bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of phospholipid, detergent and protein‐protein interaction on stability and phosphoenzyme isomerization of soluble sarcoplasmic reticulum Ca‐ATPase
Authors: VILSEN Bente , ANDERSEN Jens P. 
Published: March 2005

The purpose of the present study was to elucidate the separate roles of lipid, detergent and protein‐protein interaction for stability and catalytic properties of sarcoplasmic reticulum Ca‐ATPase solubilized in the non‐ionic detergent octa(ethylene glycol) monododecyl ether (C12E8). The use of large‐zone high‐performance liquid chromatography permitted us to define the self‐association state of Ca‐ATPase peptide at various detergent, phospholipid and protein concentrations, and also during enzymatic turnover with ATP. Conditions were established for monomerization of Ca‐ATPase in the presence of a high concentration of phospholipid relative to detergent. The lipid‐saturated monomeric preparation was relatively resistant to inactivation in the absence of Ca2+, whereas delipidated enzyme in monomeric or in oligomeric form was prone to inactivation.Kinetics of phosphoenzyme turnover were examined in the presence and absence of Mg2+. Dephosphorylation rates were sensitive to Mg2+, irrespective of whether the peptide was present in soluble monomeric form or was membrane‐bound. C12E8‐solubilized monomer without added phospholipid was, however, characterized by a fast initial phase of dephosphorylation in the absence of Mg2+. This was not observed with monomer saturated with phospholipid or with monomer solubilized in myristoylglycerophosphocholine or deoxycholate. The mechanism underlying this difference was shown to be a C12E8‐induced acceleration of conversion of ADP‐sensitive phosphoenzyme (E1P) to ADP‐insensitive phosphoenzyme (E2P).The phosphoenzyme isomerization rate was also found to be enhanced by low‐affinity binding of ATP, This was demonstrated both in membrane‐bound and in soluble monomeric Ca‐ATPase.Our results indicate that a single peptide chain constitutes the target for modulation of phosphoenzyme turnover by Mg2+ and ATP, and that detergent effects, distinct from those arising from disruption of protein‐protein contacts, are the major determinants of kinetic differences between C12E8‐solubilized and membrane‐bound enzyme preparations.
Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme
Authors: WILLING Andreas , FOLLMANN Hartmut , AULING Georg 
Published: March 2005

Ribonucleotide reduction and not DNA replication is the site for the specific manganese requirement of DNA synthesis and cell growth in the coryneform bacterium Brevibacterium ammoniagenes. To characterize the metal effect we have isolated and purified ribonucleoside‐diphosphate reductase from overproducing bacteria that were first deprived of and then reactivated by manganese ions. Purification on columns of Sephacryl S400, DEAE‐cellulose and hydroxyapatite provided an apparently homogeneous enzyme consisting of two protein subunits. These were characterized by affinity chromatography on 2′,5′‐ADP‐Sepharose as nucleotide‐binding protein B1 (Mr= 80000) and catalytic protein B2 (M r= 100000, composed of two Mr= 50000 polypeptides), which were both necessary for activity. In vitro the purified enzyme does not require added metal ions except for an unspecific, twofold activity increase observed in the presence of Mg2+ and other divalent cations. Enzyme activity is inhibited by hydroxyurea (I50= 2.5 mM). The electronic spectrum with maxima around 455 nm and 485 nm closely resembles that of manganese(III)‐containing pseudocatalase and of oxo‐bridged binuclear Mn(III) model complexes. Denaturation of the enzyme in trichloroacetic acid liberated an equimolar amount of Mn(II) which was detected by EPR spectroscopy. It was not possible to remove and reintroduce metal ions without loss of enzyme activity.Manganese‐deficient cell cultures were also grown in the presence of 54MnCl2. Ribonucleotide reductase activity and radioactivity cochromatographed in several systems. Non‐denaturing polyacrylamide gel electro‐phoresis showed that protein subunit B2 was specifically 54Mn‐labeled.All these properties suggest that the ribonucleotide reductase of B. ammoniagenes is a manganese‐containing analog of the non‐heme‐iron‐containing reductases of Escherichia coli and eukaryotes.
Elongation factor 1βγ from Artemia
Authors: JANSSEN George M. C. , MÖLLER Wim 
Published: March 2005

