bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Functional and Fluorescence Properties of Escherichia coli RNA Polymerase Reacted with Fluorescamine
Authors: DAMJANOVICH Sándor , BÄHR Wolfgang , JOVIN Thomas M. 
Published: March 2005

Fluorescamine (4‐phenylspiro[furan‐2,(3)1′‐phthalan]‐3,3′‐dione) reacts rapidly with Escherichia coli RNA polymerase and produces a fluorescent derivative which is inactivated to an extent dependent upon reagent concentration. Excess fluorescamine is rapidly hydrolysed. Reaction is with ɛ‐amino groups of lysine residues in all subunits as revealed by gel electrophoresis and fluorescence scanning.
The extent of inactivation and fluorescence yield are diminished in the presence of added template, a finding which provides evidence for the existence of reactive and essential amino groups which can be at least partially shielded by DNA in the binary complexes. The relative decrease of fluorescence is greatest in the ββ′ subunits. Holoenzyme and core enzyme show essentially the same behavior.
The inactivation of activity by fluorescamine is primarily at the level of initiation. Template binding and chain propagation are less affected.
The enzyme derivatized by fluorescamine shows an intense fluorescence with a peak at 490 nm and an excitation maximum at 390 nm. The fluorescence lifetime is in the range of 3–8 ns and the emission is highly polarized. In reactions carried out at high ionic strength the fluorescence yield is approximately double that at low ionic strength and insensitive to the presence of template.
Energy transfer is observed between the derivatized enzyme as donor and ethidium bromide as acceptor in the presence of template to which both the enzyme and intercalating dye are bound. The transfer efficiency is a function of the relative concentrations and of the conditions of reaction with fluorescamine. An average transfer distance of approx. 4–5 nm has been calculated suggesting a close proximity between bound polymerase and helical regions of the template.
Affinity Labeling of Rat‐Kidney γ‐Glutamyl Transpeptidase
Authors: INOUE Masayasu , HORIUCHI Seikoh , MORINO Yoshimasa 
Published: March 2005

The reaction of γ‐glutamyl transpeptidase from rat kidney with a glutamine analog, 6‐diazo‐5‐oxo‐L‐norleucine, resulted in irreversible inactivation of the enzyme. The concentration of this reagent giving a half‐maximum rate of inactivation was 6 mM at pH 7.5. The inactivation was prevented by the presence of reduced glutathione in a competitive fashion, which indicates the active‐site‐directed nature of this reagent. The rate of inactivation was greatly accelerated in the presence of maleate, which is known to enhance the glutaminase activity of this enzyme. The presence of maleate increased the maximum velocity of the inactivation, but did not affect the affinity of the enzyme for 6‐diazo‐5‐oxo‐L‐norleucine.Inactivation of the enzyme with 6‐diazo‐5‐oxo‐L‐[6‐14C]norleucine as well as with 6‐diazo‐5‐oxo‐L‐[1,2,3,4,5‐14C]norleucine resulted in a stoichiometric incorporation of radioactivity into the enzyme protein via covalent linkage. The amount of radioactivity incorporated was 1 mol 14C label 248 000 g enzyme protein.A native enzyme preparation showing a single protein band on polyacrylamide gel electrophoresis gave four distinct bands upon sodium dodecylsulfate/polyacrylamide gel electrophoresis.Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis of the 14C‐labeled enzyme, only the band moving the fastest towards the anode was found to contain radioactivity. This finding indicates that this protein band represents the catalytic component of the enzyme.
Amino‐Acid Sequences of Heme‐Linked, Histidine‐Containing Peptides of Five Peroxidases from Horseradish and Turnip
Authors: WELINDER Karen Gjesing , MAZZA Gilbert 
Published: March 2005

