bluuebunny/crossref_metadata_2025_split huggingface dataset:
Die hämolytischen Eigenschaften der membranmodifizierenden Peptidantibiotika Alamethicin, Suzukacillin und Trichotoxin
Authors: Irmscher G. , JUNG Günther 
Published: March 2005

The Hemolytic Properties of the Membrane Modifying Peptide Antibiotics Alamethicin, Suzukacillin and TrichotoxinThe membrane exciting peptide antibiotics alamethicin F‐50, suzukacillin A and B, and trichotoxin A‐40 can be isolated from different strains of the fungus Trichoderma viride. Cultivation, isolation and purification procedures are described in detail for alamethicin F‐50. At micromolar concentrations the peptide antibiotics cause hemolysis in human erythrocytes, while at nanomolar levels they induce the formation of voltage‐dependent pores of multi‐state properties in black lipid membranes.A detailed study of the hemolytic action with variations of concentration, temperature and incubation time revealed characteristic differences in the mode of hemolysis between the peptide antibiotics, which can be correlated with structural differences between these natural analogs. The addition of bovine serum albumin reduces the hemolytic activity. The lytic action increases considerably from hypertonic to hypotonic media. For comparison the bee venom constituent melittin and sodium dodecylsulfate have been investigated under the same conditions. The lipophilic peptide antibiotics constitute a new class of membrane disrupting agents with particular properties not known from other lytic compounds.Assuming that the lytic activity is proportional to the concentration of the peptide antibiotic, the temperature dependence of the concentration/activity curves allows the calculation of activation energies of 62 kJ/mol for alamethicin, 67 kJ/mol for suzukacillin A, and 79 kJ/mol for trichotoxin A‐40. However, melittin revealed the very low activation energy of 49 kJ/mol. These values may be correlated with results from circular dichroism and 13C nuclear‐magnetic‐reonance experiments which indicate conformational changes of the peptides aggregating at the membrane/water interphase.
Immobilization of Yeast Microbodies by Inclusion with Photo‐crosslinkable Resins
Authors: TANAKA Atsuo , YASUHARA Shigeki , OSUMI Masako , FUKUI Saburo 
Published: March 2005

Yeast microbodies containing FAD‐dependent alcohol oxidase, catalase and D‐amino acid oxidase were isolated from methanol‐grown cells of Kloeckera sp. 2201 and immobilized intact in matrices formed by a short‐time illumination of photo‐crosslinkable resin oligomers. The relative activities of catalase, alcohol oxidase and D‐amino acid oxidase of the gel‐entrapped microbodies were 36, 76 and 31% respectively as compared with those of free microbodies.Immobilization enhanced the stability of catalase to a certain degree, but not that of alcohol oxidase. The pH/activity profiles of catalase and alcohol oxidase of the entrapped organelles showed more narrow pH optima than those of the free counterparts. D‐Amino acid oxidase in immobilized microbodies showed a somewhat higher Km value for D‐alanine than that in free ones. Immobilized microbodies oxidized two moles of methanol to form two moles of formaldehyde with consumption of one mole of molecular oxygen. Addition of 3‐amino‐1,2,4‐triazole, an inhibitor of catalase, reduced the formation of formaldehyde to half the amount without change in the amount of oxygen consumed, indicating the synergic action of alcohol oxidase and catalase in methanol oxidation in the microbodies of living yeast cells.
Properties of Human Erythrocyte Catalases after Crosslinking with Bifunctional Reagents Symmetry of the Quaternary Structure
Authors: HAJDU János , WYSS Sonja R. , AEBI Hugo 
Published: March 2005

Normal erythrocyte catalase, the enzyme present in the blood of Swiss acatalasemic heterozygotes, and their hybrid produced in vitro, were studied after crosslinking with bifunctional reagents.On theoretical grounds [cf. Hajdu, J., Bartha, F. & Friedrich, P. (1976) Eur. J. Biochem. 68, 373–383] it is inferred from the dodecylsulphate gel electrophoretic patterns obtained after treating catalase with diimidates of various chain lengths that the enzyme is an isologous tetramer (D2 symmetry). The minimal distances between crosslinkable primary amino groups across the three domains of bonding are different.Reaction with diimidates causes a moderate loss of enzyme activity in all three enzyme types due to amidination rather than crosslink formation. On the other hand, crosslinking stabilizes the enzyme against urea and heat inactivation. This is most prominent with heterozygote acatalasemic catalase.Crosslinking markedly prevents the development of peroxidase activity that can be elicited in catalases by urea treatment. The role of the quaternary structure of the protein in the relationship between catalase and peroxidase activities is discussed.
Development of Escherichia coli Virus T1: Repression of Host Gene Expression
Authors: WAGNER Erwin Friedrich , PONTA Helmut , SCHWEIGER Manfred 
Published: March 2005