The guanine nucleotide exchange factor, elongation factor 1βγ (EF‐1βγ) has been purified from Artemia cysts using an improved method. The protein consists of two distinct polypeptides with relative molecular masses of 26000 (EF‐1β) and 46000 (EF‐1γ). A nucleoside diphosphate phosphotransferase activity often found in EF‐1βγ preparations has been completely separated from the actual guanine nucleotide exchange stimulatory activity of EF‐1βγ, thus indicating that nucleotide diphosphate phosphotransferase is not an intrinsic property of EF‐1β. Both EF‐1βγ and EF‐1β have been shown to stimulate the following three reactions to a comparable degree: (a) exchange of GDP bound to EF‐1α with exogenous GDP; (b) EF‐1α‐dependent binding of Phe‐tRNA to ribosomes; (c) poly(U)‐dependent poly(phenylalanine) synthesis. However, a significantly higher nucleotide exchange rate was observed in the presence of EF‐1βγ compared to EF‐1β alone.Concerning elongation factor 1γ (EF‐1γ) the following observations were made. In contrast to EF‐1β, pure EF‐1γ is rather insoluble in aqueous buffers, but the tendency to precipitate can be partially suppressed by the addition of detergents. In particular, EF‐1γ partitions solely into the detergent phase of Triton X‐114 solutions. EF‐1γ is also more susceptible to spontaneous, specific fragmentation. It is remarkably that about 5% of the cellular pool of EF‐1βγ was found to be present in membrane fractions, under conditions where no EF‐1α was detectable in these fractions. Furthermore it was noted that EF‐1βγ copurified strongly with tubulin on DEAE‐cellulose. Moreover, it was observed that from a mixture of EF‐1βγ and tubulin, EF‐1γ coprecipitates with tubulin using a non‐denaturating immunoprecipitation technique. These findings suggest that EF‐1γ has a hydrophobic domain and interacts with membrane and cytoskeleton structures in the cell.
Expression in Escherichia coli and characterization of human growth‐hormone‐releasing factor
Authors: VILLA Stefano , DE FAZIO Gabriele , DONINI Silvia , TARCHI Giulio , CANOSI Umberto 
Published: March 2005

A chemically synthesized DNA sequence, coding for the 44 amino acid residues of human growth‐hormone‐releasing factor (GRF) preceded by a tryptophan codon, was cloned in frame with Escherichia coli trpE gene within a pBR322‐derived plasmid. GRF was expressed in E. coli as a fused polypeptide chain (TrpE‐GRF) and then the GRF amino acid sequence was released from the fused protein by specific chemical cleavage at the tryptophan residue using o‐iodosobenzoic acid. The thioether group of the methionine residue of GRF was converted in the sulfonium salt derivative, in order to prevent irreversible oxidation of methionine to the sulfone derivative by the o‐iodosobenzoic acid reagent.GRF was purified by HPLC and characterized in terms of amino acid composition after acid hydrolysis, protein sequencing and gel electrophoretic behaviour. These data clearly established that the biosynthetic GRF was identical to the natural one, except for the lack of amidation at the carboxyl‐terminal amino acid. Far‐ultraviolet circular dichroism measurements established that both biosynthetic and natural GRF are devoid of secondary structure in aqueous solution at neutral pH, whereas both peptide samples achieve a high percentage of helical structure in the presence of trifluoroethanol.
Electrogenic steps in the redox reactions catalyzed by photosynthetic reaction‐centre complex fromRhodopseudomonas viridis
Authors: DRACHEVA Stella M. , DRACHEV Lel A. , KONSTANTINOV Alexander A. , SEMENOV Alexey Yu. , SKULACHEV Vladimir P. , ARUTJUNJAN Alexander M. , SHUVALOV Vladimir A. , ZABEREZHNAYA Svetlana M. 
Published: March 2005

