bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of human α‐thrombin and γ‐thrombin with antithrombin III, protein C and thrombomodulin
Authors: BEZEAUD Annie , DENNINGER Marie‐Hélène , GUILLIN Marie‐Claude
Published: March 2005

Conversion of human α‐thrombin to γ‐thrombin by limited proteolysis resulted in a decrease in the inactivation rate of the enzyme by antithrombin III. The second‐order rate constants were similar but significantly different: 11 ± 1.7 × 103 and 7 ± 0.5 × 103 M−1 s−1 for α‐ and γ‐thrombin respectively. This difference is probably related to a slight change in reactivity of the catalytic site, rather than to a structural alteration of the recognition site for antithrombin III.

The rate of protein C activation, measured in the absence of thrombomodulin, was greatly reduced by conversion of α‐thrombin to γ‐thrombin. In addition, γ‐thrombin failed to displace α‐thrombin from its complex with thrombomodulin, as demonstrated by measuring either the rate of protein C activation by thrombin‐thrombomodulin, or the fibrinogen clotting activity of thrombin‐thrombomodulin, in the presence of competing diisopropylphospho‐thrombin. It is concluded that the recognition sites involved in protein‐C–thrombin and thrombomodulin–thrombin interactions are both dramatically affected by the loss of peptide material occurring during the conversion of α‐thrombin to γ‐thrombin and/or by the resulting conformational changes.
Chlortetracyclin‐mediated continuous Ca2+ oscillations in mitochondria of digitonin‐treated Tetrahymena pyriformis
Authors: KIM Yuri V. , KUDZINA Ludmila Yu. , ZINCHENKO Valeri P. , EVTODIENKO Yuri
Published: March 2005

Ca2+ transport in mitochondria was studied in situ using digitonin‐permeabilized cells of the ciliate protozoan Tetrahymena pyriformis GL. In the presence of oxidizable substrates and inorganic phosphate, mitochondria were able to accumulate a large amount of the added Ca2+ without subsequent uncoupling and mitochondrial damage. However, the maximal Ca2+ uptake dramatically decreased in the presence of micromolar concentrations of the fluorescent calcium indicator, chlortetracyclin, which in aerobic conditions caused an uncoupling of the respiration in Ca2+‐loaded mitochondria. Moreover, on reaching hypoxia, when the rate oxygen diffusion from the air to the stirred incubation medium became a limiting factor, continuous Ca2+ oscillations were observed. Ca2+ fluxes were synchronous with the cyclic changes of the membrane potential and were followed with a significant delay by the changes of the membrane‐associated fluorescence of Ca‐chlortetracyclin complexes. Both the chlortetracyclin‐induced uncoupling of the respiration and the oscillations were prevented by either EGTA or ruthenium red. It is suggested that in conditions of the limited rate of respiration the oscillations are generated as a result of the functioning of the two Ca2+‐transport pathways: a Ca2+ uniport and a chlortetracyclin‐mediated electroneutral Ca2+ efflux.
Partial purification and some properties of a latent CO2reductase from green potato tuber chloroplasts
Authors: ARORA Sangeeta , RAMASSWAMY N. Krishnan , NAIR P. Madhusudanan
Published: March 2005

We have partially purified the CO2reductase, present in green potato tuber chloroplasts, as a latent form. Illumination of the chloroplasts in the absence of substrate, bicarbonate, activated the enzyme, which could then be obtained in soluble forms. Purification of the enzyme was achieved by (NH4)2SO4fractionation (0–30%) and adsorption and elution from a DEAE‐Sephadex A‐50 column. The final preparation showed 15‐fold purification and 50% recovery of the activity. The pH optimum for CO2reductase was 8.0. Hepes and Tricine buffers showed maximum activity whereas Tris/phosphate or borate failed to show any activity. The enzyme reaction was sensitive to the presence of metal ions like Fe3+, Hg2+, Cu2+, Mo6+and Zn2+, however, a threefold activation was observed with Fe2+. The metal requirement for CO2reductase was evident from the observed inhibition by metal chelators likeo‐phenanthroline α,α′‐dipyridyl, bathocuproine, 8‐hydroxyquinoline etc. Out of theseo‐phenanthroline was the strongest inhibitor and its concentration for 50% inhibition was 40 μM. The presence of Fe2+ions in the reaction mixture protected the enzyme from heat denaturation upto 50°C. Maximum enzyme activity was observed at 15°C. The enzyme activity showed a 30‐s lag period and the maximum was reached in 90s. Supplementation of sodium dithionite in the reaction activated enzyme threefold, suggesting involvement of dithiol groups in the catalytic activity. There was strong inhibition by – SH inhibitors like 5,5′‐dithiobis(2‐nitrobenzoic acid) andN‐ethylmaleimide and – SH reagents like dithiothreitol, 2‐mercaptoehanol and cysteine. Various nucleotide coenzyme tried inhibited the enzyme strongly.
Glutamate dehydrogenase (NADP‐dependent) mRNA in relation to enzyme synthesis in Euglena gracilis
Authors: PARKER Jane E. , JAVED Qamar , MERRETT Michael J.
Published: March 2005

Cells of Euglena gracilis Klebs strain z Pringsheim had high NADP‐dependent glutamate dehydrogenase activity when grown on glutamate as nitrogen source but activity was completely repressed in cells grown on ammonium (NH+4). A 120‐fold purification of NADPH‐glutamate dehydrogenase (subunit Mr= 45000) was achieved from glutamate‐grown cells by affinity chromatography on blue Sepharose CL‐6B. Antisera raised against the homogeneously pure protein were used to demonstrate that increase in NADPH‐glutamate dehydrogenase activity on transfer from NH+4‐grown cells were labelled with L‐[35S]methionine and anti‐(NADPH‐glutamate dehydrogenase) used to immunoprecipitate the dehydrogenase from cell extracts. NADPH‐glutamate dehydrogenase protein was detected in glutamate‐grown but not NH+4‐grown cells.

