bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Electrochemical Proton Gradient Génerated by Light in Membrane Vesicles and Chromatophores from Rhodopseudomonas sphaeroides
Authors: MICHELS Paul A. M. , KONINGS Wil N. 
Published: March 2005

The electrochemical proton gradient, , generated upon illumination of membrane vesicles and chromatophores from Rhodopseudomonas sphaeroides. has been determined. The components, the transmembrane pH gradient, ΔpH, and the membrane potential, ΔΨ, were measured with several methods. The ΔpH, determined from the pH changes in the membrane suspensions, is in good agreement with the ΔpH obtained from the distribution of acetate or methylamine. The ΔpH values obtained from the fluorescence quenching of 9‐aminoacridine in chromatophores are more than two‐fold larger. The ΔΨ was determined from the distribution of the permeant ions triphenylmethylphosphonium and thiocyanate. The ΔΨ values in chromatophores obtained from the absorbance changes of carotenoids were several‐fold larger.Illumination of membrane vesicles results in a ΔpH, inside alkaline and a ΔΨ inside negative. The ΔpH, determined from the distribution of acetate, depends on the composition of the medium and is optimally 0.6 at an external pH 7; the ΔΨ, measured from the distribution of triphenyl‐methylphosphonium, is about 70 mV. The in membrane vesicles is thus at the most 110 mV.Illumination of chromatophores results in a ΔpH, inside acid, and a ΔΨ inside positive. The ΔpH, calculated from the distribution of methylamine, depends on the external pH and increases from 0.98 at pH 6 to 1.36 at pH 8. The ΔΨ is not affected and remains about 50 mV. The in chromatophores is thus at the most 140 mV.The ΔpH and ΔΨ are in both membrane preparations strongly dependent on the composition of the medium. So is for instance the ΔΨ decreased by the presence of chloride ions and the ΔpH increased. Phosphate ions decrease the ΔpH in membrane vesicles. The ΔpH is generated at a lower rate than the ΔΨ; once the has reached a maximum the ΔpH continues to increase at the expense of the ΔΨ.
Isolation of Soluble Proteins Capable of Stimulating Aerobic Plasmalogen Biosynthesis
Authors: PALTAUF Fritz 
Published: March 2005

The terminal step during aerobic plasmalogen biosynthesis is catalyzed by a microsomal desaturase system which converts 1‐O‐alkyl‐2‐acyl‐sn‐glycerophosphoethanolamine to 1‐O‐alk‐1′‐enyl‐2‐acyl‐sn‐glycerophosphoethanolamine (ethanolamine plasmalogen). The reaction depends on oxygen and NAD(P)H and is stimulated 3–10‐fold by soluble activating factors contained in the 100 000 X g supernatant. Two stimulating proteins have been isolated from pig kidney; the partially purified proteins have identical molecular weights (27000) but differ in their respective isoelectric points (protein 1, 5.1 and protein 11, 4.9). Both proteins behave identically in the biochemical studies conducted. Exogenous substrate binds to the stimulating proteins; the transfer of ethanolamine, but not of choline phospholipids, from liposomes to microsomes is enhanced by the stimulating proteins. They stimulate plasmalogen synthesis from either exogenous or endogenous substrate (synthesized from alkylglycerophosphoethanolamine by microsomal transacylases).The stimulating proteins have no enzymatic activity themselves; it is suggested that they affect events within the membrane and function as specific mediators between the membrane‐bound enzyme system and the lipophilic substrate.
Ferredoxins from Nitrogen‐Fixing Bacteria
Authors: YATES M. Geoffrey , O'DONNELL Michael J. , LOWE David J. , BOTHE Hermann 
Published: March 2005