Host protein synthesis, measured either as amino acid incorporation into proteins or as enzyme synthesis, is inhibited rapidly after infection of Escherichia coli with T1. Analysis of this inhibition, using a technique which distinguishes between translation and transcription, revealed that translation of host mRNA is specifically blocked. Comparison of the time course of T1‐induced host repression with inhibition by the drugs rifampicin, nitrofurantoin and chloramphenicol showed that T1 affects the initiation step of host translation.Intact membranes are apparently essential for host repression, suggesting a membrane‐mediated process. Concomitant viral protein synthesis is not required. The membrane‐altering principle is a constituent of the viral particle.
Purification and Properties of Citrate Lyase Ligase from Streptococcus diacetilactis
Authors: BOWIEN Susanne , GOTTSCHALK Gerhard 
Published: March 2005

Citrate lyase ligase (acetate: SH‐[acyl‐carrier protein] enzyme ligase (AMP)) from Streptococcus diacetilactis was purified 920‐fold with a yield of 6.3%. The molecular weight of the enzyme was estimated to be 41000; the ligase consisted of one polypeptide chain.The acetylation of 1 mol of deacetyl‐citrate lyase to enzymatically active citrate lyase required 6 mol ATP. The formation of AMP and pyrophosphate in the acetylation reaction was demonstrated.Citrate lyase ligase was specific for the lyase from S. diacetilactis and did not acetylate lyases from Rhodopseudomonas gelatinosa and Enterobacter aerogenes. The substrates acetate and ATP could be replaced by propionate and dATP, respectively.The reaction rates for ATP, acetate and deacetyl‐citrate lyase followed Michaelis‐Menten kinetics (Km values: 26 μM for ATP, 25 mM for acetate and 38 nM for deacetyl‐citrate lyase).
Mechanism of the Prolyl Hydroxylase Reaction
Authors: TUDERMAN Leena , MYLLYLÄ Raili , KIVIRIKKO Kari I. 
Published: March 2005

The co‐substrate requirements of prolyl hydroxylase were studied with pure enzyme from chick embryos. No hydroxylation occurred without added Fe2+, indicating that the enzyme does not retain iron sufficiently to catalyze any reaction. Zn2+ was an effective competitive inhibitor with respect to Fe2+, but was noncompetitive with respect to the polypeptide substrate and 2‐oxoglutarate, suggesting that it replaced iron in the active site of the enzyme.The enzyme catalyzed the uncoupled decarboxylation of 2‐oxoglutarate at a rate of about 4 mol CO2 formed (mol enzyme)−1 min−1 in the presence of Fe2+, O2, and ascorbate but in the absence of the polypeptide substrate. This rate was about 1/80 of that observed in the presence of the substrate. Several compounds inhibited the enzyme competitively with respect to 2‐oxoglutarate but non‐competitively with respect to Fe2+. It seems that these two co‐substrates become bound at separate sites on the enzyme, and additional data suggested that these are distinct from the binding site of the polypeptide substrate.The reaction was completely dependent on O2. Nitroblue tetrazolium was a competitive inhibitor with respect to O2, but noncompetitive with respect to the polypeptide substrate and all other co‐substrates. Epinehrine also inhibited the enzyme, but this inhibition was competitive with respect to Fe2+. The results suggest that nitroblue tetrazolium consumed an activated form of oxygen, whereas epinephrine acted primarily by binding Fe2+.The reaction was completely dependent on ascorbate, and in contrast to previous data, this could not be significantly replaced by tetrahydrofolic acid or dithiothreitol. Dehydroascorbate replaced ascorbate in the presence of dithiothreitol but not in its absence. The results also indicate that ascorbate is not stoichiometrically consumed during the reaction.
The Use of Liver Transglutaminase for Protein Labeling
Authors: IWANIJ Victoria 
Published: March 2005