Electrogenic and redox events in the reaction‐centre complexes fromRhodopseudomonas viridishave been studied. In contrast to the previous points of view it is shown that all the four hemes of the tightly bound cytochromechave different Emvalues (‐60, +20, +310 and +380 mV). The first three hemes reveal α absorption maxima at 554 nm, 552 nm and 556 nm respectively. The 380‐mV heme displays a split α band with a maximum at 559 nm and a shoulder at 552 nm. Such a splitting is due to non‐degenerated Qxand Qytransitions in the iron‐porphyrin ring as demonstrated by magnetic circular dichroism spectra. Fast kinetic measurements show that, at redox potentials when only high‐potential hemesc‐559 andc‐556 are reduced, hemec‐559 appears to be the electron donor to P‐960+(τ= 0.32 μs) whereas hemec‐556 serves to rereducec‐559 (τ= 2.5 μs). Upon reduction of the third heme (c‐552), the P‐960+reduction rate increases twofold (τ= 0.17 μs) and all photoinduced redox events within the cytochrome appear to be complete in less than 1 μs after the flash. The following sequence of the redox centers is tentatively suggested:c‐554,c‐556,c‐552,c‐559, P‐960.To study electrogenesis, the reaction‐centre complexes fromRps. viridiswere incorporated into asolectin liposomes, and fast kinetics of laser flash‐induced electric potential difference has been measured in proteoliposomes adsorbed on a phospholipid‐impregnated film. The electrical difference induced by a single 15‐ns flash was found to be as high as 100 mV. The photoelectric response has been found to involve four electrogenic stages associated with (I) QAreduction by P‐960+(II) reduction of P‐960+by hemec‐559; (III) reduction ofc‐559 byc‐556 and (IV) protonation of Q2−B. The relative contributions of stages I, II, III and IV are found to be equal to 70%, 15%, 5% and 10%, respectively, of the overall electrogenic process. At the same time, the first three respective distances along the axis normal to the membrane plane covered by electrons, calculated from X‐ray data of Deisenhofer et al. [J. Mol. Biol. 180, 385–398 (1984)], are 22%, 18.5% and 26%. This indicates that the efficiency of electrogenic phases depends first of all upon the value of the dielectric constant of the respective membrane regions rather than upon the distance between the redox groups involved. The efficiency is higher, the deeper these groups are immersed in the membrane.
Association of glyceraldehyde‐3‐phosphate dehydrogenase with mono‐and polyribosomes of rabbit reticulocytes
Authors: RYAZANOV Alexey G. , ASHMARINA Lyudmila I. , MURONETZ Vladimir I. 
Published: March 2005

It has been shown recently that glyceraldehyde‐3‐phosphate dehydrogenase (GAPD) is one of the three major RNA‐binding proteins of rabbit reticulocytes [Ryazanov, A. G. (1985) FEBS Lett. 192, 131–134]. It was suggested that, due to its RNA‐binding capacity, GAPD can form loose dynamic complexes with polyribosomes.This communication reports that a considerable amount of GAPD activity can be found in the mono‐ and polyribosome fraction after sucrose gradient centrifugation of rabbit reticulocyte lysate. An increase of ionic strength, as well as the addition of exogenous RNA to the extract, result in the removal of GAPD from the complex with mono‐ and polyribosomes. It appears that GAPD forms the complex with polyribosomes due to the interaction with some exposed RNA regions of these structures. Although the interaction of GAPD with ribosomes is weak, it can be detected under physiological ionic conditions by the difference boundary sedimentation velocity technique.Association of GAPD with mono‐ and polyribosomes can be prevented by a low concentration (10 μM) of NADH, but not NAD+. A nitrocellulose filter binding assay also shows that NADH has a stronger inhibitory effect on the enzyme‐RNA complex formation, as compared with NAD+.We propose that the RNA‐mediated association of GAPD with mono‐and polyribosomes can provide compartmentation of the energy‐supplying system on these structures within the cell. This can maintain a high local concentration of ATP and GTP near the sites of protein synthesis.
Inhibition of mitochondrial F1 ATPase and sarcoplasmic reticulum ATPase by hydrophobic molecules
Authors: DE MEIS Leopoldo , TUENA DE GÓMEZ PUYOU Marietta , PUYOU Armando GÓMEZ 
Published: March 2005

The hydrophobic nature of the active site of two energy‐transducing ATPases was explored by comparing interactions between Pi and each of three hydrophobic drugs in the absence and presence of organic solvents. The drugs tested were the Fe · bathophenanthroline complex and the anticalmodulin drugs, calmidazolium and trifluoperazine. All inhibit the Pi⇆ ATP exchange reaction catalyzed by submitochondrial particles and the ATPase activity of both submitochondrial particles and soluble F1 ATPase. The inhibition by the three drugs is reversed by either raising the Pi concentration or by adding organic solvent (dimethylsulfoxide, ethyleneglycol or methanol) to the medium. The inhibition of the Pi⇆ ATP exchange by trifluoperazine becomes more pronounced when the electrochemical proton gradient formed across the membrane of the submitochondrial particles is decreased by the addition to the medium of the proton ionophore carbonylcyanide p‐trifluoromethoxy‐phenylhydrazone.The ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum vesicles are inhibited by the Fe · bathophenanthroline complex, calmidazolium and trifluoperazine. Phosphorylation of the ATPases by Pi, synthesis of ATP from ADP and Pi and the fast efflux of Ca2+ observed during reversal of the Ca2+ pump are inhibited by the three drugs. The inhibition is reversed by raising the concentration of Pi or dimethylsulfoxide. The three drugs tested appear to compete with Pi for a common binding site on the Ca2+‐ATPase.The data presented are interpreted according to the proposal that the catalytic site of an enzyme involved in energy transduction undergoes a hydrophobic–hydrophilic transition during the catalytic cycle.
Stress response of Tetrahymena pyriformis to arsenite and heat shock: Differences and similarities
Authors: AMARAL Margarida D. , GALEGO Lisete , RODRIGUES‐POUSADA Claudina 
Published: March 2005

