bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stereoselective effects in the interactions of pterin cofactors with rat‐liver phenylalanine 4‐monooxygenase
Authors: HAAVIK Jan , DØSKELAND Anne P. , FLATMARK Torgeir 
Published: March 2005

The (6R) and (6S) epimers of l‐erythro‐tetrahydrobiopterin (BH4) and some of its structural analogs, were tested as cofactors and non‐covalent effectors in the phenylalanine 4‐monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1) reaction. The oxidation‐reduction potentials (Em,7) of the free (not enzyme‐bound) form of the (6R) and (6S) epimers were rather similar (range 174–184 mV) for the oxidation of tetrahydropterins to quinonoid dihydropterins. Rapid‐mixing kinetic experiments were performed at 20°C under conditions which allow only a few turnover reactions of the enzyme. Three main oxidation products were identified spectroscopically at pH 6.8 for all three tetrahydropterins tested: (a) the C(4a)‐hydroxy derivatives, (b) the quinonoid dihydropterins, and (c) the stable 7,8‐dihydropterins (in that sequence). The formation of the C(4a)‐hydroxy forms closely paralleled that of tyrosine, and supports the proposal that this covalent adduct is formed as an immediate product on completion of the catalytic cycle. Assay of the initial rate of C(4a)‐hydroxy derivative formation represents a new approach in kinetic studies of this enzyme, and the kinetic parameters obtained for the phenylalanine‐activated enzyme are presented.The affinity of binding of (6R)‐BH4 and (6S)‐BH4 to phenylalanine hydroxylase was also estimated on the basis of their quenching of the intrinsic tryptophan fluorescence of the enzyme. The apparent affinities were found to correspond well to the Km values estimated in kinetic studies of the hydroxylation reaction with the phenylalanine activated enzyme, i.e. higher for (6R)‐BH4 than for (6S)‐BH4. The lower V value observed for the native enzyme with the (6R) epimer in steady‐state kinetics is explained by its higher potency as a negative effector, since the oxidation‐reduction potentials of the two diastereomers were similar.Dihydrobiopterin (BH2) was found to inhibit the hydroxylation reaction and quenched the intrinsic tryptophan fluorescence of the enzyme with the same concentration dependence as that observed with (6S)‐BH4.
Flavin‐dependent alcohol oxidase from yeast
Authors: GEISSLER Johanna , GHISLA Sandro , KRONECK Peter M. H. 
Published: March 2005

The kinetic course of the reaction of methanol and deutero‐methanol with FAD‐dependent alcohol oxidase was investigated under single‐turnover conditions [ked∼ 15000 min−1 (1H3COH) and ∼4300 min−1 (2H3COH)] and multiple‐turnover conditions [TNmax∼6000 min−1 (1H3COH) and ∼3100 min−1 (2H3COH)]. A kinetic scheme for the overall catalytic mechanism is proposed, which is characterized by (1) formation of a Michaelis complex between enzyme and substrate, (2) the reductive step involving partly rate‐limiting scission of the substrate C‐H bond, (3) reaction of the complex of reduced enzyme and aldehyde with dioxygen, and (4) a significant contribution of the dissociation rate of product from its complex with reoxidized enzyme to the overall rate.Prolonged turnover of various alcohols, including methanol, results in progressive inactivation of the enzyme by two processes. In the absence of catalase the inactivation rate increases with time due to accumulation of hydrogen peroxide, which is a potent inactivator (Kd∼ 1.6 mM; kinact∼ 0.55 min−1). In the presence of catalase inactivation during turnover is much slower, the process showing pseudo‐first‐order kinetics (Kinact∼ 0.6 mM; kinact∼ 0.005 min−1 with methanol). The ratio kcat/kinact varies with different alcohols but is always > 105. Propargyl alcohol and methylenecyclopropyl alcohol cannot be considered as suicide substrates, as compared to analogous substrates of other flavin oxidases.
Structure and expression of the chicken epidermal growth factor receptor gene locus
Authors: OLOFSSON Birgitta , PIZON Véronique , ZAHRAOUI Ahmed , TAVITIAN Armand , THERWATH Amu 
Published: March 2005

