐phenylalanyl ligand will bind the light enzyme and the l‐propyl, l‐valyl, l‐ornithyl or l‐leucyl ligand, the heavy enzyme. These columns permitted a study of the interaction of the light and heavy enzyme. The results show that the area of the light enzyme which has affinity for the heavy enzyme is that part which activates phenylalanine. The area of the heavy enzyme which interacts with the light enzyme is not that part of the enzyme which activates proline. However, it is probably located close to the activation sites for proline and leucine.

The columns, in particular with proline as ligand, provide a quick and convenient method for the separation of the light and heavy enzyme of gramicidin S synthetase.
Differently Labelled Glucosamine‐Precursor Pooles for the Biosynthesis of Hyaluronate and Heparan Sulfate
Authors: von Figura Kurt , Kiowski Wolfgang , Buddecke Eckhart
Published: March 2005

14C‐labelled hyaluronate and heparan sulfate were isolated from bovine arterial tissue following incubation in vitro in the presence of [U‐14C]glucose and [1‐14C]glucosamine, respectively. The specific radioactivity of hyaluronate was higher than that of heparan sulfate, but the ratio of their specific radioactivities (hyaluronate/heparan sulfate) was 2.5 or 1.2, depending on whether [14C]glucose or [14C]glucosamine was used as the metabolic precursor.

In the [14C]glucose experiments the proportion of radioactivity incorporated into the glucosamine and glucuronic acid moiety of hyaluronate was about 1:1, while in the case of heparan sulfate the corresponding ratio was 1:1.7. Values of 3.3 and 1.1 were calculated for the specific radioactivity ratio of the glucosamine moiety of hyaluronate and heparan sulfate (glucosamine‐hyaluronate/glucosamine‐heparan sulfate) following labelling with [14C]glucose and [14C]glucosamine, respectively.

The results are consistent with the assumption that two topographically different pools of immediate metabolic precursors exist in bovine arterial cells, one being for the biosynthesis of hyaluronate and the other for heparan sulfate.
The Specificity of Escherichia coli Endonuclease I
Authors: Ehrlich Stanislav D. , Bertazzoni Umberto , Bernardi Giorgio
Published: March 2005

Degradation of different DNA's by E. coli Endonuclease I was studied. Hyperchromicity and acid solubility of the digest as well as the average chain length (P̄n) of oligonucleotides released by the enzymes were followed during degradation; relationships between these parameters were established.

The specificity of the E. coli Endonuclease I was determined by analysing 3′‐terminal, 5′‐terminal and 5′‐penultimate nucleotide of oligonucleotides formed by the enzyme from calf thymus DNA. The compositions found differ from those expected on the basis of random breakage of DNA; this indicates that E. coli Endonuclease I recognizes sequences at least three nucleotides long.

The 3′‐terminal nucleotide composition was determined over a wide range of P̄n (150–4) and was found to be invariant, indicating that the specificity of E. coli Endonuclease I does not change during digestion.

Composition of 3′‐terminal nucleotides released from different bacterial DNA's varied linearly with the DNA (dG + dC) contents; those released from the calf thymus DNA did not fit that relationship.
Intracellular Titration of Cyclic AMP Bound to Receptor Proteins and Correlation with Cyclic‐AMP Levels in the Surviving Rat Diaphragm
Authors: Do Khac Lien , Harbon Simone , Clauser Hubert J.
Published: March 2005

Extracts prepared from rat diaphragms incubated with or without theophylline and/or epinephrine have been tested for their total cyclic AMP content and for their ability to bind exogenously added cyclic [3H]AMP. Less cyclic [3H]AMP can be bound inthe extracts after theophylline and/or epinephrine treatment indicating that the rise in cyclic AMP level was accompanied by an increase in the quantity of cyclic AMP bound intracellularly to the cyclic AMP‐dependent protein kinases. Maximum cyclic AMP binding capacities, as measured by total cyclic AMP exchanges, were however identical in all cases. Accurate estimations of intracellular binding of cyclic AMP have been correlated with the level of cyclic AMP in the tissue: the reaction seems to obey simple saturation kinetics, and apparent intracellular Kd for cyclic AMP has been evaluated as 330 nM. The findings are consistent either with a real difference in the intracellular binding constant as compared to that measured in vitro (28 nM) or with the fact that the cyclic nucleotide in the cell may not all be available for the kinase protein receptors. They also suggest that the method described may prove useful for studying any possible intracellular control beyond the step of cyclic AMP synthesis.
Transglycosylic Reactions of Nucleotides of Deoxy‐Sugars
Authors: Zemek Jiří , Kučár Štefan , Bauer Štefan
Published: March 2005