Two ferredoxins have been purified from N2‐fixing Mycobacterium flavum. Ferredoxin I had a minimum molecular weight (by amino acid analysis) of 11230 and contained 7.4 mol Fe and 7.0 mol S2− per mol. The ratio of the absorbance at 400 nm to that at 275 nm was 0.7. Ferredoxin II had a minimum molecular weight of 13478 and contained 3.4 mol Fe and 3.2 mol S2− per mol. The ratio of the absorbance at 400 nm to 268 nm was 0.42.Both ferredoxins were paramagnetic when oxidised by potassium ferricyanide and diamagnetic when reduced by sodium dithionite. This suggests that, like Azotobacter ferredoxin, they operate solely between the – 2 and – 1 oxidation states. A ferredoxin fraction of similar properties was isolated from Spirillum lipoferum. The electron paramagnetic resonance (EPR) spectra of both ferredoxins had features at g= 2.023, 2.017 and 2.00. The signals were affected by pH: below pH 6.0 that at g= 2.023 predominated, whereas above pH 6.0 the g= 2.017 features was clearly present. The g= 2.00 features was broader between pH 6.0 and 9.0 than outside this pH range. The change to the low‐pH form was not reversible.Ferredoxin I had redox centres with mid‐point potentials at – 420 mV and + 230 mV; ferredoxin II had one redox centre of mid‐point potential at approximately – 420 mV. Above + 300 mV ferredoxin I suffered irreversible oxidative damage and lost the g= 2.023 feature of the EPR spectrum. Both ferredoxins were damaged by prolonged exposure to potassium ferricyanide. Ferredoxin I took up 1.8 electrons per molecule when reduced by sodium dithionite but the EPR signal of the oxidised form integrated to only 0.35 electron per molecule. Ferredoxin II took up 0.9 electron per molecule on reduction and the EPR signal of the oxidised form integrated to 0.66 electron per molecule.It was concluded that M. flavum ferredoxin I resembles Azotobacter ferredoxin and is thus the second ferredoxin isolated that has two redox centres with mid‐point potentials approximately 700 mV apart. Ferredoxin II has no exact counterpart in other systems. Aerobic nitrogen‐fixing bacteria apparently contain ferredoxins which operate between the – 2 and – 1 oxidation states.
Study of the Sulfhydryl Groups of β‐d‐Xylosidase from Bacillus pumilus
Authors: SAMAN Eric , CLAEYSSENS Marc , DE BRUYNE Clement K. 
Published: March 2005

β‐d‐Xylosidase from Bacillus pumilus contains one intramolecular disulfide bridge and four cysteinyl residues per monomer (Mr 60000). Under denaturing conditions two of these ‐ SH residues react with 5.5′‐dithiobis(2‐nitrobenzoic acid) (Nbs2) with concomitant formation of a second cystine.
Under native conditions one cysteinyl group per polypeptide chain reacts with Nbs2 or p‐chloromercuribenzoate (ClHgBzOH) and this results in complete loss of enzymic activity. This inactivation can completely be reversed by reductive treatment (dithiothreitol) of the β‐d‐xylosidase.
Kinetic analysis of the inactivation reaction points to a saturation effect, occurring when the concentration of ‐ SH reagent is increased. A reaction model, which includes prior association of the regent with the enzyme, is proposed.
Competitive inhibitors protect against inactivation of the enzyme with Nbs2. These experiments prove that two classes of ligands, with two different binding modes, exist.
Inactivation with Nbs2 and iodoacetamide as a function of pH reveals the presence of two ionizing groups on the enzyme; their pK values (6.0 and 9.0 respectively) are consistent with those of a protein‐imidazol and protein‐cysteine group.
Cumulative evidences obtained with other ‐ SH reagents, such as 5,5′‐dithio bis(2‐nitro‐N‐trimethylbenzylammonium iodide) and iodoacetate, indicate a negatively charged reaction site. Identity with the active site or direct participation of a histidine and/or a cysteinyl group in the enzymic proces can only be suggested.
Anion Binding to Yeast Phosphoglycerate Kinase
Authors: WROBEL Jean A. , STINSON Robert A. 
Published: March 2005

The single thiol of yeast phosphoglycerate kinase was labelled with the chromophoric sulfhydryl reagent, 2‐chloromercuri‐4‐nitrophenol. Sequential additions of individual anions to this modified enzyme brought about a decrease in absorbance at 410 nm that reflected the degree of saturation of the enzyme with anion. The binding curves were analyzed to determine the dissociation constants of a number of anions with charges varying from – 1 to – 4.1. A linear relationship was found between the charge of the anion and the negative logarithm of the dissociation constant for the labelled enzyme‐anion complex. The highly charged anions, such as ATP, bound more tightly than did anions with less charge, such as Cl−. The average number of binding sites for those anions for which accurate results could be obtained was 1.06 mol per 47000g of enzyme. Several lines of evidence suggested that titration of the active center was not being monitored.Anions bound to phosphoglycerate kinase decreased the rate of reaction between the enzyme thiol and 5,5′‐dithiobis(2‐nitrobenzoic acid). The relationship between the degree of saturation of the anion binding site and the reaction rate constant was used to calculate the dissociation constant between anion and enzyme. Dissociation constants determined in this manner were in good agreement with those determined by titration of the enzyme‐mercurial complex.
Studies on the Control of Development
Authors: RHAESE Hans‐Jürgen , GROSCURTH Reinhard 
Published: March 2005