Similarity between the carboxyl‐terminal portion of the human epidermal growth factor (EGF) receptor and the deduced protein sequence of the chicken‐derived oncogene v‐erbB, of avian erythroblastosis virus strain H, has suggested that the chicken cellular erbB locus, c‐erbB, might be part of a longer EGF‐receptor gene in the chicken, whose entire coding capacity remained to be defined. The c‐erbB locus spans more than 20 × 103 base pairs (20 kbp) of DNA and contains at least 1.8 kbp homologous to the v‐erbB oncogene. We show here that human EGF receptor cDNA and chicken genomic DNA share homology not only within the c‐erbB locus but also within a 25.1‐kbp DNA region situated 5′ to this locus. The 3′ region of the EGF receptor overlaps, in sequence homology, the c‐erbB locus. The EGF receptor/c‐erbB locus in chicken generates six related but distinctly different mRNAs of sizes 12, 9, 5, 3.6, 3.2 and 2.6 kb. The transcripts of 12, 9, and 3.6 kb contain sequences coding for both the extracellular EGF‐binding domain of the receptor and the intracellular tyrosine kinase domain. The 12‐kb and 9‐kb transcripts, which have already been shown to contain the sequences coding for the v‐erbB, were found to possess, in addition, sequences that encode the entire chicken EGF receptor. The 3.2‐kb and 2.6‐kb mRNAs are homologous only to the 5′ portion of the EGF receptor gene.These results therefore indicate that the c‐erbB locus, initially defined by homology to the viral transforming gene, corresponds to the 3′ region of the EGF receptor gene in the chicken genome. The multiple, related, chicken EGF receptor RNA transcripts reported here are reminiscent of the various human EGF receptor RNA transcripts observed in normal and transformed cells.
New thiol inhibitor of Achromobacter iophagus collagenase
Authors: YIOTAKIS Athanasios , DIVE Vincent 
Published: March 2005

New synthetic mercaptotripeptides (HS‐CH2‐CH2‐CO‐Pro‐Yaa) which inhibit Achromobacter iophagus collagenase were produced in order to obtain more powerful bacterial collagenase inhibitors than currently available, and to investigate the specificity of the S3′ subsite of the enzyme.Since similar binding constants were found for inhibitors carrying uncharged residues of various sizes in the P3′ position (Yaa = Ala, Leu, Phe, Pro, Hyp) steric hindrance at the collagenase S3′ appears relatively limited.The compound (HS‐CH2‐CH2‐CO‐Pro‐Arg), which carries an arginine residue in the position P3′ and had the highest inhibition constant of the series tested (Ki= 0.5 μM), proved to be the strongest inhibitor so far reported in the literature. The weakest in the present series was the compound (HS‐CH2‐CH2‐CO‐Pro‐Asp) which carries an aspartic residue in position P3′ and had a Ki= 70 μM.The present work revealed that the charged groups in the P3′ position play a key role in the interaction of the inhibitors with the enzyme.
Glutamate decarboxylase side reactions catalyzed by the enzyme
Authors: CHOI Soo Young , CHURCHICH Jorge E. 
Published: March 2005