Anti‐(NADPH‐glutamate dehydrogenase) was used to detect NADPH‐glutamate dehydrogenase resulting from the translation of total polyadenylated RNA from Euglena in a cell‐free rabbit reticulocyte lysate system. NADPH‐glutamate dehydrogenase mRNA was present in glutamate NH+4‐grown cells, there being no apparent difference in mRNA abundance between cells showing a tenfold difference in NADPH‐glutamate dehydrogenase specific activity. These results indicate that the synthesis of this dehydrogenase is regulated primarily at the post‐transcriptional level.
Localization of the tubulin binding site for tau protein
Authors: SERRANO Luis , MONTEJO DE GARCINI Esteban , HERNÁNDEZ María Angeles , AVILA Jesús
Published: March 2005

Limited proteolysis of tubulin with subtilisin resulted in the removal of the carboxyl‐terminal moiety of tubulin subunits.

The remaining peptides from both α and β tubulin lacking the carboxyl terminal did not bind to tau factor nor to MAP2 or MAP1. The carboxyl‐terminal fragments bind to tau factor and MAP2 and both compete for the same binding sites in the tubulin molecule. Our results suggest that the carboxyl‐terminal region of tubulin is a regulatory domain for the assembly of tubulin and the site for interaction with MAPs.
Substrate‐level phosphorylation in isolated yeast mitochondria
Authors: RIGOULET Michel , VELOURS Jean , GUERIN Bernard
Published: March 2005

The activity and the control of substrate‐level phosphorylations in isolated yeast mitochondria were investigated.

The oligomycin‐insensitive ATP synthesis rate linked to 2‐oxoglutarate oxidation is of similar order of magnitude to the observed in oxidative phosphorylation. The flux control coefficients of respiratory chain activity, translocase and phosphate carrier activities were close to zero. Kinetic control was confined to 2‐oxoglutarate supply and 2‐oxoglutarate dehydrogenase complex activity.

The study of endogenous nucleotide phosphorylation showed that ADP is the first phosphate acceptor during this process. Moreover, the comparison between the whole intramitochondrial content of nucleotides and phosphate to the fraction involved in substrate‐level phosphorylation indicated a metabolic compartmentation of nucleotides and phosphate.

A linear relationship was observed between the 2‐oxoglutarate‐liked ATP synthesis rate and the internal phosphate potential: ΔG′p=ΔG′o+RT In ([ATP]/[ADP] [Pi]).

The fact that the substrate‐level phosphorylation process alone was able to maintain high internal phosphate potential has an important bioenergetic consequence, particularly for yeast grown on fermentable substrates.
Rat epidermal growth factor: complete amino acid sequence
Authors: SIMPSON Richard J. , SMITH John A. , MORITZ Robert L. , O'HARE Michael J. , RUDLAND Philip S. , MORRISON John R. , LLOYD Christopher J. , GREGO Boris , BURGESS Antony W. , NICE Edouard C.
Published: March 2005

Epidermal growth factor (EGF) isolated from the submaxillary gland of the rat (rEGF) is missing the COOH‐terminal five residues present in both mouse and human EGF. rEGF competes for the binding of 125I‐labelled mEGF to human carcinoma cells with the same affinity as mEGF. rEGF and mEGF have identical mitogenic activities on mouse 3T3 fibroblasts, thus the C‐terminal region of the sequence is not necessary for the in vitro activity of EGF. Using reversed‐phase high‐performance liquid chromatography, four molecular forms of EGF have been extracted from rat submaxillary glands. These forms represent rEGF, rEGF(2–48), rEGF(3–48) and rEGF(4–48); all forms appear to be equipotent in both the receptor binding and mitogenic assays. The isoelectric points of these rEGFs are in the range of pH 5.1 to 5.2. The primary structure of rEGF was determined from approximately 10μg protein by sequence analysis of the intact molecule and fragments obtained from the reduced and alkylated protein by chemical cleavage with CNBr and enzymic cleavage with chymotrypsin and a proline‐specific endopeptidase. Subnanomole amounts of generated peptides were purified to homogeneity by reversed‐phase microbore high‐performance liquid chromatography and analysed by automated Edman degradation in a gas‐phase sequencer. There are 48 amino acid residues in the complete polypeptide chain which lacks alanine, phenylalanine, lysine and tryptophan. The amino acid sequence of rat epidermal growth factor is: Asn‐Ser‐Asn‐Thr‐ Gly‐Cys‐Pro‐Pro‐Ser‐Tyr‐Asp‐Gly‐Tyr‐Cys‐Leu‐Asn‐Gly‐Gly‐Val‐Cys‐Met‐Tyr‐Val‐Glu‐Ser‐Val‐Asp‐Arg‐Tyr‐Val‐Cys‐Asn‐Cys‐Val‐Ile‐Gly‐Tyr‐Ile‐Gly‐Glu‐Arg‐Cys‐Gln‐His‐Arg‐Leu‐Arg. The calculated relative molecular mass from the sequence analysis is 5377.
Lipopolysaccharide‐sensitive serine‐protease zymogen (factor C) found in Limulus hemocytes
Authors: NAKAMURA Takanori , MORITA Takashi , IWANAGA Sadaaki
Published: March 2005

