bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Phospholipid Composition on the Surface Potential of Liposomes and the Activity of Enzymes Incorporated into Liposomes
Authors: NAŁȨCZ Maciej J. , ZBOROWSKI Józef , FAMULSKI Konrad S. , WOJTCZAK Lech 
Published: March 2005

Microsomes of rat liver and brain and mitochondria of rat liver and guinea‐pig brown adipose tissue were solubilized with the nonionic detergent Lubrol‐WX and the solubilized material was incorporated into liposomes of various phospholipid composition. In proteoliposomes thus formed the kinetics of arylsulphatase, glycerol‐3‐phosphate dehydrogenase, monoamine oxidase and acetylcholinesterase were measured.
It was shown that the apparent Km values of arylsulphatase and glycerol‐3‐phosphate dehydrogenase were higher in liposolnes prepared with negatively charged phospholipids and lower in liposomes containing positively charged organic amines, as compared with the Km value of enzymes incorporated into liposomes prepared from phosphatidylcholine alone. The opposite was true for monoamine oxidase and acetylcholinesterase, i.e. enzymes possessing cationic substrates. Phospholipid composition did not essentially influence the activity of the enzymes extrapolated for infinite substrate concentration (V values).
As comparcd with proteoliposomes made from phosphatidylcholine, the binding constant (Ka) of 8‐anilino‐1‐naphthalene sulphonate was higher when the vesicles contained acidic phospholipids or bis(hexadecanyl)phosphate and lower when they contained organic amines.
A correlation between changes of the surface potential calculated from Ka values of anilinonaphthalene sulphonate and variations in apparent Km values of the four enzymes under investigation indicates that the activity of membrane‐bound enzymes may be modulated by charged phospholipids due to decreasing or increasing substrate concentration in the unstirred layer, as predicted from the Boltzmann distribution.
Purification and Properties of a Mouse‐Cell DNA‐Repair Endonuclease, which Recognizes Lesions in DNA Induced by Ultraviolet Light, Depurination, γ‐Rays, and OsO4 Treatment
Authors: NES Ingolf F. 
Published: March 2005

A DNA‐repair endonuclease has been purified 117‐fold from mouse plasmacytoma cells (line MPC‐11) by gel filtration, followed by ion‐exchange and affinity chromatography. Its molecular weight was determined by gel filtration to be 28000 ± 2000. The enzyme recognizes apurinic and apyrimidinic sites induced by acid and γ‐rays in DNA, as well as another type of lesion(s) which is introduced into DNA by both ultraviolet irradiation and OsO4. Quantitative measurements of the number of nicks the purified DNA‐repair endonuclease makes in DNA treated with various amounts of OsO4 and ultraviolet light suggests that the endonuclease may act on 5,6‐dihydroxydihydrothymine lesions.The endonuclease activity was sensitive to the ionic strength and was most active in the presence of 100 mM KCl, whereas the presence of divalent cations did not stimulate the activity.
Cross‐linking of mRNA to Proteins by Irradiation of Intact Cells with Ultraviolet Light
Authors: WAGENMAKERS Anton J. M. , REINDERS Rita J. , VAN VENROOIJ Walther J. 
Published: March 2005

Ehrlich ascites tumor cells were irradiated with ultraviolet light to cross‐link intracellular RNAs to their tightly bound proteins. The efficiency of such cross‐linking in vivo was measured by two independent methods, namely by phenol/chloroform extraction and by isopycnic centrifugation. After short irradiation times more than 70% of the ribosomal RNAs and more than 80% of the mRNA were found to be cross‐linked to protein. The size of the cross‐linked RNAs was essentially unchanged, indicating that the irradiation does not lead to extensive fragmentation of the RNA.Covalent poly(A)‐containing mRNA‐protein complexes were isolated by oligo(dT)‐cellulose chromatography in the presence of sodium dodecylsulphate. The complexed proteins were freed from RNA by extensive nuclease degradation and analysed by polyacrylamide gel electrophoresis. The results show that some specific proteins are associated with poly(A)‐containing RNA in vivo.
The Core Subunit Structure in RNA Polymerase Holoenzyme Determined by Neutron Small‐Angle Scattering
Authors: STÖCKEL Peter , MAY Roland , STRELL Irmtraud , CEJKA Zdenka , HOPPE Walter , HEUMANN Hermann , ZILLIG Wolfram , CRESPI Henry 
Published: March 2005