Ribosomes from vegetative cells (vegetative ribosomes) of Bacillus subtilis synthesize guanosine 3′(2′)‐diphosphate 5′‐diphosphate (p2Gp2) and guanosine 3′(2′)‐diphosphate 5′‐triphosphate (p3Gp2). In contrast, ribosomes isolated from sporulating cells (sporulation ribosomes) synthesize adenosine 3′(2′)‐diphosphate 5′‐diphosphate (p2Ap2) and adenosine 3′(2′)‐diphosphate 5′‐triphosphate (p3Ap2). The products obtained with the aid of vegetative ribosomes were characterized by comparison with authentic substances in two chromatographic systems. The reaction mechanism is identical with that found in Escherichia coli, since [3H]GTP or GDP is pyrophosphorylated by ATP to form p2Gp2 and p3Gp2. Omission of ATP completely stops the formation of any reaction products. Furthermore, no reaction products are observed when GTP or GDP are omitted in a reaction mixture containing vegetative ribosomes. In contrast, incubation of sporulation ribosomes with ATP alone or ADP and ATP results in the formation of two products identified as p2Ap2 and p3Ap2 by their mechanism of synthesis as well as chemical and enzymatic degradation. Sporulation ribosomes (isolated at least an hour after the end of logarithmic growth) synthesize drastically less (by a factor of 100) p2Gp2 and p3Gp2. Vegetative ribosomes, on the other hand, synthesize only 1/100 the amount of p2Ap2 and p3Ap2 compared to sporulation ribosomes when isolated at least an hour before the end of logarithmic growth. By using washed ribosomes and ribosomal wash from vegetative and sporulation ribosomes, we were able to show that changes in the composition of ribosomes (probably in ribosomal proteins and/or ribosome‐associated factors) must occur during the transition from vegetative to sporulating cells. Washed ribosomes from vegetative cells cannot replace washed ribosomes from sporulating cells to synthesize p2Ap2 and p3Ap2 in the presence of ribosomal wash from sporulating cells and vice versa. During the transition from one type of ribosomes to the other, as far as the unusual nucleotide‐synthesizing activity is concerned, both types of nucleotides can be synthesized. While the ability to synthesize p2Gp2 and p3Gp2 decreases approximately at the end of logarithmic growth, the ability to synthesize p2Ap2 and p3Ap2 increases at the same time. The reason behind this obvious change in ribosomal structure and associated factors is discussed in the light of our finding that these changes are not observed in some early blocked asporogenous mutants.The observation that ribosomes from sporulating cells synthesize traces of p2Gp2 and p3Gp2 (by a factor of almost 100 less than vegetative ribosomes) is probably due to the presence of some non‐sporulating cells in any B. subtilis culture. Likewise, there are always some spores contained in a vegetatively growing culture, which are probably responsible for the observed small amounts of p2Ap2 and p3Ap2 synthesized.
An Adenosine 3′:5′‐Monophosphate/Adenosine Binding Protein from Mouse Liver
Authors: UELAND Per Magne 
Published: March 2005

Synthetic and naturally occurring adenosine derivatives were tested for their ability to inhibit the binding of cyclic [3H]AMP and [3H]adenosine to the activated cyclic AMP/adenosine binding protein from mouse liver [Ueland, P. M. and Døskeland, S. O. (1977) J. Biol. Chem. 252, 677–689]. The structural requirements for inhibition of the binding of the two ligands were different, as demonstrated by the existence of adenosine derivatives that inhibited the binding of [3H]adenosine but not the binding of cyclic [3H]AMP and vice versa. The interaction of the binding protein with AMP, ADP and adenine was investigated in more detail. The binding of radioactive AMP and ADP to the protein was determined under equilibrium conditions. Competition studies indicate that AMP binds to the cyclic AMP binding site with an apparent dissociation constant of 2 × 10−6 M and to a low‐affinity site. The double‐reciprocal plot for the binding of ADP deviated slightly from linearity suggesting the possibility of heterogeneity also for the ADP binding sites. Competition studies indicate that this nucleotide binds mainly to the cyclic AMP binding site.Cyclic AMP has previously been shown to activate its own sites under certain conditions (30°C, in the presence of KCl and Mg2+). This phenomenon has been termed homologous activation of binding sites [Ueland, P. M. and Døskeland, S. O. (1978) J. Biol. Chem., in the press]. Similar results were obtained with AMP and ADP.Displacement of [3H]adenosine binding from the sites specific for this adenine derivative was observed with unlabelled adenosine, adenine, AMP and ADP with decreasing efficiency in the order mentioned. This indicates that adenine, AMP and possibly ADP interact with the adenosine binding sites. Adenine and AMP inhibited the activation of the cyclic AMP binding capacity by ATP as previously shown for adenosine [Ueland, P. M. and Døskeland, S. O. (1978) Arch. Biochem. Biophys. 185, 195–203]. The inhibitory power closely parallelled the displacement of [3H]adenosine binding. Adenine was also demonstrated to inhibit the homologous activation of the cyclic AMP binding site.
Chorismate Mutase/Prephenate Dehydratase from Escherichia coli K12
Authors: GETHING Mary‐Jane , DAVIDSON Barrie E. 
Published: March 2005

