bluuebunny/crossref_metadata_2025 huggingface dataset:
The Dolichol Pathway of Protein Glycosylation in Rat Liver
Authors: GODELAINE Danièle , BEAUFAY Henri , WIBO Maurice , AMAR‐COSTESEC Alain
Published: March 2005

Incorporation of N‐acetylglucosamine into endogenous lipid and protein acceptors was investigated on heavy microsomes from rat liver, incubated with UDP‐N‐acetyl[14C]glucosamine and GDP‐mannose in the absence of detergent. This subcellular preparation derived for 95% or more from the rough endoplasmic reticulum and was devoid of Golgi components which contain the enzyme that adds the peripheral N‐acetylglucosamine units to glycoproteins.

The label was found almost exclusively in dolichyl diphosphate N‐acetylglucosamine, except when the subcellular preparation was treated with pyrophosphate and subsequently incubated with the nucleotide sugars in the presence of GTP. Then, the incorporation of N‐acetylglucosamine was considerably enhanced, and the additional label was associated with dolichyl diphosphate N,N′‐diacetylchitobiose, with dolichyl diphosphate oligosaccharides and with proteins. The time‐course of N‐acetylglucosamine incorporation in these products was compatible with the pathway of dolichyl diphosphate glycoconjugates for the biosynthesis of the core portion of saccharide chains linked to asparagine residues of glycoproteins. The addition of GDP‐mannose to the incubation medium was required to produce labeled dolichyl diphosphate oligosaccharides, but not to incorporate N‐acetylglucosamine in protein.

It is concluded that rough microsomes are capable of assembling dolichol‐linked oligosaccharides from exogenous nucleotide precursors and of transferring N,N′‐diacetylchitobiose, or its mannosylated derivatives, from the lipid intermediate to endogenous proteins. However, these metabolic activities are hindered in the original subcellular preparation, and in the absence of GTP. Although the earliest perceptible effect produced jointly by the treatment with pyrophosphate and by GTP was the synthesis of dolichyl diphosphate N,N′‐diacetylchitobiose, the primary action of these factors remains uncertain. They may stimulate directly the reaction forming dolichyl diphosphate N,N′‐diacetylchitobiose from dolichyl diphosphate N‐acetylglucosamine, or activate the synthesis of this latter intermediate from a particular pool of dolichyl monophosphate which is readily converted afterwards into disaccharide and oligosaccharide derivatives and glycosylates protein. The requirement for GTP might have a functional meaning, for GTP acted maximally at a concentration distinctly lower than its actual concentration in liver. The detachment of ribosomes from rough vesicles was the major alteration induced by treatment with pyrophosphate. It is suggested that the removal of ribosomes unmasks the membrane sites where GTP acts.
Methionyl‐tRNA Synthetase from Escherichia coli
Authors: FAYAT Guy , HOUNTONDJI Codjo , BLANQUET Sylvain
Published: March 2005

Both the aminoacylation and isotopic ATP‐PPi exchange activities of native and trypsin‐modified methionyl–tRNA synthetases from Escherichia coli are specifically inactivated by incubation in the presence of periodate‐treated initiator tRNAMet.

The inactivation proceeds through the formation of a reversible Schiff's base between the ɛ‐amino group of a lysine within the catalytic center of the enzyme and the 2′,3′‐aldehyde groups created at the 3′‐terminal ribose of tRNA. The Schiff's base may be stabilized by reduction with sodium borohydride.

Intact tRNAfMet competes with the inactivation by its dialdehyde. It has been verified in the case of the modified enzyme that the protection is afforded according to an equilibrium constant identical to that for tRNAfMet binding at the active site of the enzyme.

Finally it is shown that the incorporation of one molecule of the dialdehyde of [14C]tRNA completely destroys the activity of the monomeric trypsin‐modified methionyl‐tRNA synthetase.
Kinetics of the Iodination and the Coupling Reaction in Thyroglobulin Catalyzed by Lactoperoxidase and Chloramine T
Authors: LAMAS Luis
Published: March 2005

The kinetics of the iodination reaction and the coupling reaction in thyroglobulin catalyzed by lactoperoxidase and chloramine T have been compared in order to elucidate whether or not the enzyme is necessary for these reactions once the active iodine has been generated by the peroxidase system.

In the presence of added iodide, lactoperoxidase and chloramine T catalyze both reactions. Lactoperoxidase does so in a gradual fashion, while chloramine T acts almost instantaneously. Chloramine T is more rapid but less efficient than lactoperoxidase in both iodotyrosine and iodothyronine formation. At short time intervals the difference is very small, but it becomes larger at longer reaction time intervals.

We were able to study the coupling reaction independently of the iodination reaction by using chemically prelabeled thyroglobulin in the absence of added iodide. Lactoperoxidase catalyzes the reaction much more efficiently than chloramine T. Chemically prelabeled thyroglobulin releases iodide during incubation in the presence of the peroxidase system, but not in the presence of chloramine T. Thus iodide might play a role in the coupling reaction as already suggested by Dème et al. [Eur. J. Biochem. 70, 435–440 (1976)] for thyroid peroxidase‐catalyzed iodination of thyroglobulin.

From the present results it appears that peroxidase is a more potent catalytic agent than chloramine T for both the iodination and the coupling reactions. This suggests that the enzyme is involved in both reactions beyond simply providing the active form of iodine, though further work is needed to clarify the mechanisms involved.
Calf Spleen NAD Glycohydrolase
Authors: TRAVO Pierre , MULLER Hélène , SCHUBER Francis
Published: March 2005

The catalytic properties of membrane‐bound calf spleen NAD glycohydrolase were studied in comparison with previous data obtained with a solubilized hydrosoluble form of the enzyme. When the hydrolysis of NAD catalyzed by membrane‐bound NAD glycohydrolase was studied at pH values below 7.5, only insignificant interference by other NAD‐hydrolyzing enzymes was detected, and no proton‐diffusional inhibition was observed. The kinetics could, therefore, be followed using a titrimetric assay for NAD glycohydrolase activity.

