bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Substrates on the Heat Stability and on the Reactivities of Thiol Groups of 3‐Phosphoglycerate Kinase
Authors: CSERPÁN Imre , VAS Mária 
Published: March 2005

Pig muscle 3‐phosphoglycerate kinase contains seven cysteine residues/molecule enzyme. Two of them react with Ellman's reagent (Nbs2) in a second‐order reaction [k= (1.1 ± 0.1) × 103 M−1 s−1]; the reaction of the other five thiols are limited by a first‐order protein structural change [k= (2.0 ± 0.4) × 10−4 s−1] in 0.1 M Tris/HCl buffer, pH 7.5 at 20°C.Blocking the rapidly reacting thiols with Nbs2 inactivated the enzyme (these two –SH groups are not equivalent in this respect), but it does not abolish substrate‐binding ability.The rapidly reacting thiol groups readily participate in intermolecular disulfide formation following their partial blocking with Nbs2. This type of aggregation of 3‐phosphoglycerate kinase molecules also leads to inactivation.The order of effectivity of substrates in inhibiting the reaction of the slowly reacting thiols is very similar to the order of their protective effect against heat inactivation. Both phenomena presumably reflect the structure‐stabilizing effect of substrates.
Protein Synthesis Directed by Polyadenylated and Non‐polyadenylated RNA
Authors: BARAHONA Isabel , GALEGO Lisete , RODRIGUES‐POUSADA Claudina 
Published: March 2005

Free and membrane‐bound polysomes were prepared from the protozoa Tetrahymena pyriformis using a procedure which gives good recovery and practically no cross‐contamination. Polysomes are intact as analysed by sedimentation analysis. Poly(A)‐rich RNA and poly(A)‐free RNA, isolated from both populations of polysomes, show similar electrophoretic patterns. These RNAs were translated in the rabbit reticulocyte lysate cell‐free system and the translation products were analysed by one‐dimensional and two‐dimensional gel electrophoresis. The most striking differences were found in the two‐dimensional electrophoretic analysis namely: (a) a group of polypeptides (10) is synthesized mainly on membrane‐bound polysomes, (b) a second abundant group is synthesized mainly in free polysomes (c) and a third class of polypeptides is synthesized on both kinds of polysomes.Poly(A)‐free RNAs, isolated from free polysomes, are also able to promote synthesis of some polypeptides.The results are discussed taking into account the fact that T. pyriformis is a non‐secretory cell.
An Electron Microscopic Approach to the Quaternary Structure of Mitochondrial F1‐ATPase
Authors: TIEDGE Henri , SCHÄFER Günter , MAYER Frank 
Published: March 2005

The three‐dimensional structure of F1‐ATPase from beef heart mitochondria was investigated by electron microscopic techniques. The presence of high concentrations of nucleotides is essential for preservation of the quaternary structure. When in vistigated under such coinditions, monodisperse F1‐ATPase could not be distinguished from the membrane‐bound enzyme. At low resolution, the particle shape resembles an oblate ellipsoid of revolution with an axial ratio of about 2:1.From several lines of evidence (including field micrographs at higher magnifications, Markham rotational analysis, and tilting experiments), two conclusions may be drawn concerning the three‐dimenwsional fine structure of F1‐ATPase.1. At the periphery of the molecule, six globular protein masses are orientated in a way similar to the chair conformation of cyclohexane. This array is interpretated to be made up of an alternating sequence of α and β subunits.2. Part of the central space is occupied by a seventh protein mass, protrusions of which are likely to be in contact with some of the outer subunits. A γ subunit is supposed to be constituent part of this central protein mass.As a consequence, this model favours a stoichiometry oif
Binding of MgATP to the Nitrogenase Proteins from Azotobactor vinelandii
Authors: CORDEWENER Jan , HAAKER Huub , VEEGER Cees 
Published: March 2005

