bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reversible Chemical Modification of the Tryptophan Residues of Thioredoxin from Escherichia coli B
Authors: Holmgren Arne 
Published: March 2005

Treatment of thioredoxin from Escherichia coli with formic acid saturated with HCl by the method of Previero et al. (1967), resulted in a redshift of the ultraviolet spectrum of the protein consistent with chemical modification of both tryptophans to 1‐formyl‐tryptophanyl residues. Concomitantly with the formylation the protein lost its redox activity in the enzymatic reactions with thioredoxin reductase and ribonucleotide reductase. Incubation of thioredoxin in formic acid alone had no effect on the spectrum or biological properties of the protein.Gel chromatographic experiments on Sephadex G‐50 and G‐75 showed that the formylated thioredoxin was eluted well apart from native thioredoxin and existed in the form of high molecular weight aggregates. This indicated that major conformational changes of thioredoxin accompanied the conversion of the hydrophobic tryptophan residues into the more hydrophilic 1‐formyl‐tryptophans.Treatment of 1‐formyl‐tryptophanyl thioredoxin at pH 9.5 in 6 M guanidine hydrochloride for 16 h resulted in almost complete regeneration of the ultraviolet spectrum of native thioredoxin. Also regenerated was more than 50% of thioredoxin activity in the reaction with thioredoxin reluctase. The results of this reversible chemical modification strongly indicate that the two tryptophan residues play a major role in maintaining the biologically active conformation of thioredoxin from E. coli.
Effects of Aldosterone and Other Steroids in vivo on Rat‐Kidney Cortex
Authors: Trachewsky Daniel , Majumdar Adhip. P. Nandi , Congote L. Fernando 
Published: March 2005

An investigation of the effects of aldosterone, deoxycorticosterone, 9α‐fluorocortisol, and 17β‐estradiol administration, respectively, to adrenalectomized male rats on the polypeptide synthesis by and poly(U)‐directed binding of phenylalanyl‐tRNA to isolated renal cortical ribosomes has revealed several interesting facts. As well as causing an increase in the ability of the ribosomes to synthesize polyphenylalanine in the presence of poly(U), the three mineralcorticoids also increased the poly(U)‐directed binding of phenylalanyl‐tRNA to the ribosomes. 17β‐Estradiol treatment, however, caused a decrease in the ability of the ribosomes to incorporate phenylalanine into polyphenylalanine when compared to the untreated controls. There were also no significant differences in the amount of poly(U)‐directed binding of phenylalanyl‐tRNA to the ribosomes of the estrogen‐treated rats when compared to the untreated animals. Consequently, it was concluded that all four steroids had induced some alterations in the properties of the renal cortical ribosomes.The effect of aldosterone and 17β‐estradiol on the thiol content of the nuclear proteins from rat kidney cortex showed an interesting correlation with the translational effects of both steroids. Thus aldosterone increased, and 17β‐estradiol slightly decreased, the thiol content of these proteins, measured as the incorporation of radioactive iodoacetate in whole nuclei in vitro. Under the experimental conditions used, the reaction was especially concentrated in discrete protein fractions of molecular weights of about 14000 and 42000. The effect of the hormones on the thiol content was not specifically concentrated in any of these protein fractions.
Kinetic Study of the Tricarboxylate Carrier in Rat Liver Mitochondria
Authors: Palmieri Ferdinando , Stipani Italo , Quagliariello Ernesto , Klingenberg Martin 
Published: March 2005

The kinetics of citrate uptake by malate‐loaded mitochondria were measured using the inhibitor stop method and analysed for possible carrier mechanisms.