The effect of pH, ionic strength on the kinetic parameters, and shifts in binding constants for several ligands of the membrane‐bound enzyme indicate that the NAD glycohydrolase activity is influenced by an electrostatic potential due to negative charges (polyelectrolyte effect). No significant changes in kinetic mechanism could be found between both NAD glycohydrolase forms. The association of the enzyme with the membrane results in a remarkably increased thermal stability, in changes in binding properties of the active site and in the emergence of new inhibitor binding sites; e.g. adenosine 3′:5′‐monophosphate (cyclic AMP) and adenosine, which do not inhibit the hydrosoluble form of NAD glycohydrolase, are good inhibitors (respectively competitive and mixed) of the membrane‐bound enzyme. These data (i.e. allotopic changes) probably can be ascribed to enzyme conformational changes induced and stabilized by interaction with membrane constituents.
Computer Simulation of the Fructose Bisphosphatase/Phosphofructokinase Couple in Rat Liver
Authors: GARFINKEL Lillian , KOHN Michael C. , GARFINKEL David
Published: March 2005

Recycling of fructose 6‐phosphate and fructose 1,6‐bisphosphate in the rat liver under gluconeogenic and glycolytic conditions was investigated with a computer model containing representations of the kinetic properties of phosphofructokinase and fructose 1,6‐bisphosphatase under realistic physiological conditions. The two enzyme submodels were constructed from data for the isolated enzymes in vitro by formal optimization. Tissue metabolite concentrations were corrected for cytosolic/mitochondrial compartmentation and effects of chelation and protonation equilibria. This model, which mostly considers the behavior of livers from starved rats, predicts negligible recycling under physiologically realistic conditions. Metabolic regulation of fructose 6‐phosphate, the magnesium ion concentration and the distribution of adenine nucleotides appear to prevent operation of a ‘futile cycle’in vivo. Rate‐limiting chemical species were identified by sensitivity analysis.
The Causes of Sharply Bent or Discontinuous Arrhenius Plots for Enzyme‐Catalysed Reactions
Authors: LONDESBOROUGH John
Published: March 2005

Two models are proposed to account for the abrupt changes in the enthalpy of activation (ΔH*) that are sometimes observed, without corresponding abrupt changes in the maximum velocity (V), at apparent transition temperatures in the Arrhenius plots of enzyme‐catalyzed reactions. Both models predict that a small but observable discontinuity in V will usually occur, which distinguishes such plots from the sharply bent but continuous plots that result when the individual rate constants contributing to V have widely different temperature dependencies. One model applies to membrane‐bound enzymes only, and predicts that ΔH* will always be smaller above the transition. The other applies to both soluble and membrane‐bound enzymes, and can account for either an increase or a decrease in ΔH* as the temperature is raised.
Analysis of the Sea‐Urchin Genome by Homologous RNA Polymerase II Binding
Authors: Di MAURO Ernesto , PEDONE Francesco , BALLARIO Paola
Published: March 2005

We observe that Paracentrotus lividus RNA polymerase II can form stable complexes with homologous DNA at sites from which it can start RNA synthesis in vitro. The sea‐urchin genome can therefore be described in terms of the distribution of the DNA sites that form such stable complexes with the homologous RNA polymerase II [class A sites as defined in a prokaryotic homologous system by D. Hinkle and M. J. Chamberlin (1972) J. Mol. Biol. 70, 157‐185].

We describe the properties of the complexes that P. lividus RNA polymerase II forms with P. lividus DNA: resistance to heparin, dissociation kinetics, temperature and time dependence of their formation, range of RNA polymerase/DNA weight/weight ratios that give rise to the stable binding events.

The amount and the distibution of the sites that form stable complexes with P. lividus RNA polymerase II were determined by the analysis of the dissociation of the complexes formed by the enzyme on DNA fragments of various lengths.

P. lividus appears to form 4.5 × 104 stable (t1/2≥ 30 min) complexes/haploid genome. A part of these complexes has a distribution corresponding to values between 2000 and 4500 base pairs; the rest of the complexes is more widely spaced (up to a measured interval of 30000 base pairs).
Structural Studies on Yeast 3‐Phosphoglycerate Kinase
Authors: DESVAGES Gisèle , ROUSTAN Claude , FATTOUM Abdellatif , PRADEL Louise‐Anne
Published: March 2005

3‐Phosphoglycerate kinase is inactivated by 1‐cyclohexyl‐3‐(2‐morpholinoethyl)carbodiimide metho‐p,‐toluenesulfonate and nitrotyrosine ethyl ester. The coupling of 1 mol nitrotyrosine/mol enzyme is sufficient to inactivate the protein completely. A weak protection against inactivation is observed with each substrate added separately. In contrast, the complex ATP – 3‐phosphoglycerate‐enzyme or ATP–Mg–3‐phosphoglycerate–enzyme affords a considerable protection. The critical residue is identified as a glutamyl residue after isolation by immuno‐affinity chromatography of nitrotyrosyl peptide resulting from exhaustive proteolytic digestion of the modified protein. In addition, the determination of the primary sequence of the C‐terminal part of the protein leads to the location of the glutamyl residue at position eight from the C‐terminus. We conclude that this glutamyl residue is situated in the domain which does not bind the nucleotide substrates [Bryant, T. N., Watson, H. C. and Wendell, P. L. (1974) Nature (Lond.) 247, 14‐ 17]. Its role in the catalysis process is discussed.
Incorporation of the High‐Molecular‐Weight Microtubule‐Associated Protein 2 (MAP2) into Microtubules at Steady State in vitro
Authors: MANSO‐MARTINEZ Rafael , VILLASANTE Alfredo , AVILA Jesus
Published: March 2005