Guinea‐pig transglutaminase catalyzes the formation of an amide bond between the γ‐carboxyl group of peptide‐bound glutamine and a variety of amines, including fluorescent or spin‐labeled derivatives. A systematic study was conducted with this enzyme in order to establish its usefulness as a protein‐labeling technique. Transglutaminase‐catalyzed amine incorporation was tested with isolated proteins, intact cells, membrane preparations, and virus particles. It was found that, due to its large molecular weight and its specificity, the transglutaminase reaction is limited to highly exposed glutamine residues. For example, proteins such as tubulin and α2‐macroglobulin were modified while immunoglobulins (IgG) from a variety of sources were not, despite their high glutamine content. The peptide hormone glucagon was modified by transglutaminase, which resulted in the loss of hormonal activity.Most of the intact cells were not labeled with transglutaminase. Only in the case of human fibroblasts the labeling of a few heavy‐molecular‐weight proteins, including large external transformation‐sensitive (LETS) protein, occurred. However, isolated membrane preparations, such as erythrocyte ghosts, sarcoplamic reticulum and retinal disc membranes, were significantly labeled.Intact influenza virions were susceptible to the transglutaminase‐catalyzed reaction; however, only hemaglutinin but not neuraminidase was labeled.The transglutaminase reaction was found useful when specific and limited labeling is required. This technique should be particularly amenable for the introduction of fluorescent probes into membrane proteins and for elucidation of the structure‐function relationship of the proteins.
Characterization of the Cellular Binding of Exogenous Gangliosides
Authors: CALLIES Richard , SCHWARZMANN Günter , RADSAK Klaus , SIEGERT Rudolf , WIEGANDT Herbert 
Published: March 2005

Experiments were performed to characterize the accumulation of different exogenous sialoglycolipids by cells.A biphasic kinetics of accumulation appeared to be characteristic for intact primary and permanent monolayer cells and chicken erythrocytes whereas that of chicken erythrocyte ghosts was monophasic. Saturation levels could be reached with Galβ1→3 GalNAcβ1→4 Gal[3←2αNeuAc]‐β1→4 Glcβ1–1′Cer and Galβ1→3 GalNAcβ1→4 Gal[3←2αNeuAc8←2αNeuAc]β1→4 Glcβ1 – 1′Cer with concentrations of 100 and 200 nmol per ml culture medium, respectively. Under these conditions the amount of 1.3–1.4 nmol of sialoglycolipids per 100 μg of cellular protein were found to be cell‐bound after approximately two hours. In case of the ganglioside NeuAcα2→3Galβ1→4 Glcβ1 – 1′Cer no saturation could be reached.The intermediate plateau of accumulation was abolished by the agents colchicine and sodium fluoride which on the other hand had no influence on the level of saturation. In contrast the presence of cytochalasin B, 2‐deoxy‐D‐glucose plus sodium azide, and glutardialdehyde as well as low temperature decreased the amount of incorporated gangliosides drastically. The effect of cytochalasin B was limited to monolayer cultures.Mild trypsinisation liberated some 70–85% of cell‐associated gangliosides even after drug‐inhibited accumulation of gangliosides. Also some 87–95% of the cell‐bound ganglioside NeuAcα2→3 Galβ1–4 Glcβ1–1′Cer remained accessible to the action of exogenous sialidase, thus excluding gross internalisation.
Characterization of the Isolated Transferase Subunit of Citrate Lyase as a CoA‐Transferase. Evidence against a Covalent Enzyme‐Substrate Intermediate
Authors: DIMROTH Peter , LOYAL Rosemarie , EGGERER Hermann 
Published: March 2005

1. The largest subunit of citrate lyase from Klebsiella aerogenes represents an acetyl‐thio‐acyl carrier protein: citrate acyl carrier protein transferase but is shown to be also an acetyl‐CoA: citrate CoA‐transferase.2. The enzyme catalyzes an exchange of acetyl residues between acetyl‐thio‐acyl carrier protein or acetyl‐CoA and acetate. The reaction occurs in the presence and, at a much lower rate, also in the absence of acetate. The citrate‐independent reaction was attributed to a substitution of citrate by acetate as a substrate analogue.3. Additionally, the isolated enzyme, in the presence of acetyl‐CoA and citrate, catalyzed the formation of (3S)‐citryl‐CoA and was thus unequivocally shown to be a CoA‐transferase.4. The catalytic efficiency of the enzyme in the CoA‐transfer reaction is similar to that of other CoA‐transferases. It was concluded that the transferase subunit of citrate lyase and the classical CoA‐transferases could belong to one family to enzymes.5. Attempts to demonstrate this relationship were based on a comparison of the mechanism of action of the transferase subunit of citrate lyase with that established for CoA‐transferases. These form enzyme‐CoA and ‘anhydride’ intermediates.6. Evidence is presented for the generation of ‘anhydride’ intermediates on the transferase subunit of citrate lyase. The ‘anhydrides’ are ‘citric acetic anhydride’ and ‘acetic anhydride’ in the acetyl exchange reactions with and without citrate, respectively.7. In contrast to other CoA‐transferases, however, the transferase‐catalyzed CoA‐transfer from acetyl‐CoA to citrate, apparently involves no enzyme‐CoA intermediate. Neither the results of several different chemical approaches nor the kinetics were compatible with the formation of this intermediate.8. An explanation is proposed and the consequences are discussed.
β‐D‐Galactoside Transport in Escherichia coli: Substrate Recognition
Authors: SANDERMANN Heinrich 
Published: March 2005