The core subunit arrangement α2‐β‐β′ within DNA‐dependent RNA polymerase holoenzyme α2ββ′σ from Escherichia coli was investigated by neutron small‐angle scattering using label triangulation. The quaternary structure of multisubunit biomolecules can be studied by this new method if total reconstitution works in a quantitative way and if extensive replacement of C‐bound hydrogen (H) by deuterium (2H) is possible. A substitution of the selected subunits by their fully deuterated analogues was used for the analysis of the overall shapes of the core subunits α2, β and β′in situ and for the determination of the intersubunit centre‐to‐centre distances. The contrast between the buffer and the remaining ‘hydrogenated’ enzyme vanishes if the buffer contains 42%2H2O (matching of scattering length densities). The isotopic hybridization of the enzyme fulfils the conditions of isomorphous replacement as required: molecular functions, like enzyme activity, were completely preserved.The orientations of the core subunits within the holoenzyme were derived by comparing theoretical and experimental pair distance distribution functions, P(r), obtained from the scattering in tensity differences of the pair‐labelled (e.g. both β and β′ labelled) and both mono‐labelled molecules by direct Fourier transformations. Additional, the subunit shapes were refined by P(r) analyses. The arrangement of the stable core structure within the holoenzyme, which contains σ as a dissociable factor, is presented in a three‐dimensional model.
Uniformity of Carbohydrate Chains within Molecular Variants of Rat α1‐Fetoprotein with Distinct Affinity for Concanavalin A
Authors: BAYARD Bernard , KERCKAERT Jean‐Pierre 
Published: March 2005

Three rat α1‐fetoprotein fractions were obtained by chromatography on concanavalin‐A‐Sepharose : one non‐reactive, one weakly reactive and one reactive to concanavalin A. The non‐reactive and reactive variants were found to vary in the structure of their carbohydrate chains while the conformation of the weakly reactive form may modulate the accessibility of these chains to the lectin. N‐Glycosidically linked glycans from unfractionated α1‐fetoprotein were isolated and chemically characterized. Particular attention was paid to develop sensitive methods based upon hydrazinolysis, quantitative re‐N‐acetylation of glycans with [14C]acetic anhydride and thin‐layer chromatography of labeled compounds. With the aid of these methods two main kinds of glycans (1a and 2a) were obtained and fractionated on concanavalin‐A‐Sepharose into non‐reactive (1a) and reactive (2a) molecules. Moreover it was demonstrated that each α1‐fetoprotein variant contained either two glycans 1a or two glycans 2a, not randomly, but a pair of the identical carbohydrate chains at the two glycosylation sites.
Neocarratetraose 4‐O‐Monosulphate β‐Hydrolase from Pseudomonas carrageenovora
Authors: McLEAN Maitland W. , WILLIAMSON Frank B. 
Published: March 2005

Neocarratetraose 4‐O‐monosulphate β‐hydrolase has been enriched 73‐fold from the cell‐free extract of Pseudomonas carrageenovora. This enzyme catalyses the hydrolysis of the β‐glycosidic linkage of neocarratetraose 4‐O‐monosulphate [3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐1‐d‐galactopyranosyl‐(1–>4)‐3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐ d‐galactose 4‐O‐sulphate]. The products of hydrolysis are respectively neocarrabiose [3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐ d‐galactose] and neocarrabiose 4‐O‐sulphate [3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐ d‐galactose 4‐O‐sulphate]. During purification the χ‐carrageenase and 4‐sulphatase activities present in the bacterial extract were resolved from the title enzyme. From dodecylsalphate/polyacrylamide gel electrophoresis the enzyme has a molecular weight of 94000. Calcium stimulated the activity, Km 0.50 mmol dm−3. Optimal activity was found at pH 7.0 and the Km determined for the nominal substrate was 68 μmol dm−3.[14C]Neocarratetraose 4‐O‐monosulphate was prepared from [14C]carrageenan (Chondrus crispus) and utilized in a radiochemical assay for neocarratetraose 4‐O‐monosulphate β‐hydrolase. Synthesis of [1‐3H]neocarratetraitol 4‐O‐[35S]monosulphate yielded another substrate. This alditol was hydrolysed to yield a single radioactive species, thus supporting the position of sulphation of neocarratetraose 4‐O‐monosulphate deduced from 13C NMR studies. Neocarratetraose 4‐O‐disulphate [3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐4‐O‐sulphato‐β‐d‐galactopyranosyl‐(1–>4)‐3,6‐anhydro‐α‐d‐galactopyranosyl‐(1–>3)‐d‐galactose 4‐O‐sulphate] was not hydrolysed by the purified glycosidase. Tentative evidence for transglycosylation was obtained with both monosulphated substrates. A pathway is described for the sequential degradation of neocarratetraose 4‐O‐disulphate to neocarrabiose.
The Electrochemical Proton Gradient in Mycoplasma Cells
Authors: BENYOUCEF Mohammed , RIGAUD Jean‐Louis , LEBLANC Gerard 
Published: March 2005