Binding of MgATP to the MoFe and Fe proteins from Azotobacter vinelandii has been studies, By means of the flow dialysis technique it was demonstrated that one molecule of reduced Fe peotein binds one molecule of MgATP, with a dissociation ocnstant of 0.56 ± 0.11 mM. The oxidized binds two molecules of MfATP, with identical intrinsic dissociation constants of 0.29 ± 0.05 mM. The binding of MgATO to the Fe protein was also studied by equilibrium dialysis. It was found that during dialysis of reduced Fe protein in the presence of MgATP, dishionite was oxidized. Moreover, in the presence of MgATP both reduced and oxidized Fe protein were inactivated during the dialysis. These observations demonstrate that binding of MgATP to the Fe protein can only be measured by a relatively fast method.With the same methods as used for the Fe protein, no binding of MgATP to the MoFe protein of A. Vinelandii could be demonstrated.The redox properties of the Fe protein in the presence and absence of MgATP are sicussed with respect to the observed binding properties of MgATP for the Fe protein. The implications of these results are discussed with respect tothe present models for the inhteractins between the Fe and MoFe proteins of nitrogenase.
B‐Z Transition in Methylated DNA
Authors: VAN LEIR John J. C. , SMITS Marieke T. , BUCK Henk M. 
Published: March 2005

On the basis of modified neflect of differntial overlap (MNDO) quantum‐chemiocal calculations on nucleoside systems, we descrive the effect of methylation on the energies calculated for the rotation around the glycosidic C(1′)‐N bond. We found a high anti‐syn activation energy in the case of the pyrimidine nucleosides C and m5C, Whereas for the purine nucleosides G, m6G, m7 and m8 only moderate anti‐syn energetic barriers were claculated. This result is conssitent with the experimentally observed preference for d(G‐C)3 and d(G‐m5C)3 duplexes to adopt Z‐DNA stuctures, in which the syn conformation of guanine is favoured. Enhanced anti‐syn activation energy with respect to the unmethylated derivative was calculated in the cases of m5G. This result is rationalized on the basis of steric and electronic factors. In addition, an increased stabilization of the syn conformer due to selective methylation of guanine was calculated. The data obtained are in good corrspondence with the experimentally observed B‐Z transition iun synthetic methylated DNA duplexes with alternating dC‐dG sequence.Our work concerning the initiating step in the B‐Z transistion which involves rotation around the C(4′)‐C(5′) bond induced by P(V) trigonal bipyramidal intermediates is discussed. In combination with the rotation around the glycosidic C(1′)‐N bond, it can be shown that the phosphate withinh thge dpC structure is selectively sctivated.
Baker's Yeast Adenlate Kinase
Authors: TOMASSELLI Alfredo G. , NODA Lafayette H. 
Published: March 2005