Incorporation of 3‐deoxy‐, 4‐deoxy‐, and 6‐deoxy‐analogues of d‐glucose into glycogen was investigated in a reaction catalyzed with yeast UDPglucogen glucosyl transferase. It was found that hydroxyl groups at positions C‐3, C‐4 and C‐6 in the glucosyl moiety of the UDPG molecule are not essential for preserving its substrate properties in this transglycosylic reaction.

The kinetics of 3‐deoxy‐, 4‐deoxy‐ and 6‐deoxy‐glucose incorporation into glycogen was compared with that of d‐glucose and 2‐deoxy‐glucose. It was found that the affinity of the enzyme and the rate of hexose incorporation into glycogen decreases in following sequence: d‐glucose, 2‐deoxy‐, 6‐deoxy‐, 4‐deoxy‐ and 3‐deoxyglucose.

The deoxyglucosyl analogues of UDPG act as competitive inhibitors on the reaction of glycogen biosynthesis.
Cycloheximide‐Resistant Synthesis of Mitochondrial‐Membrane Components in Aspergillus nidulans
Authors: Turner Geoffrey
Published: March 2005

In the presence of cycloheximide, Aspergillus nidulans mycelium incorporated radioactive leucine preferentially into mitochondrial protein. Although cytoplasmic protein synthesis was not totally abolished in the presence of the drug, mitochondrially synthesized components could be identified by a double‐labelling procedure. Labelled mitochondria were purified by centrifugation on sucrose gradients, and electrophoresis of mitochondrial membrane proteins on dodecyl‐sulphate‐polyacrylamide gels resulted in the identification of four mitochondrially synthesized components with molecular weights of 13000, 18000, 27000 and 40000.
Effects of Yeast Proteinase and Its Inhibitor on the Inactivation of Tryptophan Synthase from Saccharomyces cerevisiae and Neurospora crassa
Authors: Tsai Hsin , Tsai Jane H. J. , Yu Peter H.
Published: March 2005

This paper reports the isolation of a proteinase and a proteinase inhibitor from Saccharomyces cerevisiae S288C (α). The purified proteinase catalyzes the hydrolysis of casein at neutral pH. It inactivates the holo‐tryptophan synthase from Neurospora crassa but does not inactivate the holo‐ornithine aminotransferase from pig kidney. This proteinase can be inhibited either by phenylmethanesulfonyl fluoride (a specific inhibitor for serine proteases), the sulfhydryl reagent, p‐mercuribenzoate, or the endogenous proteinase inhibitor present in yeast. The yeast proteinase inhibitor is a heat‐stable material with a molecular weight of approximately 13000. This proteinase inhibitor can protect both yeast and Neurospora tryptophan synthase against proteolytic inactivation. The proteinase and its inhibitor coexist in the yeast extracts. The cellular level of the proteinase and the inhibitor activities varies with the composition of the medium in which the cells are grown. Since some yeast proteinases have been shown to form inactive complexes with their respective inhibitors, it is proposed that proteinase‐inhibitor interaction is a possible mechanism for regulation of intracellular proteolytic activity.
Investigation of Nucleotide · Ribonuclease‐A Complexes with High‐Resolution 31P‐Nuclear‐Magnetic‐Resonance Spectroscopy
Authors: Haar Wolfgang , Maurer Wolfgang , Thompson John C. , Rüterjans Heinz
Published: March 2005