In a previous paper we have characterized five plant peroxidases, P1, P2, P3 and P7 of turnip and horseradish isoperoxidase C by peptide mapping studies, and only found two highly homologous sequences present in all. Both contained histidine. The findings supported previous suggestions of two histidine sequences near the peroxidase heme prostetic group. In the present paper we present the amino acid sequences around the histidine residues of all four turnip peroxidases, i. e. of 25 residues around the histidine proximal to heme, and 34 residues around the probably distally located histidine, and compare them with the histidine‐containing sequences of the complete amino acid sequence of horseradish isoperoxidase C. Substitutions of residues are rare close to these histidines, but more abundant with greater distances. The probably distal sequences of P1, P2, P3 and horseradish peroxidase C all contain two histidine residues, at positions 40 and 42. In P7, however, residue 40 is phenylalanine, a substitution presumably important to its abnormal physico‐chemical and enzymic properties.Gel filtration profiles of tryptic digests of the turnip isoperoxidases confirm their previous classification into a P1, P2 and P3 group and a distinct P7 enzyme, but further prove the presence of several sites of carbohydrate attachment in P1, P2 and P3 peroxidases, like in horeseradish peroxidase C which has eight sites. P7 has one such site.
A Mutant ATP Synthetase of Escherichia coli with an Altered Sensitivity to N,N′‐Dicyclohexylcarbodiimide: Characterization in Native Membranes and Reconstituted Proteoliposomes
Authors: FRIEDL Peter , SCHMID Brigitte I. , SCHAIRER Hans U. 
Published: March 2005

Dicyclohexylcarbodiimide‐resistant mutants of Escherichia coli were isolated and characterized. In one mutant the mutation is closely linked to the unc genes and affects the membrane‐integrated part of the ATP synthetase. The sensitivity of ATP synthetase functions to N,N′‐dicyclohexylcarbodiimide was compared in wild‐type and mutant membranes.The membrane‐integrated part of the wild‐type ATP synthetase is highly sensitive to 50 μM dicyclohexylcarbodiimide, as measured by inhibition of ATPase activity, ATP‐dependent membrane energization and restoration of lactate‐dependent energization of ATPase‐depleted membranes. In mutant membranes this concentration has only a slight effect on these activities whereas a severe inhibition is obtained at 200 μM. Using the highly water‐soluble 1‐ethyl‐3(3‐dimethylaminopropyl)‐carbodiimide the activities of wild‐type and mutant membranes are inhibited to the same extent.The ATP synthetase of wild‐type and mutant was partially purified and incorporated into liposomes. These showed an uncoupler‐sensitive ATP‐32Pi exchange and ATP‐dependent quenching of acridine‐dye fluorescence. The activities of mutant and wild‐type proteoliposomes exhibit the same pattern of sensitivity to dicyclohexylcarbodiimide as the corresponding membranes.
Phospholipid Binding and Self‐Association of the Major Apoprotein of Human and Baboon High‐Density Lipoproteins
Authors: ROSSENEU Maryvonne , BLATON Victor , VERCAEMST Rafaël , SOETEWEY Frederik , PEETERS Hubert 
Published: March 2005

The purpose of this study was to establish a relationship between self‐association and phospholipid binding of the human and the baboon apoA‐I protein.The enthalpy changes on binding dimyristoyl lecithin and lysolecithin to either the human or the baboon native apoA‐I protein were measured in a microcalorimeter. An endothermal process, most pronounced for the human apoprotein, was observed at low phospholipid levels. At higher phospholipid to protein ratios the binding was exothermal. Gel filtration experiments on Sephadex G‐200 showed that the native apoprotein of both species consists of dimers and tetramers. The baboon native apoA‐I protein contained a higher amount of dimers. After preincubation of the apoA‐I protein with lysolecithin, the enthalpy changes measured on subsequent binding of dimyristoyl lecithin were shifted towards more exothermal values compared to the curve for the native apoprotein. The amplitude of this shift corresponds to that of the endothermal process observed on binding dimyristoyl lecithin to the native apoprotein. This process was attributed to a phospholipid‐induced disaggregation of the apoA‐I protein.Gel filtration data showed a decreased extent of aggregation in the apoA‐I protein preincubated with lysolecithin. This sample consisted exclusively of dimers. Ultracentrifugal flotation of the complexes formed between the apoA‐I protein, and respectively dimyristoyl lecithin and sphingomyelin indicated that preincubation with lysolecithin increased the extent of complex formation.These results suggest that the dimeric form of the apoA‐I protein possesses the highest affinity for phospholipids. Any dissociation of higher polymers enhances the phospholipid‐binding capacity of the human and the baboon apoA‐I protein.
Purification and Properties of Pyrazon Dioxygenase from Pyrazon‐Degrading Bacteria
Authors: SAUBER Klaus , FRÖHNER Christoph , ROSENBERG Gideon , EBERSPÄCHER Jürgen , LINGENS Franz 
Published: March 2005