The citrate exchange is found to follow a first order reaction with a constant k of 1.18 min−1 and the corresponding rate of citrate uptake of 13.4 μmol × min−1× g protein−1 (at 0.5 mM citrate and 9 °C). The half time is 34 sec.
The temperature dependence of citrate exchange was measured in the range between 0 and 14 °C. An activation energy of 20.1 kcal and a Q10 of 3.6 can be calculated from the Arrhenius plot.
The concentration dependence of citrate exchange reveals hyperbolic saturation characteristics. The Km and V values for the rate of citrate uptake are 0.12 ± 0.01 mM and 22.5 ± 1.8 μmol citrate × min−1× g protein−1 respectively at 9 °C in 11 experiments.
The rate of citrate uptake has a pH optimum of about 7. The inhibition by higher pH is competitive with citrate. At pH lower than 7, V is decreased, indicating that the citrate transporting system is inactivated by acid pH.
The rate of citrate exchange is inhibited in a competitive manner by cis‐aconitate, threo‐Ds‐isocitrate, 1,2,3‐propanetricarboxylate, 1,2,3‐benzenetricarboxylate, citrate and propylcitrate. Other tricarboxylates, however, such as trans‐aconitate and 1,3,5‐pentanetricarboxylate have no effect on citrate exchange, even when added in large excess.
Succinate, malonate, oxaloacetate and oxomalonate inhibit the rate of citrate uptake. In contrast, glutarate, adipate, pimelate, 2‐oxoglutarate, aspartate and glutamate do not. Maleate, but not the trans isomer fumarate, also inhibits citrate uptake. The inhibition of citrate uptake is greater with malate than with succinate.
The rate of citrate uptake is also inhibited by the nonpenetrant anions phenylsuccinate, butylmalonate, benzylmalonate and pentylmalonate, previously thought to be specific inhibitors of the dicarboxylate carrier.
The inhibition of the rate of citrate uptake by dicarboxylates and their analogues is found to be competitive. The affinity for dicarboxylates is lower than for tricarboxylates (K1 is 0.7 mM for malate).
Phosphoenolpyruvate strongly inhibits the rate of citrate uptake, while P1 and pyruvate have only a slight effect. The inhibition by phosphoenolpyruvate is shown to be competitive (K1 is 0.11 mM).
It is concluded that the tricarboxylate carrier has a single binding site for tricarboxylates, phosphoenolpyruvate and dicarboxylates. The implications of these findings are discussed.
Isolation of Antitransaminase Antibodies with Selective Reactivity against Transaminase or Its Tryptic Hydrolysate
Authors: Katsiris Katherine D. , Dimitropoulos Constantin G. , Patramanis Iphigenia M. , Pavlatos Mary P. , Evangelopoulos Athanasios E. 
Published: March 2005

Fractionation of antitransaminase or anti‐whole‐tryptic transaminase hydrolysate antibodies was accomplished by using solid conjugates of the enzyme or its whole‐tryptic hydrolysate with albumin. By mixing in different orders, the solid complexes with antiserums prepared after immunization of experimental animals with transaminase or its hydrolysate, four antibody fractions (A, B, C and D) were isolated.
The antibody reactivity of the isolated fractions was tested by micro‐complement fixation, inhibition of the catalytic activity of the enzyme and haemagglutination. It was found that fractions B and C exhibited more reactivity with the intact transaminase molecule, while A and D reacted more specifically with the various fragments of the enzyme hydrolysate.
After fractionation of the proteolytic hydrolysate through a Sephadex G‐25 column, it was found that high molecular weight peptides were more reactive against the antibody fractions A and D, which reacted specifically with the hydrolysate. On the contrary, low molecular weight peptides reacted more intensively with the antibody fractions B and C, specific for the intact transaminase molecule.
Heterogeneity of the isolated antibody fractions, as well as of the total population of antibodies against transaminase or its whole tryptic hydrolysate, has been demonstrated by analytical acrylamide disc electrophoresis and isoelectric focussing.
DNA Methylases of Escherichia coli K12
Authors: Bouché Jean‐Pierre , Dubert Jean‐Marie 
Published: March 2005

The overall DNA methylating activity of Escherichia coli K 12 has been purified about 1500‐fold. This procedure involves the preservation of high ionic strength during successive steps to keep the activity fairly stable. Both adenine and cytosine activities are purified in these conditions although their ratio varies greatly. Thermodenaturation and sedimentation studies on the most purified fraction show that the adenine and cytosine methylating enzymes are unrelated single protein species.The sedimentation profile of the most purified activity is different from that observed with crude extracts or partially purified fractions. When heat inactivated, these fractions added to the purified enzyme restore the activity profile which they showed when native. Thus changes in sedimentation properties are due to a factor present in these fractions and lost in the course of the purification.
The Complete Amino‐Acid Sequence of Human α‐Lactalbumin
Authors: Findlay John B. C. , Brew Keith 
Published: March 2005

α‐Lactalbumin was isolated from human milk in a yield of 1.8 mg/ml of milk. The purification procedure involved ammonium sulphate fractionation (30% to 80% saturation) and pH‐4.0 precipitation, followed by gel filtration with Sephadex G‐100. A final purification stage using DEAE‐cellulose was necessary in some preparations.Peptides derived from the reduced, S‐aminoethylated protein by treatment with cyanogen bromide and digestion of these CNBr fragments with trypsin, chymotrypsin or thermolysin, were purified by gel filtration, ion exchange chromatography and high‐voltage paper electrophoresis.From the sequences of these peptides it has proved possible to deduce unambiguously the complete primary structure of the protein. Comparison with bovine α‐lactalbumin shows an identity in 72% of the residues with a further 6% being chemically similar amino acids. The corresponding figures for the human α‐lactalbumin/human lysozyme comparison, are 39% and 12%, respectively. The significance of some of the amino acid replacements is discussed.
Degradation of the Murein · Lipoprotein Complex of Escherichia coli Cell Walls by Dictyostelium Amoebae
Authors: Braun Volkmar , Hantke Klaus , Wolff Helga , Gerisch Günther 
Published: March 2005