The chemical shift of the phosphorus resonance of various nucleotides has been studied as a function of pH in the absence and presence of ribonuclease A. The 31P‐signal of the nucleotides shifts upfield upon protonation of the phosphate moiety. Thus titration curves and pK values are obtained when the 31P chemical shift is plotted against the pH value. The titration curves are analyzed in terms of interactions of the phosphate group with the base or ribose moieties of the nucleotide itself or with amino‐acid side chains of the active site of ribonuclease A. The chemical shift of the phosphorus magnetic resonance of cytidine 2′‐phosphate when this nucleotide is bound to ribonuclease A resmebles that of the dianionic form of the free compound. Proton magnetic resonance studies indicated that the histidine residue 119 of ribonuclease A is bound to the phosphate group in enzyme‐nucleotide complexes [4]. This is now confirmed, since the apparent pK value of the phosphate group of these complexes is the same as the pK value of histidine‐119 determined by proton magnetic resonance spectroscopy. Possible arrangements of the hydrogen bonds between the imidazole group of histidine‐119 and the phosphate group are disscussed. From linewidth broadeining due to chemical exchange, an average life time of the complex of cytidine 2′‐phosphate with ribonuclease A is derived.
Initiation Factor‐Dependent Binding of MS2 RNA to 30‐S Ribosomes and the Recycling of IF‐3
Authors: Vermeer Cees , van Alphen Wim , van Knippenberg Peter , Bosch Leendert
Published: March 2005

The binding of washed 30‐S ribosomal subunits of Escherichia coli to MS2 RNA in the absence of fMet‐tRNA requires the presence of the initiation factor IF‐3. No binding is observed with IF‐1, IF‐2 or a combination of these two initiation factors.

The IF‐3‐dependent binding is stimulated about two‐fold by IF‐2. IF‐1 has no effect in this respect. Optimal binding occurs at about 7 mM Mg2+.

Upon incubation of [35S]IF‐3, MS2 [3H]RNA and 30‐S subunits, complexes are formed which contain the three components in a 1:1:1 ratio. These ternary complexes have a sedimentation coefficient of about 40 S and a buoyant density in CsCl of about 1.74 g/ml.

The ternary complexes formed in the absence of IF‐2 and IF‐1 are rather labile and readily dissociate into MS2 RNA and IF‐3‐containing ribosomes. Their half‐life at 0°C is about 40 min. In the presence of IF‐2 and IF‐1, complexes are formed which remain stable for at least 6 h. Unwashed native 30‐S subunits also form stable complexes with MS2 RNA.

Binding of washed 30‐S subunits to unfolded MS2 RNA (MS2 RNA treated with formaldehyde) does not require initiation factors. Complexes containing more than one ribosomal particle per messenger can be formed. Attachment of fMet‐tRNA to these complexes requires IF‐2 and IF‐1, but is optimal in the presence of all three initiation factors.

Release of [35S]IF‐3 from the MS2 RNA · 30‐S subunit complexes occurs upon attachment of fMet‐tRNA. In the case of unwashed native 30‐S ribosomal subunits, recycling of IF‐3 occurs at the same stage as was demonstrated by a direct determination of the stoichiometry of unlabeled IF‐3 on the ribosomal complexes.
Separation and Characterization of Polypeptides from the Venom of Dendroaspis viridis
Authors: Shipolini Rudolph A. , Bailey Graham S. , Edwardson James A. , Banks Barbara E. C.
Published: March 2005

The venom of Dendroaspis viridis has been fractionated by chromatography on Sephadex G‐50 and SE‐Sephadex. Thirteen polypetides containing either 59–63 amino acid residues and four disulphide bridges or 71–73 amino acid residues and five disulphide bridges have been isolated and characterized by amino‐acid analysis and N‐terminal residue determination. The compositions of the polypeptides show many similarities but the toxicities vary in excess of 100‐fold, upwards from an LD50 of 0.08 mg/kg in mice.

