bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oxidation‐reduction potentials of ferredoxin‐NADP+ reductase and flavodoxin from Anabaena PCC 7119 and their electrostatic and covalent complexes
Authors: PUEYO José J. , GOMEZ‐MORENO Carlos , MAYHEW Stephen G. 
Published: March 2005

The oxidation‐reduction potentials of ferredoxin‐NADP+ reductase and flavodoxin from the cyanobacterium Anabaena PCC 7119 were determined by potentiometry. The potentials at pH 7 for the oxidized flavodoxin/flavodoxin semiquinone couple (E2) and the flavodoxin semiquinone/hydroquinone couple (E1) were – 212 mV and –436 mV, respectively. E1 was independent of pH above about pH 7, but changed by approximately –60 mV/pH below about pH 6, suggesting that the fully reduced protein has a redox‐linked pKa at about 6.1, similar to those of certain other flavodoxins. E2 varied by – 50 mV/pH in the range pH 5–8. The redox potential for the two‐electron reduction of ferredoxin‐NADP+ reductase was –344 mV at pH 7 (ΔEm=– 30 mV/pH).In the 1:1 electrostatic complex of the two proteins titrated at pH 7, E2 was shifted by + 8 mV and E1 was shifted by – 25 mV; the shift in potential for the reductase was + 4 mV. The potentials again shifted following treatment of the electrostatic complex with a carbodiimide, to covalently link the two proteins. By comparison with the separate proteins at pH 7, E2 for flavodoxin shifted by – 21 mV and E1 shifted by + 20 mV; the reductase potential shifted by + 2 mV. The potentials of the proteins in the electrostatic and covalent complexes showed similar pH dependencies to those of the individual proteins. Qualitatively similar changes occurred when ferredoxin‐NADP+ reductase from Anabena variabilis was complexed with flavodoxin from Azotobacter vinelandii.The shifts in redox potential for the complexes were used with previously determined values for the dissociation constant (Kd) of the electrostatic complex of the two oxidised proteins, in order to estimate Kd values for the interaction of the different redox forms of the proteins. The calculations showed that the electrostatic complexes, formed when the proteins differ in their redox states, are stronger than those formed when both proteins are fully oxidized or fully reduced.
Properties to two high‐molecular‐mass forms of glyceraldehyde‐3‐phosphate dehydrogenase from spinach leaf, one of which also possesses latent phosphoribulokinase activity
Authors: CLASPER Steven , EASTERBY John S. , POWLS Roy 
Published: March 2005

Two High‐Mr forms of chloroplast glyceraldehyde‐3‐phosphate dehydrogenase from spinach leaf can be separated by DEAE‐cellulose chromatography. One form, the high‐Mr glyceraldehyde‐3‐phosphate dehydrogenase, resembles an enzyme previously described [Yonuschot, G. R., Ortwerth, B. J. & Koeppe, O. J. (1970) J. Biol. Chem. 245 4193–4198]. The other, a glyceraldehyde‐3‐phosphate dehydrogenase/phosphoribulokinase complex, is characterised by possession of latent phosphoribulokinase activity, only expressed following incubation with dithiothreitol.This complex is composed not only of subunits A (39.5 kDa) and B (41.5 kDa) characteristic of the high‐Mr glyceraldehyde‐3‐phosphate dehydrogenase, but also of a thirt subunit, R (40.5. kDa) comigrating with that from the active phosphoribulokinase of spinach.Incubation of the complex with dithiothreitol markdely stimulated both its phosphoribulokinase and NADPH‐dependent dehydrogenase activities. This dithiothreitol‐induced activation was accompained by depolymerisation to give two predominantly NADPH‐linked tetrameric glyceraldehyde‐3‐phosphate dehydrogenases (the homotetramer, A4, and the heterotetramer, A2B2) as well as the active dimeric phosphoribulokinase. Incubation of the high‐Mr glyceraldehyde‐3‐phosphate dehydrogenase with dithiothreitol promoted complete depolymerisation yielding only the heterotetramer (A2B2).Possible structures suggested for the glyceraldehyde‐3‐phosphate dehydrogenase/phosphoribulokinase complex are (A2B2)2A4R2 or (A2B2)(A4)2R2.
Inhibition of glucose production during hepatic nerve stimulation in regenerating rat liver perfused in situ
Authors: IWAI Masaru , MIYASHITA Tetsukazu , SHIMAZU Takashi 
Published: March 2005

