bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metabolic Resistance: The Protection of Enzymes against Drugs which are Tight‐Binding Inhibitors by the Accumulation of Substrate
Authors: CHRISTOPHERSON Richard I. , DUGGLEBY Ronald G. 
Published: March 2005

Blockade of a metabolic pathway by interaction of a drug with a particular ‘target enzyme’ results in depletion of essential end‐products of the pathway and accumulation of intermediates prior to the blockade. Metabolic resistance to a particular drug can arise if the substrate of the inhibited enzyme accumulates to levels sufficiently high to compete effectively with the inhibitor, leading to restoration of full activity of the metabolic pathway after a transitory delay.Such resistance has recently been demonstrated in vitro for the interaction of the tight‐binding inhibitor N‐phosphonacetyl‐l‐aspartate (PAcAsp) with the aspartate transcarbamoylase activity of the trifunctional protein which initiates pyrimidine biosynthesis in mammals [Christopherson, R. I. and Jones, M. E. (1980) J. Biol. Chem. 255, 11381–11395]. Carbamoyl phosphate, the product of the carbamoyl phosphate synthetase activity of this trifunctional protein, accumulates to a sufficiently high concentration that the inhibitory effect of PAcAsp is effectively abolished. We have developed a theoretical model for metabolic resistance which quantitatively accounts for these experimental data. This model has been used to simulate the interaction between the following potential or proven anti‐cancer drugs and their target enzyme, under conditions similar to those which would occur in vivo: PAcAsp with aspartate transcarbamoylase; various OMP analogues [the 5′‐monophosphates of 6‐azauridine, pyrazofurin and 1‐(β‐d‐ribofuranosyl)‐barbituric acid] with OMP decarboxylase; 5‐fluorodeoxyUMP with thymidylate synthase; methotrexate with dihydrofolate reductase; and deoxycoformycin with adenosine deaminase.
Forskolin regulation of liver membrane adenylyl cyclase
Authors: BIRNBAUMER Lutz , STENGEL Dominique , DESMIER Martine , HANOUNE Jacques 
Published: March 2005

The effects of forskolin on rat liver plasma membrane adenylyl cyclase were studied. The diterpene stimulated the Vmax of the enzyme system with apparent Km values of 3–5 μM. Stimulations were marked both in the absence (20‐fold over control) as well as in the presence of various stimulators such as GTP, GuoPP[NH]P, NaF alone or in combination with glucagon. Except with GTP, where stimulations of activities by forskolin and the nucleotide were synergistic (more than additive), stimulations of combinations of GuoPP[NH]P, NaF or glucagon with forskolin were additive.Forskolin did not alter significantly the apparent Km values of the enzyme for MgATP or MnATP or the apparent Ka values (concentrations giving stimulations that are 50% of maximum) for Mg or Mn ions, GTP, GuoPP[NH]P or NaF. Forskolin caused a decrease in the concentration of glucagon required for half‐maximal stimulation from 5 μM to 1.5 μM. Except for this effect on the Ka for the glucagon, the only kinetic parameter altered was the Vmax under all conditions tested.Although proteolysis stimulated liver membrane adenylyl cyclase under control conditions, it did not enhance forskolin‐stimulated activities. More extensive proteolysis, which resulted in decreased activities in the absence of forskolin, also resulted in reduced forskolin‐stimulated activities.‘Uncoupling’ of the guanine‐nucleotide‐binding regulatory component, that mediates guanine nucleotide stimulation by addition of 30 mM MnCl2, did not result in ‘uncoupling’ of forskolin stimulation. The data indicate that the diterpene forskolin stimulates adenylyl cyclase activity by a novel mechanism that differs from that by which NaF or guanylyl nucleotides affect this membrane‐bound system and that the diterpene should be a useful tool with which to explore as yet unrecognized modes of regulation of cyclic AMP production.
The stoichiometry of proton translocation through H+‐ATPase of rat‐liver mitochondria
Authors: SHOLTZ Khristophor F. , GORSKAYA Inna A. , KOTELNIKOVA Alla V. 
Published: March 2005