The diastereomers of adenosine 5′‐O‐(1‐thiotriphosphate) ATPαS) and adenosine 5′‐O‐(2‐thiotriphosphate) (ATPβS) were used to determine the stereochemical mode of coordination of Mg2+ to ATP when bound in the yeast adenylate kinase complex. ATPαS isomer A is the preferred substrate either in the presence of Mg2+ or Cd2+; ratios of the rate of reaction at infinite concentration of substrate, V, for A to B isomers of ATPαS with Mg2+ were 457/51.4 = 8.9 and with Cd2+ 124/12.8 = 9.7. Thus there is no reversal of stereospecificity on substituting Cd2+ for Mg2+ and this supports the view that metal does not chelate with the α‐P. In contrast the V ratios of ATPβS(A) to ATPβS(B) with Mg2+ were 1.33/0.15 = 8.9 and with Cd2+ 0.18/5.1 = 0.035. Thus this reversal of stereospecificity when Cd2+ is substituted for Mg2+ (in view of the fact that Mg2+ binds to O and Cd2+ to S) leads to the conclusion that a metal chelation must occur via the β‐phosphate. Since the enzyme prefers MgATPβS(A) and CdATPβS(B) (both isomers have Δ‐screw sense) to MgATPβS(B) and CdATPβS(A) (both isomers have Δ‐screw sense) and Mg2+ also coordinates with the γ‐phosphate (by analogy with other adenylate kinase isozymes), the interpretation is that baker' yeast adenylate kinase binds MgATP as Δ, β, γ. The fluorescence of the two residues of yeast adenylate kinase has been used to investigate conformational changes of the enzyme on binding its substrates. Fluorescene is maximal at 334 nm (λexx 295 nm, pH 7.5) and is reduced 10% with no shift in λmax in the presence of ATP and reduced a further 15% on the addition of Mg2+ or other metal acitvators to E‐ATP. In contrast non‐activating metals such as Ba2+ and Sr2+ increase quenching only slightly. With ITP, UTP, CTP, GTP the quenching is 4.1%, 5.2%, 8.7% and 6.6% with or without Mg2+. Differential conformational changes of yeast adenylate kinase on binding MgATP, ATP, and other nucleotides are shown by (a) fluorescence changes, (b) previously reported kinetic and binding studied and (c) by the fact that the Km for AMP in the presence of MgATP is 17 times less than in the presence of MgGTP. These differential conformational changes result in differing tendencies of AMP to bind to the enzyme. Ultraviolet difference spectra obtained on binding of ATP to the enzyme (shoulder at 271 nm and peak at 276 nm) have been compared with the difference spectra of the interaction of ATP, adenosine and 1‐methyl adensoine respectiively with methyl mercuric hydroxide and formaldehyde. On the basis of these comparisons, the shoulder at 271 nm reflects the interaction of N(1) and the peak at 276 nm the interaction of tthe 6‐amino group of ATP with the enyzme.The following mechanism of reaction is proposed: upon binding MgATP the enzyme undergoes a conformational change which closes the active‐site crevice by bringing the two lobes closer, expelling water from the crevice. AMP is bound with higher affinity and the α‐oxygen and the α‐oxygen attacks the γ‐phosphate of ATP in a nucleophilic reaction.
Post‐Translational Fate of Variant MOPC 315 λ Chains in Xenopus Oocytes and Mouse Myeloma Cells
Authors: VALLE Giorgio , BESLEY Jayne , WILLIAMSON Alan R. , MOSMANN Timothy R. , COLMAN Alan 
Published: March 2005

The post‐translational fates of three immunoglubulin λ chain variants of MOPC 315 were investigated in mouse plasmactyoma cell lines and in mRNA‐microinjected Xenopus oocytes.Quite unexpectedly we found that one non‐secretory variant chain (λ‐43) underwent extensive post‐translational N‐glycosylation; however the presence of the oligosaccharide moiety did not account for the non‐secretory phenotype nor did it affect the rate of degradation of this λ chain. Another variant chain (λ‐47) at first believed to be non‐secretory, was found to be secreted from oocytes at a very low level, but mostly as a λ‐λ dimer. In myeloma cells a low level of λ‐47 chain was secreted and again λ‐λ dimers were the favoured secretory form. The secretory λ‐48 chain also formed λ‐λ dimers, whereas λ‐43, which was never secreted, was only found as a monomeric λ chain in both oocytes and myeloma cells.A similar relationship between assembly and secretion was found when oocytes were coinjected with MOPC 21 heavy (γ1) chain mRNA and MOPC 315 λ chain mRNAs. The wild type λ chain (λ‐48) was able to assemble with the γ chain in a covalently bound tetramer (γγλλ). The variant λ‐47 chain was also able to form γγλλ tetramers, whereas the λ‐43 was nott, even when glycosylation was prevented by tunicamycin. Both types of tetramer were secreted.These data reinforce the idea that conformational changes play a major role in the routing of secretory proteins and that the cellular mechanisms by which these changes are recognized are not cell‐type specific.
Spin‐Label Study of Histone H1‐DNA Interaction
Authors: GURARDET Jean‐Luc , LAWRENCE Jean‐Jacques 
Published: March 2005