Chromatography on DEAE‐cellulose and gel filtration on Sephadex revealed that pyrazon dioxygenase from pyrazon‐degrading bacteria consists of three different enzyme components. No component alone oxidizes the phenyl moiety of pyrazon, only when the three components are combined can oxidation be detected. Following electron paramagnetic resonance and ultraviolet measurements the protein nature of the three components was determined: component A1 (molecular weight about 180000, red‐brown in colour) is an iron‐sulphur protein. The existence of approximately two moles of iron and two moles of inorganic sulphur per mole of protein was demonstrated. This enzyme component was purified to homogeneity in disc electrophoresis. Component A2 is a yellow protein of a molecular weight of about 67000. FAD was shown to be the prosthetic group of this protein. Component B (molecular weight about 12000, brown in colour) is a protein of the ferredoxin type, which was purified to homogeneity, as demonstrated by disc electrophoresis.A hypothetical scheme for the cooperation of the three components is proposed: component A2 accepts as cosubstrate NADH and functions as a ferredoxin reductase. The ferredoxin, component B, has the function of an electron carrier. The conversion of the substrates is effected by component A1, the terminal dioxygenase.
The “b5‐like” Domain from Chicken‐Liver Sulfite Oxidase: A New Case of Common Ancestral Origin with Liver Cytochrome b5 and Bakers' Yeast Cytochrome b2 Core
Authors: GUIARD Bernard , LEDERER Florence 
Published: March 2005

Limited chymotryptic digestion of chicken‐liver sulfite oxidase destroys its ability to oxidize sulfite. From the digest can be isolated a heme‐binding fragment of molecular weight about 11000. Its purification is described, as well as its characterization by a number of methods (absorption spectroscopy, circular dichroism, electrophoretic mobility, immunochemical reactivity, amino acid analysis). The heme spectrum shows no detectable difference with that of the native enzyme.The N‐terminal sequence of this sulfite oxidase core is reported (34 residues). It shows a strong similarity to that of liver microsomal cytochrome b5 and bakers' yeast cytochrome b2 core. The sequence comparison is discussed in terms of structural similarity to cytochrome b5. Our data suggest a common evolutionary origin for the three b‐type cytochromes.
Antibodies and Autoantibodies of Glycogen Phosphorylase b: Inactivation of Pig and Rabbit Enzymes
Authors: TZARTOS Socrates J. , EVANGELOPOULOS Athanasios E. 
Published: March 2005

Pig skeletal muscle glycogen phosphorylase b was purified using ammonium sulfate fractionation, DEAE‐Sephadex A‐50 and Sephadex G‐200 column chromatography. The purified enzyme was used to immunize rabbits in the presence or in the absence of complete Freund adjuvant.Antibodies against pig phosphorylase in pure form were isolated from rabbit antisera using insoluble immunoadsorbents of pig phosphorylase. Autoantibodies against the rabbit enzyme were obtained from the same antisera using insoluble immunoadsorbents of rabbit phosphorylase. Complete inactivation of pig phosphorylase was accomplished by an antibody/enzyme molar ratio equal to 4 and autoantibody/enzyme molar ratio equal to 130. Complete inactivation of rabbit phosphorylase was accomplished by an antibody enzyme molar ratio equal to 250 and auto‐antibody/enzyme molar ratio equal to 160. Passive haemagglutination technique gave positive results with minimum amounts of 0.02 μg/ml and 0.8 μg/ml for pig and rabbit phosphorylase respectively.Kinetic experiments have shown that antibodies and autoantibodies act as noncompetitive inhibitors of both enzymes with respect to AMP and glucose 1‐phosphate but exhibit a mixed type of inhibition with respect to glycogen. When glycogen hydrolysates were used as substrate in place of intact glycogen molecules a pronounced decrease in the inhibitory capacity of antienzyme on the enzyme was demonstrated.
A Nuclear Elongation Factor of Transcription from Physarum polycephalum in vitro
Authors: ERNST Gerold Hans , SAUER Helmut Wilhelm 
Published: March 2005

Homogenates of Physarum plasmodia contain a factor which stimulates UMP incorporation on native DNA by solubilized homologous RNA polymerases in vitro. The factor is a heat‐sensitive protein and has been located in nuclei. It does not alter the template activity of DNA nor the initiation frequency of transcription. The factor interacts with free or bound RNA polymerase molecules (only at 37 °C and at low ionic strength) and yields larger transcripts in vitro.The level of the factor in vitro fluctuates: it is gradually reduced during spherulation and reaches its maximum in mid S phase of the cell cycle of Physarum.
The Molecular Defect in a Case of (Cystathionine β‐Synthase)‐Deficient Homocystinuria
Authors: GRIFFITHS Roger , TUDBALL Norman 
Published: March 2005