Molecular and cellular events associated with the response of Tetrahymena pyriformis to stress induced by sodium meta‐arsenite, have been examined by pulse‐labelling experiments. This stress agent induces the synthesis of two main groups of proteins, with molecular masses in the ranges 70–75 kDa and 25– 29 kDa, together with other proteins of molecular masses 92, 83, 46, 42 (two species), 36 and 35 kDa.Comparison of the results with those of a previous study concerning the response of T. pyriformis to heat‐shock, shows that the two main groups of proteins, as well as the 92‐kDa and 35‐kDa species, which seem to be similarly induced by both types of stress, display similar or identical peptide maps. Other stress proteins seem to be either heat‐shock‐specific or arsenite‐specific.Studies using actinomycin D suggest that the response to arsenite is controlled mainly at the transcriptional level, for the 70–75‐kDa group and 92‐kDa proteins, but it seems that the other arsenite‐induced proteins are subjected to transcriptional/translational control. In fact, results obtained by northern blotting, show that the mRNA coding for the 70‐kDa stress protein is present only in stressed cells, whereas the 27‐kDa‐coding mRNA is present both in stressed and in unstressed cells. Inhibition of translation by cycloheximide has shown that heat‐shock‐induced‐messengers are conserved under heat to be immediately translated upon removal of that inhibitor. Qualitatively similar results are obtained after prolonged treatments of T. pyriformis with arsenite and cycloheximide.The most striking difference between the responses of T. pyriformis to these two stress conditions is that arsenite does not repress normal protein synthesis so drastically as heat shock. In addition, our results suggest that some arsenite‐induced messengers may be more stable than the corresponding heat‐shock‐induced messengers.
Identification of cytoplasmic nodule‐associated forms of malate dehydrogenase involved in the symbiosis between Rhizobium leguminosarum and Pisum sativum
Authors: APPELS Michiel A. , HAAKER Huub 
Published: March 2005

The malate dehydrogenase activity (EC 1.1.1.37), present in the cytoplasm of Pisum sativum root nodules, can be separated by ion‐exchange chromatography into four different fractions. Malate dehydrogenase activity present in the cytoplasm of roots elutes mainly as a single peak.During nodule development an increase in malate dehydrogenase activity per gram of material was observed. This increase occurred concomitantly with the increase in nitrogenase activity.The kinetic properties of the separated malate dehydrogenases of root nodule cytoplasm and root cytoplasm were studied. The Km values for malate (2.6 mM), NAD+ (27 μM), oxaloacetate (18 μM) and NADH (13 μM) of the dominant form of the root nodule cytoplasm are much lower than those of the dominant malate dehydrogenase root form (64 mM, 4.4 mM, 89 μM and 70 μM respectively). Binding of malate by the enzyme‐NADH complex from root nodules results in an abortive complex, thereby blocking the further reduction of oxaloacetate by NADH. The dominant root malate dehydrogenase does not form the abortive complex.From the kinetic data it is concluded, first, that the root nodule forms of the enzyme are capable of catalysing at a high rate the reduction of oxaloacetate, to meet the demands for malate governed by the bacterioid and the infected plant cell. The second conclusion, drawn from the kinetic data, is that under physiological conditions the conversion of oxaloacetate can be controlled just by the malate concentration.Consequently the major root nodule forms of malate dehydrogenase are able to allow a high flux of malate production from oxaloacetate but also to establish a sufficient oxaloacetate concentration necessary for the assimilation and transport of fixed nitrogen.
Structure function studies on the lipoate‐acetyltransferase–component‐X‐core assembly of the ox heart pyruvate dehydrogenase complex
Authors: HODGSON Jeffery A. , DE MARCUCCI Olga G. L. , LINDSAY J. Gordon 
Published: March 2005