Bacterial endotoxin (lipopolysaccharide, LPS) induces coagulation of horseshoe crab hemolymph. Our previous studies had demonstrated that a hemolymph factor, designated factor B, was associated with the LPS‐mediated activation of the Limulus clotting system [Ohki et al. (1980) FEBS Lett. 120, 318–321]. On further purification of factor B we found that an additional component, designated factor C, was required to generate factor B activity in the presence of LPS in order to activate the proclotting enzyme. To elucidate the role of factor C in the LPS‐mediated reaction, factor C was isolated and characterized from the hemocyte lysate under sterile conditions. The preparation exhibited a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS‐PAGE) in the absence of 2‐mercaptoethanol, while two protein bands on SDS‐PAGE were observed after reduction. Thus, factor C had a Mr of 123000 consisting of a heavy chain of Mr= 80000 and a light chain of Mr= 43000. Factor C was converted to an activated form in the presence of LPS with a Mr= 123000, designated factor C̄. Upon activation, cleavage of the light chain occurred resulting in the accumulation of two new fragments of Mr= 34000 and 8500 on reduced SDS‐PAGE. A diisopropylfluorophosphate‐sensitive active site was localized in the light chain (Mr= 34000) of factor C̄. The reconstitution experiments, using factor C, factor B, proclotting enzyme and LPS, demonstrated that all of these proteins are essential for the endotoxin‐mediated coagulation system. On the basis of these results we propose that a cascade pathway of LPS‐induced activation of the Limulus clotting system consists of three sequential activations of hemolymph serine protease zymogens.
Poly(ADP‐ribosyl)ation of terminal deoxynucleotidyl transferase in vitor
Authors: TANAKA Yasuharu , ITO Kimihiko , YOSHIHARA Koichiro , KAMIYA Tomoya
Published: March 2005

The activity of purified bovine thymus terminal deoxynucleotidyl transferase was markedly inhibited when the enzyme was incubated in a poly(ADP‐ribose)‐synthesizing system containing purified bovine thymus poly(ADP‐ribose) polymerase, NAD+, Mg2+ and DNA. All of these four components were indispensable for the inhibition. The inhibitors of poly (ADP‐ribose) polymerase counteracted the observed inhibition of the transferase. Under a Mg2+ ‐depleted and acceptor‐dependent ADP‐ribosylating reaction condition [Tanaka, Y., Hashida, T., Yoshihara, H. and Yoshihara, K. (1979) J. Biol. Chem. 254, 12433–12438], the addition of terminal transferase to the reaction mixture stimulated the enzyme reaction in a dose‐dependent manner, suggesting that the transferase is functioning as an acceptor for ADP‐ribose. Electrophoretic analyses of the reaction products clearly indicated that the transferase molecule itself was oligo(ADP‐ribosyl)ated. When the product was further incubated in the Mg2+ ‐fortified reaction mixture, the activity of terminal transferase markedly decreased with increase in the apparent molecular size of the enzyme, indicating that an extensive elongation of poly(ADP‐ribose) bound to the transferase is essential for the observed inhibition. Free poly(ADP‐ribose) and the polymer bound to poly(ADP‐ribose) polymerase were ineffective on the activity of the transferase. All of these results indicate that the observed inhibition of terminal transferase is caused by the poly(ADP‐ribosyl)ation of the transferase itself.
C5a fragment of bovine complement
Authors: GENNARO Renato , SIMONIC Tatjana , NEGRI Armando , MOTTOLA Cristina , SECCHI Camillo , RONCHI Severino , ROMEO Domenico
Published: March 2005

1. C5a and des‐Arg‐C5a have been purified from bovine serum in milligram amounts. The progress of the purification was followed by measuring the chemotactic activity of the complement fragments. 2. The two polypeptides induce activation of neutrophil‐oriented locomotion and secretion with very similar dose/response effects. 3. After preparing a rabbit antiserum to bovine C5a/des‐Arg‐C5a, a competitive enzyme‐linked immuno sorbent assay (ELISA) was set up for the detection of C5a from 5 ng/mol to 1 μg/ml. 4. The complete primary structure of bovine C5a, which consists of 74 amino acids, has been determined by sequence analysis of the tryptic peptides, aligned by peptides derived from a chymotryptic digest, and by partially sequencing the intact molecule. 5. Bovine C5a has a sequence homology of 78% and 70% with porcine and human C5a, respectively, reacts with an antiserum to porcine C5a and is recognized by cell surface receptors on human neutrophils. 6. Finally, the secondary structure of bovine C5a was investigated by circular dicroic spectroscopy and predicted from the amino acid sequence. A comparison of the content and distribution of α‐helical and/or hydropathic regions, suggests that the three‐dimensional structure of C5a might be modeled from the known crystal structure of the homologous C3a molecule.
Chemical modification of sheep‐liver 6‐phosphogluconate dehydrogenase by diethylpyrocarbonate
Authors: TOPHAM Christopher M. , DALZIEL Keith
Published: March 2005

Sheep liver 6‐phosphogluconate dehydrogenase is shown to be inactivated by diethylpyrocarbonate in a biphasic manner at pH 6.0, 25°C. After allowing for the hydrolysis of the reagent, rate constants of 56 M−1 s−1 and 11.0 M−1 s−1 were estimated for the two processes. The complete reactivation of partially inactivated enzyme by neutral hydroxylamine, the elimination of the possibility that modification of cysteine or tyrosine residues are responsible for inactivation, and the magnitudes of the rate constants for inactivation relative to the experimentally determined value for the reaction of diethylpyrocarbonate with Nα‐acetylhistidine (2.2 M−1 s−1), all suggested that enzyme inactivation occurs solely by modification of histidine residues. Comparison of the experimental plot of residual fractional activity versus the number of modified histidine residues per sububit with simulated plots for three hypothetical models, each predicting biphasic kinetics, indicated that inactivation results from the modification of at most one essential histidine residue per subunit, although it appears that other (non‐essential) histidines react independently. This histidine is thought to be His‐242 and is present in the active site. Evidence in support of its role in catalysis is briefly discussed.