Enzymic and nerve growth factor activities have been located in protein fractions associated with higher molecular‐weight material.
Active Subunits of Transaldolase Bound to Sepharose
Authors: Chan William W.‐C. , Schutt Hermann , Brand Karl
Published: March 2005

Individual subunits of yeast transaldolase were studied by the method of matrix‐bound derivatives. Native dimeric transaldolase was bound to Sepharose activated with low amounts of cyanogen bromide to give matrix‐bound transaldolase in which the majority of molecules were bound covalently via only one subunit. Exhaustive washing with 6 M guanidinium chloride and renaturation in triethanolamine‐EDTA buffer resulted in a derivative (bound‐subunit transaldolase) which retained 65% of the original protein and 63% of the original activity. This active form of transaldolase is more sensitive to 2 M urea than bound‐dimeric transaldolase. Bound‐subunit transaldolase is able to associate with “nascent” soluble transaldolase subunits generated in situ by diluting a small aliquot of guanidinium‐chloridedenatured transaldolase into a much larger volume of buffer. The ability of bound‐subunit derivative to pick up soluble “nascent” transaldolase subunits is highly specific and reaches a plateau level close to the original protein and activity content of bound transaldolase. The product of such treatment (bound‐renatured transaldolase) has properties virtually indistinguishable from those of bound transaldolase. These results indicate that individual subunits of transaldolase are catalytically active.

Bound‐subunit transaldolase has Km values similar to those of the bound‐dimeric derivatives and can be inactivated with sodium borohydride in the presence of the substrate fructose 6‐phosphate. However, the pH profile of the subunit form is substantially altered suggesting a shift in the pKa of an essential protonated group from 9.2 to 8.6.
Studies of Glutamate Dehydrogenase
Authors: Koberstein Rudolf , Krause Jobst , Sund Horst
Published: March 2005

Specific interaction between α‐NADH and glutamate dehydrogenase is demonstrated by difference spectroscopy, circular dichroism and fluorescence measurements. Quantitative binding studies in the preparative ultracentrifuge yield six identical α‐NADH binding sites per oligomer with a dissociation constant of 20 μM. Evidence for six to eight additional, very weak α‐NADH binding sites is presented. Excess ADP prevents the binding of α‐NADH to the tight binding sites, indicating competition of the two nucleotides. In the enzymatic reaction, α‐NADH is inactive as a coenzyme; however, like ADP, it is an activator of the reductive amination reaction of 2‐oxoglutarate. α‐NADH reduces the self‐inhibition of high β‐NADH concentrations and thus agin parallels the effects caused by ADP. Contrary to β‐NADH or β‐NADH plus GTP, α‐NADH or α‐NADH plus GTP does not affect the dissociation‐association equiilibrium of glutamate dehydrogenase. The results are consistent with the assumption of six binding sites for α‐NADH, which are identical with the ADP binding sites and most probably the six non‐active β‐NADH binding sites.
Primary Structure of a Small Glycopeptide Isolated from Human Glomerular Basement Membrane and Carrying a Major Antigenic Site
Authors: Mahieu Philippe M. , Maghuin‐Rogister Guy R. , Lambert Paul H.
Published: March 2005

The glycopeptides containing either the disaccharide or the heteropolysaccharide chains were purified, after enzymatic digestion of glomerular basement‐membrane antigens, by gel filtration and ion‐exchange chromatography. Their antigenicity was investigated in seven patients with anti‐glomerular‐basement‐membrane antibody‐mediated glomerulonephritis, by the leukocyte migration test and radioimmunoassay.

The leukocyte migration of the seven patients is inhibited in presence of membrane antigens with both types of oligosaccharide chains. The disaccharide‐containing glycopeptide inhibits the leukocyte migration of these patients using 10‐times less antigenic material; the heteropolysaccharide‐containing glycopeptide does not modify the leudkocyte migration of the same patients. Similarly, only the disaccharide‐containing glycopeptide decreases the binding of labelled membrane antigens with anti‐glomerular‐basement‐membrane antibodies, using the radioimmunoassay. This glycopeptide carries therefore the major antigenic determinants of humoral and cellular anti‐basement‐membrane auto‐immunization.

