bluuebunny/crossref_metadata_2025_split huggingface dataset:
The ATP/ADP‐antiporter is involved in the uncoupling effect of fatty acids on mitochondria
Authors: ANDREYEV Alexander Yu. , BONDAREVA Tatjana O. , DEDUKHOVA Vera I. , MOKHOVA Elena N. , SKULACHEV Vladimir P. , TSOFINA Lilya M. , VOLKOV Nickolaj I. , VYGODINA Tatjana V. 
Published: March 2005

The ATP/ADP‐antiporter inhibitors and the substrate ADP suppress the uncoupling effect induced by low (10–20 μM) concentrations of palmitate in mitochondria from skeletal muscle and liver. The inhibitors and ADP are found to (a) inhibit the palmitate‐stimulated respiration in the controlled state and (b) increase the membrane potential lowered by palmitate. The degree of efficiency decreases in the order: carboxyatractylate (CAtr) > ADP > bongkrekic acid, atractylate. GDP is ineffective, Mg > ADP is of much smaller effect, whereas ATP is effective at much higher concentration than is ADP. Inhibitor concentrations, which maximally suppress the palmitate‐stimulated respiration, correspond to those needed for arresting the state 3 respiration. The extent of the CAtr‐sensitive stimulation of respiration by palmitate has been found to decrease with an increase in palmitate concentration. Stimulation of the controlled respiration by p‐trifluoromethoxycarbonylcyanide phenylhydrozone (FCCP) and gramicidin D at any concentrations of these uncouplers is CAtr‐insensitive, whereas that caused by a low concentrations of 2,4‐dinitrophenol and dodecyl sulfate is inhibited by CAtr.The above effect of palmitate develops immediately after addition of the fatty acid. It is resistant to EGTA as well as to inhibitors of phospholipase (nupercain) and of lipid peroxidation (ionol). Moreover, palmitate accelerates spontaneous release of the respiratory control, developing in rat liver mitochondria under certain conditions. This effect takes several minutes, being sensitive to EGTA, nupercain and ionol. Like the fast uncoupling, this slow effect is inhibited by ADP but CAtr and atractylate are stimulatory rather than inhibitory.In artificial planar phospholipid membrane, palmitate does not increase the membrane conductance, FCCP increases it strongly and dinitrophenol only slightly.In cytochrome oxidase proteoliposomes, FCCP, gramicidin and dinitrophenol (less effectively) lower, whereas palmitate enhances the cytochrome‐oxidase‐generated membrane potential. In this system, monensin substitutes for palmitate.It is concluded that the ATP/ADP antiporter is somehow involved in the uncoupling effect caused by low concentrations of palmitate and, partially, of dinitrophenol, whereas uncoupling produced by FCCP and gramicidin is due to their action on the phospholipid part of the mitochondrial membrane. A possible mechanism of this effect is discussed.
Purification and partial characterization of 1‐aminocyclopropane‐1‐carboxylate synthase from tomato pericarp
Authors: van der STRAETEN Dominique , van WIEMEERSCH Luc , GOODMAN Howard M. , van MONTAGU Marc 
Published: March 2005

1‐Aminocyclopropane‐1‐carboxylate synthase was purified 5000‐fold from LiCl‐induced tomato fruit slices by conventional and high‐performance liquid chromatography. The final preparation was estimated to be between 25% and 50% pure. Two‐dimensional gel electrophoresis indicates that 1‐aminocyclopropane‐1‐carboxylate synthase activity is associated with a 45‐kDa polypeptide, with a pI of 5.8 ± 0.2. The enzyme is inactivated both by its substrate, S‐adenosyl‐l‐methionine (AdoMet) and by one of its products, 1‐aminocyclopropane‐1‐carboxylate.Due to the extremely low abundance of the protein it was necessary to scale up the extraction in order to obtain reasonable amounts for sequence analysis. Therefore, 200 kg tomatoes were extracted on semi‐industrial scale and 1‐aminocyclopropane‐1‐carboxylate synthase purified. This yielded approximately 150 μg enzyme.
Glutamate:4,5‐dioxovaleric acid transaminase from Euglena gracilis
Authors: LOMBARDO María Elisa , ARAUJO Lidia Susana , JUKNAT Adela Ana , BATLLE Alcira M. del C. 
Published: March 2005