As shown in a previous paper [J. J. Lawrence et al. (1980) Eur. J. Biochem. 107, 263–269], convalent spin labeling of basis residues in histone H1 allows the study of the interaction of this protein with DNA. Using a step gradient dialysis procedure to reconstitute chromatin from labeled H1 and stripped chromatin, it is shown that the process of interaction of the lysine residures and DNA is the same whether histone H1 is bound to linear purified DNA or to H1‐depleted chromatin.In contrast, spin labeling of the unique tyrosine of histone H1 located in the globular part of the molecule shows that this part is more involved in the interaction with chromatin than it is with linear DNA (as judged from the lengthening of the rotational correlation time).These data are interpreted as reflecting defferent roles for th N and C termini of the molecule of H1 and the central globular part. A model, based on these observations together with examination of the primary structures of histones H1, is proposed which accounts for the H1 involvement in the chromatosome structure.
Phosphorylated Intermediate of a Transport ATPase and Activity of Protein Kinase in Membranes from Corn Roots
Authors: SCALLA René , AMORY Antoine , RIGAUD Jacqueline , GOFFEAU André 
Published: March 2005

A maize‐root microsomal fraction was enriched in ATPase by treatment with Triton X‐100. This activity, which reached 1.2–2.0/μmol Pi× min−1× gm protein−1, was specific for ATP, very slightly stimulated by K+, inhibited by orthovanadate and diethylstibestrol, resistant to oligomycin and azide, and had a Km of 1.2 mM MgATP.Incubation of th emicrosomal fraction with [y32‐P]ATP followed by electrophoresis in acid conditions revealed the presence of several phosphoproteins. The phosphorylation of a 110000‐M4 polypeptide reached the steady‐state level in less than 5 s and rapidly turned over the phosphate group. The phosphorylation level was an hyperbolic function of the [ATP] with a Km of 0.6 mM, suggesting that the rat e of Pi production was proportional to the phosphoprotein concentration. The extent of phosphoprotein was decreased by vanadate and diethylstilbestrol. The phosphorylation level was 30% decreased by 50 mM K+ or Na+ while the ATPase activity was slightly stimulated (12% and 5%. respectively). The polypeptide could not bephosphorylated in reverse by Pi. This phosphorylated intermediate from maize‐root microsomes exhibits molecular properties characteristic of transport ATPases such as the yeast plasma membrance H+ ‐translocating ATPase. This similarity indicates existence of a transport ATPase in plant plasma membranes.Three other plant microsomal polypeptides (Mr= 52000, 17000 and 16000) and a low molecular weight component (Mr < 1000) were phosphorylated much more slowly, were not undergoing a rapid turnover and were not hydrolysed by hydroxylamine. These phosphoproteins and the Mr < 1000 phosphorylated component were inhibited by vanadate and diethylstilbestrol. These properties are similar to those of the protein kinase activity recently described in yeast plasma membranes.
Interaction of tRNAPhe and tRNAVal with Aminoacyl‐tRNA Synthetases
Authors: VLASSOV Valentin V. , KERN Deniel , ROMBY Pascale , GIEBÉ Richrd , EBEL Jean‐Pierre 
Published: March 2005