Cystathionine β‐synthase activity isolated from fibroblast cultures obtained from the skin of a normal and a homocystinuric individual were both cross‐reactive with normal human liver cystathionine β‐synthase antibody.
Isoelectric focusing revealed a substantial difference in the isoelectric points of the normal and abnormal fibroblast enzymes.
Treatment of purified samples of normal and abnormal fibroblast enzymes with sodium dodecylsulphate followed by polyacrylamide gel electrophoresis indicated that both normal and abnormal enzymes were composed of two sub‐units of molecular weights 53000 and 70000.
A combination of urea and sodium dodecylsulphate treatment revealed that the respective 53000 molecular weight sub‐units were different.
It has been concluded that the molecular defect in the case of pyridoxine non‐responsive homocystinuria examined in the present investigation arises as a result of an alteration in the structural gene which codes for the lower molecular weight sub‐unit of cystathionine β‐synthase.
The Kinetics of Reoxidation of Reduced Benzylamine Oxidase
Authors: OLSSON Bengt , OLSSON Jadwiga , PETTERSSON Gösta 
Published: March 2005

The mechanism of reoxidation of reduced benzylamine oxidase has been investigated at different pH between 6 and 10 by steady‐state and transient‐state kinetic methods.
The reoxidation process involves minimally a second‐order interaction between reduced enzyme and oxygen leading to the formation of a spectrally modified enzyme intermediate, and a subsequent first‐order step converting this intermediate into free enzyme. The variation with pH of rate constants according to such a reaction scheme is reported.
Under aerobic conditions the oxygen‐independent reaction represents the main rate‐limiting step in the catalytic process at alkaline pH. At neutral or acid pH the interaction between reduced enzyme and oxygen becomes mainly rate‐limiting, indicating that the concentration of oxygen may be a critical factor controlling enzyme activity under physiological conditions.
The spectrally modified intermediate formed during the reoxidation process exhibits a dif‐ference‐absorption band centered around 290 nm in comparison to free enzyme, and an additional difference‐absorption band at 470 nm in comparison to reduced enzyme. These data indicate that formation of the intermediate, besides leading to a reappearance of the 470‐nm absorption band dis‐appearing on reduction of the enzyme, results in a spectral perturbation of one or several aromatic amino‐acid residues in the protein. This perturbation could possibly reflect a conformational change of the enzyme.
The Immunochemistry of Peptidoglycan
Authors: SEIDL Peter H. , SCHLEIFER Karl H. 
Published: March 2005

An albumin‐peptide conjugate was synthesized, which carries pentaglycine peptides with C‐terminal glycine residues as found in the interpeptide bridges of the peptidoglycan of many staphylococci. Immunization of rabbits with this synthetic immunogen yielded antisera containing predominantly antibodies against the peptide moiety of the conjugate. The quantitative precipitin and the Ouchterlony agar gel reaction with several synthetic protein‐peptide‐conjugates, immunoaffinity chromatography of the antisera on Sepharose‐(Gly‐Gly‐Gly‐Gly‐Gly)n and hapten inhibition studies with several synthetic peptides and peptide derivatives demonstrated that the antibodies were highly specific for oligoglycine peptides with C‐terminal glycine. These antibodies also reacted strongly with staphylococcal peptidoglycans with an interpeptide bridge composed of pentaglycine peptides or of pentaglycine peptides in which part of the glycine residues were replaced by L‐serine. In contrast, all the peptidoglycans lacking interpeptide bridges composed of glycine residues gave no precipitin reaction at all. The final proof for identical determinant groups of albumin‐(CH2CO‐Gly‐Gly‐Gly‐Gly‐Gly)31 and the staphylococcal peptidoglycans applied in the precipitin reaction was furnished by double gel diffusion studies and by hapten inhibition of the precipitin reaction between antisera to albumin‐(CH2CO‐Gly‐Gly‐Gly‐Gly‐Gly)31 and the corresponding peptidoglycans.For rapid screening of the different peptidoglycans, a latex agglutination test was elaborated. Purified antibodies were adsorbed to latex particles, and the titers with the particular peptidoglycans were then determined. The test was highly sensitive, in that 10 nanograms of peptidoglycan could still be detected.
Studies of Glutamate Dehydrogenase
Authors: DAVID Michael , RASCHED Ihab R. , SUND Horst 
Published: March 2005