The kinetic properties of the enzyme l‐glutamate:4,5‐dioxovaleric acid aminotransferase (Glu: DOVA transaminase) from Euglena gracilis have been studied. 5‐Aminolevulinic acid formation was linear with time for at least 45 min at 37°C and l‐glutamate was the most effective amino‐group donor. Lineweaver‐Burk double‐reciprocal plots suggested a ping‐pong reaction mechanism, with Km values for l‐glutamate and DOVA of 1.92 mM and 0.48 mM respectively. Competitive parabolic substrate inhibition by DOVA at concentrations greater than 3.5–4.5 mM was observed. Glyoxylate (4–10 mM) was found to be a competitive inhibitor with respect to DOVA, whereas at low concentrations (0–4 mM) noncompetitive plots were obtained.An analysis of the possible enzyme forms involved, was carried out. In more crude preparations most of the enzyme is found to be in the form of an enzyme‐glutamate complex.
A B‐type PDGF receptor lacking most of the intracellular domain escapes degradation after ligand binding
Authors: SEVERINSSON Liv , CLAESSON‐WELSH Lena , HELDIN Carl‐Henrik 
Published: March 2005

The characteristics of the human B‐type platelet‐derived‐growth‐factor (PDGF) receptor expressed in Chinese hamster ovary (CHO) cells, were compared with those of a mutant receptor lacking all but 19 amino acids of the intracellular domain. The transfected wild‐type receptor was synthesized as a 160‐kDa precursor that was processed to 190 kDa. Each CHO cell expressed 30000–100000 receptors which bound PDGF‐BB with a Kd of about 0.5 nM. Analysis of PDGF‐AB binding yielded non‐linear Scatchard plots; the major part of the binding sites had a Kd of 6 nM. PDGF‐AA was not bound. The receptors expressed in CHO cells were down‐regulated after binding of PDGF‐BB, and mediated degradation of 125I‐PDGF‐BB with similar efficiency as PDGF‐B‐type receptors in human fibroblasts. The transfected receptor also transduced a mitogenic signal.The mutant receptor was synthesized as a 90‐kDa precursor and was processed to 120 kDa with a slightly faster rate than the wild‐type receptor. Cells expressing the mutant receptor generally had around 106 ligandbinding sites/cell, with a Kd for binding of PDGF‐BB of 3 nM. The mutant receptor, which did not transduce a mitogenic response, mediated degradation of 125I‐PDGF‐BB, albeit less efficiently compared to the wild‐type receptor. In contrast to the wild‐type receptor, it was down‐regulated only to a limited extent and not degraded in response to ligand binding. These findings indicate a role for the intracellular part of the receptor, not only in mitogenic signaling, but also in receptor internalization and intracellular routing.
DNA polymerase α from normal rat liver is different than DNA polymerases α from Morris hepatoma strains
Authors: POPANDA Odilia , THIELMANN Heinz Walter 
Published: March 2005

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase α complex, was partially purified from a slow‐growing (TC5123) and a fast‐growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase α complexes contained RNA primase. DNA polymerase α activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values.The sedimentation coefficients of native DNA polymerases α from Morris hepatoma cells were found to be lower than that of polymerase α from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl‐dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino‐dATP were lower for DNA polymerase α from the fast‐growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base‐pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis‐Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single‐stranded DNA as a template was found to be lower for DNA polymerase α from the fast‐growing hepatomal MH3924 than for that from normal rat liver. Thus, the DNA‐polymerizing complex from MH3924 combines both higher binding strength to single‐stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.
Evidence for at least four different inositol bisphosphatases in bovine brain
Authors: HOWELL Steven , BARNABY Robert J. , ROWE Tony , RAGAN C. Ian , GEE Nicolas S. 
Published: March 2005