The binding of phenylalanine to the allosteric site of chorismate mutase/prephenate dehydratase has been studied by steady‐state dialysis.Under most of the experimental conditions examined positive co‐operativity was observed for the binding of ligand up to 50% saturation and negative co‐operativity above 50% saturation.In the presence of 0.4 M NaCl at pH 8.2 the co‐operativity was positive at all phenylalanine concentrations and the maximal stoichiometry of 1 mol of phenylalanine/mol of enzyme subunit was observed. It was concluded that there is a single phenylalanine‐binding site per subunit which is associated with the regulation of each of the mutase and dehydratase activities.The effects of enzyme concentration, NaCl, temperature and pH on the binding of phenylalanine have been investigated. Neither tyrosine nor tryptophan bound to the allosteric site of the enzyme. Enzyme that was desensitized to inhibition by phenylalanine following modification of three sulphydryl groups with 5,5′‐dithio‐bis (2‐nitrobenzoic acid) did not bind phenylalanine.The mechanism of co‐operativity, the binding of the enzyme to Sepharosyl‐phenylalanine and the physiological significance of the inhibition of the enzyme by phenylalanine are discussed in terms of the results obtained.
On the Significance of the Prosthetic Group Composition of Citrate Lyase
Authors: BAYER Ernst , EGGERER Hermann 
Published: March 2005

Klebsiella areogenes contains two different acyl carrier proteins, one specific for citrate lyase, the other for fatty acid synthetase.
The acyl carrier protein of fatty acid synthetase from K. aerogenes was isolated and compared with the corresponding protein from Escherichia coli and with the acyl carrier protein of citrate lyase from K. aerogenes.
As judged from prosthetic group compositions as well as amino acid and fingerprint analyses, the acyl carrier proteins of the two fatty acid synthetases are nearly identical but different from that of citrate lyase from K. aerogenes.
Therefore, the different prosthetic groups alone cannot be responsible for the different specificities of the acyl carrier proteins of fatty acid synthetase and citrate lyase in K. aerogenes.
The prosthetic group of citrate lyase, phosphoribosyl dephospho‐CoA, apparently represents no incidental, phosphopantetheine‐replacing aberration. The requirement of citrate lyase for the CoA‐like prosthetic group may arise from the substrate requirement of both subunit enzymes of the enzyme complex.
Oxidation of Sulfhydryl Groups in Lactate Dehydrogenase under High Hydrostatic Pressure
Authors: SCHMID Gerhard , LÜDEMANN Hans‐Dietrich , JAENICKE Rainer 
Published: March 2005

Lactate dehydrogenase from rabbit skeletal muscle in the presence of substrate exhibits irreversible deactivation at hydrostatic pressures beyond 1 kbar [Schmid. G., Lüdemann, H.‐D. & Jaenicke, R. (1975) Biophys. Chem. 3, 90–98]. In the absence of substrate and coenzyme the lability towards pressure is enhanced. The pH dependence of the effect and its inhibition by SH‐protecting agents suggest the oxidation of sulfhydryl groups to be involved in the mechanism of deactivation. Partial deactivation observed at pH 5.5–7.0 becomes complete in the range of the intrinsic pK of cysteine; addition of dithiothreitol and/or EDTA protects the enzyme from complete deactivation, and leads to the residual enzymatic activity observed at pH 7.0. Incubation of the enzyme with dithiothreitol after pressure deactivation at pH 8.5 causes partial reactivation.From pressure‐dependent measurements of the kinetics of deactivation an activation volume of δV‡=−285 ± 30 cm3· mol−1 is calculated, which exceeds numerical data reported for typical reactions in organic chemistry. Therefore, the assumption can be made that the oxidation of sulfhydryl groups is connected with structural changes in the enzyme in the rate‐determining step of the deactivation. The proposed mechanism may contribute to the toxicity of oxygen towards bacteria under high hydrostatic pressure.
Antibodies against a Synthetic Peptidoglycan‐Precursor Pentapeptide Cross‐React with at least Two Distinct Populations of Uncross‐linked Soluble Peptidoglycan Secreted by Micrococcus luteus Cells
Authors: ZEIGER Allen R. , EATON Stephen M. , MIRELMAN David 
Published: March 2005