The alkylation by ethylnitrosourea of phyosphodiester bonds in tRNAPhe from veast and in tRNAVal from yeast and from rabbit liver and that by 4‐N‐2‐chloroethyl‐n‐methylamino)‐benzylamine of N‐7 atoms of guanosine residues in yeast tRNAVal have been used to study the interaction of these tRAAs with anminoacyl‐tRNA synthetases. The modifications occurring at low yield were carried out on 3′ and'or 5′ end‐labelled tRNAs either free or in the presence of cognate or non‐cognate synthetases. After splitting of the tRNAs at the alkylated positions, the position of the modification sites in the tRNA sequences were detected by acrylamide gel electrophoresis. It was found that the synthetases protect against alkylation certain phosphate or guanosine residues in their cognate tRNAs. Non‐cognate synthetases failed to protedct efficiently specific positions in tRNA against modification.In yeast tRNAPhe the cognate phenylalanyl‐tRNA synthetase protects certain phosphates located in all four stems and in the anticodon and extra‐loop of the tRMA. Particularly strong protections occur on phosphate 34 in the anticodon loop and on phosphates 23, 27, 28, 41 in the D and anticodon stems.In yeast tRNAVal complexed with yeast valyl‐tRNA synthetase the protected phosphates are essentialy located in the tRNA. Three guanosine residues, located in the D stem, and another one in the 3′ part of the anticodon stem were also found protected by the synthetase. In mammalian tRNAVal, complexed with the congnate but heterologous yeasst valyl‐tRNA synthetase, the protected phosphates lie in the anticodon stem, in the extra‐loop andd in the T ψ arm.The location of the protected residues in the structure of three tRNAs suggests some common features in the binding of tRNAs to aminoacyl‐tRNA synthetases. These results will be discussed in the light of infsormations of interaction sites obtained by nucleases digestion and ultravilet cross‐linking methods.
Chemical Modification of the Mitochodrial bc1 by N,N′ ‐Dicyclohexylcarbodiimide Inhibits Proton Translocation
Authors: PRICE Brendan D. , BRAND Martin D. 
Published: March 2005

We report here that N,N/i′‐dicyclohexylcarbodiimide (DCCD) decreases the H2e stoichiometry of the mytochrome bc1 complex from 308 ± 0.2 (10) to 201 ± 0.1 (8) but has only a minimal effect on the H2e ratio of cytochrome oxidase under the relatively mild condition used. The veffect on the bc1 complex connot be explained by uncoupling, by inhibition of electron transport or by selective mitochondrial damage.We conclude that DCCD is an inhibitor of proton translocation within ther bc1 complex. There are three possible explanation of this effect: (a) DCCD could alter the pathway of electron flow, (b) DCCD could prevent one of the proton translocation reations but not electrton transport, (c) DCCD could prevent the couduction of the translocated proton to the external phase.
A New Myofibrillar Protein Characteristic of Type I Human Skeletal Muscle Fibres
Authors: HEIZMANN Claus W. , CELIO Marco R. , BILLETER Rudolf , REIST Ursula , MORET Maria‐Luisa , ARNOLD Eva M. 
Published: March 2005

Analysis of a single human type I (slow‐twitch) skeletal muscle fibres by two‐dimensional gel electrophoresis shows that they contain a specific protein of subunit M, 34 × 103 and PI 6.0,6.1 and 6.2 (three spots) which is absent from type IIA and IIB muscle fibres. This type I protein is located in the I‐band of isolated myofibrils as demonstrated by the indirect immunofluorescence using specific antisera against the denatured form of type I protein. Type I protein could only be extracted from myofibrils with high salt solutions (0.6 ‐ 1 M KCl), conditions which also coextract actin and myosin, demonstrating its firm attachment.Rat and cat type I muscle fibres as well as the slow‐tonic fibres from chicken anterior latissimus dorsi also contained a protein homologous to the human type I protein.The human type I protein was found to be different from the known regulatory and contractile muscle proteins by electrophoretic and immunological criteria, and may therefore be a new myofibrillar protein in type I muscle fibres.
Activity of Human Blood Platelets in Prothrombin and in Factor X Activation Induced by Ionophore A23187
Authors: VAN RIJN Jan , ROSING Jan , VAN DIEIJEN Gerbrand 
Published: March 2005

