bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Mechanism of Porcine Pancreatic α‐Amylase
Authors: Alkazaz Modhafar , Desseaux Véronique , Marchis‐Mouren Guy , Payan Françoise , Forest Eric , Santimone Marius 
Published: July 2004

Kinetics of inhibition of the two porcine pancreatic α‐amylase components (PPA I and PPA II) by acarbose were performed using reduced DP18‐maltodextrin and amylose as substrates. Similar Line‐weaver‐Burk primary plots were obtained. Two mixed non‐competitive models are proposed. X‐ray crystallographic data [Qian, M., Buisson, G., Duée, E., Haser, R. & Payan, F. (1994)Biochemistry 33, 6284–6294] are in support of the mixed non‐competitive inhibition model which involves abortive complexes. Secondary plots are different; inhibition of reduced DP18‐maltodextrin hydrolysis gives straight‐lines plots while amylose gives parabolic curves. These results, confirmed by Dixon‐plot analyses, allow us to postulate that, in inhibition of reduced DP18‐maltodextrin hydrolysis, one molecule of acarbose is bound amylase molecule. In contrast, using amylose as a substrate, two molecules of acarbose are bound. These kinetically determined binding sites might correspond to surface sites found by X‐ray crystallography [Qian, M., Haser, R. & Payan, F. (1995)Protein Sci. 4, 747–7551; the glucose site close to the active site and the maltose site, 2 nm away.In conclusion, no significant difference between PPA I and PPA II has been observed, either from molecular mass or from kinetic behaviours; this suggests multiple forms of the enzyme. A general mechanism of PPA action is proposed; in addition to the active site, long‐chain substrate hydrolysis requires the glucose‐binding site and the maltose‐binding site, while only one site is necessary for the hydrolysis of short chain substrate.
Redox Control of RNA Synthesis in Potato Mitochondria
Authors: Wilson S. Brian , Davidson Gary S. , Thomson Laura M. , Pearson Colin K. 
Published: July 2004

This study shows that the incorporation of radiolabelled UTP into RNA in Percoll‐gadient‐purified potato mitochondria is regulated by the redox state of the mitochondrial electron transport chain. An early indication that there might be a redox effect on RNA synthesis was a decrease in UTP incorporation in incubates containing an oxidisable substrate, such as succinate or malate. Subsequent use of a variety of electron transport inhibitors acting at different points in the electron transport chain established that the redox state of the Rieske iron‐sulphur protein was the major determinant of UTP incorporation. Inhibitors acting on the substrate side of the Rieske iron‐sulphur protein, and causing oxidation of components on the oxygen side of their site of action, increased UTP incorporation into RNA. These included antimycin A, myxothiazole, and undecylhydroxydioxobenzothiazole at 500 nM. Inhibitors acting on the oxygen side of the Rieske iron‐sulphur protein, and causing a reduction of components on the substrate side of the block, decreased UTP incorporation. These inhibitors were undecylhydroxydioxobenzothiazole at 25 nM and KCN. When phenazine methosulphate was present as an auto‐oxidisable electron sink the effect of KCN was diminished.The conclusion from the inhibitor experiments that the redox state of the Rieske iron‐suphur protein was important was supported when RNA synthesis was measured at a range of redox potentials. This gave a measured redox potential for the control of UTP incorporation into RNA of +270 mV and the slope of the curve indicated an n= 1 carrier. This value is close to the reported value of the Rieske iron‐suphur protein.UTP incorporation was decreased by some 50% in the presence of low concentrations of okadaic acid (5 nM), an inhibitor of protein phosphatases PP1 and PP2A, and α‐naphthyl acid phosphate, a broad‐spectrum phosphatase inhibitor, indicating that the redox effect on RNA synthesis may be mediated via protein phosphorylation. We did not, however, detect an expected increase in RNA synthesis when protein kinase inhibitors were used, so the involvement of protein phosphorylation in the redox regulation of RNA synthesis is as yet uncertain.
Characterization of the murine plasminogen/urokinase‐type plasminogen‐activator system
Authors: Lijnen H. Roger , Van Hoef Berthe , Collen Désiré 
Published: July 2004

