bluuebunny/crossref_metadata_2025_split huggingface dataset:
Affinity Labelling of the Estradiol‐17β Dehydrogenase from Human Placenta with Substrate Analogs
Authors: PONS Michel , NICOLAS Jean‐Claude , BOUSSIOUX Anne‐Marie , DESCOMPS Bernard , CRASTES de PAULET André 
Published: March 2005

Affinity labelling oft lie estradiol‐l7β dehydrogenase of human placenta has been performed using derivatives of estradiol‐17β carrying alkylating groups in nine different positions on the steroid nucleus.The active‐site‐directed character of the inhibitions is confirmed by the following observations: the affinity labels are substrates or competitive inhibitors, the enzyme is protected against inactivation and alkylation by the substrate and by the coenzyme, the stoichiometry of the alkylation is two moles of inhibitor per 68000 g of enzyme (dimer).The alkylation of a histidine residue which is fast and extensive when the alkylating side chain is on the C‐3 carbon atom, is dramatically decreased when the alkylating side chain is shifted towards rings B and D. These results allow the location of this histidine in the vicinity of ring A and probably on the β face of the steroid nucleus.The reactivity of a cysteine located on the active site was quite different, showing increasing alkylation when the alkylating substituent of the affinity labels was shifted from C‐3 to C‐16 of the steroid nucleus.The correlation of this result and that obtained using an alkylating analog of NAD (3‐chloroacetylpyridine‐adenine dinucleotide) indicates that this cysteine is located in the catalytic region of the active‐site, at the junction of the ring D of the steroid nucleus with the nicotinamide moiety of the coenzyme
Affinity Chromatography of Acetylcholinesterase
Authors: MASSOULIÉ Jean , BON Suzanne 
Published: March 2005

An easily prepared affinity column for acetylcholinesterase is described, which may be operated at ionic strength high enough to prevent aggregation of the asymmetric forms of the enzyme. Specific elution by tetraethylammonium or decamethonium was quantitative. The performance of this column is comparable to that of the column described by Dudai and Silman.It is shown that the hexyl “spacer arm” strongly participates in the enzyme binding and that its replacement with the more hydrophilic spermine chain lowers the affinity. The hexyl chain itself is shown to bind acetylcholinesterase, although with lower affinity and capacity than the complete column. This binding is also partly reversed by inhibitors. Such hydrophobic columns bind the native asymmetric forms of the enzyme more strongly than the lytic globular ones.The aromatic quaternary ligand inhibits Electrophorus but not Torpedo acetylcholinesterase; therefore the column does not retain the Torpedo enzyme. Differences in Km between acetylcholinesterases of the two species also point to differences in their active sites.
Membrane‐Bound dd‐Carboxypeptidase and Transpeptidase Activities from Bacillus megaterium KM at pH 7
Authors: MARQUET Alberto , NIETO Manuei , DIAZ‐MAURIÑO Teresa 
Published: March 2005

The membranes from Bacillus megaterium KM contained a DD‐carboxypeptidase with optimum activity under the following conditions: pH 7; ionic strength, 1.3 M; temperature, 40 °C and below 20 °C. It did not require any divalent cation, but was inactivated by Cu2+ and Hg2+. It was stimulated by 2‐mercaptoethanol and low concentrations of p‐chloromercuribenzoate.
The membrane preparation also catalyzed a simple transpeptidation reaction using as carboxyl acceptors d‐alanine or glycine.
The conditions for optimum activity, temperature‐inactivation, temperature‐dependence of the activity, carboxyl donor specificity, sensitivity to β‐lactam antibiotics, and insensitivity to potential peptide inhibitors of both enzyme activities, was identical. The dd‐carboxypeptidase showed inhibition by d‐alanine and Ac2‐l‐Lys‐d‐Ala.
The inhibition by β‐lactam antibiotic was reversible for both enzymic activities and the time dependence for their recovery was identical.
The dd‐carbozypeptidase was very sensitive to changes in the configuration and size of the side‐chains of the C‐terminal dipeptide of the substrate. Amino acid residues at the C‐terminus that precluded the peptide from being a dd‐carboxypeptidase substrate were not acceptors in the transpeptidation reaction. Dipeptides were not acceptors for the ‘model transpeptidase’.
It is suggested that both activities are catalysed by the same enzyme molecule, whose physiological role is not the formation of peptide crosslinks during peptidoglycan biosynthesis.
New Methods for the Study of Complex Enzyme Kinetics Illustrated by Analysis of the Wavy Curves of νversus [S] and Non‐linear Double‐Reciprocal Plots for Human‐Placental 15‐Hydroxyprostaglandin Dehydrogenase
Authors: BARDSLEY William G. , CRABBE M. J. C. 
Published: March 2005