Antibodies elicited by a synthetic immunogen, (Glu60 Ala40)n [Ala‐dGlu(Lys‐dAla‐dAla)]5.1, related to a major class of peptidoglycan precursors, were purified by an affinity column of Sepharose 4B, to which the hapten, Ala‐dGlu(Lys‐dAla‐dAla), was covalently attached and were then coupled to Sepharose 4B. Low‐molecular‐weight [14C]alanine‐labeled products secreted by Micrococcus luteus cells grown in the presence of penicillin G were applied to the antibody‐coupled gel. The bound material, which should be univalent to the antibody, was eluted completely by 0.02 mM α‐tert‐butyl‐oxycarbonyl‐lysyl‐d‐alanyl‐d‐alanine. High‐molecular‐weight uncross‐linked soluble peptidoglycan secreted by M. luteus cells grown in the presence of penicillin G was completely bound to the affinity column, regardless of whether the soluble peptidoglycan was labeled in its alanine moieties or in its glucosamine and muramic acid residues. A minor fraction of the bound soluble peptidoglycan was released with 0.02 mM tripeptide (‘paucivalent’ fraction). The remainder of the bound material was eluted with 2 mM tripeptide, indicating that a major portion of the high‐molecular‐weight material was ‘multivalent’.When glycan‐labeled soluble peptidoglycan was stored in buffered saline at – 5°C for several months, a fraction of the labeled material was no longer bound to the affinity gel. Digestion of this non‐binding fraction, as well as the paucivalent and multivalent fractions with hen egg‐white lysozyme, was consistent with the affinity chromatographic data. These results indicate that an amidase present in these preparations cleaved different amounts of peptide from the soluble peptidoglycan preparations, producing strands which are polydisperse with respect to their peptide substitution.
Control of Enzymic Hydrolysis of Polyadenylate Segment of Messenger RNA: Role of Polyadenylate‐Associated Proteins
Authors: MÜLLER Werner E. G. , ARENDES Josef , ZAHN Rudolf K. , SCHRÖDER Heinz C. 
Published: March 2005

The role of poly(A)‐associated proteins in the breakdown of poly(A) sequences in both mammalian polyribosomes and in isolated poly(A) · protein complexes has been studied on an enzymic level. Two nucleases (alkaline exoribonuclease and endoribonuclease IV; both isolated from eukaryotic tissue), which preferentially hydrolyze poly(A) sequences, have been applied to determine the susceptibility of poly(A) in dependence on the presence of poly(A) · protein(s).Polysomes, isolated from L5178y mouse lymphoma cells, do not contain endogenous poly(A) nuclease activity.The poly(A) segment in polysomes is hydrolyzed by the exoribonuclease, irrespective of the preincubation conditions used. Pretreatment of the polysomes with 1 M urea increases the rate of hydrolysis considerably. In contrast to susceptibility of poly(A) towards the exoribonuclease, the endoribonuclease IV does not hydrolyze poly(A) in mammalian polyribosomes under low salt conditions (140 mM KCl). However pretreatment of the polysomes with 1 M urea renders the poly(A) susceptible to endoribonuclease IV.Similar results were obtained using poly(A) · protein complex, which has been isolated from mRNA · protein by ribonuclease digestion; two polypeptide species were found to be present in this complex, one with a molecular weight of 79000 and a second one with a molecular weight of 51000. While the poly(A) segment is hydrolyzed by exoribonuclease both after low and 1 M urea salt pretreatment, the homopolymer can be degraded by endoribonuclease IV only after a pretreatment with urea.In control experiments it was established that under incubation conditions of 1 M urea a poly(A)‐associated protein is dissociated from the homoribopolymer. Therefore we conclude that the susceptibility of poly(A) to nucleolytic enzymes is determined by the interaction with a poly(A)‐associated protein.