The murine plasminogen/urokinase‐type plasminogen‐activator (u‐PA) system was studied using purified proteins, plasma and endothelioma cells. Recombinant murine u‐PA was obtained as a single‐chain molecule of 45 kDa which was converted to two‐chain u‐PA with plasmin by cleavage of the Lys159‐Ile1 60 peptide bond. Murine plasminogen, purified from plasma as a single‐chain protein of 95 kDa, was resistant to quantitative activation with murine recombinant two‐chain u‐PA: only 15% activation within 1 h at 37°C was obtained in mixtures of 1 μM plasminogen and 5 nM recombinant two‐chain u‐PA, whereas quantitative activation was observed in the autologous human system. Addition of 6‐aminohexanoic acid to native murine plasminogen resulted in quantitative activation within 1 h. In murine plasma in vitro, plasminogen was also resistant to quantitative activation with u‐PA (50% activation within 1 h at 37°C with 50 nM recombinant two‐chain u‐PA, whereas in the human system nearly quantitative activation was obtained). Murine plasma clots submerged in murine plasma were resistant to lysis with u‐PA; ≤2% clot lysis in 2 h was obtained with 80nM recombinant two‐chain u‐PA in the autologous murine system whereas 50% clot lysis in 2 h required only 15 nM recombinant two‐chain u‐PA in the autologous human system.Saturable binding of murine recombinant two‐chain u‐PA was observed to murine endothelioma cells that are genetically deficient in u‐PA (u‐PA−/− End cells). Binding was characterized by a Kd of 5.5 nM and 800000 binding sites/cell. However, u‐PA−/− End cells did not significantly stimulate the activation rate of murine plasminogen by murine recombinant two‐chain u‐PA and did not enhance the plasmin‐mediated conversion rate of murine recombinant single‐chain u‐PA to its two‐chain derivative. Murine recombinant two‐chain u‐PA bound to murine endothelioma cells was quantitatively inhibited by murine plasminogen‐activator inhibitor‐1 (PAI‐1).Thus, the interactions between murine plasminogen, u‐PA and PAI‐1 are qualitatively similar to those between their human counterparts. However, quantitative differences were observed both in the presence of cells and in plasma which may contribute to a reduced u‐PA‐mediated fibrinolytic activity in the murine systems.
Expression of the ADP/ATP Carrier and Expansion of the Mitochondrial (ATP + ADP) Pool Contribute to Postnatal Maturation of the Rat Heart
Authors: Schönfeld Peter , Schild Lorenz , Bohnensack Ralf 
Published: July 2004

The role of the ADP/ATP carrier (AAC), a key protein of the mitochondrial ATP‐generating system, is not clear during postnatal rat heart development. To elucidate this role, the phosphorylating respiration (state 3), the activity and the content of AAC, the size of the exchangeable mitochondrial (ATP + ADP) pool and the control of AAC over respiration at state 3 were measured in mitochondria isolated from rat hearts at various postnatal ages.There was a 5‐fold increase in the AAC activity from newborn to aged rat hearts, which was paralleled by a 1.5‐fold increase in state 3 respiration. At birth, the AAC and the F0F1‐ATP synthase exerted about 80% of the control over phosphorylating respiration (state 3: flux control coefficients 0.39 ± 0.04 and 0.38 ± 0.08). The strong increase in the AAC activity was partly caused by the doubling of the protein content. In addition, the turnover number of AAC increased by a factor of 2.5 due to the expansion of the (ATP + ADP) pool from 3.4 ± 0.9 to 10.6 ± 1.5 nmol · mg protein−1. The data strongly indicate that the increase in the AAC activity is an essential step in the postnatal maturation of rat heart mitochondria.
In vivo Phosphorylation of Actin in Physarum Polycephalum
Authors: De Corte Veerle , Gettemans Jan , Waelkens Etienne , Vandekerckhove Joël 
Published: July 2004

