bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Use of Affinity Chromatography in Determining the Sites of Protein‐Protein Interaction Relative to the Binding Sites of Substrates in Gramicidin S Synthetase
Authors: Pass Lidia , Zimmer Trine‐Lise , Laland Søren G. 
Published: March 2005

Columns containing one of the amino acids in gramicidin S bound through its carboxyl group to the amino group of amino‐alkyl‐Sepharose (CNBr‐activated Sepharose + 3,3′‐diaminodipropylamine) have been prepared and were found to exhibit affinity for the corresponding enzymes (light or heavy) of gramicidin S synthetase. The d‐phenylalanyl ligand will bind the light enzyme and the l‐propyl, l‐valyl, l‐ornithyl or l‐leucyl ligand, the heavy enzyme. These columns permitted a study of the interaction of the light and heavy enzyme. The results show that the area of the light enzyme which has affinity for the heavy enzyme is that part which activates phenylalanine. The area of the heavy enzyme which interacts with the light enzyme is not that part of the enzyme which activates proline. However, it is probably located close to the activation sites for proline and leucine.The columns, in particular with proline as ligand, provide a quick and convenient method for the separation of the light and heavy enzyme of gramicidin S synthetase.
Differently Labelled Glucosamine‐Precursor Pooles for the Biosynthesis of Hyaluronate and Heparan Sulfate
Authors: von Figura Kurt , Kiowski Wolfgang , Buddecke Eckhart 
Published: March 2005

14C‐labelled hyaluronate and heparan sulfate were isolated from bovine arterial tissue following incubation in vitro in the presence of [U‐14C]glucose and [1‐14C]glucosamine, respectively. The specific radioactivity of hyaluronate was higher than that of heparan sulfate, but the ratio of their specific radioactivities (hyaluronate/heparan sulfate) was 2.5 or 1.2, depending on whether [14C]glucose or [14C]glucosamine was used as the metabolic precursor.
In the [14C]glucose experiments the proportion of radioactivity incorporated into the glucosamine and glucuronic acid moiety of hyaluronate was about 1:1, while in the case of heparan sulfate the corresponding ratio was 1:1.7. Values of 3.3 and 1.1 were calculated for the specific radioactivity ratio of the glucosamine moiety of hyaluronate and heparan sulfate (glucosamine‐hyaluronate/glucosamine‐heparan sulfate) following labelling with [14C]glucose and [14C]glucosamine, respectively.
The results are consistent with the assumption that two topographically different pools of immediate metabolic precursors exist in bovine arterial cells, one being for the biosynthesis of hyaluronate and the other for heparan sulfate.
The Specificity of Escherichia coli Endonuclease I
Authors: Ehrlich Stanislav D. , Bertazzoni Umberto , Bernardi Giorgio 
Published: March 2005

Degradation of different DNA's by E. coli Endonuclease I was studied. Hyperchromicity and acid solubility of the digest as well as the average chain length (P̄n) of oligonucleotides released by the enzymes were followed during degradation; relationships between these parameters were established.The specificity of the E. coli Endonuclease I was determined by analysing 3′‐terminal, 5′‐terminal and 5′‐penultimate nucleotide of oligonucleotides formed by the enzyme from calf thymus DNA. The compositions found differ from those expected on the basis of random breakage of DNA; this indicates that E. coli Endonuclease I recognizes sequences at least three nucleotides long.The 3′‐terminal nucleotide composition was determined over a wide range of P̄n (150–4) and was found to be invariant, indicating that the specificity of E. coli Endonuclease I does not change during digestion.Composition of 3′‐terminal nucleotides released from different bacterial DNA's varied linearly with the DNA (dG + dC) contents; those released from the calf thymus DNA did not fit that relationship.
Transglycosylic Reactions of Nucleotides of Deoxy‐Sugars
Authors: Zemek Jiří , Kučár Štefan , Bauer Štefan 
Published: March 2005