Bovine brain supernatant contains at least four enzymes capable of hydrolysing inositol bisphosphates. These activities may be distinguished on the basis of their metal, salt and pH dependence, sensitivity to Li+ ions and thiol‐modification reagents, and on their molecular sizes. In addition to Li+‐sensitive Ins(1,4)P2/Ins(1,3,4)P3 1‐phosphatase [Gee et al. (1988) Biochem. J. 253, 777–782] which has an absolute requirement for Mg2+, two (Li+‐insensitive and Mg2+‐independent) phosphatases, capable of hydrolysing Ins(3,4)P2 and Ins(1,3)P2, respectively, have been identified. Both enzymes were inhibited by only moderate concentrations of salt, although for the former there was no obvious correlation between inhibitory potency and either the nature of the anion/cation or the ionic strength of the buffer. Ins(3,4)P2 phosphatase had a pH optimum of 7.6 and this activity could be resolved on gel‐filtration columns into a two overlapping peaks of molecular mass 170 kDa and 450 kDa.Mg2+‐independent Ins(1,3)P2 phosphatase had a pH optimum of 7.1 and displayed a single broad activity peak on gel‐filtration columns. However, if assays were performed in the presence of Mg2+, a second Ins(1,3)P2 phosphatase was revealed (35 kDa), which had a pH optimum of 8.8. Ins(1,4)P2/Ins(1,3,4)P3 1‐phosphatase, Ins(3,4)P2 phosphatase, Mg2+‐independent Ins(1,3)P2 phosphatase and inositol monophosphatase were all inhibited by 5,5′‐dithiobis(2‐nitrobenzoic acid) with IC50 values of 34 μM, 65 μM and 560 μM and 1100 μM, respectively. The metabolism of Ins(1,3,4)P3 by brain supernatant was also examined. Product specificity was shown to be entirely dependent on the buffer conditions employed. In Mg2+‐containing buffers, Ins(1,3,4)P3 was hydrolysed predominantly to Ins(3,4)P2, consistent with hydrolysis by Ins(1,4)P2/Ins(1,3,4)P3 1‐phosphatase. In the presence of EDTA, Ins(1,3,4)P3 was degraded exclusively by a 4‐phosphatase enzyme generating Ins(1,3)P2. Under these conditions, high concentrations of Ins(3,4)P2 blocked the hydrolysis of Ins(1,3,4)P3.
Shift to the Na+ from of Na+/K+‐transporting ATPase due to modification of the low‐affinity ATP‐binding site by Co(NH3)4ATP
Authors: SCHEINER‐BOBIS Georgios , ESMANN Mikael , SCHONER Wilhelm 
Published: March 2005

Inactivation of purified Na+/K+‐transporting ATPase by the MgATP complex analogue Co(NH3)4ATP, which binds to the low‐affinity ATP‐binding site, results in the concomitant inhibition of the K+‐activated p‐nitrophenylphosphatase, which is considered to be a partial reaction catalyzed by the enzyme in the E2 conformational state.
Complete inactivation of Na+/K+‐transporting ATPase by Co(NH3)4ATP does not alter the ADP/ATP exchange reaction which is considered to be part of the catalytic activity in the E1 conformation.
The enzyme binds cosin at the high‐affinity ATP‐binding site as measured by the change in eosin fluorescence. Eosin binding to the Co(NH3)4ATP‐inactivated enzyme is, in contrast to the untreated enzyme, not stimulated by Na+. Inactivation by Co(NH3)4ATP increased the half‐maximal opposing effect of K+ on eosin binding from 1.1 mM in the control to 43.2 mM in the almost completely inactive enzyme. No eosin fluorescence changes were observed when the Co(NH3)4ATP‐inactivated enzyme was treated subsequently with CrATP. This MgATP complex analogue forms a stable complex at the high‐affinity ATP‐binding site. CrATP thus abolishes eosin binding.
It is concluded, that Co(NH3)4ATP interacts with Na+/K+‐transporting ATPase in the E2 conformation and arrests it there. This affects eosin binding to the high‐affinity ATP‐binding site, since the K+ sensitivity is lost. A possible interpretation of these differing effects of Co(NH3)4ATP on partial reactions of Na+ K+‐transporting ATPase is that the sodium pump works as an (α,β)2 diprotomer. It is likely that the arrest of one α,β protomer in the E2 conformational state by occupancy of the low‐affinity ATP‐binding site with Co(NH3)4ATP induces the Na+ form (E1 form) in the corresponding α,β protomer, as is indicated by the unaffected ADP ATP exchange and the response of the eosin fluorescence on Na+ and K+.
On the mechanism of inhibition of methanol dehydrogenase by cyclopropane‐derived inhibitors
Authors: FRANK Johannes , van KRIMPEN Simon H. , VERWIEL P. Eugène J. , JONGEJAN Jacob A. , MULDER Antonia C. , DUINE Johannis A. 
Published: March 2005