Purified high‐molecular‐weight microtubule‐associated protein 2 (MAP2) labelled with [32P]‐phosphate has been obtained and used as a marker to study the incorporation of this protein into micfotubules at steady state in vitro. The incorporation kinetics of protein MAP2 show two different mechanisms of addition of this protein into microtubules. A fast incorporation, which proceeds essentially independently of microtubule assembly, indicates its addition into binding positions which may be available along the structure. Once the microtubule has been saturated with the protein, its incorporation proceeds from one end of the microtubule, in a polymerization‐dependent manner, at a slower rate. Loss of the protein from microtubules at steady state proceeds fundamentally at the opposite end.
Purification of Two DD‐Carboxypeptidases/Transpeptidases with Different Penicillin Sensitivities from Proteus mirabilis
Authors: SCHILF Wolfgang , MARTIN Hans Herbert
Published: March 2005

Two membrane‐bound enzymes of Proteus mirabilis with the dual functions of peptidoglycan DD‐carboxypeptidase and transpeptidase (named DD‐carboxypeptidase/transpeptidase H and L) were isolated and purified by selective solubilization with the nonionic detergent Genapol X‐100, affinity chromatography on matrix‐bound ampicillin, and preparative isoelectric focusing in the presence of detergent.

Purified enzymes H and L were, respectively, penicillin‐binding proteins 4 and 5 among seven major penicillin‐binding proteins present in P. mirabilis.

The enzymes differed in the following properties. Enzyme H had an Mr of 49000; isoelectrie point at pH 8.1; high sensitivity to benzylpenicillin and permanent inactivation because of high stability of the enzyme‐antibiotic complex El* (half‐life 300 min); fragmentation of benzylpenicillin with formation of phenylacetylglycine during the slow decay of El*; it functioned as an endopeptidase on peptide‐crosslinked side chains of peptidoglycan. Enzyme L had an Mr of 43000; isoelectrie point at pH 5.9; low sensitivity to benzylpenicillin and low stability of El* (half life 7.2 min) with rapid recovery of enzyme activity; no function as an endopeptidase.

The properties of enzyme L were identical with those of the single active DD‐carboxypeptidase found previously in the spheroplast L‐form of P. mirabilis grown in the presence of benzylpenicillin. We conclude that the partial penicillin resistance of P. mirabilis, with growth as L‐form and synthesis of peptide‐crosslinked peptidoglycan, depends on the continuing function of enzyme L as a DD‐carboxypeptidase and transpeptidase in the presence of the antibiotic.
Bovine Kidney Pyruvate Dehydrogenase Complex
Authors: KRESZE Georg‐B. , RONFT Hedwig , DIETL Brigitte
Published: March 2005

Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated rapidly by papain. However, none of the component activities of the complex is destroyed during inactivation of the overall reaction.

The core component, lipoate acetyltransferase, is cleaved by papain to give principal fragments with Mr 26500 and 26000 (as determined by dodecylsulfate gel electrophoresis). Much more slowly, the α chain of the pyruvate dehydrogenase component is attacked.

Fragmented lipoate acetyltransferase retains its complete enzymatic activity and remains of high molecular weight. It is unable, however, to bind the other component enzymes, pyruvate dehydrogenase and lipoamide dehydrogenase. Therefore, the multienzyme complex is disassembled when treated with papain.

A method is described which allows the rapid and convenient isolation of nicked lipoate acetyltransferase as well as unfragmented pyruvate dehydrogenase and lipoamide dehydrogenase from papain‐treated complex under very mild conditions. The two uncleaved component enzymes have identical properties and similar specific activities as enzyme preparations obtained by other, more laborious procedures.
Degradation of Basement‐Membrane Collagen by Neutral Proteases from Human Leukocytes
Authors: UITTO Veli‐Jukka , SCHWARTZ David , VEIS Arthur
Published: March 2005

A neutral extract from human leukocytes was shown to have proteolytic activity which could degrade triple‐helical basement membrane collagen from bovine lens capsules into specific triple‐helical sub‐fragments. The enzyme responsible is not identical to the leukocyte collagenase active against interstitial collagen types I, II and III.

After denaturation, the neutral protease reaction products showed three major peptides with molecular weights of 70000, 50000 and 30000, by dodecylsulphate gel electrophoresis analysis. Electron microscopic observation and viscosity measurements showed the initial twice‐pepsinized collagen to be comprised of intact rod‐like molecules about 300 nm long. The fragments produced by the protease were also triple‐helical and were resistant to the action of trypsin at 20°C. The fragments were completely degraded by purified bacterial collagenase or by raising the reaction temperature above the melting temperature of the basement membrane collagen during incubation with leukocyte extract.

Enzyme activity has its pH optimum between 7 and 9, EDTA, EGTA and 1, 10‐phenanthroline (metal‐chelating agents) completely inhibited enzyme activity, as did serum. Partial inhibition of the activity was obtained with phenylmethylsulfonyl fluoride and soybean trypsin inhibitor.
Effect of ATPase Inhibitors on the Proton Pump of Respiratory‐Deficient Yeast
Authors: SERRANO Ramón
Published: March 2005

Diethylstilbestrol and dicyclohexylcarbodiimide inhibit the ATPase of the plasma membranes and the proton‐pumping activity of the cells in a respiratory‐deficient mutant of Saccharomyces cerevisiae. The effects of the inhibitors in vivo seem to be specific because neither the proton permeability nor the ATP levels of the cells are affected. These results indicate that the yeast plasma‐membrane ATPase corresponds to the proton pump of the cells. The fact that both inhibitors of the ATPase delay the fall of ATP levels which follows a block of fermentation indicates that ATPase function is one of the major ATP‐consuming pathways in yeast. In addition, diethylstilbestrol prevents the fall of ATP levels produced by dinitrophenol, suggesting that this fall was caused by partial dissipation of the proton gradient and consequent stimulation of the proton‐pumping ATPase.
On the Number of Steroid‐Binding Sites of Δ5‐3‐Oxosteroid Isomerase
Authors: PENNING Trevor M. , WESTBROOK Edwin M. , TALALAY Paul
Published: March 2005