To explore the possible role of gap junctions in neural regulation of hepatic glucose metabolism, the effects of hepatic nerve stimulation on metabolic and hemodynamic changes were examined in normal and regenerating rat liver which was perfused in situ at constant pressure via the portal vein with a medium containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate.1. The content of connexin 32, a major component of gap junctions in rat liver, decreased transiently to about 25% of the control level in regenerating liver 48–72 h after partial hepatectomy and recovered to normal by the 11th day after the operation.2. In normal liver, electrical stimulation of the hepatic nerves (10 Hz, 20 V, 2 ms) and infusion of noradrenaline (1 μM) both increased glucose and lactate output and reduced perfusion flow.3. In early stage of regenerating liver 48 h and 72 h after partial hepatectomy, the increase in glucose output in response to nerve stimulation was almost completely inhibited, whereas the change in lactate balance was partially suppressed and the reduction of flow rate was retained. The response of glucose output to nerve stimulation recovered by the 11th day after partial hepatectomy. In contrast, exogenous application of noradrenaline increased glucose output even in the early stage of regenerating liver.4. The increase in noradrenaline overflow during hepatic nerve stimulation in the early stage of regenerating liver was approximately the same as in normal liver. Liver glycogen was sufficiently preserved in the early stage of regenerating liver. However, noradrenaline infusion could no more increase glucose output both in normal and in regenerating livers after 24 h of fasting that depleted liver glycogen.These results suggest that the impaired effects of sympathotic nerve stimlation on glucose metabolism observed in regenerating liver are derived neither from reduced release of noradrenaline nor from depletion of liver glycogen, but rather from transient reduction of gap junctions which assist signal propagation of the nerve action through intercellular communication in rat liver.
Regulation of transcription of the gene coding for peroxisomal 3‐oxoacyl‐CoA thiolase of Saccharomcyes cerevisiae
Authors: EINERHAND Alexandra W. C. , VOORN‐BROUWER Tineke M. , ERDMANN Ralf , KUNAU Wolf‐H. , TABAK Henk F. 
Published: March 2005

Transferring Saccharomyces cerevisiae cells from glucose‐ to oleate‐containing growth media results in a significant increase in the number and volume of peroxisomes. To investigate this proliferation process we studied the transcriptional regulation of the gene coding for peroxisomal 3‐oxoacyl‐CoA thiolase (EC 2.3.1.16) in response to the switch in carbon source. Expression was proved to be repressed during growth on glucose, derepressed during growth on glycerol, and induced during growth on oleate as the sole carbon source. By deletion and mutational analysis of sequences upstream of this gene, we have identified a region which is involved in the regulation of transcription. It is contained within a 52‐base‐pair sequence, UAST52 (upstream activation sequence thiolase 52), located between 203 and 151 nucleotides upstream of the translational initiation codon. This sequence proved to be required for repression, derepression and induction of transcription, and was able to activate transcription from the truncated version of the heterologous iso‐1‐cytochrome‐c (CYCI) promoter in a similar way as in the wild‐type promoter context.Sequence comparison revealed that the UAST52 contained a sequence motif (‘β‐oxidation box’, that is very similar to sequences located in the 5′‐upstream regions of the genes coding for two other β‐oxidation enzymes of S. cerevisiae: the peroxisomal acyl‐CoA oxidase and the peroxisomal trifunctional β‐oxidation enzyme of S. cerevisiae. Mutational analysis of the ‘β‐oxidation box’ indicates that this sequence motif acts as a UAS in vivo.Sequence comparison also revealed that just upstream of the ‘β‐oxidation box’, between positions – 213 and – 201, a potential binding site occurred for the yeast multifunctional autonomously replicating sequence binding factor ABF1. Gel‐retardation‐competition experiments indicate that ABF1 binds specifically to this sequence.
Purification and characterization of phosphatidylinositol 4‐kinase from human erythrocyte membranes
Authors: WETZKER Reinhard , KLINGER Reinhard , HSUAN Justin , FRY Michael J. , KAUFFMANN‐ZEH Andrea , MÜLLER Elke , FRUNDER Horst , WATERFIELD Michael 
Published: March 2005