Incorporation of 3‐deoxy‐, 4‐deoxy‐, and 6‐deoxy‐analogues of d‐glucose into glycogen was investigated in a reaction catalyzed with yeast UDPglucogen glucosyl transferase. It was found that hydroxyl groups at positions C‐3, C‐4 and C‐6 in the glucosyl moiety of the UDPG molecule are not essential for preserving its substrate properties in this transglycosylic reaction.The kinetics of 3‐deoxy‐, 4‐deoxy‐ and 6‐deoxy‐glucose incorporation into glycogen was compared with that of d‐glucose and 2‐deoxy‐glucose. It was found that the affinity of the enzyme and the rate of hexose incorporation into glycogen decreases in following sequence: d‐glucose, 2‐deoxy‐, 6‐deoxy‐, 4‐deoxy‐ and 3‐deoxyglucose.The deoxyglucosyl analogues of UDPG act as competitive inhibitors on the reaction of glycogen biosynthesis.
Initiation Factor‐Dependent Binding of MS2 RNA to 30‐S Ribosomes and the Recycling of IF‐3
Authors: Vermeer Cees , van Alphen Wim , van Knippenberg Peter , Bosch Leendert 
Published: March 2005

The binding of washed 30‐S ribosomal subunits of Escherichia coli to MS2 RNA in the absence of fMet‐tRNA requires the presence of the initiation factor IF‐3. No binding is observed with IF‐1, IF‐2 or a combination of these two initiation factors.
The IF‐3‐dependent binding is stimulated about two‐fold by IF‐2. IF‐1 has no effect in this respect. Optimal binding occurs at about 7 mM Mg2+.
Upon incubation of [35S]IF‐3, MS2 [3H]RNA and 30‐S subunits, complexes are formed which contain the three components in a 1:1:1 ratio. These ternary complexes have a sedimentation coefficient of about 40 S and a buoyant density in CsCl of about 1.74 g/ml.
The ternary complexes formed in the absence of IF‐2 and IF‐1 are rather labile and readily dissociate into MS2 RNA and IF‐3‐containing ribosomes. Their half‐life at 0°C is about 40 min. In the presence of IF‐2 and IF‐1, complexes are formed which remain stable for at least 6 h. Unwashed native 30‐S subunits also form stable complexes with MS2 RNA.
Binding of washed 30‐S subunits to unfolded MS2 RNA (MS2 RNA treated with formaldehyde) does not require initiation factors. Complexes containing more than one ribosomal particle per messenger can be formed. Attachment of fMet‐tRNA to these complexes requires IF‐2 and IF‐1, but is optimal in the presence of all three initiation factors.
Release of [35S]IF‐3 from the MS2 RNA · 30‐S subunit complexes occurs upon attachment of fMet‐tRNA. In the case of unwashed native 30‐S ribosomal subunits, recycling of IF‐3 occurs at the same stage as was demonstrated by a direct determination of the stoichiometry of unlabeled IF‐3 on the ribosomal complexes.
Separation and Characterization of Polypeptides from the Venom of Dendroaspis viridis
Authors: Shipolini Rudolph A. , Bailey Graham S. , Edwardson James A. , Banks Barbara E. C. 
Published: March 2005

The venom of Dendroaspis viridis has been fractionated by chromatography on Sephadex G‐50 and SE‐Sephadex. Thirteen polypetides containing either 59–63 amino acid residues and four disulphide bridges or 71–73 amino acid residues and five disulphide bridges have been isolated and characterized by amino‐acid analysis and N‐terminal residue determination. The compositions of the polypeptides show many similarities but the toxicities vary in excess of 100‐fold, upwards from an LD50 of 0.08 mg/kg in mice.Enzymic and nerve growth factor activities have been located in protein fractions associated with higher molecular‐weight material.
Active Subunits of Transaldolase Bound to Sepharose
Authors: Chan William W.‐C. , Schutt Hermann , Brand Karl 
Published: March 2005

