bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Pathway of Exogenous and Endogenous Carbohydrate Utilization in Escherichia coli: A Dual Function for the Enzymes of the Maltose Operon
Authors: Palmer T. Norman , Wöber Günter , Whelan William J. 
Published: March 2005

The components of the maltose‐utilizing system in Escherichia coli ML, i.e. maltose permease, amylomaltase and maltodextrin phosphorylase, were re‐investigated, with the following findings:

The concentrating mechanism (permease) is the rate‐limiting step in the maltose‐utilizing system. Maltose and higher maltodextrins, up to an average chain length of at least 10 glucosyl units, can be taken up at the same rate. Maltodextrins of an average chain length of 20 glucosyl units are restricted in their uptake, as reflected in the growth‐rate of the organism.
Growth studies with extensively purified maltose show that this sugar, in contrast to maltotriose and higher maltodextrins, is a poor inducer of the system.
Pullulanase is co‐induced by maltodextrins, along with the other enzymes. The pullulanase seems to be bound to the cell wall and has properties very similar to those of the well‐studied pullulanase of Aerobacter aerogenes.
The complete system, now referred to as the maltodextrin‐utilizing system, comprises the enzymes pullulanase, maltodextrin permease, (a name replacing maltose permease) amylomaltase and maltodextrin phosphorylase.
This group of enzymes was also demonstrated to be present in Streptococcus mutans and A. aerogenes. In addition, A. aerogenes possesses an inducible α‐amylase. This last enzyme permits a wider range of branched 1,4‐α‐glucans to be utilized by the latter organism.
The enzymes of glycogen degradation in E. coli NCTC 5928 were investigated. This organism utilizes isoamylase to debranch the reserve polysaccharide, liberating the component chains. These maltodextrin chains are subsequently metabolized by the enzymes of the maltodextrin‐utilizing system.
We suggest that E. coli uses pullulanase for the debranching of extracellular carbohydrates, while isoamylase is presumed to function intracellularly in the degradation of glycogen.
Maltodextrins are provided for intracellular utilization in E. coli by either of two routes. Pullulanase and permease function in the degradation and uptake of extracellular branched α‐glucans. Isoamylase produces maltodextrins during the debranching process of intracellular glycogen. Maltodextrins arising by either route are metabolized to glucose and glucose 1‐phosphate by the concerted actions of amylomaltase and maltodextrin phosphorylase.

A. aerogenes and S. mutans probably have similar enzymic machinery, the former also having an extracellular α‐amylase to assist exogenous glucan degradation.9. A general scheme for the metabolism of branched 1,4‐α‐glucans in bacteria and plants is proposed.
d‐Galactose Dehydrogenase from Pseudomonas saccharophila
Authors: Wengenmayer Friedrich , Ueberschär Karl‐Heinz , Kurz Gerhart , Sund Horst 
Published: March 2005

d‐Galactose dehydrogenase from Pseudomonas saccharophila has been purified over 700‐fold with an over‐all yield of 27%. The purified enzyme exhibits a specific activity of about 200 μmol NADH formed × min−1× mg protein−1 and has been shown to be homogeneous by ultracentrifugation and by gel electrophoresis.
The amino acid composition of the enzyme has been determined.
The native enzyme has a sedimentation coefficient (S20,w0) of 5.94 S and a diffusion coefficient (D20,w0) of 5.60 F. The experimentally determined partial specific volume (v̄) is 0.744 ml/g. The molecular weight calculated from these data is 103000, in good agreement with the value of 102000 estimated by gel permeation chromatography.
The enzyme dissociates into polypeptide chains in the presence of 5 M guanidine hydrochloride or 0.1% sodium dodecylsulfate. The molecular weight of the polypeptide chains has been found to be 25000 by ultracentrifugation (S20,w0= 1.35 S, D20,w0= 5.28 F) and by dodecylsulfate‐polycrylamide electrophoresis. The native enzyme is composed of four polypeptide chains.
The pI of d‐galactose dehydrogenase is 5.1. The pH‐optimum is between pH 8–9 in Tris‐HCI buffer. AT PH 8.6 and 30°C the limiting michaelis constant for NAD+ is Ka= 0.14 mM, the limiting michaelis constant for d‐galactose is Kb= 1.0 mM and the dissociation constant for NAD+ is Kia= 0.48 mM.
The enzyme is specific for NAD and uses NADP only to a minor extent as a coenzyme. It oxidizes the following sugars arranged according to relative rates, in decreasing order: d‐FUCOSE > d‐galactose > 3‐deoxy‐d‐galactose > 2‐deoxy‐d‐galactose > 4‐deoxy‐d‐galactose > l‐arabinose > 2‐deoxy‐2‐amino‐d‐galactose.
The Specificity of Pancreatic Deoxyribonuclease
Authors: Ehrlich Stanislav D. , Bertazzoni Umberto , Bernardi Giorgio 
Published: March 2005