The electrochemical proton gradient, generated upon glycolysis by Mycoplasma mycoides var. Capri cells has been determined. The components, the transmembrane pH gradient, ΔpH, and the membrane potential, ΔΨ, were measured using several methods.The determination of the ΔpH was conducted by measuring the transmembrane distribution of weak acids (acetate and butyrate) and of a weak base (methylamine), using flow dialysis and filtration techniques. The transmembrane electrical potential was determined from the distribution of the lipophilic cation Ph3MeP+ and of Rb+ or K+ in the presence of valinomycin. At extracellular pH 7.2, glycolyzing Mycoplasma cells maintain an internal pH more alkaline (0.5 pH unit) than that of the milieu and an electrical potential of–85 mV, interior negative. The in M. mycoides var. Capri cells is thus about–115 mV.When the external pH was altered from 7.7 to 5.7, ΔΨ decreased from–90 mV to–60 mV. On other hand although the internal pH decreased, ΔpH was found to increase from 0.2 to 1.0 pH unit. Since the changes in ΔΨ were largely compensated by the changes in ΔpH, remained practically constant at about–115 mV throughout the pH range tested.Finally, inhibition of ΔpH by N,N′‐dicyclohexylcarbodiimide, carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone or nigericin confirmed that chemiosmotic phenomena contribute to energy transduction across the membranes of M. mycoides var. Capri cells.
Gradation of the Magnitude of the Electrochemical Proton Gradient in Mycoplasma Cells
Authors: BENYOUCEF Mohammed , RIGAUD Jean‐Louis , LEBLANC Gerard 
Published: March 2005

The results presented show that in Mycoplasma mycoides var. Capri, regulation of glucose uptake by its non‐metabolizable analogue methyl α‐d‐glucoside, can be used to control intracellular ATP content. This in turn leads to a control of the rate of proton extrusion catalysed by the Mg2+‐dependent ATPase () and the respective amplitudes of the components of . When Mycoplasma cells are incubated with 10 mM methyl α‐d‐glucoside, the amplitude of of the electrical potential ΔΨ and of the chemical gradient ΔpH become continuous functions of external glucose concentration within the limits of the non‐energized and fully energized states.Analysis of the relationships between graduated amplitudes of ΔΨ, ΔpH and () show that the primary form of energy stored by a generator is the electrical potential.
Interaction of Clonidine and Clonidine Analogues with α‐Adrenergic Receptors of Neuroblastoma × Glioma Hybrid Cells and Rat Brain
Authors: ATLAS Daphne , SABOL Steven L. 
Published: March 2005

Clonidine and several analogues of clonidine are shown to be useful probes for α2‐adrenergic receptors in a comparative study of ligand binding and inhibition of adenylate cyclase. The α‐adrenergic properties of a new potential probe, N‐(4‐hydroxyphenacetyl)‐4‐aminoclonidine hydrochloride, are described. [3H]Clonidine binds to α‐receptors of NG108‐15 neuroblastoma × glioma hybrid cell membranes with Kd values of 1.7 and 33 nM for putative high‐affinity and low‐affinity sites, respectively. p‐Aminoclonidine and hydroxyphenacetyl aminoclonidine displace [3H]clonidine from the high‐affinity sites with Kd values of 2.3 and 5.8 nM, respectively. Rat brain α2‐receptors also exhibit high affinity toward clonidine, p‐aminoclonidine, and hydroxyphenacetyl aminoclonidine, as determined by displacement of specifically bound [3H]clonidine. Clonidine, p‐aminoclonidine, and hydroxyphenacetylaminoclonidine elicit modest inhibition (up to 24%) of NG108‐15 adenylate cyclase by interaction with α2‐receptors (Kd,app 300, 50, and 130 nM, respectively); these compounds also partially reverse the inhibition elicited by (–)‐norepinephrine.Components of the adenylate cyclase assay mixture, particularly ATP, GTP, sodium ions, and a nucleoside‐triphosphate‐regenerating system, decrease the high‐affinity [3H)clonidine binding to NG108‐15 membranes; in the presence of these components, α‐receptors possess only low affinity (Kd 43 nM) for [3H]clonidine. These results are consistent with the concept that certain components required for the receptor‐mediated inhibition of adenylate cyclase convert α2‐receptors from a high‐affinity inactive state to a low‐affinity active state.
An Analysis of Activity Determinations in a Series of Coupled Redox Reactions with Special Reference to Hydrogenase
Authors: van DIJK Cees , MAYHEW Stephen G. , VEEGER Cees 
Published: March 2005