Amoebae of the cellular slime mold Dictyostelium discoideum can be grown in submerged culture on Escherichia coli W7. This strain is deficient in the synthesis and decarboxylation of diaminopimelate, the amino acid specific for murein. With [3H]diaminopimelate‐labeled E. coli W7, it was found that the murein · lipoprotein complex (rigid layer) of the bacterial cell wall is degraded completely by the amoebae, and diaminopimelate is converted to lysine which is then incorporated into protein. Cleavage of the bond between N‐acetylmuramic acid and l‐alanine and release of alanine from the murein peptide side chain is shown in vitro. Several glycosidases which degrade the polysaccharide chains of the murein were separated and both muramidase and N‐acetyl‐β‐d‐glucosaminidase activity could be demonstrated. The amoebae also contain proteolytic and lipolytic enzymes which degrade the lipoprotein in vitro. From the lipid which is covalently bound to the protein, palmitic acid was released. All these hydrolases have an optimum of pH 3 which suggests a lysosomal origin.Amoebae of Dictyostelium discoideum strain ax‐2, grown in a bacteria‐free medium take up isolated murein · lipoprotein complex. These amoebae contain, without prior growth on E. coli cells, the hydrolases which are responsible for the degradation of the murein · lipoprotein complex and for the conversion of diaminopimelate into lysine.
Medium Hypertonicity and Polyribosome Structure in Hela Cells
Authors: Wengler Gerd , Wengler Gisela 
Published: March 2005

The influence of hypertonicity of the growth medium on polyribosome structure in Hela cells was measured after double labelling of the cells under conditions where the ribosomal RNA and the polyribosomal messenger RNA were labelled with [14C]uridine and [3H]uridine, respectively.Increase of the tonicity of the growth medium by addition of NaCl or sucrose leads to a total disaggregation of the cellular polyribosomes within less than 10 min. The process is fully reversible, even if RNA synthesis is blocked by high doses of actinomycin D, and can be repeated at least twice under these conditions.This reaction is probably caused by a specific block of initiation of protein synthesis under these conditions.The ribosomes are set free as 80‐S monoribosomes free of messenger RNA, which is found in the lysate in the form of ribonucleoprotein complexes of widely varying sedimentation velocities.Some possible implications of these findings are discussed.
Sequence of the Murein · Lipoprotein and the Attachment Site of the Lipid
Authors: Braun Volkmar , Bosch Valerie 
Published: March 2005

The primary structure of a covalent lipoglycoprotein (murein · lipoprotein) from the cell wall of Escherichia coli is presented. The amino acid sequence consists of 57 residues. The lipid is attached to the N‐terminal serine and the murein is bound to the 6‐amino group of the C‐terminal lysine of the lipoprotein. The repetitive design of the sequence suggests a very conservative evolution in which a structural gene coding for 15 amino acids was duplicated once and then only half of this gene, thus coding for 7 amino acids was fused four times with the first 29 amino acids. Some deletions but only few exchanges of amino acids apparently occurred.This structural membrane lipoprotein aggregates strongly when released from the murein by lysozyme digestion. The molecular weight was therefore determined in 2%, 0.5% and 0.1% dodecylsulfate by polyacrylamide gel electrophoresis and gel chromatography. The molecular weight of 13000 thus found deviated from that of 10000 calculated from the sum of the amino acids, the lipid as known so far and the part of the murein remaining bound to the lipoprotein. This was not due to a different amount of dodecylsulfate binding by the lipoprotein compared with proteins containing no lipid but may be due to a more spherical shape of the small molecule in contrast to the rod‐like shape of standard proteins in dodecylsulfate.
The Structural Subunit of Amyloid
Authors: Zuckerberg Avraham , Gazith Joseph , Rimon Abraham , Reshef Tamar , Gafni Joseph 
Published: March 2005