The kinetics of degradation of calf thymus DNA by pancreatic DNAase has been investigated with the main purpose of providing the background for studying the specificity of the enzyme and its use in investigations of nucleotide sequences in DNA. The relationships between the hyperchromic shift or acid solubility and the reciprocal average degree of polymerization (P̄n−1) of the hydrolysate have been established; these can be used in order to prepare oligonucleotides of desired average chain length simply by measuring the increase of absorbance or of acid solubility of the digest during DNA degradation.The specificity of the pancreatic DNAase was studied by determining the base compositions of 3′‐terminal, 5′‐terminal and 5′‐penultimate nucleotides of oligonucleotides released by the enzyme. The 3′‐terminal nucleotide composition was found not to vary in the P̄n, or average size, range 150 to 8 and 30 to 8 for digestions of calf thymus DNA in the presence of magnesium and manganese ions, respectively, indicating that no change in the specificity takes place during digestion. The compositions of 3′‐terminal, 5′‐terminal and 5′‐penultimate nucleotides differ from those expected on the basis of random splitting of DNA. The conclusion is drawn that pancreatic DNAase recognizes sequences at least three nucleotides long.
Rate‐Limiting Factors in Ethanol Oxidation by Isolated Rat‐Liver Parenchymal Cells
Authors: Grunnet Niels , Quistorff Bjørn , Thieden Herluf I. D. 
Published: March 2005

Isolated rat‐liver parenchymal cells oxidized ethanol at a rate of 1.4, 1.7, 1.9 and 2.5μmol/min per ml packed cells at 4, 20, 40 and 65 mM ethanol, respectively. Between 40 and 65 mM ethanol an abrupt 30% increase in the ethanol oxidation rate was observed.
The activity of the NAD‐dependent alcohol dehydrogenase in a homogenate of isolated cells corresponded to 1.25–5 U/ml packed cells, depending on the assay method used.
Fructose or pyruvate enhanced the oxidation rate of ethanol by 1.4–2.0 μmol/min per ml packed cells independent of the ethanol concentration applied. No additive effect of the two compounds upon ethanol oxidation was observed.
Pyrazole inhibited the NAD dependent alcohol dehydrogenase activity in a homogenate of isolated cells as well as the unstimulated and the fructose‐stimulated ethanol oxidation with a Ki‐value of 9–13 μM.The “fructose effect” was inhibited 100% by pyrazole concentrations, which inhibited the unstimulated ethanol oxidation only 30%, indicating that the “fructose effect” is mediated via alcohol dehydrogenase.
mM pyrazole only slightly inhibited that part of the ethanol oxidation which was not catalyzed by alcohl dehydrogenase, whereas 18 mM pyrazole inhibited also this pathway significantly.
50 μ pyrazole had no effect at all upon the basal ethanol oxidation rate wheras the fructose‐stimulated ethanol oxidation was 30% inhibited.
The results concerning the effect of fructose and pyruvate upon ethanol oxidation are interpreted in terms of the mechanism for the reaction catalyzed by alcohol dehydrogenase. At low concentrations of ethanol and in the absence of fructose, the rate‐limiting step in ethanol oxidation appears to be dissociation of the enzyme‐NADH complex, whereas, in the presence of fructose, the maximal activity of ethanol dehydrogenase may be rate‐limiting for the oxidation of ethanol.
The results also suggest that enzyme systems other than alcohol dehydrogenase participate in ethanol oxidation at high concentrations of ethanol.
Binding of the Initiation Factor IF‐3 to Escherichia coli Ribosomes and MS2 RNA
Authors: Vermeer Cees , Boon Jan , Talens Anneke , Bosch Leendert 
Published: March 2005