Extraction of cyclopropanol‐inactivated methanol dehydrogenase (MDH) gave a mixture of two interconverting compounds. The same compounds could be prepared from 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐f]quinoline‐4,5‐dione (PQQ) and cyclopropanol using a metal oxide (e.g. Ag2O) as a catalyst. Structure elucidation revealed that a C5 3‐propanal adduct of PQQ is formed which is present in the extract as a diastereoisomeric mixture of the ring‐closed form. Cyclopropanone gave an analogous product, while cyclopropylmethanol behaved as a substrate and was oxidized by the enzyme without ring‐opening.From the work described, several arguments can be derived to reject the idea that inactivation proceeds via formation of a pair of free radicals. The mechanism probably consists of a concerted proton abstraction, rearrangement of the cyclopropoxy anion to a ring‐opened carbanion and attack of the latter on the electrophilic C5 of PQQ. The measured rate of inactivation (3.7 s−1) is in agreement with such a mechanism. The role of the metal oxide and the enzyme in this process is the catalysis of the addition step and possibly a positioning of the reactants. As only a sole type of quinoprotein alcohol dehydrogenase becomes inhibited, the cyclopropane derivatives studied here can be regarded as mechanism‐based inhibitors.The modified PQQ in cyclopropanone‐inactivated MDH is fluorescent. A fluorescent intermediate was also observed in the catalytic cycle of MDH with methanol as a substrate. Its rate of formation and decay and the strongly decreased level of fluorescence in the presence of activator are in accordance with the view that the fluorescing species is the previously found oxidized‐MDH · substrate (MDHox· S) complex. Since the decomposition of this complex requires activator and model studies have failed so far to mimic the enzyme, it seems that the combination of enzyme and activator is essential for the oxidation of the alcohol substrate.
Guanylate kinase from Saccharomyces cerevisiae
Authors: BERGER Albin , SCHILTZ Emile , SCHULZ Georg E. 
Published: March 2005

This paper describes a large‐scale purification of guanylate kinase (ATP + GMP · ADP + GDP) from Saccharomyces cerevisiae, the crystallization of the enzyme and preliminary X‐ray investigations. Furthermore the complete amino acid sequence of the enzyme has been determined and was compared to adenylate kinase sequences.

Guanylate kinase was purified in five steps to homogeneity: crude extract, ion‐exchange chromatography, affinity chromatography and gel filtration twice.
The enzyme was crystallized to single octahedral bipyramids with sizes up to 500 × 200 × 150 μm3. Preliminary X‐ray results are given.
The final sequence shows 186 amino acids (Mr= 20 548), containing one cysteine and one tryptophan. It was determined from peptides of five cleavages of the whole protein. Three cleavages were used for determination of the whole polypeptide chain. From the other two, only some peptides were used to secure overlaps and the cysteine position. The N‐terminal blocking group was identified by 1H‐NMR spectroscopy.
Since guanylate kinase shows the mononucleotide binding pattern GXXGXGK, it was compared to other proteins containing this pattern. But no further homology signal could be detected. A comparison with adenylate kinases revealed significant similarity in another chain segment. This led to the conclusion that guanylate kinase is at least partially homologous to the adenylate kinases.
Nematocysts (stinging capsules of Cnidaria) as Donnan‐potential‐dominated osmotic systems
Authors: WEBER Jakob 
Published: March 2005

Isolated nematocysts (capsules of stinging cells in Cnidaria) from the freshwater polyp Hydra vulgaris [Weber, J., Klug, M. & Tardent, P. (1987) Comp. Biochem. Physiol. 88B, 855–862] are under a constant internal osmotic pressure of 12.5 MPa. The capsular wall which withstands this considerable pressure has an average elasticity modulus of approximately 1 GPa, enabling the cyst to swell from its relaxed state to more than double its volume. If the large concentrations of Mg2+ and Ca2+ within isolated nematocysts are substituted by alkali ions, the capsular volume increases by 15% and the final osmotic pressure rises to 15.3 MPa whereas after substitution by Ca2+ a decrease to 90% of the volume and 6.5 MPa is observed.Evidence obtained from the osmotic behavior of nematocysts, as well as data from in vitro exchange of their cations, are consistent with a physicochemical model in which the internal osmotic pressure of nematocysts and thus their ability to discharge is governed by the cationic composition of the content and the salt concentrations in the environment.
Enantiotopic differentiation in horse‐liver alcohol‐dehydrogenase‐catalyzed oxidoreduction studied with novel substrates having organometallic moieties
Authors: YAMAZAKI Yoshimitsu , UEBAYASI Masami , HOSONO Kuniaki 
Published: March 2005