“Water‐soluble” amyloid, prepared from three organs obtained from two patients (one with genetic and one with secondary amyloidosis), was purified by sequential treatment with 10% NaCl and 90% acetone. After dissociation in 5 M guanidine‐HCl, ultrafiltration isolated a low molecular weight component which we designated F‐protein. On polyacrylamide‐dodecylsulfate electrophoresis, F‐protein proved to be essentially homogeneous and had a mobility of 1.07 relative to bromophenol blue marker. In all three preparations, the amino acid composition, the 16.4–17.1% nitrogen content, the < 0.25% neutral sugars and the glycine amino terminal indicate a single polypeptide in pure state. This polypeptide, in contrast to the parent material, showed neither green birefringence after Congo red staining nor fibrillar structure on electron microscopy.Reorganization into fibrils and recovery of staining properties identifying amyloid was obtained by solution of the isolated polypeptide in 8M urea and sequential exchange of the solvent by 0.05 M NaHCO3 and distilled water.These observations suggest that F‐protein is the structural subunit of the amyloid fibril.
Galactose Transport in Escherichia coli and the Galactose‐Binding Protein
Authors: Vorisek Josef , Kepes Adam 
Published: March 2005

Galactose uptake and galactose‐binding protein have been examined in a galactokinase‐deficient mutant of Escherichia coli K108.

[14C]Galactose was found to be accumulated up to one‐hundred‐thousand‐fold in the cytoplasm and to be subject to permanent turnover with unchanged velocity.
The concentration‐dependence curve exhibits sigmoïdicity and reveals a very high affinity of the system for galactose. The spectrum of specificity was explored by competition.
Metabolic inhibitors such as fluoride and uncouplers have weak or moderate effects, only a combination of several inhibitors can cause an efflux at a rate comparable to the rate of uptake.
Among inactivating agents N‐ethylmaleimide, parachloromercuribenzoate and dinitrofluorobenzene have no short term effects. Mercuric chloride and formaldehyde are strong inactivators. The effect of HgCl2 is reversible by excess thiol.
Only small amounts of barium‐precipitable derivatives of galactose (e.g. galactose 6‐phosphate) are observed and these do not exhibit turnover.
The concentrated shock fluid binds [14C]galactose in equilibrium dialysis experiments. The binding curve is sigmoïdal with respect to galactose.
The binding is not abolished by HgCl2. The implications of these findings on possible transport mechanisms are discussed.
Resolution of the Hemolytic and the Hydrolytic Activities of Phospholipase‐C Preparation from Clostridium perfringens
Authors: Sabban Esther , Laster Yehudith , Loyter Abraham 
Published: March 2005

Phospholipase‐C preparations from Clostridium perfringens can induce hemolysis of chicken erythrocytes in the presence of EDTA. Under these conditions hydrolysis of membrane phospholipids does not take place. When Ca2+ is added to the incubation media both hemolysis and hydrolysis of phospholipids occur. However, when Mn2+ or UO22+ is added, both the hemolysis and hydrolysis of membrane phospholipids are strongly inhibited.When phospholipase‐C (Cl. perfringens) preparations are heated at 56°C in the presence of Ca2+, the hemolytic activity is inhibited while the hydrolytic activity of the enzyme is retained. Such non‐hemolytic phospholipase‐C barely hydrolyse phospholipids from intact chicken red‐blood cells. However, it readily hydrolyses phospholipids from erythrocyte ghosts.Thus it appears that membrane phospholipids are unavailable to the enzyme unless they are exposed by hemolysis.
An Alkaline Protease from Acremonium kiliense
Authors: VAN Heyningen Simon 
Published: March 2005

A protease, purified from the growth medium of Acremonium kiliense Grütz, has a pH optimum of 10.5 for the hydrolysis of casein.
It hydrolyzes the reduced, carboxymethylated B‐chain of bovine insulin at 13 points, principally those next to the carboxyl group of aromatic and hydrophobic amino acids, but not at the basic amino acid residues.
It does not hydrolyze dipeptides, benzoyl arginine ethyl ester or glutaryl‐l‐phenylalanine‐p‐nitroanilide.
The enzyme can be titrated with N‐trans‐cinnamoyl imidazole and assayed with the p‐nitrophenyl esters of benzoyloxycarbonyl amino acids.
The enzymic hydrolysis of N‐acetyl phenylalanine ethyl ester can be followed by an absorbance change at 221 nm or by means of a pH‐stat. Analysis of the variation of Km and V with pH for this substrate suggests that there is a group involved in the activity which has a pK of 6.6 in the free enzyme and in the active enzyme · substrate complex.
It is inhibited by diisopropylphosphofluoridate and phenylmethylsulphonyl fluoride, suggesting that it is a serine protease. It is unaffected by metals, metal‐chelating agents, iodoacetate or 2‐mercaptoethanol.
Thermochemistry of the Avidin‐Biotin Reaction
Authors: Suurkuusk Jaak , WadsÖ Ingemar 
Published: March 2005