The binding of IF‐3 to 70‐S, 50‐S and 30‐S ribosomes and subunits of Escherichia coli and to MS2 RNA was studied employing Sephadex G‐200 chromatography and sucrose‐gradient centrifugation to recover the complex formed. In the latter case, reaction mixtures were fixed with 0.25% glutaraldehyde prior to centrifugation. Total reaction mixtures were also analyzed by polyacrylamide‐gel electrophoresis.
Highly purified IF‐3, either unlabeled or labeled with 35S was employed and consistent results were obtained with both factor preparations.
Binding of IF‐3 to 30‐S subunits was independent of the Mg2+ concentration in the range 1–14 mM. The binding to 50‐S subunits and MS2 RNA was counteracted by increasing the Mg2+ concentration in the same range.
Binding at 0°C and at 37°C occurred to the same extent both with 30‐S and 50‐S ribosomal subunits.
The number of IF‐3 molecules bound per 30‐S subunit approached unity, that bound per 50‐S subunit exceeded unity when the IF‐3 input was sufficiently raised.
At low factor/ribosome input ratios IF‐3 binds preferentially to 30‐S particles. When the ratio is increased binding to 50‐S subunits and 70‐S ribosomes also occurs. The IF‐3‐mediated dissociation of 70‐S ribosomes is strongly inhibited by adding increasing amounts of 30‐S subunits.
At all Mg2+ concentrations studied the relative extent of binding can be expressed as: 30‐S > 70‐S, 50‐S > MS2 RNA.
Binding to all types of ribosomes and subunits and to MS2 RNA is not stimulated by the addition of IF‐1 and IF‐2. The latter factors stabilize the 30‐S · IF‐3 complexes, however.
Purification and Properties of d‐Gluconate Dehydratase from Clostridium pasteurianum
Authors: Bender Rudolf , Gottschalk Gerhard 
Published: March 2005

d‐Gluconate dehydratase from Clostridium pasteurianum has been purified by heat treatment, chromatography on DEAE‐Sephadex and gel filtration to about 80% homogeneity. The overall procedure resulted in a 33‐fold purification with a yield of 16%. Further purification by electrophoresis yielded preparations homogeneous by electrophoretic and ultracentrifugal criteria. Analysis of the enzyme by sedimentation equilibrium experiments, gel‐filtration and sucrosegradient centrifugation gave a molecular weight of 131 000, 130 000 and 118 000, respectively. Treatment with sodium dodecylsulfate and mercaptoethanol dissociated the enzyme into two subunits (Mr= 64000).
The enzyme is inactivated by exposure to oxygen and the activity can be restored by incubation with sulfhydryl compounds such as mercaptoethanol, dithiothreitol, reduced glutathione and cysteine in conjunction with ferrous ions for 2 h at 35°C.
The reactivated enzyme dehydrates gluconate in the presence of divalent metal ions only. Ferrous ions (2 mM) are most effective but can be partially replaced by Mn2+, Co2+ and Mg2+. EDTA inhibits the reaction completely.
d‐Gluconate dehydratase exhibits maximum activity in the pH region of 7.3 to 7.8 Among several compounds tested only d‐gluconate is dehydrated (Km value = 5.5 mM).
Human α2‐Macroglobulin, Characterization and Trypsin
Authors: Hamberg Ulla , Stelwagen Patrick , Ervast Hanna‐Stina 
Published: March 2005

The trypsin binding was compared with preparations of α2‐macroglobulin obtained from human plasma, serum or Cohn's fraction III by ammonium sulphate precipitation. Determination of the binding ratio at various purification steps showed close to 1:1 molecular proportion, regardless of the degree of purity of the preparations. The α‐macroglobulin · trypsin complex was separated by gel chromatography on Sephadex G‐200, using standardized conditions and estimating the bound and free typsin in the eluate fractions. It was found that activated plasmin interfered with trypsin binding of α2‐macroglobulin from Cohn's fraction III.Immunologically pure α2‐macroglobulin was obtained in six preparative steps from whole plasma after applying zone electrophoresis in polyvinylchloride. The protein was characterzed by sedimentation rate, immunochemical analyses, isoelectric focusing, amino acid analysis and trypsin binding.Decreased binding capacity towards trypsin and plasmin could be detected after lyophylization of α2‐macroglobulin. Comparison with low‐temperature denaturation in sedimentation studies suggests that the functionally intact protein was not restored in spite of 90% reaggregation of the 11–12‐S subunit. The structure dependence of the enzyme binding is discussed.
Chick‐Erythrocyte Chromatin as a Template for RNA Synthesis in vitro
Authors: Bolund Lars , Johns Ernest W. 
Published: March 2005