Two species of PtdIns 4‐kinase with molecular masses of 50 kDa and 45 kDa were detected in human erythrocyte membranes using SDS/PAGE. These enzymes were purified to near homogeneity and found to display very similar enzymatic characteristics. The purification scheme consisted of solubilization from erythrocyte membranes in the presence of Triton X‐100, followed by Cibacron‐blue–Sephadex, phosphocellulose and Mono Q anion‐exchange chromatography. The final step in the purification protocol was preparative SDS/PAGE, followed by electroelution and renaturation of the enzyme. This procedure afforded an about 4000‐fold purification of the enzyme from erythrocyte membranes.Characterization of the [32P]PtdInsP products formed by the purified PtdIns kinases indicated that these enzymes specifically phosphorylated the D‐4 position of the inositol ring.The Km values of both PtdIns 4‐kinase species for PtdIns and ATP were found to be 0.2 mM and 0.1 mM, respectively. the enzymes are both activated by Mg2+. and inhibited by Ca2+ and by adenosine. The potential importance of these effectors for the regulation of PtdIns phosphorylation in cells is discussed.
An oxidized analog of α‐human atrial natriuretic polypeptide is a selective agonist for the atrial‐natriuretic‐polypeptide clearance receptor which lacks a guanylate cyclase
Authors: KOYAMA Satoshi , TERAI Takao , INOUE Takeshi , INOMATA Kaoru , TAMURA Kouichi , KOBAYASHI Yuji , KYOGOKU Yoshimasa , KOBAYASHI Masakazu 
Published: March 2005

The differences in biological functions between α‐human atrial natriuretic polypeptide (α‐hANP) and its oxidized analog, MetSO‐α‐hANP, have been investigated. Analysis of the ANP receptor subtypes by affinity labeling has shown that a bovine pulmonary aortic endothelial cell line (CPAE cells) primarily expresses ANP‐R1 (R, receptor) coupled to particulate guanylate cyclase, while Hela cells from human cervical carcinoma predominantly express ANP‐R2, which lacks a guanylate cyclase. α‐hANP could bind to both ANP receptor subtypes with high affinity, while MetSO‐α‐hANP showed more selective binding to ANP‐R2 than to ANP‐R1. The activity of MetSO‐α‐hANP for stimulation of guanylate cyclase coupled to ANP‐R1 was about 520‐fold less than that of α‐hANP (median effective dose = 2.5 nM for α‐hANP, 1.3 μM for MetSO‐α‐hANP), indicating that MetSO‐α‐hANP was a partial agonist for this receptor. While this oxidized analog could inhibit the cAMP production through ANP‐R2, with 0.15 times the activity of α‐hANP (median concentration = 0.31 nM for α‐hANP, 2.0 nM for MetSO‐α‐hANP). In in vivo studies, the diuretic activity of MetSO‐α‐hANP was 25–100‐fold less than that of α‐hANP. In addition, MetSO‐α‐hANP could potentiate the diuretic activity of α‐hANP that was also caused by C‐ANF4–23, a specific agonist for ANP‐R2.These results demonstrate that MetSO‐α‐hANP can act as an agonist more selective for ANP‐R2 than for ANP‐R1, both in vivo and in vitro. The relationship between receptor selectivities and the conformation of α‐hANP or MetSO‐α‐hANP was also discussed.
The piscine plasma retinol‐binding protein
Authors: BERNI Rodolfo , STOPPINI Monica , ZAPPONI Maria Carla 
Published: March 2005