The ratio of the number of protons transported directly by H+‐ATPase of intact mitochondria and that of the hydrolyzed ATP (the proton translocation quotient) is determined. A special kinetic method which makes possible determination of ATPase and H+‐translocase activities in the same experiment has been used to estimate the proton translocation quotient for mitochondrial H+‐ATPase. The quotient is found to be, on average, equal to 3.31 mol/mol.It is shown that the protonophore 3,5‐di‐tert‐butyl‐4‐hydroxybenzylidenemalononitrile can greatly decrease the proton translocation quotient. This supports our assumption concerning a labile coupling between H+‐translocase and ATPase in mitochondria [I. A. Gorskaya, K. F. Sholtz, S. A. Moreva, A. V. Kotelnikova (1979) Biochemistry (Engl. Transl. Biokhimiya) 44, 765–770]. A decrease in the translocation quotient in the presence of the protonophore is likely to be due to the ability of this uncoupler to take back the translocated protons into the mitochondrial matrix before their release into the medium.An electrostatic model of the molecular mechanism of H+‐translocase and ATPase coupling in the H+‐ATPase complex is discussed. The model is in agreement with the results obtained: transport of more than two protons per each hydrolyzed ATP molecule, and variable efficiency of the process.
Coupling between fatty acid binding and sulfhydryl oxidation in bovine serum albumin
Authors: TAKABAYASHI Kenji , IMADA Teruaki , SAITO Yuji , INADA Yuji 
Published: March 2005

Albumin with and without a free sulfhydryl group (mercapt‐albumin and nonmercapt‐albumin) has low and high amounts of fatty acids, respectively. It was found that this difference was brought about by the following two mechanisms: (a) the binding of fatty acids increases the rate of oxidation of the sulfhydryl group, (b) the oxidation of the sulfhydryl group of the protein enhances the binding of fatty acids.The incubation of the protein with fatty acids enhanced reversibly the reaction rate of the free sulfhydryl group with 5,5′‐dithiobis(2‐nitrobenzoic acid). This effect of fatty acids depends on the kind of fatty acid added. Among saturated long‐chain fatty acids tested, the shortest fatty acid, lauric (dodecanoic) acid, had the strongest effect. Oleic (cis‐9‐octadecenoic) acid, which has a long chain but also one cis‐double bond, however, was as effective as lauric acid in enhancing the reactivity of the sulfhydryl group on the protein. Analyses with palmityl‐Sepharose and equilibrium partitioning of palmitic acid between heptane and aqueous protein solutions have shown that nonmercapt‐albumin has higher affinity to the fatty acid than mercapt‐albumin.These results imply that the binding of fatty acids and the oxidation of sulfhydryl groups of the protein are intimately coupled.
Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart
Authors: SÜMEGI Balázs , ALKONYI István 
Published: March 2005

In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2–1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady‐state reaction rate following the lag phase.The pre‐steady‐state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 × 104 M−1s−1 to 1.3 × 105 M−1s−1 and decreases the rate constant of dissociation from 2.3 × 10−2s−1 to 4.1 × 10−3s−1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin‐diphosphate‐independent pyruvate‐binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex.Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine‐labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.
Phosphorolytic activity of Escherichia coli glycyl‐tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P‐NMR spectroscopy and thin‐layer chromatography
Authors: LED Jens J. , SWITON Werner K. , JENSEN Kaj F. 
Published: March 2005

The catalytic activity of highly purified Escherichia coli glycyl‐tRNA synthetase has been studied by 31P‐NMR spectroscopy and thin‐layer chromatography on poly(ethyleneimine)‐cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses of adenosine(5′)tetraphospho(5′)adenosine (Ap4A) and adenosine(5′)triphospho(5′)adenosine (Ap3A), also catalyzes the formation of ADP from inorganic phosphate and the enzyme‐bound glycyl adenylate. Accordingly it was shown that E. coli glycyl‐tRNA synthetase, in the presence of inorganic phosphate, glycine, and Mg2+ ions, catalyzes the synthesis of ADP from three different substrates which all lead to enzyme‐bound glycyl adenylate, that is, ATP, adenosine 5′‐[β,γ‐methylene]triphosphate and Ap4A. It was furthermore demonstrated that the only pathway by which a synthetase‐catalyzed degradation of Ap4A can occur is through the reaction between inorganic phosphate and the enzyme‐bound glycyl adenylate, synthesized from Ap4A. Likewise a 20‐fold increase of the phosphorolytic activity of the investigated synthetase was observed when Mg2+ was replaced by Mn2+.Besides establishing the phosphorolytic activity of the applied enzyme, the study also showed that the preparation catalyzes a glycine‐independent transfer of the γ‐phosphate group from ATP to nucleoside 5′‐diphosphates.The importance of the observed reaction between inorganic phosphate and enzyme‐bound aminoacyl adenylate in relation to the remaining catalytic activities of aminoacyl‐tRNA synthetases is discussed, as well as the biological significance of the reaction.
The calmodulin‐dependent glycogen synthase kinase from rabbit skeletal muscle
Authors: WOODGETT James R. , DAVISON Michael T. , COHEN Philip 
Published: March 2005