A new method for discovering the minimum degree of rate equations using only the experimental graphs and a straight‐edge (transparent ruler) is presented.This method is then illustrated by an analysis of the wavy ν vs [S] curves and non‐linear double‐reciprocal plots reproducibly given by NAD‐dependent 15‐hydroxyprostaglandin dehydrogenase for which a new improved purification is described.It is shown by this analysis that the enzyme has a complex mechanism involving cooperatively linked dependent sites and requiring a rate equation of at least fourth degree in prostaglandin and NAD+ with no kinetically significant dead‐end complexes.
Active Transport by Membrane Vesicles from Anaerobically Grown Escherichia coli Energized by Electron Transfer to Ferricyanide and Chlorate
Authors: BOONSTRA Johannes , SIPS Herman J. , KONINGS Wil N. 
Published: March 2005

Active transport of amino acids by membrane vesicles from Escherichia coli, grown anaerobically on glucose in the presence of nitrate, can be energized under anaerobic conditions by electron transfer in the nitrate respiration system with formate as electron donor and nitrate as acceptor. A high rate of amino acid transport is also obtained under anaerobic conditions by electron transfer from formate to the nitrate analogue chlorate or to the membrane‐impermeable electron acceptor ferricyanide. Electron transfer from formate to nitrate results in the generation of an electrical potential as is indicated by the uptake of the lipophilic cation triphenylmethylphosphonium.Ferricyanide accepts electrons from at least two sites of the nitrate respiration system. One of these sites appears to be nitrate reductase, because cytochrome b, reduced by formate, is completely reoxidized by ferricyanide and glutamate transport energized by formate plus ferricyanide and formate plus nitrate are affected by the same electron transfer inhibitors. A second site of electron transfer to ferricyanide appears to be located prior to nitrate reductase in the nitrate respiration system, since formate is oxidized at a higher rate in the presence of ferricyanide than with nitrate while formate/ferricyanide energizes transport of amino acids at a lower rate than formate/nitrate. Moreover, electron transfer inhibitors block electron transfer from formate to nitrate to a significantly higher extent than from formate to ferricyanide. The effects of irradiation of the membrane vesicles with near ultra‐violet light suggest that quinones play an essential role in the electron transfer from formate to nitrate or ferricyanide. Irradiation blocks completely formate‐dependent nitrate and ferricyanide reduction and active transport driven by formate/nitrate and formate/ferricyanide, but has hardly any effect on the activity of formate dehydrogenase and on ascorbate/phenazine methosulphate/oxygen‐driven transport. Similar effects of ferricvanldc have been observed in membrane vesicles from E. coli, grown anaerobically in the presence of fumarate. In these membrane vesicles a high rate of lactose and triphenylmethylphosphonimm uptake under anaerobic conditions is obtained by electron transfer from glycerol 1‐phosphate to fumarate and also to ferricyanide and evidence has been presented for the involvement of cytochromes in these electron transfers.
Reiteration Frequency of the Histone Genes in the Genome of the Amphibian, Xenopus laevis
Authors: JACOB Elard , MALACINSKI George , BIRNSTIEL L. 
Published: March 2005

Radioactive RNA was obtained from Xenopus laevis gastrula‐stage embryos which had been labelled by microinjection of [3H]uridine. A 9–12‐S RNA fraction was isolated by phenol extraction of whole embryos followed by sucrose gradient centrifugation of the RNA and polyacrylamide gel electrophoresis. This RNA was identified as histone mRNA on the basis of its chemical and physical properties. On polyacrylamide gels the labelled RNA could be subfractionated into different components with electrophoretic mobilities similar to those of histone mRNAs of the sea urchin. 9 ‐ 12‐S RNA lacked poly(A) sequences. In a protein synthesis system in vitro the RNA fraction directed the synthesis of the histones H4, H2A, H2B, H3 and, possibly, of H 1 protein.The Xenopus histone mRNA was challenged with Xenopus DNA under conditions of vast DNA excess. A cot1/2 of between 110 and 250 mol · s · 1−1 was obtained from the RNA trace curve, indicating that the histone genes were 20–50‐fold reiterated in the haploid genome of the species. This value indicates that although amphibians have several embryological and morphological features in common with sea urchins, the reiteration frequency for Xenopus histone genes is closer to the value of 10–20 established for mammals than the value of 400–1000 determined for sea urchins.
Heterogeneity of Native Ribosomal 60‐S Subunits in Ehrlich Ascites Tumor Cells Cultured in vitro
Authors: VAN VENROOIJ Walther J. , JANSSEN Albert P. M. 
Published: March 2005