A homogeneous glutamate decarboxylase isolated from pig brain contains 0.8 mol of tightly bound pyridoxal 5‐phosphate/enzyme dimer. Upon addition of exogenous pyridoxal 5‐phosphate (pyridoxal‐5‐P), the enzyme acquires maximum catalytic activity.Preincubation of the enzyme with l‐glutamate (10 mM) brings about changes in the absorption spectrum of bound pyridoxal‐5‐P with the concomitant formation of succinic semialdehyde. However, the rate of this slow secondary reaction, i.e. decarboxylative transamination, is 10−4 times the rate of normal decarboxylation. It is postulated that under physiological conditions enzymatically inactive species of glutamate decarboxylase, generated by the process of decarboxylative transamination, are reconstituted by pyridoxal‐5‐P produced by the cytosolic enzymes pyridoxal kinase and pyridoxine‐5‐P oxidase.The catalytic activity of resolved glutamate decarboxylase is recovered by preincubation with phospho‐pyridoxyl‐ethanolamine phosphate. The experimental evidence is consistent with the interpretation that the resolved enzyme binds the P‐pyridoxyl analog, reduces the stability of the covalent bond of the phospho‐pyridoxyl moiety, and catalyzes the formation of pyridoxal‐5‐P.
Dynamic interactions of enzymes involved in triosephosphate metabolism
Authors: OROSZ Ferenc , OVÁDI Judit 
Published: March 2005

A steady‐state kinetic analysis of the coupled reactions catalysed by the three‐enzyme system, aldolase, glyceraldehyde‐3‐phosphate dehydrogenase and triosephosphate isomerase, was performed. The kinetic parameters of the progress curves of end‐product formation calculated for noninteracting enzymes were compared with those measured in the two‐enzyme and three‐enzyme systems. Changes in the fluorescence anisotrophy of labelled dehydrogenase upon addition of aldolase and/or isomerase were also measured.Glyceraldehyde‐3‐phosphate oxidation catalysed by glyceraldehyde‐3‐phosphate dehydrogenase in the presence of isomerase (which ensures rapid equilibration of the triosephosphates) follows single first‐order kinetics. The rate constant depends simply on the concentration of the dehydrogenase, indicating no kinetically significant isomerase‐dehydrogenase interaction. Fluorescence anisotropy measurements also fail to reveal complex formation between the two enzymes.The steady‐state velocity of 3‐phosphoglycerate formation from fructose 1,6‐bisphosphate in the reactions catalysed by aldolase and dehydrogenase is not increased twofold on addition of the isomerase, even though a 1:2 stoichiometry of fructose 1,6‐bisphosphate/glyceraldehyde 3‐phosphate is expected. In fact, by increasing the concentration of the isomerase, the steady‐state velocity actually decreases. This effect of the isomerase may be a kinetic consequence of an aldolase‐isomerase interaction, which results in a decrease of aldolase activity. Furthermore, the fluorescence anisotropy of labelled dehydrogenase, measured at different aldolase concentrations, is significantly lower when the sample contains isomerase.The decrease in the steady‐state velocity of the consecutive reactions caused by the elevation of isomerase concentration could be negated by increasing the dehydrogenase concentration in the three‐enzyme system. All of these observations fit the assumption that the amount of aldolase‐dehydrogenase complex is reduced due to competition of isomerase with dehydrogenase. The alternate binding of dehydrogenase and isomerase to aldolase may regulate the flux rate of glycolysis.
Effects of long‐chain fatty acids on the inhibition by antimycin of respiration in hepatocytes and isolated mitochondria from rat liver
Authors: GREGORY Roland B. , BERRY Michael N. 
Published: March 2005