Individual subunits of yeast transaldolase were studied by the method of matrix‐bound derivatives. Native dimeric transaldolase was bound to Sepharose activated with low amounts of cyanogen bromide to give matrix‐bound transaldolase in which the majority of molecules were bound covalently via only one subunit. Exhaustive washing with 6 M guanidinium chloride and renaturation in triethanolamine‐EDTA buffer resulted in a derivative (bound‐subunit transaldolase) which retained 65% of the original protein and 63% of the original activity. This active form of transaldolase is more sensitive to 2 M urea than bound‐dimeric transaldolase. Bound‐subunit transaldolase is able to associate with “nascent” soluble transaldolase subunits generated in situ by diluting a small aliquot of guanidinium‐chloridedenatured transaldolase into a much larger volume of buffer. The ability of bound‐subunit derivative to pick up soluble “nascent” transaldolase subunits is highly specific and reaches a plateau level close to the original protein and activity content of bound transaldolase. The product of such treatment (bound‐renatured transaldolase) has properties virtually indistinguishable from those of bound transaldolase. These results indicate that individual subunits of transaldolase are catalytically active.Bound‐subunit transaldolase has Km values similar to those of the bound‐dimeric derivatives and can be inactivated with sodium borohydride in the presence of the substrate fructose 6‐phosphate. However, the pH profile of the subunit form is substantially altered suggesting a shift in the pKa of an essential protonated group from 9.2 to 8.6.
Primary Structure of a Small Glycopeptide Isolated from Human Glomerular Basement Membrane and Carrying a Major Antigenic Site
Authors: Mahieu Philippe M. , Maghuin‐Rogister Guy R. , Lambert Paul H. 
Published: March 2005

The glycopeptides containing either the disaccharide or the heteropolysaccharide chains were purified, after enzymatic digestion of glomerular basement‐membrane antigens, by gel filtration and ion‐exchange chromatography. Their antigenicity was investigated in seven patients with anti‐glomerular‐basement‐membrane antibody‐mediated glomerulonephritis, by the leukocyte migration test and radioimmunoassay.The leukocyte migration of the seven patients is inhibited in presence of membrane antigens with both types of oligosaccharide chains. The disaccharide‐containing glycopeptide inhibits the leukocyte migration of these patients using 10‐times less antigenic material; the heteropolysaccharide‐containing glycopeptide does not modify the leudkocyte migration of the same patients. Similarly, only the disaccharide‐containing glycopeptide decreases the binding of labelled membrane antigens with anti‐glomerular‐basement‐membrane antibodies, using the radioimmunoassay. This glycopeptide carries therefore the major antigenic determinants of humoral and cellular anti‐basement‐membrane auto‐immunization.The amino acid sequence of the glycopeptide is Hyl‐Gly‐Glu‐Asp‐Gly, the disaccharide prosthetic group being linked to the hydrolysine residue of the peptidic chain; its immunodominant antigenic site is formed by the Galp‐Hyl unit.
Pulse‐Recovery Studies on Cycloheximide‐Insensitive Protein Synthesis in Neurospora
Authors: Lansman Robert A. , Rowe Mark J. , Woodward Dow O. 
Published: March 2005

The effects of cycloheximide on cytoplasmic protein synthesis in Neurospora are reversible. In these experiments, the products of mitochondrial protein synthesis were labeled in vivo in the presence of cycloheximide; analyses were made of the size distribution and enzyme association of these products as the cells recovered from cycloheximide inhibition.In cells harvested immediately after a 20‐min cycloheximide pulse, four protein species of high specific activity were resolved in polyacrylamide gels of mitochondrial protein electro‐phoresed in the presence of 1% sodium dodecylsulfate. Only small amounts of labeled protein were found to co‐purify with cytochrome oxidase isolated from these cells. In cells harvested after two to three hours of recovery from cycloheximide inhibition, three of the four original labeled peaks remain in whole mitochondrial protein. These peaks correspond precisely in gel mobility to the three highest‐molecular‐weight protein species associated with cytochrome oxidase, species which are heavily labeled in enzyme preparations from recovery cultures. These gel patterns can be obtained only when the preparations are treated with phenylmethylsulfonyl fluoride, an inhibitor of protease activity. The results obtained are discussed in terms of an assembly process in which the major stable products of mitochondrial protein synthesis become integrated into the cytochrome oxidase complex only when cytoplasmically‐made components are available for assembly.
Elongation Factor 1 from Krebs II Mouse Ascites Cells
Authors: Drews Jürgen , Bednarik Karl , Grasmuk Hans 
Published: March 2005