Horse‐liver alcohol‐dehydrogenase‐catalyzed oxidation of 1,2‐bis(hydroxymethyl)ferrocene (1) gave (1R)‐(+)‐1‐formyl‐2‐hydroxymethylferrocene (3) (86 ± 2% enantiomeric excess, e.e.), while the reduction of the corresponding dialdehyde 1,2‐diformylferrocene (2) gave the antipode (1S)‐(–)‐3 (94 ± 2% e.e.). This fact indicates that the pro‐R group in both 1 and 2 was preferentially converted by the enzyme.When one of two substituents on the substrate was replaced by a methyl group or moved to the β‐site, the stereoselectivity in the reaction decreased as evidenced by the enantiomeric purity of the products (5–64% e.e.). Treatment of racemic 1‐hydroxyethylferrocene (14) with horse‐liver alcohol dehydrogenase (HLADH) gave optically pure (R)‐(–)‐14together with acetylferrocene. The reduction of 2 with HLADH, NAD and (2H6)ethanol gave (–)‐(1S,2R)‐1‐formyl‐2‐[(R)‐hydroxy(2H1)methyl]ferrocene and that of 1,2‐di[(2H)formyl]ferrocene with HLADH, NAD and ethanol gave (–)‐(1S,2R)‐1‐(2H)formyl‐2‐[(S)‐hydroxy(2H1)methyl]ferrocene. These configurations indicate that the enzymic reduction occurred on the re‐face of pro‐R formyl group. The re‐face selectivity was also found in the enzymic reduction of (6‐benzaldehyde)tricarbonylchromium and its (2H)formyl analogue.Docking of 2 into the active site of HLADH was examined using computer graphics. It has been suggested that the enantioselectivity to the pro‐R side in the oxidoreduction of 1 and 2 by HLADH is a natural consequence of the re‐face selectivity, which is caused by a steric interaction between the ligand and the side chain of Phe‐93 or the Zn complex and strengthened by an interaction between the unreactive polar α‐substituent and the protein, probably by hydrogen bond formation.
Isolation and characterization of different activated forms of factor VIII, the human antihemophilic A factor
Authors: BIHOREAU Nicolas , SAUGER Annick , YON Jeannine M. , van de POL Hendrik 
Published: March 2005

Factor VIII was purified 1200‐fold from commercial concentrates (Centre National de Transfusion Sanguine) by immunoaffinity chromatography using an anti‐(80‐kDa light chain) monoclonal antibody. The different molecular forms isolated were subsequently separated and analyzed using Fast Protein Liquid Chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis. The different factor‐VIII forms obtained, consisted of 80‐kDa light chains, each being associated with one more‐or‐less fragmented heavy chain ranging over 90–210 kDa. The specific activity of these different forms was 7000 U/mg.At differnt stages of activation of factor VIII by thrombin, various forms were separated and identified. Activated complexes were found to result from the association of the 70‐kDa light chain (generated from the 80‐kDa light chain) with heavy chains ranging over 90–210 kDa. Two different thrombin activation steps were characterized. The first step corresponding to the cleavage of the 80‐kDa light chain led to a sixfold increase in the procoagulant activity, and yielded a stable activated intermediate form. Compared with normal factor VIII, the ratio of von Willebrand activity to factor‐VIII activity, measured in the activated fractions, decreased, indicating that von Willebrand factor dissociates from factor VIII after proteolysis of the light chain by thrombin.In the second step, the 90‐kDa heavy chain was cleaved into two polypeptides of 45 kDa and 50 kDa, which were associated with the 70‐kDa proteolyzed light chain, generating the final activated complex (45–50–70 kDa).The new intermediate forms we described imply a new scheme for the multistep activation process of factor VIII.
Reconstitution of a light‐stimulated adenylate cyclase from retina and Neurospora crassa preparations
Authors: MUSCHIETTI Jorge P. , BIANCHINI Graciela M. , MARTINETTO Horacio E. , CARRICARTE Valentina C. , GIUSTO Norma , FARBER Débora B. , TORRES Héctor N. , FLAWIA Mirtha M. 
Published: March 2005

Adenylate cyclase catalytic subunits from Neurospora crassa membranes may interact with regulatory factors from membranes of bovine retinal rod outer segments (pretreated with N‐ethylmaleimide), reconstituting a heterologous system which, in the presence of light, is catalytically active in assay mixtures containing MgATP. Maximal activation was observed at 550 nm. Transducin‐depleted retinal membranes were not capable of reconstituting the heterologous light‐stimulated adenylate cyclase system. Addition of a transducin preparation to depleted membranes restored the reconstitution capacity of these membranes.A similar heterologous adenylate cyclase system was reconstituted with Neurospora and mouse retinal whole membranes (pretreated with N‐ethylmaleimide). Membranes from mice suffering photoreceptor degeneration (rd homozygotes) did not reconstitute an heterologous adenylate cyclase system.