bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a cysteine‐containing peptide after affinity labelling of Ca2+‐ATPase of sarcoplasmic reticulum with the disulfide of 3′(2′)‐O‐biotinyl‐thioinosine triphosphate
Authors: KISON Ralf , MEYER Helmut E. , SCHONER Wilhelm 
Published: March 2005

3′(2′)‐O‐Biotinyl‐thioinosine triphosphate is a substrate of the Ca2+ pump of sarcoplasmic reticulum. Its disulfide inactivates the Ca2+‐ATPase with two different velocities. The rapidly inactivated sulfhydryl group cannot be protected by ATP and is therefore considered to be outside the ATP binding site. The slowly reacting sulfhydryl group interacts with the disulfide of 3′(2′)‐O‐biotinyl‐thioinosine triphosphate with a dissociation constant of Kd== 137 μM and an inactivation velocity constant of 1.7×10−3 s−1. It is protected by ATP with two different dissociation constants of the enzyme‐ATP complex of Kd== 221 μM and 1130 μM. The slowly reacting sulfhydryl group is therefore considered to be part of the ATP binding site.Since it was impossible to isolate a tryptic peptide by affinity purification on matrix‐bound avidin after affinity labelling with the disulfide of 3′(2′)‐O‐biotinyl‐thioinosine triphosphate, differential labelling with iodo[2‐14C]acetic acid after affinity labelling with the disulfide of 3′(2′)‐O‐biotinyl‐thioinosine triphosphate was carried out. Tryptic digestion and FPLC purification led to the isolation of a radioactive carboxymethyl derivative of the cysteine‐containing peptide ANACNSVIR.This peptide is equivalent to the cDNA‐derived sequence 468–476 of Ca2+‐ATPase [Brandl et al. (1986) Cell 44, 597–607] and is located between the phosphorylation site, Asp351, and Lys515, a part of the putative purine binding subsite of ATP. Although the carboxymethylation of Cys471 is hindered by (biotinyl‐s6ITP)2, the strong dilution of the specific radioactivity of iodo[2‐14C]acetic acid in the isolated peptide 468–476 argues against its direct interaction with the ATP analogue. It is therefore proposed that Cys471 undergoes ATP‐dependent conformational changes.
Characterization of four different mammalian‐cell‐derived recombinant human interferon‐β1s
Authors: UTSUMI Jun , MIZUNO Yasuko , HOSOI Kazuo , OKANO Kiyoshi , SAWADA Ritsuko , KAJITANI Masayuki , SAKAI Ikuo , NARUTO Masanobu , SHIMIZU Hirohiko 
Published: March 2005

In order to evaluate the structural identification of various recombinant human interferon‐β1s, the recombinant proteins were produced in four different mammalian cells (human PC12 and PC8 lung adenocarcinoma cells, Chinese hamster ovary cells and mouse C127 cells) and characterized. Each mammalian‐cell‐derived recombinant human interferon‐β1 represented a single band of 23 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, the same molecular mass as fibroblast‐derived natural human interferon‐β1. Specific activities, amino acid compositions, amino‐terminal sequences, peptide maps on C18 reversed‐phase high‐performance liquid chromatography and circular dichroic spectra of recombinant proteins were in good agreement with natural ones.On the other hand, the patterns of isoelectric focusing were different between mammalian‐cell‐derived recombinant human interferon‐β1s and natural human interferon‐β1. Sugar composition analysis revealed that the recombinant protein from Chinese hamster ovary cells has a similar sugar composition to that of natural protein and the other recombinant proteins have increased amounts of galactose and glucosamine in comparison to the natural protein. Furthermore, there is no galactosamine in the natural protein, while small amounts of galactosamine were detected in the oligosaccharides released from PC8‐ and C127‐derived recombinant proteins by N‐glycanase.These results indicate that mammalian‐cell‐derived recombinant human interferon‐β1s have identical polypeptides to those of natural human interferon‐β1 but their carbohydrate moieties, including unusual N‐linked oligosaccharides, are individually different from natural ones and depend on the host cell.
The effect of caldesmon on assembly and dynamic properties of actin
Authors: GAŁA̧ZKIEWICZ Barbara , BELAGYI Joseph , DA̧BROWSKA Renata 
Published: March 2005