The enthalpy for the binding of biotin to avidin has been determined at 25, 34 and 43°C (pH 9, ammonium acetate buffer): ΔH(25°C) =–22.5 ± 0.1 kcal/mol biotin; ΔCp=–237 ± 12 cal × mol biotin−1× K−1.Solubilities and enthalpies of solution of biotin in water and in ethanol were determined at 25°C. The transfer process for biotin between water and ethanol was considered as a simple model for the protein binding process.
The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K12
Authors: Véron Michel , Falcoz‐Kelly Françoise , Cohen Georges N. 
Published: March 2005

Aspartokinase I‐homoserine dehydrogenase I from Escherichia coli K 12 was subjected to mild proteolysis. A fragment carrying only the desensitized homoserine dehydrogenase activity was purified. It is a dimer having a subunit molecular weight of 55000 as opposed to 86000 for the subunit of the native tetrameric enzyme.On the other hand, a threonine sensitive aspartokinase devoid of homoserine dehydrogenase activity was extracted from the mutant Gif 108. The purified protein is shown to have subunits shorter than those of the wild‐type enzyme (molecular weight 47000).It was shown that the two catalytic activities of aspartokinase I‐homoserine dehydrogenase I are sequentially distributed on the single polypeptide chain: the aspartokinase activity is located in the amino‐terminal section and the homoserine dehydrogenase in the carboxyl‐terminal section.The results are discussed in relation to the configuration of the native enzyme and with the possible origin of the bifunctional protein.
Characterization of Chicken Red‐Cell RNAase
Authors: Sánchez De Jiménez Estela , Léon De Miles Ma. de la Paz , Ruth Román P. 
Published: March 2005

RNAase from 13‐day‐old chicken‐embryo red cells was purified. Nuclei and cytoplasmic fractions were used as source of the enzyme. The nuclear enzyme was purified 1440‐fold and the one from cytoplasm, 1683‐fold.Both enzyme preparations showed the same optimum pH and ion specificity, but different ionic strength dependence and temperature stability.Kinetic studies with these RNAase fractions indicated that both of them are endonucleases with specificity for pyrimidine nucleotide bases in the substrate, hydrolyzing poly(U) or poly‐(A,U,G) faster than poly(C) or poly(A,C,G). Both enzyme fractions showed strong dependence on the sedondary structure of RNA, and did not hydrolyze single stranded DNA. The implications of these characteristics on the enzyme role are discussed.
Oxidation‐Reduction Potentials and Ionization States of Two Turnip Peroxidases
Authors: Ricard Jacques , Mazza Gilbert , Williams Robert J. P. 
Published: March 2005

In the present paper, differences in the environment of the iron atom of two turnip isoperoxidases, called P1, and P7, have been established. This study is performed by way of spectrophotometric titrations, and by the analysis of oxidation‐reduction potential variations with regard to hydrogen ion concentration.Turnip peroxidases P1 and P7 exhibit near infrared bands with intensities and energies almost identical to those of metmyoglobin and horseradish peroxidase.In the case of turnip hemoprotein P1, an ionization, with an apparent pK close to 10, has been shown only in the oxidized form of the enzyme. Two other ionizations, in the ferric and the ferrous forms, respectively, occur also. They possess the same apparent pK, equal to 4.3, and seem to correspond to the same group. Another ionization is seen, with an apparent pK equal to 6.3, which specifically occurs in the reduced peroxidase. These results are very close to those obtained by various workers on horseradish peroxidase.In the case of turnip hemoprotein P7, the situation is very different. An ionization, with a pK close to 6.5, occurs in the ferric form of the enzyme. Two other ionizations, one in the oxidized form, the other in the reduced form of the enzyme, occur also. They both possess the same apparent pK equal to 8.4. A further ionization pertaining to the ferrous form only, is also seen, with an apparent pK close to 5.The above results are in agreement with the existence of the same axial ligands, namely histidine and water, in both hemoproteins. A likely interpretation of the ionizations detected in the acidic pH range is to assume that the distal groups of the proteins, for both peroxidases P1 and P7, are different. The occurrence, respectively, of a carboxyl group and a histidine residue would agree with the spectrophotometric and potentiometric titration data.The high value of the oxidation‐reduction potentials of peroxidase P7, and the unexpected stability of the oxygenated compound of its ferrous form, are also compatible with an oxygenase function of this hemoprotein.