The amino acid sequence of the glycopeptide is Hyl‐Gly‐Glu‐Asp‐Gly, the disaccharide prosthetic group being linked to the hydrolysine residue of the peptidic chain; its immunodominant antigenic site is formed by the Galp‐Hyl unit.
Pulse‐Recovery Studies on Cycloheximide‐Insensitive Protein Synthesis in Neurospora
Authors: Lansman Robert A. , Rowe Mark J. , Woodward Dow O.
Published: March 2005

The effects of cycloheximide on cytoplasmic protein synthesis in Neurospora are reversible. In these experiments, the products of mitochondrial protein synthesis were labeled in vivo in the presence of cycloheximide; analyses were made of the size distribution and enzyme association of these products as the cells recovered from cycloheximide inhibition.

In cells harvested immediately after a 20‐min cycloheximide pulse, four protein species of high specific activity were resolved in polyacrylamide gels of mitochondrial protein electro‐phoresed in the presence of 1% sodium dodecylsulfate. Only small amounts of labeled protein were found to co‐purify with cytochrome oxidase isolated from these cells. In cells harvested after two to three hours of recovery from cycloheximide inhibition, three of the four original labeled peaks remain in whole mitochondrial protein. These peaks correspond precisely in gel mobility to the three highest‐molecular‐weight protein species associated with cytochrome oxidase, species which are heavily labeled in enzyme preparations from recovery cultures. These gel patterns can be obtained only when the preparations are treated with phenylmethylsulfonyl fluoride, an inhibitor of protease activity. The results obtained are discussed in terms of an assembly process in which the major stable products of mitochondrial protein synthesis become integrated into the cytochrome oxidase complex only when cytoplasmically‐made components are available for assembly.
Measurements of the Concentration of Active Sites in Preparations of Yeast Alcohol Dehydrogenase
Authors: Dickinson Mark
Published: March 2005

Studies on the inactivation of yeast alcohol dehydrogenase by iodoacetate indicate that under certain conditions reaction of two equivalents of reagent produces a 90–95% loss of activity. Such observations suggest two essential thiol groups per molecule. Measurements of NADH binding using three independent methods indicate a similar number of coenzyme binding sites.
The Complete Amino‐Acid Sequence of Non‐Immunolobulin Amyloid Fibril Protein AS in Rheumatoid Arthritis
Authors: Sletten Knut , Husby Gunnar
Published: March 2005

The primary structure of a non‐immunoglobulin amyloid protein AS has been determined. The protein was found to consist of 76 amino acid residues corresponding to a molecular weight of 9145. The sequence analysis showed clearly that the protein was homogeneous. A characteristic distribution of hydrophobic amino acids was observed and suggested as being of importance for the ability of this protein to form fibrils. A comparison of the protein with other amyloid protein AS showed a high degree of variability, particularly in the carboxyl‐terminal region.
The Physiological Role of the Lymphoid System
Authors: Constantopoulos Andreas , Najjar Victor A.
Published: March 2005

Blood thrombocytes are shown to bind specific fractions of autologous γ‐globulin in isotonic low‐ionic‐strength sucrose solution. Thrombocytes in freshly drawn blood that were prepared in their plasma not exposed to sucrose solution were found coated with the same specific thrombophilic γ‐globulin fractions. Judging by the active physiological role of erythrophilic and leucophilic γ‐globulin, it is proposed that thrombophilie γ‐globulin may have an equally important role, the nature of which is yet to be determined.
Elongation Factor 1 from Krebs II Mouse Ascites Cells
Authors: Drews Jürgen , Bednarik Karl , Grasmuk Hans
Published: March 2005

A procedure for the purification of elongation factor 1 (EF‐1) from Krebs II mouse ascites cells is described. Ascites EF‐1 occurs in multiple forms of different molecular weight like the corresponding enzymes from calf brain and rat liver. Biogel A‐5m chromatography of our purest EF‐1 resulted in a pattern indicating the presence of different molecular weight components. However, only a single polypeptide band was observed when this same material was analysed by electrophoresis on acrylamide gels containing sodium dodecylsulphate. With this method the molecular weight of the EF‐1 polypeptide chain was determined to be 47000. It is suggested that the occurrence of multiple forms of EF‐1 is due to the association of 47000‐mol. wt subunits to form aggregates of different sizes.