1. Retinol‐binding protein (RBP) has been isolated from the pooled plasma or rainbow trouts (Oncorhinchus mykiss) by gel filtration, hydrophobic interaction chromatography and ion‐exchange chromatography. By this procedure two forms of the protein, both with a molecular mass (approximately 20 kDa) similar to that of mammalian RBP, were purified to homogeneity. Five amino acid substitutions have been found in the partial (about 60%) sequences of the two forms of trout RBP, which are presumably acetylated at their N terminus. The apparent participation of six conserved cysteines in the formation of disulphide bridges, as in human RBP, and the similarity (about 60%) of the amino acid sequence of trout and mammalian RBPs, indicate the existence of a similar overall structure organization in evolutionary distant RBPs.2. Although the two forms of trout RBP are not physiologically involved in the formation of any protein‐protein complex in plasma, they are capable of interacting with mammalian transthyretin, albeit with a binding affinity (K′d= 15–40 μM) considerably lower than that of mammalian RBP. Our data indicate that the two forms of trout RBP also possess the region that in mammalian RBP has the functional role of binding transthyretin. It is suggested that transthyretin (or a homologous protein) was modified, during phylogenetic development of the non mammalian vertebrates, to acquire a binding site for such a region of the RBP molecule.
31P‐NMR assignment and conformational study of NADPH bound to Lactobacillus casei dihydrofolate reductase based on two‐dimensional 1H‐31P‐heteronuclear and 1H‐detected 1H‐31P‐shift‐correlation experiments
Authors: GEROTHANASSIS Ioannis P. , BIRDSALL Berry , BAUER Christopher J. , FEENEY James 
Published: March 2005

For any detailed NMR conformational study of a protein – ligand complex it is essential to have specific resonance assignments. We have now assigned the pyrophosphate 31P resonances in spectra of NADPH bound to Lactobacillus casei dihydrofolate reductase (DHFR) by using a combination of 1H‐31P‐heteronuclear shift‐correlation (HETCOR), 1H‐31P‐heteronuclear multiple‐quantum‐coherence correlation spectroscopy (HMQC‐COSY), 1H‐1H COSY, homonuclear Hartmann‐Hahn (HOHAHA) and NOE spectroscopy (NOESY) experiments.The nicotinamide pyrophosphate phosphorus, P(n), has been unequivocally assigned to a signal (−14.07 ppm) which shows a large 3JP‐O‐C‐H coupling constant. Such a coupling constant when combined with the appropriate Karplus relationship provides conformational information about the P‐O‐C‐H torsion angle. The torsion angle changes by 65°± 10° for the binary complex compared with the value in free NADPH. The observed coupling constants for the binary (DHFR – NADPH) and ternary (DHFR – NADPH – methotrexate) complexes (12.3 and 10.5 ± 0.6 Hz, respectively) indicate that the pyrophosphate group has a similar conformation in the two complexes.
Evidence against a role for phosphorylation/dephosphorylation in the regulation of acyl‐CoA: cholesterol acyl transferase
Authors: CORTON Julia M. , HARDIE D. Grahame 
Published: March 2005