Both 6‐phosphogluconate and organic phosphate protect against inactivation, and a kinetic analysis of the protection indicated a dissociation constant of 2.1 × 10−6 M for the enzyme–6‐phosphogluconate complex. NADP+ also protected, but this might be due, at least in part, to a reduction in the effective concentration of diethylpyrocarbonate.
The protein phosphatases involved in cellular regulation
Authors: HOLMES Charles F. B. , CAMPBELL David G. , CAUDWELL F. Barry , AITKEN Alastair , COHEN Philip
Published: March 2005

The complete primary structure of inhibitor‐2, a specific inhibitor of protein phosphatase‐1, has been determined. The protein consists of a single polypeptide chain of 203 residues, and has a relative molecular mass of 22835 Da. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The threonyl residue phosphorylated by glycogen synthase kinase‐3 is located at position 72. The molecule is very hydrophilic, lacks cysteine residues and the single tryptophanyl and phenylalanyl residues are at positions 46 and 139, respectively. The N‐terminal alanyl residue is N‐acetylated. Digestion with Straphylococcus aureus V8 proteinase, trypsin, or cleavage with cyanogen bromide, destroyed the biological activity of inhibitor‐2, demonstrating that many large fragments (e.g. 1–49, 49–92, 67–101, 108–134, 142–182 and 163–197) are inactive. Digestion with clostripain generated a peptide comprising residues 25–114 which retained 2% of the inhibitory potency of the parent molecule. There is no sequence homology between inhibitor‐2 and inhibitor‐1.
Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells
Authors: NARI Johanès , NOAT Georges , DIAMANTIDIS Grégoire , WOUDSTRA Mireille , RICARD Jacques
Published: March 2005

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell‐wall growth process, for autolysis is maximum during both cell extension and cell‐wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo‐and endo‐glycosyltransferases. The optimum pH of autolysis is about 5.

Increasing the ionic strength of the bulk phase where cell‐wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at ‘low’ ionic strength, the local pH in the cell wall is lower than in the bulk phase.

One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell‐wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell‐wall autolysis was enhanced with respect to the rate of autolysis obtained with cell‐wall fragments kept at pH 5.

The pH response of the multienzyme plant cell‐wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell‐wall Donnan potential, is inhibited by protons, whereas the wall‐loosening enzymes involved in cell growth are activated by protons.
Acid Bohr effect of a monomeric haemoglobin from Dicrocoelium dendriticum
Authors: SMIT Jan Derk G. , SICK Hinrich , PETERHANS Alex , GERSONDE Klaus
Published: March 2005

The dioxygen affinity of Dicrocoelium dendriticum haemoglobin was determined as a function of pH with a thin‐layer diffusion technique. From the oxygen dissociation and association curves Hill coefficients h equal 1 were obtained throughout. Ultracentrifugation studies prove this haemoglobin to be monomeric irrespective of pH and ligation state. Thus, Dicrocoelium haemoglobin is a non‐cooperative monomer. It has the highest O2 affinity so far known for any monomeric haemoglobin: its half‐saturation pressure, p50 value, ranges at 25°C from 0.016 mm Hg to 0.15 mm Hg (2.13–20.0 Pa) dependent on pH. Dicrocoelium haemoglobin shows an acid Bohr effect only and as such it constitutes a new class of haemoglobins. Its log p50 versus pH plot (Bohr effect curve) is characterized by a large amplitude, Δlog p50= 0.96, and an inflection point (Bohr effect pK) at pH 5.0. A model for the acid Bohr effect of D. dendriticum haemoglobin is proposed. By generalization, both the alkaline and the acid Bohr effect in various monomeric haemoglobins may arise from a single Bohr group complex (salt bridge).
Further study of hydroxyapatite high‐performance liquid chromatography using both proteins and nucleic acids, and a new technique to increase chromatographic efficiency
Authors: KAWASAKI Tsutomu , IKEDA Koetsu , TAKAHASHI Shoichi , KUBOKI Yoshinori
Published: March 2005

Previously developed hydroxyapatite high‐performance liquid chromatography columns were tested further by using not only proteins but also nucleic acids. In both cases it was confirmed that the column can, in fact, discriminate subtle structural differences among molecules. Especially for protein mixtures a new technique was introduced to increase the efficiency of chromatography.
The time‐dependent inactivation of human brain dihydropteridine reductase by the oxidation products of L‐dopa
Authors: WARING Paul
Published: March 2005

Dihydropteridine reductase (DHPR) was irreversibly inactivated in a time‐dependent way by incubation with 3,4‐dihydroxyphenylalanine (L‐dopa). The inactivation was oxygen‐dependent; incubation under nitrogen gave partial protection. The inactivation was stimulated by the presence of horse‐radish peroxidase/hydrogen peroxide. L‐Dopa itself was not an inhibitor of DHPR although dopachrome, the aminochrome formed following oxidation of L‐dopa, was a reversible inhibitor of DHPR with an I50 of 0.60 mM. The quinone products of oxidation of L‐dopa were responsible for the time‐dependent inactivation of DHPR.