Component X, the recently recognised subunit of mammalian pyruvate dehydrogenase complex, was shown by immune blotting to be present in all of nine tissues dissected from rat. This finding indicated that component X was not an isoenzyme of the lipoate acetyltransferase (E2) associated with one or a limited number of tissues.Native pyruvate dehydrogenase complex was shown to bind IgG raised to isolated component X, indicating that there were at least some regions of the X subunit exposed at the periphery of the complex.Lipoyl groups of ox heart pyruvate dehydrogenase complex were specifically cross‐linked by reaction with phenylene‐o‐bismaleimide in the presence of pyruvate and the subunits contributing to the products of cross‐linking were identified by immune blotting. Species with very high Mr containing both E2 and component X, were formed in high yield, as well as apparent E2/E2 and E2/X dimers and trimers and an X/X dimer. These results showed that acetylated lipoyl groups of different E2 and X subunits were able to interact in all possible combinations.The types of cross‐linked E2 products formed suggested that two thiols, reactible with phenylene‐o‐bismaleimide, were rapidly generated in the presence of pyruvate. The results were most easily explained by the presence of two acetylatable lipoyl groups on each E2 polypeptide.
Modifications of oxidative phosphorylations in mitochondria isolated from a mutant of Saccharomyces cerevisiae
Authors: MANON Stephen , GUERIN Martine 
Published: March 2005

Mutants of Saccharomyces cerevisiae were isolated which supported two unlinked nuclear mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial one conferring paromomycin sensitivity. Mitochondria isolated from such a mutant exhibited modifications of several phosphate‐requiring functions: (a) kinetic parameters of the phosphate dependence of ATP synthesis were modified; (b) in the absence of phosphate the inner mitochondrial membrane exhibited a high proton leakage; (c) mutant mitochondria always exhibited a poor respiratory control and required tenfold more phosphate to reach a maximal state 3 of respiration; (d) phosphate transport, as measured by swelling experiments, was mersalyl‐insensitive and, consequently, state 3 of the respiration and ATP synthesis remained less mersalyl‐sensitive than in wild‐type mitochondria.Analysis of the mitochondrial metabolism of diploid and segregant strains indicates that these modifications are related to the cryosensitive phenotype; however, at present, a cooperative effect of the mitochondrial mutation cannot be eliminated. It is proposed that the phosphate carrier itself or a regulatory element was modified.
Regulation of angiotensin II receptors and steroidogenic responsiveness in cultured bovine fasciculata and glomerulosa adrenal cells
Authors: PENHOAT Armelle , JAILLARD Christine , CROZAT Anne , SAEZ José M. 
Published: March 2005

The aim of the present study was to investigate the effect of several effectors on angiotensin II (A‐II) receptors and steroidogenic responsiveness in cultured bovine fasciculata cells. Treatment of adrenal cells for 24 h with A‐II (0.1 μM), corticotropin (1 nM), phorbol ester (PMA 0.1 μM), calcium ionophore A23187 (0.1 μM) and cyclic 8‐bromoAMP (1 mM) produced a loss of A‐II receptors whereas the A‐II antagonist [Sar1‐Ala8]A‐II (0.1 μM) led to a small but significant increase. The extent of the down‐regulation of receptors following maximal concentrations of A‐II was greater than that produced by the other agents. The effects of A‐II were dose‐dependent with a ID50 of 3 nM. Since cycloheximide and actinomycin blocked the down‐regulation of receptors, it seems likely that the effectors lead to the synthesis of certain proteins which inhibit the recycling of internalized receptors.Pretreatment of adrenal cells with A‐II induced both homologous (90% decrease) and heterologous (corticotropin 83, PMA and ionophore 76% decrease) steroidogenic desensitization. However, the cAMP response to corticotropin of A‐II‐pretreated cells was higher (P < 0.001) than for control cells. Pretreatment with PMA and A23187 also resulted in both homologous and heterologous steroidogenic refractoriness but to a lesser degree than that induced by A‐II. In contrast, corticotropin‐pretreated cells responded normally to further stimulation with corticotropin or A‐II. Similarly pretreatment of bovine adrenal glomerulosa cells with A‐II (1 nM and 0.1 μM) and corticotropin (1 nM) also induced A‐II receptor loss and steroidogenic refractoriness.The present findings indicate that, in contrast to the results reported in vivo in the rat, where A‐II leads to up‐regulation of its own receptors on glomerulosa cells and increases steroidogenic responsiveness, this peptide results in both down‐regulation and desensitization in cultured bovine fasciculata and glomerulosa cells. Our results also emphasize the absence of correlation between A‐II receptor loss and steroidogenic responsiveness.