Native large ribosomal subunits in cultured Ehrlich ascites honor cells analyzed by high‐resolution CsCl isopycnic centrifugation consist of at least two classes of particles with densities of 1.57 g/cm3 (L1) and 1.59 g/cm3 (L11), respectively.A wash with 0.5 M KCI converts L1 into particles with the density of L11 particles. Incubation of derived large subunits (density 1.59 g/cm3) with 0.5 M KCI wash of reticuiocyte ribosomes leads to the formation of particles with the density of L1 particles.A protein with a molecular weight of 57000 present in the high‐KCl wash of 60‐S native subunits was virtually absent in the KCI wash of 40‐S subunits or polyribosomes suggesting that specific protein factors may be present on some native 60‐S subunits. Possible functions of these protein factors are discussed.
Isolation and Partial Characterization of Elastase from Dog Granulocytes
Authors: DELSHAMMAR Magnus , OHLSSON Kjell 
Published: March 2005

An elastolytic enzyme has been isolated from dog granulocyte leukocytes. The purification procedure included preparation of the granula fraction, chromatography on Sephadex G‐75 and ion‐exchange chromatography on SP‐Sephadex C‐50 at pH 6.0.
The elastase isolated was homogeneous in analytical disc electrophoresis and showed in sodium dodecylsulfate electrophoresis a single protein component with the molecular weight of 24800. The. enzyme lacked tyrosine and lysine and the N‐terminal amino acid was phenylalanine. No carbohydrate or sialic acid were detected.
The dog granulocyte elastase showed similar activities as human granulocyte elastase on elastin and fibrin. The Km value for 3‐carboxypropionyl‐l‐alanyl‐l‐alanyl‐l‐alanyl‐p‐nitroanilide was 2.50 mM and the pH optimum 8.5. The elastase preparation obtained was 99.5%, active as judged from active‐site titration.
The enzyme is a cationic protein and shows pronounced trailing on agarose gel electrophoresis.
A monospecific antiserum against the purified enzyme was produced in rabbits.
Protease I from Escherichia coli
Authors: PACAUD Michèle , SIBILLI Lise , LE BRAS Gérard 
Published: March 2005

Protease I, a periplasmic endopeptidase from Escherichia coli has been further purified by a modified procedure. While the purified protein consists of a single polypeptide chain of about 21000 daltons, its molecular weight in dilute salt solution was estimated to be near 43000. suggesting that the enzyme has a marked tendency to dimerize. It has only one disulphide bond and is very sensitive to urea.In agreement with previous evidence of a chymotrypsin‐like specificity, hydrolytic assays of various p‐nitrophenyl esters of N‐substituted amino acids showed that phenylalanine and tyrosine derivatives are the best substrates for the enzyme. The Km (app) for N‐benzoyloxycarbonyl‐L‐tyrosine‐p‐nitrophenyl ester at pH 7.5 in 100 mM sodium phosphate buffer at 25 oC was found to be 0.2 mM. In contrast to chymotrypsin, protease I is unable to hydrolyse N‐acetyl‐L‐phenylalanine ethyl ester and its tyrosine analogue. Moreover, the enzyme appears devoid of amidase activity and exhibits a low activity upon polypeptides. At 37 oC, it cleaves the carboxymethylated B‐chain of bovine insulin at four points: Phe25‐Tyr26, Phe24‐Phe25, Leu15‐Tyr16 and Ser9‐His10. From a detailed study of peptides bonds hydrolyzed, it was concluded that protease I has a stringent requirement for both residues forming the scissile bond, and appears to possess an extended hydrophobic binding site.
Bovine Neurophysins I, II and C:
Authors: DE MEY Jan , VANDESANDE Frans 
Published: March 2005