Our earlier fluorescence measurements using N‐(1‐pyrenyl)iodoacetamide‐labeled actin revealed that caldesmon interacts with G‐actin accelerating its nucleation at low salt concentration and causing polymerization in the absence of salt [Galązkiewicz, B., Mossakowska, M., Osińska, H. & Dąbrowska, R. (1985) FEBS Lett. 184, 144–149]. In this work the caldesmon‐induced process of actin polymerization as well as the dynamic properties of the polymers formed have been investigated with the use of fluorescence, electron paramagnetic resonance (EPR) and electron microscopy techniques.Fluorescence titration of N‐(1‐pyrenyl)iodoacetamide‐labeled actin with caldesmon showed saturation of the polymerization at a 1:3 molar ratio of caldesmon/actin monomer. Parallel pelleting experiments revealed, however, that the process of polymer formation is biphasic and only at higher concentrations of caldesmon does the copolymer contain around one caldesmon/three actin monomers. At low concentration of caldesmon a complex of one caldesmon/nine actin monomers is formed. EPR spectroscopy, using maleimide spin label bound at Cys374 of actin, also indicated that one caldesmon molecule polymerizes nine actin monomers. Taken together, these results might suggest the existence of weak and strong forms of actin binding to caldesmon and detection of only the latter by the fluorescence method.Copolymers of actin and caldesmon are indistinguishable from actin polymerized by salt with respect to their appearance in the electron microscope and their ability to interact with heavy meromyosin, although they are characterized by lower torsional flexibility as indicated by immobilization of spin labels attached to actin.
Preferential induction of the rat hepatic P450 I proteins by the food carcinogen 2‐amino‐3‐methyl‐imidazo[4,5‐f]quinoline
Authors: RODRIGUES A. David , AYRTON Andrew D. , WILLIAMS Elisabeth J. , LEWIS David F. V. , WALKER Ronald , IOANNIDES Costas 
Published: March 2005

Administration of the food carcinogen, 2‐amino‐3‐methyl‐imidazo[4,5‐f]quinoline (IQ) to rats gave rise to significant dose‐dependent increases in the microsomal O‐deethylations of ethoxycoumarin and ethoxyresorufin but had no effect on the O‐dealkylation of pentoxyresorufin and the NADPH‐dependent reduction of cytochrome c, and decreased the N‐demethylation of dimethylnitrosamine. Microsomal cytochrome b5 and total cytochrome P‐450 levels decreased following the administration of the carcinogen.
Hepatic microsomal preparations from IQ‐treated animals were much more efficient than control in activating the premutagen 2‐amino‐6‐methyldipyrido[1,2‐a:3′,2′‐d]imidazole to mutagenic intermediates in the Ames test.
Immunoquantification of two of the major families of cytochrome P‐450, namely P450 I and P450 II B, using ELISA techniques showed that treatment with IQ induced the apoprotein levels of the P450 I family but not of P450 II B.
Immunoblot analysis employing polyclonal antibodies against P450 I revealed that IQ induced both isoenzymes of this family, namely P450 I A1 and A2.
It is concluded that IQ is an inducer of the rat hepatic monooxygenases, selectively inducing the P450 I family as predicted by a computer‐graphic analysis of its dimensions which showed that it is a large, essentially planer, molecule.
Purification of GDP mannose: dolichyl‐phosphate O‐β‐D‐mannosyltransferase from Saccharomyces cerevisiae
Authors: HASELBECK Anton 
Published: March 2005