Actin‐fragmin is a heterodimeric protein complex from Physarum polycephalum microplasmodia that is phosphorylated in vitro at residues Thr203 and Thr202 of the actin subunit by the endogenous actin‐fragmin kinase. Following phosphorylation, the F‐actin capping activity of the complex becomes Ca2+‐dependent, suggesting a fundamental regulatory role in controlling F‐actin growth [Gettemans, J., De Ville, Y., Waelkens E. and Vandekerckhove, J. (1995)J. Biol. Chem. 270, 2644–26511.In this study we analysed actin phosphorylation in vivo. We demonstrate that the actin‐fragmin complex constitutes the only substrate of the actin‐fragmin kinase in plasmodia. Monomeric actin is not phosphorylated. Immunoprecipitation of actin‐fragmin reveals that approximately 405% of the actin sub‐unit of the complex is phosphorylated in vivo. However, using purified substrate and kinase, the complex can be quantitatively phosphorylated as judged by two‐dimensional gel electrophoresis. Through comparative phosphopeptide fingerprinting, we show that the phosphorylation sites in vivo are identical to those identified in vitro.We additionally characterized a complex of actin and the NH2‐terminal half of fragmin (residues 1–168) that is also phosphorylated by the same kinase. In contrast to actin‐fragmin, phosphorylation of the complex between actin and residues 1–168 of fragmin is independent of Ca2+ because the second Ca2+‐dependent regulatory actin‐binding domain is missing.By artificially varying the actin‐fragmin concentration or the actin‐fragmin kinase activity present in microplasmodia cytosolic extracts, we attempted to detect alternative protein substrates for the actin‐fragmin kinase. The fact that none could be identified suggests that the control and properties of actin‐fragmin phosphorylation observed in vitro may stand as a model for F‐actin growth control in Physarum cells.
Analysis of the 1H‐NMR Chemical Shifts of Cu(I)‐, Cu(II)‐ and Cd‐Substituted Pea Plastocyanin
Authors: Ubbink Marcellus , Lian Lu Yun , Modi Sandeep , Evans Philip A. , Bendall Derek S. 
Published: July 2004

To compare cadmium‐substituted plastocyanin with copper plastocyanin, the 1H‐NMR spectra of CuI‐, CuII‐ and Cd‐plastocyanin from pea have been analyzed. Full assignments of the spectra of CuI‐and Cd‐plastocyanin indicate chemical shift differences up to 1 ppm. The affected protons are located in the four loops that surround the Cu site. The largest differences were found for protons in the hydrogen bond network which stabilizes this part of the protein. This suggests that the chemical shift differences are caused by very small but extensive structural changes in the network upon replacement of CuI by Cd.For CuII‐plastocyanin the resonances of 72% of the protons observed in the Cul form have been identified. Protons within ?0.9 nm of the CuII were not observed due to fast paramagnetic relaxation. The protons between 0.9–1.7 nm from the CuII showed chemical shift differences up to 0.4 ppm compared to both CuI‐ and Cd‐plastocyanin. These differences can be predicted assuming that they represent pseudocontact shifts. When corrected for the pseudocontact shift contribution, the CuII‐plastocyanin chemical shifts were nearly all identical within error to those of the Cd form, but not of the CuI‐plastocyanin, indicating that the CuII‐plastocyanin structure, in as far as it can be observed, resembles Cd‐ rather than CuI‐plastocyanin. In a single stretch of residues (64–69) chemical shift differences remained between all three forms after correction.The fact that pseudocontact shifts were observed for protons which were not broadened may be attributable to the weaker distance dependence of the pseudocontact shift effect compared to paramagnetic relaxation. This results in two shells around the Cu atom, an inner paramagnetic shell (0–0.9 nm), in which protons are not observed due to broadening, and an outer paramagnetic shell (0.9–1.7 nm), in which protons can be observed and show pseudocontact shifts.It is concluded that Cd‐plastocyanin is a suitable redox‐inactive substitute for Cu‐plastocyanin.
Reversible Inhibition of Sheep Liver Sorbitol Dehydrogenase by Thiol Compounds
Authors: Lindstad Rune I. , Mckinley‐Mckee John S. 
Published: July 2004