1. A number of galactosides and other sugar compounds were examined as inhibitors of facilitated or active transport by the lactose permease system of Escherichia coli. Efficient inhibition required an α‐ or β‐anomeric galactopyranosyl ring of D‐configuration, a free 6‐hydroxyl group, and a certain aglycone size which was reached, for example, by monosaccharide or nitrophenyl substituents.2. Aromatic α‐D‐galactopyranosides acted as high‐affinity inhibitors (Ki, below 50 μM). At lest two of them were not transported, in contrast of α‐galactoside disaccharides and to aromatic β‐D‐galactopyranosides.3. β‐D‐galactoside transport was not significantly inhibited by specific inhibitors and transition‐state analogues of β‐galactosidase (D‐galactal, D galactonolactone).4. The β‐D‐galactopyranoside, lactitol, and the α‐D‐galactopyranoside, galactinol, were not efficiently bound by the lactose permease system, although the maximal rate of uptake of lactitol was similar to that of lactose. By comparison with several structrally related D‐galactopyranosides, the decreased affinity was attributed to an effect of the membrane/water interface. A model for substrate recognition by the lactose permease system is presented.
Physico‐chemical Properties of a DNA Binding Protein: Escherichia coli Factor H1
Authors: SPASSKY Annick , BUC Henri C. 
Published: March 2005

H1 factor is a heat‐stable protein found in large amounts in Escherichia coli. In vitro, this protein has been found to stimulate transcription of λ templates by E. coli DNA‐dependent RNA polymerase.The subunit molecular weight of this factor has been re‐estimated and found to be 15500 ± 1000 in the presence of 2‐mercaptoethanol. Crosslinking experiments performed with dimethylsuberimidate in the presence or absence of the DNA template indicate the presence of multiples of the 15500‐Mr subunit up to the tetramer, the dimeric species being predominant. One cysteinyl residue per 15000‐Mr subunit is labeled by 4‐chloro‐7‐nitrobenzofurazan. This residue is not labeled if the factor is exposed to oxidizing conditions. In this case, three lysyl residues are titrated.H1 factor behaves as a DNA‐binding protein. We have detected binding to DNA by two independent methods: displacement of a fluorescently labeled factor by the native protein and retention of radioactive DNA on millipore filters in the presence of the factor. Under our experimental conditions (high ionic strength, absence of magnesium ions), the saturation function of λplac DNA as well as of wild‐type λ DNA has been found to be non‐cooperative. Saturation is reached when 300 ± 30 molecules of dimeric factor are bound per λ molecule, the average dissociation constant of the complex being 10 nM.The dissociation time of the H1· DNA complex is less than 5 s at 37°C. The binding of this factor lowers the affinity of native DNA for ethidium bromide. In the presence of this intercalating dye, the solubility of the complex decreases drastically.
Change of Protein Reactivity in Mammalian Ribosomal Subunits as a Function of Temperature
Authors: REBOUD Anne‐Marie , BUISSON Monique , MARION Marie‐Jeanne , REBOUD Jean‐Paul 
Published: March 2005

Reductive methylation of rat liver ribosomal subunits was carried out at different temperatures and the reaction was followed as a function of time. The percentage of protein lysine residues which were methylated reached a definite plateau for each temperature, and was considerably increased by heating (four times at 40°C). This increase was not observed when free ribosomal proteins were heated under the same conditions. Ribosomal subunits kept their biological activity (elongation steps of protein synthesis) even when methylated to a high extent. Half of the subunit activity was still found when 67% of the lysine residues were methylated.Proteins were divided into different classes on the basis of their alkylation in response to temperature. Those which reacted poorly within subunits even at high temperature should interact directly with rRNA. The results suggest that the protein reactivity modifications induced by heating (up to 37°C), which were often totally or partially reversible, reflect a conformational change of the proteins themselves within the subunits. Besides protein unfolding, higher temperatures produced structural modification and inactivation of the subunits, which were restrained by the presence of aminoacyl‐tRNA.
Enzymic Synthesis, Characterisation and Nuclear‐Magnetic‐Resonance Spectra of pppA2′p5′A2′p5′A and Related Oligonucleotides: Comparison with Chemically Synthesised Material
Authors: MARTIN Edward M. , BIRDSALL Nigel J. M. , BROWN Ronald E. , KERR Ian M. 
Published: March 2005