Two reactions of the blood coagulation cascade are highly stimulated by membranes containing negatively charged phospholipids. These are the activation of prothrombin to thrombin by factor Xa with factor Va as cofactor, and the conversion of factor X into Xa by factor IXa with factor VIIIa as cofactor. It is thought that under physiological conditions these reactions take place with the clotting factors adsorbed at the surface of blood platelets.The role of blood platelets in the two reactions was studied using two assay systems consisting of highly purified clotting factors. The rates of formation of thrombin or factor Xa, measured with specific chromogenic substrates, were dependent on the quantity of negatively charged phospholipid present in the reaction mixture. Phospholipid vesicles with platelet phospholipid composition were highly active in the two reaction systems. When the purified lipids were substituted by disrupted platelets, containing the same quantity of lipids, the same high activities were found. With intact human blood platelets a low but significant activity was observed. Incubation of the platelets with the calcium ionophore A23187, resulted in a 50–60‐fold activity increase in formation of thrombin and factor Xa. Platelet disruption increases procoagulant activity, since the negatively charged phospholipids, originally situated in the inner leaflet of the lipid bilayer of the platelet membrane become exposed upon platelet disruption. Ionophore activation of platelets was however not due to lysis since discharge of platelet lactate dehydrogenase was less then 3%. The activity of A23187‐activated platelets in formation of thrombin and factor Xa was completely abolished after incubation with phospholipase A2, which is to be expected if negatively charged lipids are present at the platelet outer surface. In contrast to platelets, erythrocytes could not be activated by the ionophore.As found by other workers, A23187 induces release of ATP and factor V(a) and results in platelet aggregation. The appearance of procoagulant activity, ATP and factor V(a) release were all dependent on the A23187 concentration and were too rapid to study the time course. All platelet activities were induced between 0.2 μM and 0.7 μM A23187, suggesting a relationship between the release processes and the appearance of procoagulant activity.
Calcium‐Independent Activation of Adenylate Cyclase by Calmodulin
Authors: KILHOFFER Marie‐Claude , COOK G. HOPE , WOLFF J. 
Published: March 2005

Adenylate cyclase of Bordetella pertussis is stimulated by calmodulin by two distinct interactions. At low activator concentrations (∼ 1 nM) the process is Ca2+‐dependent (i.e. inhibited by EGTA added before calmodulin). High activator concentrations (∼ 0.1–10 μM) stimulate adenylate cyclase also in the presence of EGTA, an effect not accounted for by residual Ca2+ or low concentrations of Ca · calmodulin, which thus appears to be due to calcium‐free calmodulin. Some calmodulin dose‐response curves show both phases of stimulation, separated by a plateau of activity, and half‐maximal activating concentrations differ by 100–300‐fold. Both effects are on the V and not the Km for ATP and are not mimicked by 105‐fold greater concentrations of parvalbumin or by various polyanions.In addition, adenylate cyclase stimulation at high calmodulin concentrations is greater in the presence of EGTA than in its absence. This enhancement is also produced by 1,10‐phenanthroline and 8‐hydroxyquinoline but not by non‐chelating isomers. These compounds are poor Ca2+ chelators, stimulate at any calmodulin concentration (unlike EGTA), and suggest regulation of this adenylate cyclase by a second metal ion.
Further Characterization of the Three Polypeptide Chains of Bovine and Human Short‐Chain Collagen (Intima Collagen)
Authors: JANDER Renate , RAUTERBERG Jürgen , GLANVILLE Robert W. 
Published: March 2005

Short‐chain collagen was isolated from bovine and from human placenta after limited pepsin digestion. By reductive cleavage of disulfide bonds under non‐denaturing conditions, mainly non‐collagenous domains were cleaved off yielding a uniform component composed of three polypeptide chains with molecular masses between 37 kDa and 48 kDa in a nearly equimolar ratio. The chains (SC1*, SC2* and SC3*) were isolated and characterized. By comparison of peptide patterns obtained after various cleavage procedures, they could be identified as more or less shortened forms of SC1, SC2 and SC3, the isolation of which from bovine short‐chain collagen has been described [Jander et al. (1981) Eur. J. Biochem. 114, 17–25].The peptide patterns; as well as N‐terminal sequence determination, give evidence for the genetic individuality of the three chains; the data so far available suggest that together they form one triple‐helical structure which represents the collagenous domain of the basic molecular unit of short‐chain collagen.