Adrenochrome also demonstrated a time‐dependent inactivation of DHPR. Inactivation by adrenochrome demonstrated a saturation effect suggesting the reversible formation of a complex preceding inactivation. No radiolabel was incorporated into DHPR following inactivation by L‐[14C]‐dopa. Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS‐PAGE) demonstrated the presence of a dimer of DHPR. A mechanism of inactivation involving the oxidative coupling of essential thiol groups was proposed to explain inactivation.
On the stimulation of respiration by α‐adrenergic agonists in perfused rat liver
Authors: TAYLOR Wayne M. , VAN DE POL Erika , BYGRAVE Fyfe L.
Published: March 2005

Interactions between phenylephrine‐induced oxygen consumption, lactate and pyruvate output, and urea and glucose production were examined in perfused livers from fed or 48‐h‐fasted rats. Within 2 min of phenylephrine infusion, oxygen consumption in perfused livers was increased by approximately 40%. Increases in oxygen consumption induced by phenylephrine were essentially abolished in the presence of carboxyatractyloside, whereas those induced by dinitriphenol were still evident. Phenylephrine‐induced increases in oxygen consumption were accompanied by enhanced rates of gluconeogenesis and ureogenesis in livers from fed or 48‐h‐fasted animals. These data indicate that phenylephrine‐induced increases in respiration in perfused rat liver may result from an enhanced rate of mitochondrial oxidative phosphorylation in response to an increased cellular energy requirement.
Characterization of phosphatidylinositol kinase activity associated with the insulin receptor
Authors: SALE Graham J. , FUJITA‐YAMAGUCHI Yoko , KAHN C. Ronald
Published: March 2005

Various lipids were tested as substrates for the insulin receptor kinase using either receptor partially purified from rat hepatoma cells by wheat‐germ‐agglutinin–Sepharose chromatography or receptor purified from human placenta by insulin‐Sepharose affinity chromatography. Phosphatidylinositol was phosphorylated to phosphatidylinositol 4‐phosphate by the partially purified insulin receptor. In contrast, phosphatidylinositol 4‐phosphate and diacylglycerol were not phosphorylated. In some, but not all preparations of partially purified insulin receptor, the phosphatidylinositol kinase activity was stimulated by insulin (mean effect 33%). Phosphatidylinositol kinase activity was retained in insulin receptor purified to homogeneity. Insulin regulation of the phosphatidylinositol kinase was lost in the purified receptor; however, dithiothreitol stimulated both autophosphorylation of the purified receptor and phosphatidylinositol kinase activity in parallel about threefold. (Glu80Tyr20)n, a polymeric substrate specific to tyrosine kinases, inhibited the phosphatidylinositol kinase activity of the purified receptor by > 90% and inhibited receptor autophosphorylation by 67%. Immunoprecipitation by specific anti‐receptor antibodies depleted by > 90% the phosphatidylinositol kinase activity in the supernatant of the purified receptor and the phosphatidylinositol kinase activity was recovered in the precipitate in parallel with receptor autophosphorylation activity. These characteristics of the phosphatidylinositol kinase activity of the purified insulin receptor and its metal ion preference paralleled those of the receptor tyrosine kinase activity and differed from bulk phosphatidylinositol kinase activity in cell extracts, which was not significantly inhibited by (Glu80Tyr20)n, stimulated by dithiothreitol or depleted by immunoprecipitation with anti‐(insulin receptor) antibody. These results suggest that the insulin receptor is associated with a phosphotidylinositol kinase activity; however, this activity is not well regulated by insulin. This kinase appears to be distinct from the major phosphotidylinositol kinase(s) of cells. Its relationship to insulin action needs further study.
Proton magnetic resonance spectroscopic studies of the interaction of ubiquinone‐10 with phospholipid model membranes
Authors: ONDARROA Monica , QUINN Peter J.
Published: March 2005

Proton magnetic resonance spectra of ubiquinone‐10 and ubiquinone‐10 dispersed with dipalmitoylglycero‐phosphocholine or egg phosphatidylcholine in aqueous medium have been obtained. The dispersions are in the form of multilamellar liposomes as judged by 31P‐NMR spectra and the thermal history of the samples have ensured that ubiquinone not incorporated into the phospholipid structure only gives rise to a broad‐line NMR proton spectrum. A high‐resolution proton spectrum of ubiquinone is observed with upfield shifts of the O‐methyl protons of the benzoquinone rings, indicating close proximity of the molecules but with an arrangement different from the pure liquid ubiquinone. Spectra obtained in the presence of the lanthanide shift reagents, dysprosium fluorooctanedionate and Dy(NO3)3, which have a preferred location in the hydrophobic and hydrophilic domains, respectively, of ubiquinone/phospholipid codispersions, are consistent with the partitioning of ubiquinone into a hydrophobic phospholipid environment remote from the aqueous phase. The type of arrangements of ubiquinone that could be accomodated within bilayers of phospholipid are discussed.
Effect of polyamines on translation fidelity in vivo
Authors: McMURRY Laura M. , ALGRANATI Israel D.
Published: March 2005

Polyamines, when added to cell‐free protein‐synthesizing systems, have been shown either to reduce mistranslation or to increase it depending upon the composition of the reaction mixture. To study this question under conditions as natural as possible we investigated the effects of polyamines on the fidelity of protein synthesis in intact Escherichia coli bacterial cells, using strains which were auxotrophic for polyamine synthesis. Error was measured in two ways: (a) the incorporation of [3H]histidine into coat protein of bacteriophage MS2, the gene of which does not code for histidine, and (b) the synthesis of a basic variant of MS2 coat protein in which a lysine erroneously replaces an asparagine, causing a change in isoelectric point. We found that when cell cultures were supplemented with polyamines there was no effect on the first type of error and the second type decreased twofold. The measured errors occurred at the level of translation because their frequency increased in the presence of streptomycin and decreased in cells bearing a streptomycin‐resistance mutation known to lower the occurrence of translational misreading. The average erroneous incorporation per mol coat protein in the presence of polyamines was 1.43 ± 0.59 mmol histidine and 25–34 mmol lysine/asparagine substitution. The reason for the different effect of polyamines on the two types of error is not known but could be related to the difference between their corresponding frequencies or to codon‐specific effects.
In vitro deacylation of lipopolysaccharide of Salmonella minnesota by Acanthamoeba castellanii enzymes
Authors: DROZANSKI Wincenty , GALANOS Chris , SCHLECHT Siegfried , LÜDERITZ Otto
Published: March 2005