A procedure for the purification of elongation factor 1 (EF‐1) from Krebs II mouse ascites cells is described. Ascites EF‐1 occurs in multiple forms of different molecular weight like the corresponding enzymes from calf brain and rat liver. Biogel A‐5m chromatography of our purest EF‐1 resulted in a pattern indicating the presence of different molecular weight components. However, only a single polypeptide band was observed when this same material was analysed by electrophoresis on acrylamide gels containing sodium dodecylsulphate. With this method the molecular weight of the EF‐1 polypeptide chain was determined to be 47000. It is suggested that the occurrence of multiple forms of EF‐1 is due to the association of 47000‐mol. wt subunits to form aggregates of different sizes.The factor has essentially the same enzymatic properties which were described for EF‐1 from reticulocytes, calf brain and rat liver. The attachment of aminoacyl‐tRNA to ribosomes catalyzed by EF‐1 follows a strict 1:1 stoichiometry. EF‐2 when introduced into the ribosomal binding assay allows the recycling of EF‐1 and subsequent synthesis of polypeptide chains. The possible implications of this finding with respect to the conditions which allow stoichiometric or catalytic function of EF‐1 are discussed.
Cytochrome b2 from Bakers' Yeast (L‐Lactate Dehydrogenase)
Authors: Jacq Claude , Lederer Florence 
Published: March 2005

Bakers' yeast cytochrome b2 [lL‐(+)‐lactate dehydrogenase or l‐(+)‐lactate cytochrome c oxidoreductase], usually purified by crystallisation according to Appleby and Morton, was shown to be a degraded form of the enzyme, arising by a proteolytic modification of the species formed in vivo. We present here an improved purification procedure and some properties of the intact enzyme.This intact cytochrome b2 is shown, by cross‐linking and by gel filtration studies, to be in a tetrameric form with a molecular weight of about 230 000. The four identical subunits are composed of a single polypeptide chain associated with one haem and one flavin (FMN). The amino‐acid composition is presented. Alanine and glutamic acid are shown to be respectively C‐ and N‐terminal residues.A comparison of these results with those concerning the degraded form suggests that there is little loss of peptide material and that the gross quaternary structure of the protein is not modified by the proteolytic process. However circular dichroism studies show that the haem environment is altered.The evidence leading to propose a biglobular structure for the enzyme is discussed.
Isolation and DNA‐RNA Hybridization Properties of Small‐Molecular‐Weight Nuclear RNA Components from Baby‐Hamster‐Kidney Cells
Authors: Engberg Jan , Hellung‐Larsen Per , Frederiksen Sune 
Published: March 2005

Several small‐molecular‐weight nuclear RNA components (snRNAs) have been isolated and purified from nuclei of baby hamster kidney cells. The proportion of the cell genome complementary to the different snRNA components was investigated by DNA‐RNA hybridization. The values for saturation range between 0.4 × 10−4% and 0.8 × 10−4% which correspond to about 2000 genes for each of the snRNA components per cell. For comparison the saturation value for rRNA is 2.2 × 10−4% which corresponds to about 200 genes per cell. Saturation experiments with mixtures of the snRNA components indicate that there are separate genes for the different snRNA components.Hybridization competition experiments show that no base sequence homologies exist between the snRNAs and other RNAs such as rRNA, 5‐S RNA and tRNA.The DNA complementary to the snRNAs is distributed throughout the band of the bulk DNA of buoyant‐density fractionated DNA. This is in contrast to the DNA complementary to rRNA which bands as a heavy satellite. Hence the snRNA components can not be fragments of the non‐conserved parts of the rRNA precursor.
The Effects of Bases and Nucleosides on the Intracellular Contents of Nucleotides and 5‐Phosphoribosyl 1‐pyrophosphate in Escherichia coli
Authors: Bagnara Aldo S. , Finch Lloyd R. 
Published: March 2005