A calmodulin‐dependent glycogen synthase kinase distinct from phosphorylase kinase has been purified ∼ 5000‐fold from rabbit skeletal muscle by a procedure involving fractionation with ammonium sulphate (0–33 %), and chromatographies on phosphocellulose, calmodulin‐Sepharose and DEAE‐Sepharose. 0.75 mg of protein was obtained from 5000 g of muscle within 4 days, corresponding to a yield of ∼ 3 %. The Km for glycogen synthase was 3.0 μM and the V 1.6–2.0 μmol min−1 mg−1.The purified enzyme showed a major protein staining band (Mr 58 000) and a minor component (Mr 54 000) when examined by dodecyl sulphate polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 696 000 by sedimentation equilibrium centrifugation, indicating a dodecameric structure. Electron microscopy suggested that the 12 subunits were arranged as two hexameric rings stacked one upon the other.Following incubation with Mg‐ATP and Ca2+‐calmodulin, the purified protein kinase underwent an ‘autophosphorylation reaction’. The reaction reached a plateau when ∼ 5 mol of phosphate had been incorporated per 58 000‐Mr subunit. Both the 58 000‐Mr and 54 000‐Mr species were phosphorylated to a similar extent. Autophosphorylation did not affect the catalytic activity.The calmodulin‐dependent protein kinase initially phosphorylated glycogen synthase at site‐2, followed by a slower phosphorylation of site‐1 b. The protein kinase also phosphorylated smooth muscle myosin light chains, histone H1, acetyl‐CoA carboxylase and ATP‐citrate lyase. These findings suggest that the calmodulin‐dependent glycogen synthase kinase may be a enzyme of broad specificity in vivo.Glycogen synthase kinase‐4 is an enzyme that resembles the calmodulin‐dependent glycogen synthase kinase in phosphorylating glycogen synthase (at site‐2), but not glycogen phosphorylase. Glycogen synthase kinase‐4 was unable to phosphorylate any of the other proteins phosphorylated by the calmodulin‐dependent glycogen synthase kinase, nor could it phosphorylate site 1 b of glycogen synthase. The results demonstrate that glycogen synthase kinase‐4 is not a proteolytic fragment of the calmodulin‐dependent glycogen synthase kinase, that has lost its ability to be regulated by Ca2+‐calmodulin.
ATP‐AMP phosphotransferase from Paracoccus denitrificans
Authors: YEH Shing‐shing , TOMASSELLI Alfredo G. , NODA Lafayette H. 
Published: March 2005

Adenylate kinase has been extracted from Paracoccus denitrificans by toluene treatment and purified 370‐fold (overall yield of 44%, 554 U/mg, Mr 22 000, pI 4.7; pH optimum 7.5–8.0) by using successive column chromatography on DEAE‐cellulose DE‐52, Matrex‐Blue A and Sephadex G‐75. The enzyme was homogeneous by dodecylsulfate gel electrophoresis and constancy of specific activity across the single peak found in Sephadex G‐75 chromatography. Crystals in the form of heavy needles have been obtained in poly(ethylene glycol)‐400. The enzyme is specific for adenine (deoxyadenine) nucleotides with Km values for ATP, ADP and AMP of 340 μm, 980 μM and 93 μM respectively and dissociation constants, for ATP, MgATP and AMP of 7.3 μM, 7.1 μM and 6.9 μM respectively. The enzyme was noted to have no disulfide bond, two free sulfhydryl groups and a total of 208 residues. Aboe 50 mM KCl the enzyme activity drops off gradually. The enzyme is stable at neutral pH and below a temperature of 44°C. Adenylate kinase from P. denitrificans resembles adenylate kinases from other sources with respect to such properties as the dependence on specific divalent cations, maximal activities at a ratio of Mg2+ :ATP of about 1:1 and the ratio of Mg2+ :ADP of 1:2, in having MgATP (or MgADP) binding site and another substrate binding site for AMP (or ADP), and in requiring an intact histidine and one or more arginine residues for enzymatic activity.Adenylate kinase from P. denitrificans appears to resemble the so‐called muscle‐type (cytoplasmic) rather than the mitochondrial‐type adenylate kinase by comparisons of the following properties: low molecular weight, having — SH groups and no disulfide bonds, pH‐optimum of 8.0 for formation of ADP, positive cross‐reaction by ELISA test and in requiring a low concentration of Ap5A to inhibit 95% activity.
Isolation and characterization of the three fractions (DE‐I, DE‐II and DE‐III) of rat‐liver Z‐protein and the complete primary structure of DE‐II
Authors: TAKAHASHI Kuni , ODANI Shoji , ONO Teruo 
Published: March 2005