The template activity of chick erythrocyte chromatin was measured with bacterial RNA polymerase and found to be low in comparison with that of calf thymus chromatin. The chromatins were precipitated in a standardized way from very dilute solutions prior to the assay in order to minimize differences in solubility or physical state between the templates which might affect their ability to support transcription.Selective and quantitative removal of the unique histone F2C from chick erythrocyte chromatin dramatically increased its template activity. Calf thymus chromatin devoid of histone F1 was, however, stil a better template than chick erythrocyte chromatin depleted of both F2C and F1. Reconstitution of the depleted chromatins with the adequate amount of histone restored the original template activites whether erythrocyte F2C or calf thymus F1 was used. The results argue against a unique functional role of histone F2C as a super‐repressor.Exchange of the majority of proteins between chick erythrocyte and calf thymus chromatin caused an exchange of template properties. Non‐histone proteins extracted from calf thymus chromatin with 0.35M NaCl did not “activate” the chick erythrocyte chromatin although they were found to bind to it.
The Interaction of Chaotropic Anions with Proteins at Acid pH
Authors: Aviram Irit 
Published: March 2005

The activity of pepsin on small substrates, as well as its autodigestibility, fluorescence and circular dichroism were unaffected by the presence of “disordering” anions.Pepsin activity on hemoglobin and bovine serum albumin was inhibited by the same anions, while the hydrolysis of lysozyme was enhanced.The anions also altered the fluorescence and circular dichroism spectra of bovine serum albumin. These findings are discussed in relation to conformational changes induced by disordering ions at acid pH.
Cytochrome b in Neurospora crassa Mitochondria
Authors: Weiss Hanns , Ziganke Barbara 
Published: March 2005

Cytochrome b was purified from Neurospora crassa mitochondria by means of chromatography on oleyl‐polymethacrylic acid resin, chromatography on DEAE‐cellulose and recycling gel filtration on Sephadex G‐75, all steps being performed in a medium containing bile acids and salts.The heme content of the preparation was calculated on the basis of incorporated 59Fe and [3H]‐leucine to be 33–40 μmol/g protein, corresponding to a minimum molecular weight of 25000 to 30000.Upon gel filtration on Sephadex G‐75 in a bile acid and KCl medium the preparation migrated like a 55000‐molecular‐weight protein. Upon gel electrophoresis on polyacrylamide or upon gel filtration on Sephadex G‐100 in a dodecylsulfate medium the protein part of the preparation migrated as one band of the apparent molecular weight of approximately 30000. These findings suggest that the purified cytochrome b is a dimeric heme protein. In vivo incorporation of [3H]leucine into the protein part of the purified cytochrome b was insensitive to cycloheximide but sensitive to chloramphenicol. This indicates that the apoprotein of cytochrome b is translated on mitochondrial ribosomes.
Light‐Induced Absorbance Changes of the High‐Potential Cytochrome b559 in Chloroplasts
Authors: Ben‐Hayyim Gozal 
Published: March 2005

Light‐induced absorption changes of cytochrome b559 have been studied in isolated chloroplast preparations at room temperature.
It was shown that cytochrome b559 could undergo both photooxidation and photoreduction, both these reactions occurring to its high‐potential form.
In the presence of FCCP and DCMU, a reversible photoreduction could be detected which was not sensitive to the presence of oxygen and which was probably a result of a cyclic electron flow around photosystem 2.
It is suggested that the high‐potential form of cytochrome b559, which is the native form, is mainly photooxidized by photosystem 2. The photoreduction is also mediated by photosystem 2 and can be detected only when electrons cannot flow from photosystem 2 to photosystem 1, i.e. in the presence of DCMU, or when the carriers are fully reduced in the absence of an electron acceptor.
Comparative Studies on the Template‐Initiator Requirements of the Chick‐Embryo DNA Polymerase I and the Avian‐Myeloblastosis‐Virus DNA Polymerase
Authors: Rougeon François , Brun Gilbert , Chapeville François 
Published: March 2005

The template requirements of chick embryo DNA polymerase I devoid of nuclease activity, have been investigated. This enzyme catalyzes a repair reaction in vitro. Active templates were obtained by treatment of native DNA by DNAase I or exonuclease III. The DNA polymerase I activity requires divalent cations and is inhibited by high salt concentrations and sulfhydryl group blocking reagents.Homopolydeoxynucleotides dAn, dTn, dCn, are efficient templates in the presence of hydrogen‐bound initiator. The high efficiency of hybrid structures rAn· dTn, dAn· rUn and rIn· dCn in which RNA acts as‐initiator and DNA as template suggests an essential role of this enzyme in DNA replication. The enzyme does not catalyze RNA‐directed DNA synthesis.DNA polymerase I of chick embryo and the avian myeloblastosis virus DNA polymerase were compared on the basis of their activities in the presence of various synthetic primers. Using synthetic DNA · RNA hybrid such as rAn· dTn, the cellular enzyme elongates the RNA strand by copying the DNA template whereas the viral enzyme mainly copies the RNA strand and less efficiently the DNA strand. With polymers of the (I + C) or (G + C) series, both enzymes are equally efficient which dCn as template; the viral enzyme activity can be distinguished from cellular enzymatic activity principally by its capacity to transcribe rCn and less efficiently rIn. The relationship between the responses of these enzymes in vitro to synthetic primers and the assumed physiological function of the cellular and viral DNA polymerases are discussed.
Energetic and Spectroscopic Studies on the Interaction between Thiamine Diphosphate and Apotransketolase
Authors: Heinrich Claus P. , Schmidt Dieter , Noack Klaus 
Published: March 2005