In this paper a spectrophotometric method is described to study the effects of pH and redox potential on the hydrogen production activity of hydrogenase, which will be applied in the following paper.The theoretical part of this paper gives the mathematical description of system in which a redox mediator is continuously reduced by a redox system, in a non‐rate‐limiting way and continuously enzymatically oxidized, in a rate‐limiting way. Due to the changes in redox potential during the course of the reaction the ratio reduced oxidized redox mediator declines gradually. It is possible to relate the changes in this ratio to the actual amount of product formed.The calculation of product formation has been applied to the production of hydrogen by hydrogenase in a system with a redox mediator, whose reduced form functions as the ultimate electron donor for hydrogenate, and the dithionite/(bi)sulphite redox couple as reducing system. The changes in the ratio reduced/oxidized redox mediator as a function of pH and redox potential, followed spectrophotometrically, have been related to the hydrogen production activity of hydrogenase.The second part of the paper deals with the analysis of the theory and describes the conditions under which the method can be used with methyl viologen as redox mediator.The conditions for determining the pH dependence of the hydrogen production activity manometrically are also described.
Dexamethasone‐Binding Proteins in Cytosol and Nucleus of Rat Thymocytes
Authors: TSAWDAROGLOU Nikos G. , GOVINDAN Manjapar V. , SCHMID Wolfgang , SEKERIS Constantine E. 
Published: March 2005

Dexamethasone‐binding proteins from the cytosol and the nucleus of rat thymocytes were analyzed by ionexchange chromatography on DEAE‐cellulose. Three dexamethasone‐binding proteins were revealed in cytosol one in the flow‐through (DE‐1) and two (DE‐2 and DE‐3) eluting from the column with 0.13 M and 0.23 M NH4Cl, respectively. In nuclear extracts only one receptor fraction, present in the flow‐through, could be detected.By a combination of affinity chromatography on Cl‐Sepharose to which dexamethasone 21‐methanesulfonate was linked through a disulfide bond and DEAE‐cellulose chromatography, three receptor proteins were highly purilicd from cytosol, with molecular weights of 45000, 72000 and 90000 and one from nuclear extracts with molecular weight of 72000. Antibodies to the 45000‐Mr and 90000‐Mr proteins were elicited in rabbits. The antibodies to the 45000‐Mr protein cross‐react with the 90000‐Mr protein. Similarly, the antibodies to the 90000‐Mr protein cross‐react with the 45000‐Mr protein. Antibodies to either of the two proteins immunoprecipitate 60–70% of the dexamethasone‐binding activity of rat thymus cytosol. Immunoaffinity chromatography of cytosol and nucleosol on columns of Sepharose linked to the IgG against either the 45000‐Mr or the 90000‐Mr protein leads to binding of these proteins on the columns but not of the 72000‐Mr species. Two nuclear polypeptides with molecular weights of 36000 and 38000 remain attached to the immunoaffinity column; these polypeptides may represent degradation products of the cytoplasmic receptor upon entrance into the nucleus. Antibodies against two dexamethasone‐binding proteins from rat liver cytosol immunoprecipitate the 45000‐Mr and 90000‐Mr cytosol receptors from rat thymus.
Synthesis and Biological Properties of Enzyme‐Resistant Analogues of Substance P
Authors: SANDBERG Bengt E. B. , LEE Chi‐Ming , HANLEY Michael R. , IVERSEN Leslie L. 
Published: March 2005