In phosphate buffer at pH 7.0, 5,5′‐dithio‐bis(2‐nitrobenzoic acid), N‐ethylmaleimide or iodo‐acetamide do not alter the activity of beef liver glutamate dehydrogenase. Iodoacetate, however, inactivates the enzyme irreversibly by alkylation. Combined addition of the coenzyme NADH and the substrate 2‐oxoglutarate or the effector GTP protects against this inactivation.The alkylation reaction is independent of pH between pH 6–9 indicating that amino, imidazole or phenolic groups are probably not involved in this reaction. Titration of the thiol groups, after denaturation of the enzyme, revealed the loss of approximately one group per polypeptide chain. However, this is not due to the exclusive alkylation of a cysteine residue, since alkylation with iodo‐[2‐14C]acetic acid also labels a methionine residue. 50% of the label is incorporated into methionine‐169 and only 7% into cysteine‐115, the remaining radioactivity is distributed in minor quantities (4%) in several unidentified residues. A probable cause of the erroneous thiol groups titration is discussed.
A Mathematical Model for the Influence of Anionic Effectors on the Phosphofructokinase from Rat Erythrocytes
Authors: OTTO Matthias , HEINRICH Reinhart , JACOBASCH Gisela , RAPOPORT Samuel 
Published: March 2005

The influence of the positive effectors AMP, sulphate, glucose 1,6‐bisphosphate and the negative effector 2,3‐bisphoglycerate on rat erythrocyte phosphofructokinase has been investigated. The kinetic data have been fitted to the Monod‐Wyman‐Changeux model as well as to a model based on a closed association‐dissociation equilibrium. The application of the fitting procedure yields for both models a good correspondence between theoretical and experimental data and equal results with respect to the action of the effectors on the enzyme. The corresponding dissociation constants for the binding of the positive effectors to the active state are: AMP 35 μM, sulphate 0.43 mM and glucose 1,6‐bisphosphate 15 μM. 2,3‐Bisphosphoglycerate as an inhibitor stabilizes the inactive state (dissociation constant: 1.4 mM).A preliminary discrimination between the Monod‐Wyman‐Changeux model and the association‐dissociation model has been attempted.
Aminopeptidase N from Escherichia coli
Authors: MURGIER Maryse , PELISSIER Corinne , LAZDUNSKI Andrée , BERNADAC Alain , LAZDUNSKI Claude 
Published: March 2005

The subcellular localization of aminopeptidase N (previously called aminoendopeptidase) has been investigated. This enzyme was found to be partially released (30–40%) by osmotic shock or by converting Escherichia coli K 10 cells to spheroplasts. However, in all other E. coli strains (K12, B/r, MRE 600, ML 308) tested, this enzyme is not released at all by these procedures and thus behaves like a cytoplasmic enzyme. The crypticity of aminopeptidase N is surprisingly low, 75–85% of the enzyme activity is directly assayable in intact cells of any E. coli strain. Various inhibitors of transport systems do not interfer with this assay. Aminopeptidase activity could also be assayed in spheroplasts, even when an insolubilized substrate was used, which suggests a surface location of this enzyme. As well, N‐ethylmaleimide (0.4 mM), under conditions which do not allow penetration in the cytoplasm, caused 70% inhibition of aminopeptidase N. Binding of 125I‐labeled antiaminopeptidase N antibody to spheroplasts (from K12 strain) was used to assay the orientation of aminopeptidase N in the membrane. This enzyme is exposed on the outer surface of the cytoplasmic membrane. Confirmation of this orientation was obtained by comparing the accessibility of aminopeptidase, alkaline phosphatase and β‐galactosidase to fluorescamine in intact cells. Only 16% of the total β‐galactosidase was labeled with this fluorescent reagent whereas 44–45% of the aminopeptidase N and 59% of the alkaline phosphatase were labeled. Electron microscopic visualization of insolubilized reaction products of aminopeptidase N within the cells showed that these products are located at the poles of the cells. Neither mutant cells which were devoid of aminopeptidase N activity nor parental strains with the enzyme activity inhibited with phenylmercuric chloride contained the characteristic black caps. Thus, it appears that the periplasm is enlarged at the poles of the cells and that the reaction product is mainly located in these places.Investigation of the type of interactions of aminopeptidase N with the plasma membrane only revealed that aminopeptidase N has mainly an electrostatic interaction with the outer surface, probably mediated by magnesium ion bridges. Additional interactions are involved since disruption of the integrity of the cytoplasmic membrane is required to totally release this enzyme.