The number of steroid‐binding sites in Δ5‐3‐oxosteroid isomerase of Pseudomonas testosteroni (EC 5.3.3.1) has been determined from measurements of the red shift of the ultraviolet chromo‐phore of 19‐nortestosterone upon binding to the enzyme. The experiments include spectroscopic measurements when limiting concentrations of either 19‐nortestosterone or isomerase are titrated with varying concentrations of the complementary ligand. Analysis of the results indicates one binding site per subunit (Mr 13394). Scatchard plots indicate a single family of equivalent binding sites. 5,10‐Secoestr‐5‐yne‐3,10,17‐trione is a suicide substrate of isomerase [Batzold & Robinson (1975) J. Am. Chem. Soc. 97, 2576]. The time course for inactivation of isomerase with an excess of 5,10‐seco[7‐3H]estr‐5‐yne‐3,10,17‐trione was parallel to the covalent incorporation of steroid and gave a final stoichiometry of nearly one steroid molecule per subunit of enzyme. Alkylation of [14C]iso‐merase with excess of this 3H‐labeled steroid followed by gel‐filtration and dialysis gave an inactivated enzyme with a 3H/14C ratio that corresponds to one molecule of steroid bound per subunit; this stoichiometry was constant over a wide range of protein concentrations (0.1 − 10 mg/ml).

Diffusion of [3H]progesterone into hexagonal crystals of isomerase showed that at saturation one steroid molecule was bound per protomer. Taken together these findings strongly support the conclusion that one molecule of steroid is bound per subunit of isomerase both in solution and in the crystal state.
Deoxyribonucleic Acid Methyltransferase from the Eukaryote, Chlamydomonas reinhardi
Authors: SANO Hiroshi , SAGER Ruth
Published: March 2005

DNA methyltransferase was purified 310‐fold from a green alga, Chlamydomonas reinhardi vegetative cells. The native enzyme of molecular weight 55000‐58000 catalyzed the transfer of methyl groups from S,‐adenosylmethionine to the 5 position of cytosine in DNA. Native DNA accepted methyl groups 10‐fold more than did denatured DNA. The sequence specificity analysis of methylated deoxycytidine in vitro revealed that the enzyme introduces methyl groups preferentially into sequences containing 5'd(T‐mC‐R)3′. Kinetic analysis of the reaction indicated that the enzyme obeys a random sequential mechanism. The extent of saturation with methyl groups depends upon the species from which the DNA was obtained. Kinetic analysis of the reaction catalyzed by RNA polymerase II has indicated that DNA methylation decreases the rate of initiation of RNA synthesis, but does not affect the rate of RNA chain elongation.
The Enhancement of Cytotoxicity of N‐Methyl‐N‐nitrosourea and of y‐Radiation by Inhibitors of Poly(ADP‐ribose) Polymerase
Authors: NDUKA Nsirim , SKIDMORE Christopher J. , SHALL Sydney
Published: March 2005

Inhibitors of poly(ADP‐ribose) polymerase show a synergistic potentiation of cytotoxicity with certain DNA‐damaging agents. Non‐toxic concentrations of 5‐methylnicotinamide dramatically potentiate the cytotoxicity of N‐methyl‐N‐nitrosourea as tested by the cloning ability of mouse leukaemia (LI210) cells. A dose‐enhancement factor of about 10 is observed. This potentiation is dependent on the concentration of 5‐methylnicotinamide. The methylxanthines theobromine, theophlline and caffeine also increase the cytotoxicity of methylnitrosourea. Thymidine, in the presence of sufficient deoxycytidine to overcome the perturbation of deoxynucleotide metabolism, also potentiates the cytotoxicity of methylnitrosourea. Nicotinate, which is not an inhibitor of poly‐(ADP‐ribose) polymerase, has no effect on methylnitrosourea toxicity.

A very small, but consistent, enhancement of the toxicity of y‐radiation by the same inhibitors has been observed.

We suggest that this potentiation of cytotoxicity is mediated by inhibition of (ADP‐ribose)n biosynthesis; and that the biosynthesis is stimulated by DNA damage. We therefore propose that (ADP‐ribose)n takes part in cellular repair mechanisms, either by modifying chromatin structure or by a specific participation in DNA repair.
Formation and Dissociation of the Covalent Complexes Between Trypsin and Two Homologous Inhibitors, α1‐Antitrypsin and Antithrombin III
Authors: MAHONEY Walter C. , KURACHI Kotoku , HERMODSON Mark A.
Published: March 2005

The mechanisms by which α1‐antitypsin and antithrombin III inhibit trypsin were investigated by chemical stability studies and amino acid sequence analyses of the enzyme‐inhibitor complexes. One‐to‐one covalently linked complexes were formed between each of the inhibitors and trypsin. The complexes were stable over the course of the experiments at pH 8 or lower, and benzamidine, hydroxylamine, thiols, guanidine, or dodecylsulfate had no apparent effect on the stability of the complexes. The complexes dissociated slowly at pH 9 or greater. Neither of the inhibitors was active after dissociation from its complex with trypsin. Sequence analysis indicated that no new aminoterminus was generated when α1‐antitrypsin formed its complex with trypsin, but that two new amino termini were formed in approximately one‐to‐one ratio when the complex dissociated. This may indicate that α1‐antitrypsin contains two inhibitory sites for trypsin in close spatial proximity on the molecule.
Biosynthesis of Vitamin B12
Authors: HÖRIG Joachim A. , RENZ Paul
Published: March 2005