Bovine neurophysins were prepared by a modified method, in which a Biogel P‐60 column was used. This yielded two neurophysin fractions, the first containing neurophysin I and small quantities of the other neurophysins, the second containing neurophysin II and C, and only traces of nem‐ophysin I.
Antibodies against neurophysin I, II and C were prepared by an original method, 5 μg in 100 μl water of each of the two fractions were applied on a gel, slab and separated by iso‐electric focusing in a pH gradient 4–6.The separated bands were visualized with 8‐aniline‐l‐naphthalene sulfonic acid, magnesium salt and strips respectively containing neurophysin I, II or C were cut out. The neurophysin‐containing strips were homogenized in complete Freund's adjuvant and injected into rabbits.
The specificity of the antisera were tested by immunocytochemistry and by radioimmunoassay. By this latter method, it was determined that cross‐reactivity was less than 1%. The cross‐reaction, observed with the immunohistochernical method could be eliminated by differential absorption.
It was found that neurophysin C antisera were undistinguishable from the neurophysin II antisera, while showing little cross‐reactivity with the neurophysin I antisera. This suggests that in vivo neurophysin C is not a real neurophysin, or at least, that it is very similar to neurophysin II.
Highly purified bovine neurophysins I, II and C were prepared by an improved preparative iso‐electric focusing method. By modifying a LKB Uniphor electrophoresis apparatus, the elution technique of Svendsen could be applied. This technique makes it possible to elute the proteins without switching off the voltage. The resolution of the technique is close to that offered by analytical gel isoelectric focusing.
Identification and Some Properties of a New Fast‐Reacting Plasmin Inhibitor in Human Plasma
Authors: COLLEN Désiré 
Published: March 2005

Fresh plasma was seeded with trace amounts of highly purified biologically intact iodine‐labelled plasminogen and the plasmin · inhibitor complexes formed after activation with streptokinase or urokinase separated by gel filtration. Two radioactive peaks were observed, the first one eluted in the void volume and the second one just before the 7‐S globulin peak. In incompletely activated samples, the second peak was always predominant over the first one.Both components were purified with high yield by a combination of affinity chromatography on lysine‐agarose and gel filtration, and investigated by dodecylsulphate‐polyacrylamide gel electrophoresis and immunoelectrophoresis. Neither component reacted with antisera against α1‐antitrypsin, antithrombin III, C1‐esterase inhibitor, inter‐α‐trypsin inhibitor or α1‐antichymotrypsin. The component of the first peak appeared to be a complex between plasmin and α2‐macroglobulin which reacted with antisera against human plasminogen and against α2‐macroglobulin.The component of the second peak had a molecular weight (Mr) of 120000 ‐ 140000 by dodecylsulphate‐polyacrylamide gel electrophoresis and upon reduction displayed a doublet band with an Mr of 65000 ‐ 70000 and a band with Mr 11000. It reacted with antisera against plasminogen and with antisera raised against this complex and absorbed with purified plasminogen. The latter antisera reacted with a single component in plasma which is different from the above‐mentioned plasma protease inhibitors. Specific removal of this component from plasma by immuno‐absorption resulted in disappearance of the fast‐reacting antiplasmin activity whereas α2‐macroglobulin was found to represent the slower‐reacting plasmin‐neutralizing activity. In the presence of normal plasma levels of these proteins, the specific removal or absence of α1‐antitrypsin, antithrombin III or C1‐esterase inhibitor did not alter the inactivation rate of plasmin when added to plasma in equimolar amounts to that of plasminogen.It is concluded that only two plasma proteins are important in the binding of plasmin generated by activation of the plasma plasminogen, namely a fast‐reacting inhibitor which is different from the known plasma protease inhibitors and which we have provisionally named antiplasmin, and α2‐macroglobulin, which reacts more slowly.
Isolation of a Crosslinked Cyanogen‐Bromide Peptide from Insoluble Rabbit Collagen Tissue Differences in Hydroxylation and Glycosylation of the Crosslink
Authors: HENKEL Werner , RAUTERBERG Jürgen , STIRTZ Toni 
Published: March 2005

A radioactive peptide has been isolated from cyanogen bromide digests of sodium boro[3H]hydride‐reduced collagen from rabbit bone, tendon and skin. It was identified as a crosslinked peptide linking the short C‐terminal cyanogen bromide peptide a1‐CB6B (17 amino acid residues) to α1‐CB5 (37 residues) from the helical part of the chain of an adjacent molecule. Both peptides could be separated after cleaving the crosslink with periodate. Thus the crosslinked peptide αl‐CB5 ×αl‐CB6B originates from an intermolecular crosslink between quarter‐staggered molecules within collagen fibrils previously assigned as ‘head‐to‐tail’ link.The chemical nature of the reduced crosslinking component was identified and was shown to differ between peptides derived from different tissues: α1‐CB6B ×α1‐CB5 from bone contains hydroxylysinohydroxynorleucine [o5Lys(o5ωNle)] whereas the skin peptide contains hydroxylysinonorleucine [o5Lys(ωNle)]. The peptide derived from tendon contains both components.The relation of o5Lys(ωNle) to o5Lys(o5ωNle) in the peptides corresponds to that of the original tissue. On the other hand, histidino‐hydroxymerodesmosine which is a major reduced crosslinking component_ in skin and tendon, is completely absent in the isolated peptides.The crosslinking component in the skin peptide is completely glycosylated, mainly by glucosylgalactosyl residues and to a smaller extent by galactosyl residues. o5Lys(o5ωNle) from the bone peptide is only partly glycosylated, containing equal amounts of the disaccharide and monosaccharide. Only slight glycosylation was found in the tendon peptide.
Activities of Protein‐Deficient Particles Derived from 50‐S Ribosomal Subunits by NH4Cl/Ethanol Treatment
Authors: BERNABEU Carmelo , VAZQUEZ David , BALLESTA Juan P. G. 
Published: March 2005