1. As detailed below, we have been able to reproduce observations of time‐dependent changes in the activity of acyl‐CoA: cholesterol acyl transferase (ACAT) in rat liver microsomes, that were suggested to represent evidence of a role for reversible phosphorylation in the regulation of cholesterol ester formation.2. ACAT in washed rat liver microsomes was inactivated in a time‐dependent manner in the presence of Mg2+. However, this effect of Mg2+ appears to be caused by aggregation of microsomal vesicles rather than dephosphorylation, since it could be abolished by rehomogenization, and was mimicked by Ca2+, another agent which causes aggregation. Fluoride did not prevent this effect of Mg2+, but masked it by causing a rapid activation that appeared to be a non‐specific effect of increased ionic strength.3. Under conditions where other proteins were rapidly dephosphorylated, microsomal ACAT activity from rat liver was not affected by incubation with the purified catalytic subunits of protein phosphatases 1, 2A or 2C. Similar results were obtained using protein phosphatases 1 or 2A on microsomes from a macrophage cell line (J774.2 cells). Incubation of cultured J774.2 cells with a cell‐ permeable inhibitor of these two protein phosphatases, okadaic acid, also had no effect on cholesterol ester formation.4. A high‐speed‐centrifugation supernatant fraction (S303) from rat liver activated ACAT in the presence of MgATP. This effect was not abolished by prior heat‐treatment of the fraction, and the supernatant fraction could not be replaced by purified AMP‐activated protein kinase or a variety of other protein kinases.5. The results above were obtained using assays involving endogenous cholesterol as the substrate. The MgATP‐dependent activation by S303 was reduced or abolished when the assays were carried out in the presence of the detergent Triton WR‐1339 plus cholesterol, or detergent alone.6. These results do not support the idea that ACAT is regulated by reversible phosphorylation. The most likely explanation for the effect of S303 is that it is an artefact caused by changes in the availability of endogenous cholesterol to the enzyme.
Redox control of catalysis in ATP‐citrate lyase from rat liver
Authors: WELLS Timothy N. C. , SAXTY Barbara A. 
Published: March 2005

In thiol redox buffers at pH 8.0, rat liver ATP‐citrate lyase is in equilibrium between an oxidised inactive form and a reduced active form. The reduced enzyme is inactivated by oxidised glutathione (GSSG) at a rate of 45 min−1· M−1 and the oxidised enzyme is activated by reduced glutathione (GSH) at a rate of 3.2 min−1· M−1. At redox equilibrium, the enzyme activity depends on the ratio [GSH]2/[GSSG]. The inactivation involves formation of a protein‐protein disulphide rather than a protein‐glutathione complex. This reaction has Keq= 78 ± 7 mM for the oxidative reaction. Activity can therefore be controlled by the redox state of the cell, being more active in the fed state than in the oxidatively stressed state.This redox process is also important in the in vitro enzyme assay, where ATP‐citrate lyase is in redox equilibrium with oxygen and either dithiothreitol or 2‐mercaptoethanol. Reduction is a two‐step process, requiring high concentrations of reductant for full activation (30 mM dithiothreitol or 200 mM 2‐mercaptoethanol). The enzyme inhibitor, Medica‐16 raises the redox equilibrium constant to greater than 400 mM. It binds more tightly to the oxidised form of the enzyme, with Ki < 40 μM compared to 180 μM for the reduced form.
Purification and characterization of maize seedling casein kinase IIB, a monomeric enzyme immunologically related to the α subunit of animal casein kinase‐2
Authors: DOBROWOLSKA Grazyna , MEGGIO Flavio , SZCZEGIELNIAK Jadwiga , MUSZYNSKA Grazyna , PINNA Lorenzo A. 
Published: March 2005

Casein kinase IIB (CKIIB), a protein kinase related to animal casein kinase‐2 (CK2), has been purified to homogeneity. It appears to be a monomeric enzyme, composed by an individual 39 kDa subunit, homologous to the α/α′ subunits of animal CK2 and devoid of the autophosphorylatable 25‐kDa α subunit of animal CK2, which display an heterotetrameric α2β2/αα′β2 structure. Such a conclusion is supported by the following lines of evidence: (1) CKIIB displays an apparent 39000 Mr by gel filtration on Ultrogel AcA 34 and it gives rise to a single prominent protein band of similar Mr (38 000) upon SDS/PAGE; (2) upon incubation of the enzyme with [32P]ATP, no radiolabeled bands are detectable which might be attributable to either canonical or atypical β subunits; (3) the 39‐kDa band immunoreacts with antisera that recognize the α subunit of rat and chicken CK2; (4) conversely, no component immunologically related with the β subunit could be detected in CKIIB by Western‐blot analyses with antisera that recognize animal β subunits; (5) the recombinant β subunit of human CK2 is readily phosphorylated by CKIIB, the reaction being prevented, rather than stimulated, by polylysine, a behaviour typical of animal CK2 autophosphorylation.While the responsiveness of CKIIB to either heparin inhibition or polylysine stimulation are reminiscent of those of animal CK2, its peptide substrate specificity is significantly different and its thermolability is increased. Altogether these data would indicate that maize seedling CKIIB represents a naturally occurring monomeric form of CK2 devoid of non‐catalytic subunits. Its properties, compared to those of animal CK2, suggest that the β subunits of animal CK2 may be responsible for structural modifications conferring an altered specificity and an increased stability to the catalytic subunit.
Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense
Authors: MBAWA Zeres R. , GUMM Ian D. , SHAW Elliott , LONSDALE‐ECCLES John D. 
Published: March 2005