It has been reported that supplying preformed purine and pyrimidine bases and nucleosides to growing cells of Escherichia coli and other organisms results in the preferential utilization of these precursors for nucleotide biosynthesis, but the factors responsible for inhibition of the de novo pathways in vivo are not known. We have surveyed the effects of addition of purine and pyrimidine bases and nucleosides on the intracellular contents of nucleotides and 5‐phosphoribosyl 1‐pyrophosphate (P‐Rib‐PP) of Escherichia coli in an attempt to determine what some of these factors might be. The results show that initially adenine is converted mainly to ATP, guanine mainly to GTP and uracil mainly to UTP, while hypoxanthine is converted to both ATP and GTP. The corresponding nucleosides have similar effects except for adenosine, whose effects are similar to those of hypoxanthine and inosine. All purine precursors, except for guanosine, deplete the intracellular content of P‐Rib‐PP to 10% or less of its normal value. This finding indicates that the limitation in the concentration of P‐Rib‐PP for the first reaction of the de novo pathway is an important factor in the inhibition of that pathway when a preformed purine is available to support a salvage pathway of purine nucleotide biosynthesis. Our results also suggest that the decreased availability of P‐Rib‐PP may also cause immediate transient decreases observed in the levels of CTP and UTP, after the addition of a preformed purine precursor.Uracil as a pyrimidine precursor causes only a slight depletion of P‐Rib‐PP while uridine causes an initial increase in the P‐Rib‐PP content. It is concluded, therefore, that an increase in feedback inhibition, caused by elevated concentrations of uridine nucleotides, may be a major factor causing the inhibition of de novo biosynthesis of pyrimidine nucleotides when preformed pyrimidines are available, or that the inhibition may not be fully effective until repression and growth have diluted out the enzymes for the de novo biosynthesis.
Subunit Interactions in Enzyme Catalysis
Authors: Nari Joannès , Mouttet Christian , Fouchier Francis , Ricard Jacques 
Published: March 2005

The effects of subunit interactions on the steady‐state rate of the reaction catalyzed by the enzymel‐phenylalanine ammonia‐lyase, have been investigated using the concepts of structural kinetics developed in a previous paper. Both theoretical and experimental methods have been proposed, which allow a choice among the possible kinetic models. These methods rest on the mathematical analysis of rate data obtained in the presence of substrate analogues, used as inhibitors.In the absence of inhibitor, the reciprocal plots exhibit a significant downward curvature. If a substrate analogue is present in the reaction medium, the plots can be straight lines, or can even exhibit an upward curvature. If vs and vI are the steady‐state rate in the absence and in the presence of inhibitor, respectively, the plots (vs/vI)‐1 versus inhibitor concentration are either straight lines if the inhibitor is d‐phenylalanine, or parabolas if the inhibitor is benzoic acid.The detailed analysis of these data as well as previous information obtained on the number of polypeptide chains, suggest that the enzyme is made up of two protomers (Mr 160X103), each protomer consisting of two. polypeptide chains: an α chain (Mr 75X103) and a β chain (Mr 85X103). The kinetic data indicate the existence of a hybrid conformation of the enzyme during substrate binding. Thus, phenylalanine ammonia‐lyase appears to follow neither the classical allosteric model of Monod, nor the simple sequential model of Koshland, but a partially‐concerted mechanism of subunit interactions. The binding of the substrate induces a conformation change in the corresponding subunit as well as the appearance of a new conformation of the unliganded protomer.When d‐phenylalanine or benzoic acid are used as inhibitors, no hybrid enzyme · substrate · inhibitor complex is formed. It thus appears that the new unliganded conformation, called A′, is still able to bind the substrate, l‐phenylalanine, but cannot form a complex with d‐phenyl‐alanine or benzoid acid.The behaviour of the free enzyme appears to be different with regard to D‐phenylalanine and benzoic acid. The unliganded phenylalanine ammonia‐lyasc can bind one molecule of d‐phenylalanine only, whereas it can bind two molecules of benzoic acid. It thus appears that the binding of d‐phenylalanine on a subunit, induces a conformation change of the unliganded subunit, the new conformation (A″) being unable to form a complex with any molecule of inhibitor. In the case of benzoic acid, on the other hand, the new conformation of the unliganded protomer is still able to bind an inhibitor molecule.
Effects of Potassium Iodide on Aspartate Aminotransferase
Authors: Burridge Noranna , Churchich Jorge E. 
Published: March 2005

The absorption spectrum of the phosphopyridoxal form of the enzyme, aspartate aminotransferase, can be perturbed by the anion iodide over a wide range of pH values.The binding of this anion to the catalytic site of the enzyme brings about a decrease in the absorption at 360 nm and a concomitant increase in the absorption band centered at 430 nm corresponding to the positively charged nitrogen of the aldimine linkage of the cofactor.The absorption spectral changes are accompanied by variations in the fluorescence intensity emitted by the cofactor. The fluorescence studies reveal an efficient collisional interaction between the excited state of pyridoxal‐5‐P and the perturbing anion iodide. It is proposed that the active site of the enzyme aspartate aminotransferase is very accessible to the anion iodide.