The factor has essentially the same enzymatic properties which were described for EF‐1 from reticulocytes, calf brain and rat liver. The attachment of aminoacyl‐tRNA to ribosomes catalyzed by EF‐1 follows a strict 1:1 stoichiometry. EF‐2 when introduced into the ribosomal binding assay allows the recycling of EF‐1 and subsequent synthesis of polypeptide chains. The possible implications of this finding with respect to the conditions which allow stoichiometric or catalytic function of EF‐1 are discussed.
Synthesis and Catalytic Properties of the Heptapeptide l‐Seryl‐l‐Prolyl‐l‐Cysteinyl‐l‐Seryl‐α‐l‐Glutamyl‐l‐Threonyl‐l‐Tyrosine
Authors: Fridkin Matitahu , Goren H. Joseph
Published: March 2005

The heptapeptide L‐Ser‐l‐Pro‐l‐Cys‐l‐Ser‐l‐Glu‐l‐Thr‐l‐Tyr has a structural role in bovine carboxypeptidase A. However, from a standpoint of an enzyme model five of the seven amino acids may participate in catalysis of hydrolysis of p‐nitrophenylacetate. The heptapeptide has been synthesized from the C‐terminal to the N‐terminal using dicyclohexylcarbodiimide as the coupling agent. The amino groups were blocked by tert‐butyloxycarbonyl and the side‐chains were blocked by benzyl groups. The free heptapeptide was obtained on treatment of the blocked heptapeptide with anhydrous hydrogen fluoride. Treating the blocked heptapeptide in liquid ammonia with sodium results in a 25% cleavage of serine from the N‐terminal. The hydrolysis of p‐nitrophenylacetate was followed spectrophotometrically at 400 nm (p‐nitrophenoxide ion appearance) and at 270 nm (p‐nitrophenyl ester loss). The determination of rate constants were by the “initial slope” method. Known nucleophilic catalysts imidazole, cysteine and glutathione, gave rate constants which agreed with the literature. The synthetic heptapeptide catalyzed the hydrolysis of p‐nitrophenylacetate and its catalytic ability lay in the sulfhydryl group of the cysteine residue. The pH profile of the second‐order rate constant of heptapeptide‐catalyzed ester hydrolysis indicated that the latter molecule is 5 times more active than cysteine and 10 times more active than glutathione. An ionizable group of pKa 8.3 is involved in the catalysis. This pKa yields a second‐order rate constant approximately 10 times above the Brosted relationship for thiol‐catalyzed p‐nitrophenylacetate hydrolysis. The pKa of the cysteine residue of the heptapeptide appears to be above 10 according to a spectrophotometric titration method. A concerted acid‐base catalytic mechanism is proposed that permits the explanation of the high catalytic reactivity, the ionizable group of pKa 8.3 and the apparent high pKa of the cysteine sulfhydryl group.
Cytochrome b2 from Bakers' Yeast (L‐Lactate Dehydrogenase)
Authors: Jacq Claude , Lederer Florence
Published: March 2005

Bakers' yeast cytochrome b2 [lL‐(+)‐lactate dehydrogenase or l‐(+)‐lactate cytochrome c oxidoreductase], usually purified by crystallisation according to Appleby and Morton, was shown to be a degraded form of the enzyme, arising by a proteolytic modification of the species formed in vivo. We present here an improved purification procedure and some properties of the intact enzyme.

This intact cytochrome b2 is shown, by cross‐linking and by gel filtration studies, to be in a tetrameric form with a molecular weight of about 230 000. The four identical subunits are composed of a single polypeptide chain associated with one haem and one flavin (FMN). The amino‐acid composition is presented. Alanine and glutamic acid are shown to be respectively C‐ and N‐terminal residues.

A comparison of these results with those concerning the degraded form suggests that there is little loss of peptide material and that the gross quaternary structure of the protein is not modified by the proteolytic process. However circular dichroism studies show that the haem environment is altered.

The evidence leading to propose a biglobular structure for the enzyme is discussed.