Homogenates of Propionibacterium freudenreichii transform riboflavin into 5,6‐dimethyl‐benzimidazole. This process is stimulated by nicotinamide. Homogenates of Propionibacterium shermanii form only small amounts of 5,6‐dimethylbenzimidazole from riboflavin in the absence of nicotinamide, but also form appreciable amounts in the presence of nicotinamide. 2. The stimulation of the 5,6‐dimethylbenzimidazole‐forming system by nicotinamide shows a lag phase which is abolished by preincubation of the homogenate with nicotinamide. Since no lag phase is observed when nicotinamide is replaced by nicotinate, nicotinate seems to be the true stimulating agent. These observations are in agreement with the fact that nicotinamide is rapidly split to nicotinate in homogenates of P. freudenreichii. 3. The 5,6‐dimethylbenzimidazole‐forming homogenate system is only active at a high buffer concentration (0.3‐0.5 M) and in the presence of oxygen. The system has a pronounced oxygen optimum. 4. Flavin mononucleotide and flavin‐adenine dinucleotide are better substrates for the 5,6‐dimethylbenzimidazole‐forming homogenate system than riboflavin. But with [1′ ‐14 C] riboflavin as substrate the specific radioactivity of 5,6‐dimethylbenzimidazole is higher than the specific radioactivity of flavin ‐ adenine dinucleotide and lower than the specific radioactivity of flavin mono‐nucleotide. Therefore flavin mononucleotide is the immediate substrate for the formation of 5,6‐dimethylbenzimidazole. 5. A tentative reaction sequence for the transformation of flavin mononucleotide into 5,6‐dimethylbenzimidazole is discussed.
Interactions between Ellipticine and Phospholipids. Effect of Ellipticine and 9‐Methoxyellipticine on the Phase Behaviour of Phosphatidylglycerols
Authors: EL MASHAK El‐Sayed Mostafa , TOCANNE Jean‐Francois
Published: March 2005

Ellipticine and some of its derivatives constitute an interesting class of substances displaying an antitumoral activity against both experimental and human tumors. We have recently shown that some of these molecules were able to interact rapidly and strongly with acidic phospholipids. In the present report, amphiphilic ellipticine and 9‐methoxyellipticine were tested against synthetic dilauroyl phosphatidylglycerol and dipalmitoylphosphatidylglycerol as well as a sample of phosphatidyl‐glycerol with methyl‐branched fatty acids, to investigate the stoichiometry of these interactions and their consequences on the organization of the lipid phase. Through surface pressure and surface potential measurements, carried out at the air‐water interface, it is shown that the two drugs strongly interact with phosphatidylglycerols, whatever the film surface pressure or the nature and physical state of the lipid acyl chains may be. Such interactions bring about large film expansions and a complete modification of the phase properties of the lipids. These interactions achieve, at saturation, drug/lipid associations near the 1/1 molar ratio, stabilized by both electrostatic and hydrophobic forces. The drug molecules can be viewed as inserted between the lipid molecules, the—NH+group interacting with the phosphate function and the aromatic moiety being in contact with the lipid acyl chains. From compression isotherms of 1/1 drug/lipid mixtures, it can be inferred that the two drugs cause a large condensing effect on both the dilauroyl and dipalmitoyl derivatives of phosphatidyl‐glycerol, as compared to the behaviour of the lipids alone when ionized. In the mixed films, both lipids would exist in a very similar and rather condensed state, intermediate between the liquid‐expanded and gel states in which these two lipids can respectively exist on pure water. The possibility of membranes being one of the sites of action of ellipticines is discussed.
High Variability of the Electron Carrier Plastocyanin in Microalgae
Authors: BOHNER Herbert , MERKLE Hellmut , KRONECK Peter , BÖGER Peter
Published: March 2005

The plastocyanin content of Scenedesmus can be dramatically varied with the copper content of the culture medium. Figures as high as 7 mmol plastocyanin/mol chlorophyll are possible. Electron paramagnetic resonance (EPR) spectroscopy has been used to determine this physiological response quantitatively in intact cells having different amounts of plastocyanin. The results obtained by the EPR technique were compared with data on isolated plastocyanin determined either by spectro‐photometry or immunoelectrophoresis. Agreement was found for the amount of plastocyanin detected by the first two methods, whereas the last assay yielded data at least 25% higher on the average. Under all culture conditions a copper‐free plastocyanin precursor is present.

The EPR properties of purified plastocyanin and those of cellular plastocyanin located within the thylakoids are practically identical in terms of g‐values, hyperfine splittings, signal linewidths and saturation behavior at temperatures of 12‐15 K. Our data indicate that plastocyanin is not present in a membrane‐bound form but exists as a single soluble pool.

The studies have been extended to the algae Dunaliella parva and Bumilleriopsis filiformis. The first species exhibits a limited variation of plastocyanin with the copper content of the medium. Furthermore, no cytochrome c‐553 could be detected in Dunaliella even under conditions of copper deficiency. In contrast, Bumilleriopsis does not contain plastocyanin regardiess of the amount of copper offered.
Fragmentation of Crystalline β‐Haemocyanin of Helix pomatia with Plasmin and Trypsin. Location of the Fragments in the Polypeptide Chain
Authors: GIELENS Constant , VERSCHUEREN Laurent J. , PRÉAUX Gisèle , LONTIE René
Published: March 2005

The action of plasmin on tenth molecules of βC‐haemocyanin of Helix pomatia at pH 8.2 yielde first a three‐domain fragment P3 and a five‐domain fragment P1 (present as a dimer at pH 8.2 Fragment P1 was further split by plasmin into a four‐domain fragment P2 and a one‐domai fragment P4 (also present as a dimer at pH 8.2). Trypsinolysis of P2 yielded T2 and fragment X, which was further split into T1C and T3.