Three fractions (DE‐I, DE‐II and DE‐III) of Z‐protein (fatty acid binding protein) have been isolated from rat liver cytosol by DEAE‐cellulose chromatography and characterized. They had the same molecular weight of 14000 and essentially identical amino acid composition. However, compositions of endogenous fatty acids were found to differ strikingly from one another. Long‐chain fatty acids detected in DE‐II were palmitic, stearic, oleic, linoleic and arachidonic acids. In contrast to DE‐II, DE‐III contained mainly arachidonic acid. Molar ratios of endogenous long‐chain fatty acids to both DE‐II and DE‐III were estimated to be around 1.0. Unlike the latter two fractions, DE‐I was virtually lipid‐free.Analyses of the three fractions by polyacrylamide gel electrophoresis, electrofocusing and DEAE‐cellulose chromatography before and after delipidation suggested that the difference between DE‐I and DE‐II was in part due to fatty acids bound to DE‐II. In contrast, DE‐III appeared to be somewhat different from these forms in its protein structure, though tryptic peptide mappings of the three fractions did not reveal clear differences among them.Analysis of the primary structure was made on the most abundant fraction, DE‐II, to investigate the relationship among the three fractions and to other proteins. The protein was a single chain consisting of 127 amino acid residues and had a mostly acetylated NH2 terminus and a free sulfhydryl group. The complete sequence of Z‐protein showed striking homology to cellular retinoid binding proteins and peripheral nerve myelin P2 protein, which indicated the presence of a new family of cellular lipid‐binding proteins diverged from a common ancestor. A possible intragenic duplication of Z‐protein was also suggested.
Actions of glucagon on flux rates in perfused rat liver
Authors: KIMMIG Rainer , MAUCH Teri Jo , KERZL Willibald , SCHWABE Ursula , SCHOLZ Roland 
Published: March 2005

Changes in the rates of glucose, lactate and pyruvate production following infusion of glucagon were studied in isolated livers from fed or fasted rats perfused with non‐recirculating Krebs‐Henseleit bicarbonate buffer.

Evidence was presented that under these experimental conditions, the release of lactate plus pyruvate into the perfusate represents 80–90% of the total glycolytic flux; oxidation of pyruvate derived from glucose and/or glycogen accounts for the remaining 10–20%, whereas recycling of pyruvate to glucose is negligible.
In the presence of glucagon, rates of lactate plus pyruvate production were diminished to less than 30% in the fed state (glycogen as substrate) and to less than 10% in the fasted state (glucose as substrate). Rates of pyruvate oxidation were unchanged. Although recycling of pyruvate to glucose was enhanced, it could account for not more than 20% of the decrease in lactate plus pyruvate production. The data indicate a strong inhibition of the substrate flux through glycolysis.
The glucagon concentration for half‐maximal inhibition of glycolysis was 0.2 nM, independent of the substrate (glucose or glycogen) and the nutritional state. The effective concentrations were within the physiological range of glucagon concentrations reported for portal venous blood.
Transient state analyses indicated that the inhibition of glycolysis precedes the stimulation of glycogenolysis. When after a delay glycogenolysis was accelerated, it was followed by a transient stimulation of glycolysis. The stimulatory component in the glucagon effect on glycolysis was diminished in glycogen‐depleted livers and when glucose was the main substrate. The coordinated control of glycolysis and glycogenolysis by glucagon and the interaction of the two processes in the transient state are discussed.
Chemical structure of the lipid A component of the lipopolysaccharide from a Proteus mirabilis Re‐mutant
Authors: SIDORCZYK Zygmunt , ZÄHRINGER Ulrich , RIETSCHEL Ernst Th. 
Published: March 2005