A cysteine protease (trypanopain‐Tc) with cathepsin‐L‐like properties has been purified from Trypanosoma congolense. The enzyme has an apparent molecular mass of 31–32 kDa by SDS/ PAGE and 66 kDa by gel chromatography. It has a pI 7.4 and a high affinity for concanavalin A. Trypanopain‐Tc catalyses the limited proteolysis of a variety of protein substrates such as fibrinogen, serum albumin and trypanosome variant‐surface glycoprotein. It has minimal or no activity against casein or elastin.A variety of peptidyl amidomethylcoumarins and peptidyl diazomethanes were used to test the specificity of trypanopain‐Tc. The better substrates had Arg or Lys in P1 and hydrophobic amino acids in P2 and P3. The best substrate found for trypanopain‐Tc was Z‐Phe‐Arg‐NHMec (Z, benzyl‐oxycarbonyl; NHMec, 7‐amido‐4‐methylcoumarin). The kinetic constants for the hydrolysis of Z‐Phe‐Arg‐NHMec were kcat= 17.4 s−1, Km= 4.4 μM, kcat/Km= 4.0 μM−1· s−1, which are very similar to those of cathepsin L with this substrate. The specific substrates for cathepsin B (Z‐Arg‐Arg‐NHMec) and cathepsin H (Arg‐NHMec) were not hydrolysed by trypanopain‐Tc under the conditions tested. The pH optimum of trypanopain‐Tc against Z‐Phe‐Arg‐NHMec was pH 6.0 but it showed a broad peak of activity extending well into the alkaline region. The enzyme was activated by low‐molecular‐mass thiol compounds and inhibited by cystatin, L‐trans‐epoxysuccinyl‐4‐guanidinobutane (E‐64) and a variety of peptidyl diazomethanes. The most effective diazomethane inhibitors (Z‐Leu‐Leu‐Met‐CHN2, Z‐Leu‐Met‐CHN2 and Z‐Leu‐Lys‐CHN2, were inhibitory at nanomolar concentrations and were trypanocidal in vitro after 24–48 h incubation in ≥ 20 μM [inhibitor]. However, it is not clear whether the trypanocidal activity of these inhibitors is a consequence of the inhibition of trypanopains or of some other essential proteolytic activities within the parasites.
Evolution of ruminant hemoglobins
Authors: GIARDINA Bruno , AREVALO Felicidad , CLEMENTI Maria E. , FERRARA Lucio , Di LUCCIA Aldo , LENDARO Eugenio , BELLELLI Andrea , CONDÒ Saverio G. 
Published: March 2005

The ligand‐binding properties of hemoglobins from two homozygote phenotypes (AA and BB) of water buffalo (Bubalus bubalis) have been characterized by equilibrium and kinetic techniques. In the case of the BB phenotype, the two constituent hemoglobins have been purified and separately analysed.Buffalo hemoglobins display the reduced sensitivity to organic phosphates characteristic of ruminant hemoglobins, their physiological effector probably being the chloride ion.In contrast to the other known hemoglobins from ruminants, all the hemoglobins from the water buffalo display a significant temperature sensitivity, the ΔH for oxygen binding in the presence of physiological effectors approaching that of human hemoglobin (ΔH=−30.5 kJ/mol O2).This discrepancy with the other ruminant hemoglobins (e.g. ox, ΔH=−10.4 kJ/mol O2), whose primary structure is very similar to that of buffalo, hemoglobins might be correlated to the different habitat and phylogenetic history of the two subfamilies (Bos and Bubalus) of Bovidae.