Fragments P3 and P4 corresponded respectively to the tryptic fragments T1A and T1B, als by their circular dichroic spectra.

The determination of the N‐terminal groups and the order of splitting allowed the location of the fragments in the polypeptide chain: P3 (a–c), P2 (d–g), P4 (h); T1A (a–c), T3 (d), TIC (e–f) T2 (g), T1B (h).
Characterisation of Cucurbitin from Various Species of the Cucurbitaceae
Authors: BLAGROVE Robert J. , LILLEY Glenn G.
Published: March 2005

There have been many studies of cucurbitin, the major seed storage globulin which can be isolated in crystalline form from several genera of cucurbits. However, recent literature reports have suggested a wide range of molecular weights for oligomeric cucurbitin at neutral pH in buffered aqueous salt solutions. We have measured the molecular weight of oligomeric cucurbitin isolated from mature pumpkin seeds by several methods in the analytical ultracentrifuge. The molecular weights of the major and minor components present are 325000 and 630000 respectively. In combination with dodecylsulphate gel results, these values show that cucurbitin is a hexamer composed of subunits of molecular weight 54000, while most preparations contain several percent of a dodecamer composed of the same subunits. The molecular weight of the major component was 320000 from analytical gel filtration on Sepharose CL‐6B.

Aqueous salt solutions of cucurbitin isolated from mature seeds of marrow, cucumber, rock melon, water melon, and ornamental gourds are also composed of major (hexamer) and minor (dodecamer) components. The major fractions have similar elution volumes following chromatography over Sepharose CL‐6B, sedimentation coefficients in the range 12.7–12.9 S and molecular weights in the range 324000–327000 from meniscus depletion sedimentation equilibrium measurements. Although there is limited size heterogeneity between the cucurbitin subunits, there is considerable charge heterogeneity. This has been demonstrated by electrophoresis on cellulose acetate membranes in 8 M urea at pH 8.5 with and without reduction of disulphide bonds. The existence of two classes of subunit polypeptide (acidic Mr∼ 33000 and basic Mr∼ 22000), comparable to those for glycinin (soybean) or legumin (pea, broad bean), has been shown by two‐dimensional electrophoresis.
Hemoglobins of an Amphibia, the Neotenous Ambystoma mexicanum
Authors: BOISSEL Jean‐Paul , WAJCMAN Henri , LABIE Dominique
Published: March 2005

The primary structure of axolotl (neotenous Ambystoma mexicanum) α chain has been determined. NH2‐terminal sequence data were performed using the solid‐phase method. Complete amino acid assignments were deduced from the sequences of peptides obtained after cleavage with cyanogen bromide; the methionine‐containing peptides, isolated from α chain tryptic digest, allowing the alignment of these fragments. All overlaps have been clearly established. Axolotl α chain contains 142 residues, one extra phenylalanine residue being located at its N terminus, when compared with mammalian α chains. The amino acid sequences of this polypeptidic chain and of an other urodele, the newt Taricha granulosa, show 44 differences with only 18 non‐isopolar replacements. Homologies between various vertebrate a chains are briefly discussed.
Glycolysis in Trypanosoma brucei
Authors: VISSER Nico , OPPERDOES Fred R.
Published: March 2005

The possibility that the glycosomes present in the bloodstream form of Trypanosoma brucei [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360–364] constitute a separate pool of glycolytic intermediates within the cell was investigated.

In titrations of intact cells with digitonin, a differential activation of glycolytic enzymes was observed. Enolase, pyruvate kinase and the cell‐sap marker alanine aminotransferase were activated at 0.05 mg digitonin per mg protein. The nine glycosomal enzymes involved in the conversion of glucose and glycerol into 3‐phosphoglycerate were activated only at digitonin concentrations between 0.7 and 9.8 mg/mg protein. In subcellular fractions the activities of the latter enzymes were all latent between 70 and 92%. Latency was abolished by addition of 0.1% Triton X‐100 or partly by five cycles of freezing and thawing. We conclude that the glycosomal enzymes are surrounded by a membrane, which forms a permeability barrier to intermediates and co‐factors of glycolysis.

The concentrations of glycolytic intermediates and of adenine nucleotides were measured under aerobic conditions as well as in the presence of 1 mM salicylhydroxamic acid, a respiratory inhibitor. Addition of salicylhydroxamic acid caused the following changes: (a) The levels of almost all glycolytic intermediates measured decreased. Glycerol‐3‐phosphate, however, increased fourfold, (b) The phosphate potential was drastically lowered from 2900 to 450 M −1. (c) The trypanosomes became more reduced, as monitored by a change in the apparent redox state of the NADH/NAD+ couple from E′h= ‐189 to E′h=–219 mV.

From the high levels of metabolite concentrations found and from comparison of the apparent mass‐action ratios calculated for the separate glycolytic reactions with those for other organisms, we conclude that in bloodstream form T. brucei the glycolytic intermediates are present in the glycosomes as well as in the cytosol and that the two pools of intermediates equilibrate with each other, despite the presence of the glycosomal membrane.
Induction of Choline Transport and Its Role in the Stimulation of the Incorporation of Choline into Phosphatidylcholine by Polyamines in a Polyamine Auxotroph of Saccharomyces cerevisiae
Authors: HOSAKA Kohei , YAMASHITA Satoshi
Published: March 2005

A mutant of Saccharomyces cerevisiae, defective in ornithine decarboxylase, was isolated. A prolonged culture of the mutant in a polyamine‐free medium resulted in a great decrease in the polyamine content and in cessation of growth. The addition of polyamines to the culture induced the growth after a lag period of 5–6.5 h. The growth rate in the presence of polyamine was comparable to that of the wild‐type strain. The effectiveness of polyamines was as follows: spermidine > putrescine ∼ spermine.