Protein‐deficient ribosomal particles obtained by treatment of 50‐S subunits from Escherichia coli ribosomes with I M NH4Cl and 50% ethanol contain less than 3 % of proteins L7 and L12 and about 7 % of proteins L10 and L11. Proteins L1, L5, L8/9 and L25 are also released during the treatment but in amounts accounting for less than 40%.The particles are able to form peptide bonds in different systems, such as ‘fragment reaction’, puromycin reaction and formation of dipeptides. They also bind N‐acetylphenylalanyl‐tRNA and phenylalanyl‐tRNA non‐enzymically but are unable to support any of the elongation‐factordependent reactions tested. However, when methanol is present, they display up to 20% of the control EF‐G‐dependent GTP activities, such as GTP hydrolysis and formation of the ternary complex EF‐G. GuoPP(CH2)P · ribosome. The first activity is totally sensitive to the antibiotic thiostrepton while the formation of the ternary complex is unaffected by the drug. When measured by equilibrium dialysis the core particles are shown to be able to bind radioactive thiostrepton.The results show that protein L11 is not an absolute requirement either for peptidyl transferase activity or for the binding of thiostrepton, although in the last case the protein strongly enhances the ribosome affinity for the antibiotic.
The Uptake and Metabolism of Uridine by the Slime Mould Physarum polycephalum
Authors: BIRCH Barbara , TURNOCK Geoffrey 
Published: March 2005

Uridine is taken up by microplasmodia of Physarum polycephalum via a saturatable transport system with an apparent Km of 29.μM. An intracellular concentration significantly higher than that in the growth medium is attained, suggesting that the uptake is an active process. Both deoxyribonucleosides and ribonucleosides are competitive inhibitors of the uptake of uridine.
In contrast, the rate of entry of uridine into surface plasmodia is a linear function of the concentration of the nucleoside in the growth medium, and the uptake is not inhibited by other nucleosides.
As well as serving as a source of pyrimidine nucleotides for the synthesis of nucleic acids, uridine is also catabolised by P. polycephalum. Uracil accumulates in the growth medium and there is also significant conversion of C‐2 of the pyrimidine ring to CO2. The proportion of uridine subject to catabolism in surface plasmodia is less than that observed for microplasmodia.
Accumulation of Free Ribosomal PrÖteins S1, L7, and L12 in Escherichia coli
Authors: RAMAGOPAL Subbanaidu 
Published: March 2005

The total content of free ribosomal proteins in the cells of Escherichia coli was determined to study the nature of intracellular accumulation during growth. Labeled ribosomes and post‐ribosomal supernatant were prepared from exponentially growing and stationary‐phase cultures. The fraction of free ribosomal protein in the supernatant was estimated by resolving both the acidic and basic proteins separately with two different techniques of two‐dimensional gel electrophoresis. Free ribosomal proteins in the cell sap were identified an the basis of coelectrophoresis with authentic ribosomal protein ‐markers, molecular weights and amino acid composition. Among the acidic proteins, S1, L7, and L12 Were identified and examined in detail.All three proteins accumulated to significant levels in these cultures. Stationary‐phase cells contained 2–4 times more free S1, L7, and L12 than midlogarithmic phase cells. Moreover, free S1, L7, and L12 and ribosome‐bound forms were stable during exponential and post‐exponential growth of cultures. At this growth transition, non‐ribosomal proteins in the supernatant and those associated with the ribosomes showed different characteristics of accumulation.The ratio of L12: L7 in the supematant did not exhibit a remarkable shift during the growth cycle like the ratio of L12: L7 in ribosomes. In addition, free L12 in the supernatant was not acetylation, although there was a rapid acetylation in the cells