A previous study [Berry, M. N., Gregory, R. B., Grivell, A. R. & Wallace, P. G. (1983) Eur. J. Biochem. 131, 215–222] suggested that long‐chain fatty acid (palmitate) oxidation by hepatocytes was less sensitive than short‐chain fatty acid (hexanoate) oxidation to inhibition by a given concentration of antimycin.
Re‐examination of this phenomenon showed that palmitate oxidation by hepatocytes could be depressed by antimycin to the same degree as other NAD+‐linked substrates, only if the concentration of the inhibitor was raised 2–4‐fold. The presence of palmitate also reduced the sensitivity to antimycin of hepatocytes metabolizing lactate or pyruvate.
Over the range of fatty acids tested, butyrate (C4) to stearate (C18), only long‐chain (> C10) fatty acids endowed cells with decreased sensitivity towards antimycin.
2‐Bromopalmitate, a non‐metabolizable fatty acid, and inhibitor of fatty acid oxidation, also decreased the inhibitory effect of antimycin in cells, suggesting that long‐chain fatty acids per se rather than their metabolites, reverse the inhibition by antimycin. Moreover, another inhibitor of fatty acid oxidation, 2‐tetradecylglycidic acid, did not diminish the effects of palmitate.
Succinate oxidation in isolated mitochondria that had been inhibited by a low concentration of antimycin could be restored by subsequent addition of palmitate or other long‐chain fatty acids such as dodecanoate, tetradecanoate and oleate under conditions where fatty acid oxidation was prevented. 2‐Bromopalmitate, likewise partially restored antimycin‐depressed succinate oxidation. This amelioration of antimycin inhibition was counteracted by the addition of more antimycin and was not seen upon addition of defatted bovine serum albumin, palmitoylcarnitine or octanoate.
The total amount of antimycin bound to mitochondria was not affected by the presence of palmitate.
The data suggest that long‐chain fatty acids are able to interact with the mitochondrial inner membrane in a manner which can relieve the inhibitory effect of antimycin, whether the antimycin is added to the cell or mitochondrial suspension before or after fatty acid addition.
The interaction of phosphatidylcholine bilayers with Triton X‐100
Authors: GOÑI Félix M. , URBANEJA Maria‐Angeles , ARRONDO José Luis R. , ALONSO Alicia , DURRANI Aziz A. , CHAPMAN Dennis 
Published: March 2005

The interaction of multilamellar phosphatidylcholine vesicles with the non‐ionic detergent Triton X‐100 has been studied under equilibrium conditions, specially in the sub‐lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X‐100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X‐100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration.The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier‐transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H‐NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier‐transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel‐to‐fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P‐NMR and 2H‐NMR spectra, and a new low‐temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X‐100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one‐component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.
Characterization of the thioredoxin system in the facultative phototroph Rhodobacter sphaeroides Y
Authors: CLEMENT‐METRAL Jenny D. , HÖÖG Jan‐Olov , HOLMGREN Arne 
Published: March 2005

This paper reports the purification and characterization of a thioredoxin system (thioredoxin, thioredoxin reductase, NADPH) from the facultative phototroph Rhodobacter sphaeroides Y. Rhodobacter sph. Y thioredoxin was purified to homogeneity with an assay based on the reduction of 5,5′‐dithiobis(2‐nitrobenzoic acid) by NADPH and Escherichia coli thioredoxin reductase. Rhodobacter sph. Y thioredoxin reductase was purified with the same assay using NADPH and E. coli thioredoxin. Rhodobacter sph. Y thioredoxin contained 102 amino acid residues and had a single intrachain disulfide bond. The two half‐cystine residues are part of the active site made up of the sequence ‐Ala‐Glu‐Trp‐Cys‐Gly‐Pro‐Cys‐Arg‐ which is identical to that of E. coli thioredoxin except for the presence of an Arg instead of a Lys. Rhodobacter sph. Y thioredoxin contains two tryptophan residues. The fluorescence intensity of the tryptophan residues is quenched in oxidized thioredoxin; on reduction, a much smaller increase is observed with Rhodobacter sph. Y thioredoxin than with the E. coli protein. However, the presence of 5 M guanidine · HCl results in the complete exposure of the two tryptophan residues. Rhodobacter sph. Y thioredoxin reductase has structural and functional similarities to E. coli thioredoxin reductase: it has a molecular mass of 68 kDa, and consists of two, probably identical, subunits. Each subunit has one bound FAD molecule. The enzyme is highly specific for NADPH; it is also highly specific for Rhodobacter sph. Y thioredoxin with a Km value of 3.3±0.6 μM. A kinetic study of the two thioredoxin systems shows that they have a high degree of cross‐reactivity.
Binding of 2′(3′)‐O‐(2,4,6‐trinitrophenyl)‐adenosine 5′‐diphosphate opens the pathway for protons through the chloroplast ATPase complex
Authors: WAGNER Richard , PONSE Gudrun , STROTMANN Heinrich 
Published: March 2005