Phosphatidylcholine‐synthesizing activity during the lag phase of growth was determined by measuring the rate of incorporation of [14C]choline into phosphatidylcholine. The incorporation rate was markedly increased with time by polyamine prior to the initiation of cell division. Polyamines were effective in the following order: spermidine > putrescine ∼ spermine. Experiments whit methylgiyoxal bis(guanylhydrazone), an inhibitor of S‐adenosylmethionine decarboxylase, showed that putrescine stimulates cell growth and choline incorporation into phosphatidylcholine after it has been converted into spermidine in the cell.

The induction of the choline transport system was shown to be responsible for the increase in the rate of incorporation of [14C]choline into phosphatidylcholine effected by polyamines. A low concentration of cycloheximide completely prevented the induction of choline transport by polyamines. The levels of the CDP‐choline pathway enzymes such as choline kinase, cholinephosphate cytidyltransferase and cholinephosphotransferase were not significantly changed.
Isomers in Thioredoxins of Spinach Chloroplasts
Authors: SCHÜRMANN Peter , MAEDA Kayo , TSUGITA Akira
Published: March 2005

We have developed a method for the concomitant purification of several components of the ferredoxin/thioredoxin system of spinach chloroplasts. By applying this method to spinach‐leaf extract or spinach‐chloroplast extract we separated and purified three thioredoxins indigenous to chloroplasts. The three thioredoxins, when reduced, will activate certain chloroplast enzymes such as fructose‐1,6‐bisphosphatase and NADP‐dependent malate dehydrogenase. Fructose‐ 1,6‐bisphosphatase is activated by thioredoxin f exclusively. Malate dehydrogenase is activated by thioredoxin mb and thioredoxin mb in a similar way, and it is also activated by thioredoxin f but with different kinetics.

All three thioredoxins have very similar relative molecular masses of about 12000 but distinct isoelectric points of 6.1 (thioredoxin f), 5.2 (thioredoxin mb) and 5.0 (thioredoxin mc). The amino acid composition as well as the C‐terminal and N‐terminal sequences have been determined for each thioredoxin. Thioredoxin f exhibits clear differences in amino acid composition and terminal sequences when compared with the m‐type thioredoxins. Thioredoxin mb and thioredoxin mc, however, are very similar, the only difference being an additional lysine residue at the N‐terminus of thioredoxin mb.

Amino acid analyses, terminal sequences, immunological tests and the activation properties of the thioredoxins support our conclusion that thioredoxins mb and me are N‐terminal redundant isomers coming from one gene whereas thioredoxin f is a different protein coded by a different gene.
Properties of a Ca2+‐Activated Protease Specific for the Intermediate‐Sized Filament Protein Vimentin in Ehrlich‐Ascites‐Tumour Cells
Authors: NELSON W. James , TRAUB Peter
Published: March 2005

A Ca2+‐activated neutral protease is described which, when tested against various native proteins, appears to be specific for vimentin, the 58000‐M, subunit protein of intermediate‐sized (7–11 nm) filaments in Ehrlich‐ascites‐tumour cells. The protein subunits of other classes of intermediate‐sized filaments have been tested; neurofilament protein and glial fibrillary acidic protein are not degraded, however skeletin, the subunit protein of intermediate‐sized filaments in smooth muscle, is degraded. The protease is found associated with the detergent resistant cytoskeleton of Ehrlich‐ascites‐tumour cells: proteins, other than vimentin, present in this structure are not degraded. The protease is activated by Ca2+ and Sr2+ but not by other divalent cations tested: the Ca2+ concentration required for activation is 10 μM. The pH optimum is between pH 7.5 and 8.0, and the KCl concentration required for optimal activity is 100 mM. The protease is inhibited by 1‐chloro‐3‐tosylamido‐7‐amino‐l‐2‐heptanone hydrochloride and l‐1‐tosylamido‐2‐phenylethyl chloromethyl ketone but not by soybean trypsin inhibitor; inhibition by phenylmethylsulphonyl fluoride is moderate. The high substrate specificity of the protease suggests it may play a role in vimentin intermediate‐sized filament protein turnover in Ehrlich‐ascites tumour cells.
The Influence of Elongation‐Factor‐Tu · GTP and Anticodon‐Anticodon Interactions on the Anticodon Loop Conformation of Yeast tRNATyr
Authors: WEYGAND‐DURASEVIC Ivana , KRUSE Torben A. , CLARK Brian F. C.
Published: March 2005

The interactions of yeast tRNATyr, spin‐labelled at position i6A‐37 next to the anticodon, with EF‐Tu · GTP and with Escherichia coli tRNAVal (which has a complementary anticodon) have been studied. The immobilization of the spin label upon ternary complex formation shows a conformational change of the anticodon region, although this part of tRNATyr is not in direct contact with the protein, as indicated by RNase T1 digestion. Upon anticodon‐anticodon interaction, no conformational change of the anticodon loop of tRNATyr was observed.
Molecular Characteristics of a Non‐deletion α‐Thalassaemia of the Po River Delta
Authors: del SENNO Laura , BERNARDI Francesco , BUZZONI Daniele , MARCHETTI Giovanna , PERROTTA Carla , CONCONI Francesco
Published: March 2005

The form of α‐thalassaemia of the Po river delta presents haematological and globin biosynthetic characteristics similar to α‐thalassaemia‐1 but never gives rise to Hb H disease nor to hydrops foetalis. In α‐thalassaemic subjects originally from this region globin mRNA translation and α‐globin gene arrangement have been investigated.