Molecular complexes found between indoleacetate and various thiamine derivatives have been studied as a model system for the coenzyme‐apoenzyme interaction in transketolase from baker's yeast. Association constants, ΔH and ΔS values for these complexes were determined by ultraviolet and proton magnetic resonance spectroscopy. No remarkable differences of these constants were found for the various thiamine derivatives, except tetrahydrothiamine and 4‐methyl‐5‐(2‐hydroxyethyl)thiazole, which did not give a complex with indoleacetate. These findings prove that only the positively charged thiazolium ring system is able to form a charge transfer complex with indoleacetate. If indoleacetate was replaced by tryptophan, charge transfer complex formation with the thiamine derivatives could also be demonstrated by circular dichroism measurements, where a new band around 320 nm appeared.A negative Cotton effect around 320 nm is also observed upon addition of thiamine diphosphate to apotransketolase. The circular dichroism spectra of transketolase reconstituted with the diphosphates of thiamine, oxythiamine, pyrithiamine and tetrahydrothiamine indicate different kinds of coenzyme binding. A ΔH of 7.4 kcal/mol was found for the enthalpy of formation of holotransketolase from its components.
Fluorescence Time‐Resolved Spectroscopy and Fluorescence Anisotropy Decay of the Staphylococcus aureus Endonuclease
Authors: Brochon Jean‐Claude , Wahl Philippe , Auchet Jean‐Claude 
Published: March 2005

The intrinsic fluorescence of Staphylococcus aureus endonuclease has been studied by the time‐resolved spectroscopy method. The tyrosine emission, with a decay time of 1.8 ns, is separated from the tryptophan emission with a decay time of 5.5 ns.The binding of an inhibitor, thymidine 3′,5′‐bisphosphate (pdTp), decreases the intensity of the tyrosine spectrum.The molecular correlation time determined by the anisotropy decay measurement of the tryptophanyl residue is 8.9 ns. This value corresponds to a strongly hydrated molecule.The fixation of pdTp does not change this correlation time, showing that no gross structural modifications are induced by the inhibitor.
Studies of Glutamate Dehydrogenase
Authors: Rasched Ihab , Sund Horst , Jörnvall Hans 
Published: March 2005

Glutamate dehydrogenase was reacted with glyoxal and NaB3H4 and the labelled protein digested with trypsin. The radioactive peptides were isolated and identified in the primary structure of the enzyme.Many peptides contained small amounts of radioactivity which is probably due to unspecific reactions with glyoxal. The amino group of one lysine residue was labelled to a greater extent than any other residue and accounted for about half of the total label. This amino group is involved in the binding of the substrate 2‐oxoglutarate and was identified as lysine‐27. The lysine‐126 which reacts with pyridoxal phosphate and 4‐iodoacetamidosalicylate is not modified with glyoxal but also involved in the binding of the substrate. This leads to the conclusion that both these lysine residues, although separated by 99 amino acids in the primary structure, may be at the active site of the enzyme and hence positioned close together in the tertiary structure.
Enzymatic and Chemical Synthesis of 3‐Sulfinopropionic Acid, an Analog of Succinic Acid
Authors: JollÉS‐BERGERET Bernadette 
Published: March 2005

3‐Sulfinopropionic acid, the product of transamination and dehydrogenation of homohypotaurine (3‐aminopropanesulfinic acid), was isolated as the barium salt from incubated extracts of Pseudomonas fluorescens. The sodium and barium salts of the same compound were chemically synthetized from acrylic acid and formaldehyde sulfoxylate with the intermediary formation of bis(2‐carboxyethyl)sulfone. The identity of the compounds prepared either by enzymatic or by chemical synthesis has been demonstrated.Some physicochemical properties of 3‐sulfinopropionic acid are deseribed.