The effect of 2′(3′)‐O‐(2,4,6‐trinitrophenyl)‐adenosine 5′‐diphosphate (TNP‐ADP) on photophosphorylation and on the proton conductivity of the thylakoid membrane has been investigated. The results show that TNP‐ADP is a potent competitive inhibitor of photophosphorylation (Ki= 1–2 μM). Moreover, in the absence of ADP and Pi, TNP‐ADP accelerates basal electron transport of chloroplasts. Addition of ADP, which promotes release of the analogue from CF1, completely reverses this effect of TNP‐ADP; likewise Pi alone reverses stimulation of electron transport by TNP‐ADP. Dicyclohexylcarbodiimide treatment, which is known to close CF0 to H+, completely abolishes the effect of TNP‐ADP.The measurements of the alkalization of the medium and the acidification of the thylakoid lumen following single turnover flashes showed that binding of TNP‐ADP to CF1 increased membrane permeability for H+. Further results suggest that binding of TNP‐ADP to the catalytic site of CF1 opens the CF0‐CF1 complex for H+. Since ADP, as well as Pi alone, reverses the effect, it is concluded that TNP‐ADP induces a conformation of the CF0‐CF1 complex similar to the one triggered by simultaneous binding of ADP plus Pi. This may be achieved by interaction of the TNP residue with the Pi binding site. Thus it seems that the status of the catalytic site(s) in CF1 can be transmitted to the CF0 part to control proton flux through the ATPase complex in an economically reasonable way.
Properties of different Ca2+ pools in permeabilized rat thymocytes
Authors: GUKOVSKAYA Anna S. , ZINCHENKO Valery P. , PETRUNYAKA Victor V. , KHODOROV Boris I. , EVTODIENKO Yury V. 
Published: March 2005

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen‐induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin‐permeabilized and intact rat thymocytes using a Ca2+‐selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin‐2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non‐mitochondrial. Ca2+ uptake by the non‐mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 μM. The non‐mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 μM. At free Ca2+ concentration close to the cytoplasmic (0.1 μM) the main calcium pool in permeabilized thymocytes is localized in the non‐mitochondrial compartment.Ca2+ accumulated in the non‐mitochondrial pool can be released by inositol 1,4,5‐triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP‐dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non‐mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3‐induced Ca2+ mobilization.Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin‐2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non‐mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+‐containing an Ca2+‐free medium. This indicates a mitogen‐induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.
Ferredoxin reductase levels in the ovaries of pigs and superovulated rats during follicular cell growth and luteinization
Authors: TUCKEY Robert C. , STEVENSON Patricia M. 
Published: March 2005

The concentration of ferredoxin reductase, a component of the mitochondrial steroidogenic electron transport chain, was measured in the ovaries of pigs and superovulated rats by a protein blotting procedure using polyclonal antibodies to the purified protein.The concentration of ferredoxin reductase in porcine granulosa cells doubled during growth of follicles from small (1 – 2mm diameter) to large (6 – 12 mm diameter). The concentration doubled again during the period of luteinization. This is in contrast to the rate of cholesterol side‐chain cleavage, which showed little change during follicular growth but increased by more than tenfold during luteinization. A similar large increase in cholesterol side‐chain cleavage occurs during the period of luteinization in the ovaries of superovulated rats, but as for the pig, only a small increase in ferredoxin reductase was observed.A threefold increase in the yield of mitochondrial protein from tissue homogenates was found between the granulosa cells of small – medium follicles and the cells of the corpora luteum. The increase in ferredoxin reductase during follicular development and luteinization, therefore, correlates well with the increase in mitochondria in the cells, but does not correlate with the dramatic increase in cholesterol side‐chain cleavage activity which occurs during luteinization. Based on these results, it is unlikely that the level of ferredoxin reductase limits the expression of the full steroidogenic activity of the granulosa cells of the ovary.