The chemical structure of the lipid A component from the lipopolysaccharide of a Proteus mirabilis Re‐mutant (strain R45) was analysed. It consists of a β(1–6)‐linked d‐glucosamine disaccharide which carries two phosphate groups, one being ester‐linked to position 4′ of the nonreducing glucosaminyl residue and the other being bound to the glycosidic hydroxyl group of the reducing glucosaminyl residue. The ester‐bound phosphate group is quantitatively substituted by a 4‐amino‐4‐deoxy‐l‐arabinopyranosyl residue, the glycosidic phosphoryl group appears to be unsubstituted. Two available hydroxyl groups of the disaccharadide (probably at positions 3 and 3) are acylated by approximately 1 mol each of (R)‐3‐tetradecanoyloxytetradecanoic and (R)‐3‐hydroxytetradecanoic acid/mol. The amino group of the nonreducing glucosaminyl residue carries (R)‐3‐tetradecanoyloxytetradecanoic and that of the reducing residue (R)‐3‐hydroxytetradecanoic acid. In addition smaller amounts of (R)‐3‐hexadecanoyloxytetradecanoic acid are present in amide linkage.The attachment site of the oligosaccharide portion to lipid A was also investigated. It was found that the hydroxyl group at position 6′ of the nonreducing glucosaminyl residue carries 3‐deoxy‐d‐manno‐octulosonic acid. This indicates that the saccharide portion in this Proteus lipopolysaccharide is linked to lipid A via the primary hydroxyl group in position 6′.
Mode of action towards oligopeptides and proteins of hydrolase H, a high‐molecular‐weight aminoendopeptidase from rabbit skeletal muscle
Authors: NISHIMURA Toshihide , OKITANI Akihiro , KATAKAI Ryoichi , KATO Hiromichi 
Published: March 2005

The mode of action towards oligopeptides and proteins of hydrolase H purified from rabbit skeletal muscle was studied. The presence of protamine or α‐N‐benzoylarginine p‐nitroanilide (an endopeptidase substrate) changed both the Km and V values of the enzyme towards Leu‐β‐naphthylamide (an aminopeptidase substrate). This indicates that the binding site for an endopeptidase substrate is different from that for an aminopeptidase substrate. Hydrolase H as an aminopeptidase displayed broad specificity. The enzyme hydrolyzed various dipeptides readily except the dipeptides containing Pro or an amino acid with a hydrophobic β‐branched chain at the NH2 terminus. Pro and Val at the NH2 terminus of tripeptides were also difficult to release, whereas Ile and Val of tetrapeptides were easily released in contrast with those of dipeptides. The longer the peptide chain of Glyn (n= 2, 3,4), the more susceptible was it to hydrolase H.Hydrolase H behaved as an endopeptidase only towards protamine among the proteins tested. The other proteins, casein, bovine serum albumin, myofibrils, troponin, hemoglobin, sarcoplasmic proteins, and myoglobin were probably attacked only by the aminopeptidase activity of the enzyme.
Oxidation of glutathione by the superoxide radical to the disulfide and the sulfonate yielding singlet oxygen
Authors: WEFERS Heribert , SIES Helmut 
Published: March 2005

The reaction of superoxide with reduced glutathione (GSH) was studied with two O72‐producing systems: xanthine oxidase using xanthine or acetaldehyde as substrates and, secondly, quinol autoxidation.The capability of GSH to quench superoxide radicals was detected by lowered O72‐mediated cytochrome c3+ reduction. The formation of the oxidation products, glutathione disulfide (GSSG) and glutathione sulfonate (the latter at levels of about 6–15% compared to GSSG), was dependent on the O72‐production and was inhibited by superoxide dismutase. The presence of GSH together with an O72‐producing system led to an extra uptake of oxygen, which was also depressed by superoxide dismutase. The observed O2 uptake was accounted for by the formation of GSSG and GSO−3 from GSH; the data are in accordance with a mechanism involving thiyl radicals.Low‐level chemiluminescence measurement indicated the formation of excited oxygen species. The intensity of photoemission was dependent on the GSH concentration and on the O−·2 production rate. Chemiluminescence was inhibited by superoxide dismutase and also by glutathione peroxidase, but not by catalase or OH· quenchers. Spectral analysis and the effects of 1,4‐diazabicyclo[2.2.2]octane and sodium azide indicated the contribution of singlet molecular oxygen to the light emission. It is suggested that singlet oxygen results from an intermediate oxygen addition product such as a glutathione peroxysulphenyl radical.