Six analogues of substance P were synthesized with the aim of developing a metabolically stable peptide that would retain the biological activity of substance P. A recently isolated and characterized substance‐P‐degrading enzyme from human brain with a high specificity for substance P described in the preceding paper in this journal was used as a model for the enzymatic inactivation of substance P. The synthetic analogues were designed to protect the peptide bonds on the carboxyl side of residues 6, 7 and 8 of substance P, which represent the sites of cleavage by substance‐P‐degrading enzyme. To test for increased enzymatic resistance, the analogues were incubated with the enzyme, the digests were separated on a high‐performance liquid chromatography reverse‐phase column and the peptide fragments were collected and identified by amino acid analysis. Of the analogue, described, an heptapeptide analogue of residues 5–11. < Glu‐Gln‐Phe‐MePhe‐MeGly‐Leu‐MetNH2, showed almost complete resistance both towards substance‐P‐degrading enzyme and to degradation on exposure to rat hypothalamic slices. This analogue was about a third as potent as substance P in competing for binding to receptor sites for this peptide in rat brain membranes and a tenth as potent in eliciting contractions of the guinea pig ileum. The peptides were synthesized using the solid‐phase technique with polydimethylacrylamide as a solid support and the coupling was achieved with pre‐formed symmetrical anhydrides in dimethylacetamide.Fluorenylmethyloxycarbonyl was used as an α‐amino protecting group in conjunction with t‐butyloxycarbonyl as an ɛ‐amino protecting group. Ammoniolytic cleavage from the resin was followed by stepwise elution from an SP‐Sephadex column, deprotection with trifluoroacetic acid and chromatography on a Bio‐Rex 70 ionexchanger. The peptides were finally purified on a semi‐preparative reverse‐phase column.
Distinction between Two Subpopulations of β1‐Adrenergic Receptors in Human Adipose Cells
Authors: JACOBSSON Bo , VAUQUELIN Georges , WESSLAU Christian , SMITH Ulf , STROSBERG A. Donny 
Published: March 2005

The β‐adrenergic receptors in human adipose membranes were identified by the specific and saturable binding of the β‐adrenergic antagonist (–)‐[3H]dihydroalprenolol. The total number of sites in control membranes was 0.32 ± 0.03 pmol/mg protein and the equilibrium dissociation constant for binding (Kd) was 2.6 nM and 2.5 nM as determined by Scatchard analysis of experiments on equilibrium binding and kinetics respectively. The β1‐adrenergic nature of the receptors was derived from the order of potencies of β‐adrenergic agonists (isoproterenol > norepinephrine > epinephrine) to complete with (–)‐[3H]dihydroalprenolol for binding. Studies of saturation binding, kinetics and competition binding revealed the presence of a single class of β1‐adrenergic receptors.Prolonged incubation of human adipose cells in the presence of (–)‐norepinephrine decreases the lipolytic response to β‐adrenergic agonists, and reduces by 50% the concentration of β‐adrenergic receptors. The Kd values for (–)‐[3H]dihydroalprenolol and the β‐adrenergic agonists remain unchanged. Catecholamines also produce a rapid conformational change of approximatively 50% of the receptors in control membranes as revealed by their increased sensitivity towards inactivation by the alkylating agent N‐ethylmaleimide. This inactivation process is not observed in desensitized membranes, which indicates that desensitization and inactivation by agonists plus N‐ethylmaleimide affect the same receptor population. The β‐adrenergic receptors in human adipocytes can thus be divided into two subpopulations on the basis of the different consequences of their interaction with agonist molecules.
Dihydrolipoamide Dehydrogenase Component of the Pyruvate Dehydrogenase Complex from Escherichia coli K12
Authors: SCHMINCKE‐OTT Eva , BISSWANGER Hans 
Published: March 2005

The regulation of the biosynthesis of dihydrolipoamide dehydrogenase is dependent on the biosynthesis of the pyruvate dehydrogenase complex. The gene coding for the dihydrolipoamide dehydrogenase appears to be included in the regulation of the pyruvate dehydrogenase operon. Possibly a secondary promoter is inserted.Dihydrolipoamide dehydrogenase was purified in its free form using a dihydrolipoamide‐agarose affinity column and avoiding denaturating conditions.The enzyme shows complete cross‐reactivity with antibodies against the pyruvate dehydrogenase complex and has a higher specific activity than any preparations described thus far. The transition state activation energy of the catalytic activity is smaller for the complex‐bound enzyme than that found for the free enzyme. In its complexed form, the enzyme also proves to be somewhat more stable under alkaline conditions.The reaction catalyzed by the dihydrolipoamide dehydrogenase shows the behaviour of a ping‐pong mechanism. The Michaelis constants for the substrates NAD and dihydrolipoamide found with the free enzyme are about four times those observed with the enzyme integrated into the native complex. The catalytic reaction of both forms of the dihydrolipoamide dehydrogenase is inhibited by NADH. The mechanism of this inhibition cannot simply be explained by a product inhibition. Rather the further reduction of the catalytically active, half‐reduced enzyme form to the catalytically inactive, fully reduced form has to be considered as causing the inhibition.