Reversible inhibition of sheep liver sorbitol dehydrogenase by various thiol compounds has been studied. Most species inhibit the enzyme‐catalyzed reaction competitively with respect to sorbitol, due to the formation of ternary enzyme‐NAD‐thiol complexes. The primary interaction of thiol inhibitors with the enzyme active site involves the catalytic zinc atom, and a bidentate mode of binding to the active‐site metal is indicated for some bifunctional thiols in their ternary complexes. Enzyme‐bound thiolate facilitates NAD binding to the enzyme and vice versa, mainly due to mutual electrostatic stabilization. The aromatic thiols 1‐thio‐1‐phenylmethane and 1‐thio‐2‐phenylethane are especially potent inhibitors with an inhibition constant of 0.30 μM at pH 9.9. The inhibitory effect of aliphatic thiols, which is positively correlated with alkyl chain length, parallels that observed previously with the related enzyme horse liver alcohol dehydrogenase and indicates that interaction with an enzymic hydrophobic site is important for inhibitor binding. Several reversible inhibitors afford competitive protection against affinity labelling of the enzyme by 2‐bromo‐3‐(5‐imidazolyl) propionic acid due to the formation of binary enzyme‐thiol complexes.The present study establishes thionucleosides as a novel class of potent sorbitol dehydrogenase inhibitors. The thionucleosides 6‐thioguanosine and 6‐thioinosine gave mixed inhibition with respect to sorbitol, due to the formation of enzyme‐NAD‐inhibitor and enzyme‐NADH‐inhibitor complexes.In order to enable a correlation of the substrate and inhibitor specificities of the enzyme, the kinetic constants for several sorbitol dehydrogenase substrates were determined. l‐threitol and dl‐1–phenyl‐1, 2‐ethanediol are good substrates with, at high pH, kinetic constants similar to those of sorbitol. The potent inhibition by dithiothreitol and the aromatic thiols thus parallels the substrate specificity of the enzyme. The sorbitol competitive inhibitor I‐thiosorbitol is also a substrate with, at pH 7.4, a maximum velocity of 0.17 s‐1 and a Michaelis constant of 8.6 mM.Dithiothreitol forms a tight ternary complex with the enzyme‐NAD complex with a molar absorbance of 16.4 · 103 M−1· cm‐1 at 311 nm. A spectrophotometric titration of the enzyme with NAD in the presence of dithiothreitol is described, which enables an accurate determination of the concentration of sorbitol dehydrogenase active sites and confirms the activity assay of the enzyme.
Role of the Insulin Receptor C‐Terminal Acidic Domain in the Modulation of the Receptor Kinase by Polybasic Effectors
Authors: Baron Véronique , Gual Philippe , Alengrin Françoise , Van Obberghen Einmanuel 
Published: July 2004

Basic polymers such as polylysine have been found to activate insulin receptor autophosphorylation and kinase activity toward substrates. It was suggested that acidic receptor domains may be involved in the interaction of the receptor with these basic effectors. In a previous study, we have shown that the receptor acid‐rich C‐terminal sequence, including residues 1270–1280, is involved in the regulation of the receptor kinase activity. Moreover, this domain may be the site of interaction with histone, which is a modulator of the receptor kinase.In this study, we investigated whether the insulin receptor domain comprising amino acids 1270–1280 is involved in the interaction with polybasic effectors. We used anti‐peptide serum directed to this sequence, and basic activators such as polylysine, polyarginine and protamine sulfate. Our antibodies inhibit polylysine‐induced receptor autophosphorylation, whereas they have no effect on receptor phosphorylation stimulated by concanavalin A which is a non‐basic activator of the insulin receptor. Polylysine‐induced receptor aggregation was blocked by the antibodies (Fab fragments or whole Ig), indicating that competition occurs between the antibody and polylysine at the level of their binding site to the receptor. Finally, we observed a direct interaction of the 125I‐peptide corresponding to receptor sequence 1270–1280 with the basic polymers in dot‐blot experiments. Interestingly, the peptide did not bind spermine, a basic molecule which is not an activator of the insulin receptor kinase.Our data indicate that the insulin receptor C‐terminal acidic domain including residues 1270–1280 is involved in the interaction of polylysine and other polybasic molecules with the receptor. Since this receptor region has been implicated in the regulation of the receptor kinase activity, we propose that interaction of basic effectors with this domain may be responsible for their activating properties.
Differences in the Magnesium Dependences of the Class I and Class II Aminoacyl‐tRNA Synthetases from Escherichia coli
Authors: Airas R. Kalervo 
Published: July 2004

The magnesium dependences of the ATP/PPi exchange and tRNA aminoacylation of reactions were measured for six aminoacyl‐tRNA synthetases (isoleucyl‐, tyrosyl‐ and arginyl‐tRNA synthetases from class I, and histidyl‐, lysyl‐ and phenylalanyl‐tRNA synthetases from class II). The measured values were subjected to best‐fit analyses using sum square error calculations between the data and the calculated curves in order to find the mode of participation of the Mg2+ and to optimize the sets of the kinetic constants.The following four dependences were observed: the class II synthetases require three Mg2+ for the activation reaction (including the one in MgATP), but the class I synthetases require only one Mg2+ (in MgATP); in class II synthetases both MgPPi and Mg2PPi participate in the pyrophosphorolysis of the aminoacyl adenylate. Arginyl‐tRNA synthetase from class I also shows a better fit if also Mg2PPi reacts, but in the isoleucyl‐ and tyrosyl‐tRNA synthetases only MgPPi but not Mg2PPi is used in the pyrophosphorolysis. Different synthetases have different requirements for the tRNA‐bound Mg2+ and spermidine, independent of the enzyme class. 1–4 Mg2+ or spermidines are required in the best fit models. At the end of the reaction in all the synthetases analysed the dissociation of Mg2+ from the product aminoacyl‐tRNA essentially enhances the subsequent dissociation of the aminoacyl‐tRNA from the enzyme. The binding of ATP to the E · aminoacyl‐tRNA complex also speeds up the dissociation of the aminoacyl‐tRNA from most of these enzymes.
Cloning, Expression and Properties of the α′ Subunit of Casein Kinase 2 from Zebrafish (Danio Rerio)
Authors: Antonelli Marcelo , Daniotti José L. , Rojo Daniel , Allende Catherine C. , Allende Jorge E. 
Published: July 2004