Enzymatic deacylation of the lipopolysaccharide isolated from a Salmonella Rd mutant by a cell‐free preparation from Acanthamoeba castellanii has been studied. The degradation was found to be dependent on the presence of a surface‐active component (Triton X‐100) in the reaction mixture. The lipid A part of the lipopolysaccharide was the primary target of the enzymes, which cleaved with high efficiency the ester‐bound long‐chain non‐hydroxylated and 3‐hydroxylated acyl residues, i.e. lauric, myristic, palmitic and 3‐hydroxymyristic acid. The cell‐free preparation also exhibited amidase activity cleaving about 50% of the amide‐bound 3‐hydroxymyristic acid residues. In addition the extract proved to possess phosphatase activity liberating ester‐bound and glycosidically bound phosphate groups of lipid A. On the other hand, the glucosaminyl‐β1, 6‐glucosamine disaccharide was not degraded and remained bound to the oligosaccharide part (heptose/3‐deoxyoctulosonic acid) of the lipopolysaccharide.
Inhibition of dolichyl phosphate biosynthesis by compactin in cultured rat hepatocytes
Authors: ÅSTRAND Ing‐Mari , FRIES Erik , CHOJNACKI Tadeusz , DALLNER Gustav
Published: March 2005

Isolated rat hepatocytes were cultured in monolayer for about 24 h. During this period the cells exhibited constant protein and lipid synthesis. When the culture medium contained compactin, a competitive inhibitor of the 3‐hydroxyl‐3‐methylglutary‐coenzyme‐A reductase, dolichyl‐P synthesis was inhibited by 91% at the end of the incubation, as estimated by the incorporation of [3H]acetate and by 77% as estimated by the incorporation of 32Pi. These results indicate that in primary cultures of rat hepatocytes dolichyl monophosphate is mainly synthesized through a de novo process, while phosphorylation through the CTP‐mediated kinase is of limited functional importance.
Thiol‐dependent proteases of African trypanosomes
Authors: LONSDALE‐ECCLES John D. , MPIMBAZA George W. N.
Published: March 2005

The proteases of several species of African trypanosomes were analysed by electrophoresis in sodium dodecyl sulphate/polyacrylamide gels containing fibrinogen or collagen. After electrophoresis the gels were incubated in the presence of enzyme activators and/or inhibitors and then stained with Coomassie brilliant blue. The areas where the proteolytic activity had degraded the fibrinogen did not stain and so formed clear bands against a blue background. The proteases were found to have pH optima between 5 and 6, and required dithiothreitol or 2‐mercaptoethanol for full expression of their activity. They were inhibited by amino acid chloromethanes, iodoacetamide, p‐chloromercuribenzoate and other inhibitors of the thiol‐dependent proteases, as well as by the trypanocidal drugs berenil® (4,4′‐diamidinodiazoaminobenzene‐diacetamidoacetate) and pentamidine [1,5‐di‐(4‐amidinophenoxy)pentane‐di‐(2‐hydroxyethanesulphonate)]. Trypanosoma evansi, Trypanosoma brucei brucei and Trypanosoma brucei gambiense each have a protease with a relative molecular mass, Mr, of 28000. In addition they occasionally exhibit activity at higher Mr values (up to 105000). Trypanosoma congolense has a low‐Mr protease (31000) and may exhibit higher‐Mr proteases (up to 150000). The protease profiles of Trypanosoma vivax differ from the other species, T. brucei or T. congolense, and are present in lesser amounts. The proteases of the cultured procyclic forms are present in much smaller amounts than those of the metacyclic or mammalian blood stream forms of these parasites. The catalytic properties and inhibition characteristics of these thiol‐dependent enzymes suggest that they resemble the mammalian lysosomal cathepsins B and L.
Regulation of dehydrogenases/one‐electron transferases by modification of flavin redox potentials
Authors: TEGONI Mariella , JANOT Jean Marc , LABEYRIE Françoise
Published: March 2005

Spectroscopic and potentiometric measurements have been carried out, at room temperature, during anaerobic titrations of Hansenula anomala L‐lactate cytochrome c oxidoreductase (or flavocytochrome b2) both in the presence and in the absence of pyruvate (the physiological reaction product). Under the same conditions, the flavin spectral contribution was estimated and the flavosemiquinone proportion was directly determined by electron paramagnetic resonance measurements.