The data obtained indicate that α‐globin synthesis and reticulocyte α‐globin mRNA are reduced by one fourth; in addition, since the defect in α‐globin synthesis does not change with cell ageing, a possible instability of α‐globin mRNA is excluded. Restriction enzyme analysis of the DNA shows a normal α‐globin gene organization. This form of α‐thalassaemia is therefore of the non‐deletion type; its molecular lesion is either at the level of α‐globin mRNA transcription or processing. The fact that this, as well as other forms of non‐4eletion α‐thalassaemia, have a phenotipic expression similar to α‐thalassaemia 1 is discussed.
Binding of Sea‐Urchin RNA Polymerase II on Homologous Histone Genes
Authors: BUONGIORNO‐NARDELLI Mario , BALLARIO Paola , DI MAURO Ernesto
Published: March 2005

The binding of sea‐urchin RNA polymerase II to homologous cloned histone genes (h22) has been studied using the electron microscope. Stable binary complexes selectively form at the H4‐H2B (pre‐H213) spacer region in an apparently cooperative manner.
Spectroscopic and Photoreduction Studies of Copper Chromophores in Ceruloplasmin
Authors: HERVÉ Martine , GARNIER Arlette , TOSI Lucia , STEINBUCH Marion
Published: March 2005

Photoreduction of native ceruloplasmin, using the 454.5‐nm line of an Ar+ laser, enables the identification of type‐Ia, type‐Ib and type‐II copper. The circular dichroic spectra of N−3‐bound type‐II copper and SCN−‐bound type‐II copper are obtained by the same procedure after anionic treatment of ceruloplasmin. From circular dichroic and resonance Raman evidence it appears that some of type‐Ia and type‐Ib copper ligands differ. Type‐Ib copper ligands seem to the same as type‐I copper in plastocyanin and azurin. Even though type‐Ib copper is coordinated to one sulfur of cysteine and one sulfur of methionine (or disulfide of cystine), the methionine sulfur is not a ligand for type‐Ia copper.
Modification of the Thermodynamic Properties of the Electron‐Transferring Groups in Mitochondrial Succinate Dehydrogenase upon Binding of Succinate
Authors: BONOMI Franco , PAGANI Silvia , CERLETTI Paolo , GIORI Camillo
Published: March 2005

The redox properties of the covalently‐bound flavin and of the tetrahedral iron‐sulfur center S1 of succinate dehydrogenase were studied as a function of the binding of different ligands to the enzyme. The midpoint potential of both flavin and S1 increases by some 200 mV when protein binds succinate to a site having KSuccd= 0.8–1.0 mM, thus different from the substrate binding site. Succinate binding increases the potential of the oxidized flavin/semiquinone half‐cell more than that of the semiquinone/reduced flavin one: this results in higher semiquinone formation with increasing succinate. Malonate and fumarate appear to mimic, in this regard, the effect of succinate.

The increase in midpoint potential of S1 upon binding of dicarboxylic acid is related to an increase in hydrophobicity of the cluster environment.

The possible molecular basis for the modulation of the flavin potential is discussed together with the significance of this shift on the catalytic behaviour of the protein.
Effect of Autonomic Denervation on DNA Synthesis during Liver Regeneration after Partial Hepatectomy
Authors: KATO Hideo , SHIMAZU Takashi
Published: March 2005

The regulatory role of autonomic nerves in liver regeneration after partial hepatectomy was studied in rats by bilateral subdiaphragmatic vagotomy or splanchnicectomy.

In control rats the wet weight of the regenerating liver was restored to approximately 80% of the preoperative weight 72h after partial hepatectomy. Restoration of the liver weight was significantly impaired in vagotomy rats, but not in splanchnicectomy. Increases in the DNA and protein contents of the regenerating liver were also suppressed by vagotomy.

Hepatic DNA synthesis, measured as the incorporation of [methyl‐3H]thymidine into DNA at various times after partial hepatectomy, was significantly less in vagotomized rats, and slightly more in splanchnicectomized rats than in control rats. The onset of DNA synthesis triggered by partial hepatectomy was also delayed by vagotomy.

The increases in activities of hepatic aspartate transcarbamoylase and thymidine kinase, the key enzymes in synthesis of pyrimidine nucleotides via the de novo and salvage pathways respectively, during liver regeneration, were significantly suppressed and retarded in vagotomized rats. Conversely, splanchnicectomy tended to stimulate these enzyme inductions after partial hepatectomy.

During starvation the plasma insulin level decreased after partial hapatectomy in control and vagotomized rats, as in sham‐operated rats, but showed a transient increase 6 h after partial hepatectomy in splanchnicectomized rats.

It is concluded that vagotomy inhibits and delays DNA synthesis and proliferation of liver cells after partial hepatectomy, whereas splanchnicectomy tends to stimulate these processes. The data also suggest that para‐sympathetic innervation of the liver may play an important regulatory role in liver regeneration.
The Complete Primary Structure of the Allosteric l‐Lactate Dehydrogenase from Lactobacillus casei
Authors: HENSEL Reinhard , MAYR Ulrich , YANG Chao‐yuh
Published: March 2005

The polypeptide chain of the allosteric L‐lactate dehydrogenase (EC 1.1.1.27) of Lactobacillus casei consists of 325 amino acid residues. Despite the strikingly different enzymatic characteristics of the allosteric L‐lactate dehydrogenase of L. casei and of the non‐allosteric vertebrate enzymes, the sequence of the allosteric enzyme shows a distinet homology with that of the non‐allosteric vertebrate enzymes (average identity: 37%). An especially high sequence homology can be identified within the active center (average identity: 70%). A clear deviation of the L. casei enzyme from the vertebrate enzyme is the lack of the first 12 amino acid residues at the N terminus and an additional 7 amino acid residues at the C terminus. The localization of the binding site of the allosteric effector D‐fructose 1,6‐bisphosphate and pH and effector‐induced changes of the spectro‐scopic properties are discussed on the basis of the primary structure.