We have previously shown that a low‐molecular‐weight inhibitor (pppA2′p5′A2′p5′A) of cell‐free protein synthesis, effective at subnanomolar concentrations, is formed on incubation of extracts from interferon‐treated cells or rabbit reticulocytes with double‐stranded RNA and ATP. The inhibitors synthesised with the reticulocyte or interferon‐mediated enzymes, i.e. (2′‐5′)An synthetases, behave identically on thin‐layer chromatography and electrophoresis and have the same biological activity in the inhibition of cell‐free protein synthesis. Here we show that they also behave identically on chromatography on DEAE‐Sephadex in the presence of urea and yield identical products on enzymic and sequential degradation by periodate oxidation and β‐elimination. As previously shown with the interferon inhibitor, preparations of the reticulocyte pppA2′p5′A2′p5′A also include the corresponding biologically inactive dimer, pppA2′p5′A, and biologically active tetramer, ppp(A2′p)35′A, and occasionally higher oligomers in decreasing amounts. Further purification of the pppA2′p5′A2′p5′A yielded small amounts of the corresponding 5′‐diphosphates and 5′‐monophosphates. The 5′‐diphosphate has the same specific biological activity in the inhibition of cell‐free protein synthesis as the triphosphate. The 5′‐monophosphate is essentially inactive.Milligram quantities of the reticulocyte inhibitor can be conveniently synthesised by incubating the (2′‐5′)An synthetase from these cells bound either to poly(I) · poly(C)‐Sepharose or poly(I) · poly(C)‐paper with ATP. Simple methods for the fractionation of the resultant (2′‐5′)An, into its individual components are described. Proton and phosphorus nuclear magnetic resonance spectra were obtained for the putative pppA2′p5′A2′p5′A and the corresponding 5′‐monophosphate and 5′‐diphosphate from this material and for the A2′p5′A2′p5′A ′core′ derived from these by treatment with bacterial alkaline phosphatase. The spectra were compared with those for chemically synthesised A2′p5′A2′p5′A and commercially available A3′p5′A3′p5′A, AMP, ADP and ATP. The results are in accord with the proposed structure.Phosphorylation of chemically synthesised A2′p5′A2′p5′A yielded material with chromatographic properties and a specific biological activity in the inhibition of cell‐free protein synthesis apparently identical to those of biologically synthesised pppA2′p5′A2′p5′A.
Formate‐Nitrite Reduction in Escherichia coli K12
Authors: ABOU‐JAOUDÉ Antoine , PASCAL Marie‐Claire , CHIPPAUX Marc 
Published: March 2005

Components of the electron transfer pathway from formate to nitrite were identified and compared with those of the formate‐nitrate system. Information was derived from studies on various mutants, different electron donors, and effects of inhibitors. An explanation is proposed for the loss activity in spheroplasts or cell free extract.

Formate and d‐lactate with E′o (pH 7) values of –420 mV and –180 mV respectively reduce both nitrite and nitrate. On the contrary ascorbate/phenazine methosulfate (+ 80 mV) does not reduce nitrite.
Formate dehydrogenase and hemin‐deficient mutants lack both formate nitrite and nitrate reductase activities. Quinone‐lacking mutants lost 40% of the formate‐nitrite reduction and all of the formate‐nitrate reductase activity. Strains affected on nitrate‐reductase cytochrome b or nitrate reductase enzyme have retained a normal capacity to reduce nitrite with formate. Mutant nir197 has simultaneously lost the formate‐nitrite reductase activity and the biosynthesis of the cytochrome c552.
With HpHOQnO one inhibitory site was found. Ultraviolet irradiation revealed the presence of quinones at more than one site. Carbon monoxide inhibited formate‐nitrite reductase but not d‐lactate‐nitrite reductase activity. This gas had no effect on the formate dehydrogenase. Action of inhibitors on the redox steady state showed the presence in the electron pathway of cytochrome c552 as well as a b‐type cytochrome.
The formate‐nitrite electron pathway proposed shares with that of nitrate a membrane‐bound part constituted by formate‐dehydrogenase, cytochrome b, and quinones. It also involves a soluble specific part containing at least the cytochrome c552. It is suggested that the loss of a soluble periplasmic element or its dilution lead to the loss of the activity, as is the case in spheroplasts and osmotic‐shock‐treated cells or in cell‐free extracts and sucrose‐treated cells.