In the present study, we show the visible light absorption and paramagnetic characteristics of the flavin radical at 18°C and also the dramatic effect of pyruvate on the redox potential of each monoelectronic couple of the flavin. Thermodynamic stabilization of the semiquinone form, in the presence of pyruvate, is interpreted as a mode of regulation of flavocytochrome b2 activity. Taking into account that analogous controls have been observed with two other flavoenzymes belonging to this class of dehydrogenases/one‐electron transferases, we suggest that redox potential modulation could be a type of regulation effective for the whole class of enzymes in which a semiquinone is an obligate intermediate.
Nucleotide sequence of the promoter and amino‐terminal encoding region of the Escherichia coli pepN gene
Authors: BALLY Marc , FOGLINO Maryline , BRUSCHI Mireille , MURGIER Maryse , LAZDUNSKI Andrée
Published: March 2005

The nucleotide sequence of the region probably responsible for regulation of pepN expression and of the region encoding the amino‐terminal part of aminopeptidase N, has been determined. The transcription start site was identified by S1 nuclease mapping. All features of the promoter are those of a weak promoter and no obvious structure responsible for regulation was identified, although a possible Pho box is located 63 base pairs upstream from the Pribnow box. The reading frame was unambiguously determined by purifying the protein and by sequencing the first 21 NH2‐terminal residues. The NH2‐terminal region of aminopeptidase N does not contain any fragment resembling signal sequence and the protein is not produced in a precursor form.

A divergent promoter, which might be that of pncB, encoding the nicotinic acid phosphoribosyltransferase (P. Terpstra, personal communication), has also been identified, which allows the assignment of the gene organization in this chromosomal region as .
High expression of Bacillus licheniformisα‐amylase with a Bacillus secretion vector
Authors: SIBAKOV Mervi
Published: March 2005

A gene coding for the heat‐stable α‐amylase from Bacillus licheniformis ATCC14580 has been expressed with the aid of a B. amyloliquefaciensα‐amylase‐based expression/secretion vector by joining the structural part of the gene to a pool of vectors after the B. amyloliquefaciensα‐amylase promoter and signal sequence. The recombinant plasmids obtained were stably maintained in B. subtilis and the heat‐stable α‐amylase activity rose several hundred times from the level of the donor.

Eight different constructions were further analyzed. Each of them had an intact B. amyloliquefaciens signal sequence, the only difference being in a few nucleotides beyond the C terminus of the signal peptide. This, however, was enough to cause up to fourfold differences in protein yield. Possible reasons for the variation in the production level are discussed.
Stimulation of lecithin: cholesterol acyltransferase activity by apolipoprotein A‐II in the presence of apolipoprotein A‐I
Authors: CHEN Ching‐Hong , ALBERS John J.
Published: March 2005

Various combinations of incorporation and addition of apolipoprotein A‐I (apo A‐I) and apolipoprotein A‐II (apo A‐II) individually or together to a defined lecithin‐cholesterol (250/12.5 molar ratio) liposome prepared by the cholate dialysis procedure were used to study the effect of apo A‐II on lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity of both purified enzyme preparations and plasma. When apo A‐I (0.1–3.0 nmol/assay) alone was incorporated or added to the liposome, apo A‐I effectively activated the enzyme. By contrast, when apo A‐II (0.1–3.0 nmol/assay) alone was incorporated into or added to the liposome, apo A‐II exhibited minimal activation of LCAT activity, approximately 1% of the activity obtained by an equal amount of apo A‐I. Addition of apo A‐II (0.1–3.0 nmol/assay) together with apo A‐I (0.8 nmol/assay) to the liposome reduced the LCAT activity to approximately 30% of the level obtained with addition of apo A‐I alone. On the other hand, addition of apo A‐II (0.1–3.0 nmol/assay) or addition of lecithin‐cholesterol liposome containing apo A‐II (0.1–3.0 nmol/assay) to lecithin‐cholesterol liposome containing apo A‐I‐(0.8 nmol/assay) did not significantly alter apo A‐I activation of LCAT activity. However, when the same amounts (0.1–3.0 nmol/assay) of apo A‐II were incorporated together with apo A‐I (0.8 nmol/assay) into the liposome, apo A‐II significantly stimulated LCAT activity as compared to activity obtained with incorporation of apo A‐I alone. The maximal stimulation was obtained with 0.4 nmol apo A‐II/assay for both purified and plasma enzyme. At this apo A‐II concentration, approximately 4‐fold and 1.8‐fold stimulation was observed for purified enzyme and plasma enzyme, respectively. These results indicated that apo A‐II must be incorporated together with apo A‐I into lecithin‐cholesterol liposomes to exert its stimulatory effect on LCAT activity and that apo A‐II in high‐density lipoprotein may play an important role in the regulation of LCAT activity.
NADPH/NADH‐dependent cold‐labile glutamate dehydrogenase in Azospirillum brasilense
Authors: MAULIK Pushpita , GHOSH Sudhamoy
Published: March 2005

A cold‐labile glutamate dehydrogenase (GDH, EC 1.4.1.3) has been purified to homogeneity from the crude extracts of Azospirillum brasilense. The purified enzyme shows a dual coenzyme specificity, and both the NADPH and NADH‐dependent activities are equally cold‐sensitive. The enzyme is highly specific for the substrates 2‐oxoglutarate and glutamate. Kinetic studies with GDH indicate that the enzyme is primarily designed to catalyse the reductive amination of 2‐oxoglutarate. The NADP+‐linked activity of GDH showed Km values 2.5 × 10−4 M and 1.0 × 10−2 M for 2‐oxoglutarate and glutamate respectively. NAD+‐linked activity of GDH could be demonstrated only for the amination of 2‐oxoglutarate but not for the deamination of glutamate. The Lineweaver‐Burk plot with ammonia as substrate for NADPH‐dependent activity shows a biphasic curve, indicating two apparent Km values (0.38 mM and 100 mM) for ammonia; the same plot for NADH‐dependent activity shows only one apparent Km value (66 mM) for ammonia. The NADPH‐dependent activity shows an optimum pH from 8.5 to 8.6 in Tris/HCl buffer, whereas in potassium phosphate buffer the activity shows a plateau from pH 8.4 to 10.0. At high pH (> 9.5) amino acids in general strongly inhibit the reductive amination reaction by their competition with 2‐oxoglutarate for the binding site on GDH. The native enzyme has a Mr= 285000 ± 20000 and appears to be composed of six identical subunits of Mr= 48000 ± 2000. The GDH level in A. brasilense is strongly regulated by the nitrogen source in the growth medium.
Metabolic control analysis of moiety‐conserved cycles
Authors: HOFMEYR Jan‐Hendrik S. , KACSER Henrik , van der MERWE Kirsten J.
Published: March 2005