The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division. The holoenzyme is a tetramer composed of two catalytic subunits (α and/or α′) and regulatory subunits (β2). The α and α′ subunits are encoded by different genes but are very similar in amino acid sequence, except that α′ is normally considerably shorter. There have been extensive biochemical studies with recombinant α and β subunits of many species, but only one previous description of the activity of an isolated recombinant α′ subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J. & Pyerin, W. (1994) Recombinant human casein kinase II. A study with the complete set of subunits (α, α′, and β), site‐directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur. J. Biochem. 220, 263–273). In the present work, the isolation and bacterial expression of a cDNA coding for the α′ subunit of zebrafish (Danio rerio) is reported. The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the α′ subunits of human and chicken, and 82% identical to the sequenced portion of the CK2α subunit of zebrafish. The recombinant α′ subunit has apparent Km, values for ATP (6 μM), GTP (20 μM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant α subunit of Xenopus laevis. The α′ subunit kcat, was 7.2 min‐1 which is again similar to that of Xenopus laevisα subunit (7.5 min‐1). The α′ subunit also behaved similarly to CK2α with regard to optimal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu80Tyr20) peptide. However α′ kinase activity was less sensitive to poly(U) inhibition than α, it was more heat stable than α, and α′ was slightly more sensitive to KCl inhibition than α. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory β subunit which shifted the optimal salt concentration of the phosphorylating activity. The α′2β2 holoenzyme was inhibited by KCl concentrations above 100 mM, while the α2β2 enzyme was stimulted by KCl concentrations up to 150 mM and required 180 mM for inhibition.Another important difference between α and α′ is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory β subunit. When assayed at 100 mM KCl stoichiometric amounts of CK2β produced maximal stimulation of both α′ (D. rerio) and α (X. laevis), however the activity levels with α′ were stimulated 20‐fold by β while the addition of β stimulated α (X. luevis) only 7–8‐fold.
Mechanisms of Inhibition and Uncoupling of Respiration in Isolated Rat Liver Mitochondria by the General Anesthetic 2,6‐Diisopropylphenol
Authors: Kigoulet Michel , Devin Anne , Avéret Nicole , Vandais Bénédicte , Guérin Bernard 
Published: July 2004

We investigated the effects of 2,6‐diisopropylphenol on oxidative phosphorylation of isolated rat liver mitochondria. Diisopropylphenol strongly inhibits state‐3 and uncoupled respiratory rates, when glutamate and malate are the substrates, as a direct consequence of the limitation of electron transfer at the level of complex I. In addition, diisopropylphenol acts as an uncoupler in non‐phosphorylating mitochondria, which leads to an increase in respiratory rate and a large decrease in proton‐motive force. However, such effects cannot be due to the classical protonophoric property of this drug, since addition of ADP plus oligomycin before diisopropylphenol avoids this increase in proton permeability, and in phosphorylating mitochondria, the ATP/O ratio is not significantly affected by diisopropylphenol addition. In the absence of added ADP, diisopropylphenol modifies some mitochondrial ATPases in such a way that they become insensitive to oligomycin and unable to couple proton movement to ATP synthesis or hydrolysis. However, these modified enzymes can catalyse passive proton permeability, which leads to uncoupling. Addition of ADP before diisopropylphenol prevents these changes. We propose that ADP induces a change in conformation of ATPase, which leads to insensitivity of this complex towards diisopropylphenol.In conclusion, we show that diisopropylphenol has two main effects on rat liver mitochondria: inhibition of the respiratory chain at the level of complex I level and modification of ATPase such that, in the absence of phosphorylation, it catalyses a H+ leak, which becomes negligible when oxidative phosphorylation is functional.