1. Moiety‐conserved cycles are metabolic structures that interconvert different forms of a chemical moiety (such as ATP‐ADP‐AMP, the different forms of adenylate), while the sum of these forms remains constant. Their metabolic behaviour is treated within the framework of control analysis [Kacser, H. & Burns, J. A. (1973) Symp. Soc. Exp. Biol 27, 65–104].

2. To explain the importance of the conserved sum of cycle metabolites as a parameter of the system, the cycle is first regarded as a ‘black box’. The interactions of the cycle with the rest of the system are expressed in terms of ‘cycle elasticities’ and ‘cycle control coefficients’ by the usual connectivity properties. The conserved sum is seen to be an ‘external’ parameter in the sense that its effect is described by a combined response expression. All cycle coefficients can be written in terms of elasticities and concentrations of cycle metabolites.

3. The treatment shows how connectivity expressions should be modified when moiety‐conserved cycles are present and establishes new summation and connectivity properties. The analysis is applied to a two‐member moiety‐conserved cycle and its general application is discussed.
Magnesium uptake by pancreatic islet cells is modulated by stimulators and inhibitors of the B‐cell function
Authors: HENQUIN Jean‐Claude , NENQUIN Myriam , AWOUTERS Patricia , COGNEAU Michel
Published: March 2005

28Mg2+ uptake by rat islets was measured during incubation with various stimulators or inhibitors of insulin release. D‐Glucose induced a dose‐dependent increase in 28Mg2+ uptake after 10 min or 120 min. The threshold concentration was around 6 mM and the maximum effect was observed with 15–20 mM glucose. After 120 min 28Mg2+ uptake was also stimulated by the metabolized sugars mannose, N‐acetylglucosamine or glyceraldehyde, was unaffected by the non‐metabolized or poorly metabolized L‐glucose, galactose, 3‐O‐methylglucose, 2‐deoxyglucose, fructose or mannoheptulose and was inhibited by glucosamine. The effect of glucose was markedly impaired by mannoheptulose, glucosamine, aminooxyacetate and NH4Cl, but was only partially decreased by D600 or diazoxide, which were ineffective in a glucose‐free medium. Tolbutamide or KCl slightly increased 28Mg2+ uptake. Alanine, leucine alone or with glutamine, and ketoisocaproate also stimulated 28Mg2+ uptake, whereas arginine and lysine decreased it. These changes in 28Mg2+ uptake, brought about by various modifiers of the B‐cell function, are thus similar but not identical to the changes in Ca2+ uptake, and are not the consequence of insulin release. The stimulatory effect of glucose requires glucose metabolism by islet cells, but is only partially due to depolarization of the B‐cell membrane.
Complete amino acid sequence of plastocyanin from a green alga,Enteromorpha prolifera
Authors: SIMPSON Richard J. , MORITZ Robert L. , NICE Edouard C. , GREGO Boris , YOSHIZAKI Fuminori , SUGIMURA Yasutomo , FREEMAN Hans C. , MURATA Mitsuo
Published: March 2005

The complete amino acid sequence of the plastocyanin from the green algaEnteromorpha proliferahas been determined by Edman degradation of the intact molecule and fragments produced by enzymatic cleavage of the polypeptide chain with chymotrypsin,Staphylococcus aureusprotease, proline‐specific endopeptidase, Lys‐C endopeptidase and trypsin. The molecule consists of 98 amino acid residues with a calculated relative molecular mass of 10103. The amino acid sequence ofE. proliferaplastocyanin shows a high degree of homology with those plastocyanins from other algae and higher plants. In particular, the four residues which are copper ligands in other plastocyanins and in the bacterial electron transport protein azurin (two histidines, one cysteine and one methionine) are conserved. Five out of the six acidic amino acid side‐chains which create an ‘acidic patch’ on the surface of plastocyanin fromPopulus nigravar.italica[Colman, P. M. et al. (1978)Nature (Lond.) 272, 319–324] are conserved in the amino acid sequence ofE. proliferaplastocyanin.
Two‐dimensional gel analysis of repetitive nuclear DNA sequences in the genome of Physarum polycephalum
Authors: MEYER Georg , HILDEBRANDT Armin
Published: March 2005

The composition of repetitive sequences in restriction patterns of nuclear DNA of Physarum polycephalum was determined by high‐resolution gel analysis.

Three types of repeated DNA fragments in the size range of (0.2–2) × 103 base pairs could be identified as discrete spots on the gels and distinguished by their abundance and above‐average base composition of either guanine and cytosine (G + C) or adenine and thymidine (A + T).

On comparing the DNA composition from exponentially growing plasmodia with that of starved plasmodia, which have become competent to sporulate and have lost 80% of their nuclei, no change was detected among the (A + T)‐rich repeat fractions, whereas several of the (G + C)‐rich fractions revealed fewer copies in the DNA prepared from starved cells.

As shown by hybridization under saturating conditions, the reduction of several (G + C)‐rich repeated sequences in the restricted nuclear DNA in sporulation‐competent cells can be explained by a 64% elimination of the extrachromosomal nucleolar ribosomal DNA sequences.