The enzyme GDP mannose: dolichyl‐phosphate O‐β‐d‐mannosyltransferase (GDP‐Man: DolP mannosyltransferase) catalyzing the reaction: GDP‐man + DolP⇌ DolP‐Man + GDP has been purified from Saccharomyces cerevisiae to homogeneity. The purification was achieved using a combination of column chromatographic methods with preparative gel electrophoresis.The enzyme has an apparent molecular mass of 30 kDa on SDS/polyacrylamide gels. Enzymatic activity could be correlated directly with this band.Antibodies against the transferase were raised in rabbits. The immune serum obtained removed enzymatic activity from a detergent extract of yeast membranes and reacted specifically with the 30‐kDa band on immunoblots. Experiments addressing the orientation of this enzyme in the endoplasmic reticulum membrane are presented by using selective trypsin and N‐ethylmaleimide treatment.
Localization of adriamycin in model and natural membranes
Authors: DUPOU‐CÉZANNE Laurence , SAUFEREAU Anne‐Marie , TOCANNE Jean‐François 
Published: March 2005

The interaction of adriamycin with lipids was studied in model (monolayers, small unilamellar vesicles, large multilamellar vesicles) and natural (chinese hamster overy cell) membranes by measurement of fluorescence energy transfer and fluorescence quenching. 2‐APam, 7‐ASte, 12‐ASte and anthracene‐phosphatidylcholine were used as fluorescent probes in which the anthracene group is well located at graded depths in the membrane. Eggyolk phosphatidylcholine and a 1/1 mixture of it with bovine brain phosphatidylserine were used in model membrane systems.Large fluorescence energy transfer was observed between these molecules as donors and the drug as acceptor. With liposomes, at pH 7.4 and over an adriamycin concentration range of 0–100 μM, the efficiency of energy transfer was 12‐ASte > 7‐ASte > 2‐APam, with 100% energy transfer for 12‐ASte above a drug concentration of 30 μM. At pH 5, where the fatty acids are buried deeper (0.45 nm) in the lipid bilayer due to protonation of the carboxyl group, the order of energy transfer 7‐ASte > 12‐ASte = 2‐APam was observed. Measurements of fluorescence quenching using the non‐permeant Cu2+ ion as quencher and spectrophotometric assays indicated that around 40% of the adriamycin molecules were deeply embedded in the lipid bilayer.Adriamycin molecules thus appear to penetrate the lipid bilayer, with the aminoglycosyl group interacting with the lipid phosphate groups and the dihydroanthraquinone residue in contact with the lipid fatty acid chains.In contrast, fluorescence energy transfer and quenching studies on CHO cells showed that adriamycin penetrated the plasma membrane of theese cells to a much more limited extent than in the model membrane systems. This can be related to the squeezing out of the drug from a film of phosphatidylcholine which was observed in monolayers by means of surface pressure, potential and fluorescence experiments.These observations indicated that the penetration of adriamycin into lipid bilayers strongly depends on the molecular packing of the lipid.
Staphylococcal α‐toxin increases the permeability of lipid vesicles by cholesterol‐ and pH‐dependent assembly of oligomeric channels
Authors: FORTI Stefano , MENESTRINA Gianfranco 
Published: March 2005

α‐Toxin, a lethal hemolytic toxin secreted by Staphylococcus aureus, forms ionic channels of large size in lipid membranes. To investigate the mechanism of channel assembly we have studied the kinetics of pore formation on small unilamellar vesicles. We have used two assays of vesicle permeabilization: one is the release of a fluorescent molecule trapped in their inner compartment; the other is the dissipation of an imposed potential.Both methods indicate that the kinetics are complex consisting of an initial delay followed by a non‐linear relaxation. The dependence of the pore formation rate and the extent of permeabilization on the toxin/vesicle ratio indicates that aggregation of 4–10 preinserted toxin monomers underlies channel assembly.The pH dependence of permeabilization suggests that protonation of an acidic group of the toxin is a prerequisite to channel formation. Inclusion of cholesterol in the target vesicles potentiates α‐toxin effects, in a dose‐dependent way, possibly by facilitating its protonation.The location of the proton‐binding site on the two adjacent aspartic acid residues in positions 127 and 128 of the toxin monomer is proposed.
The antiglycogenolytic action of 1‐deoxynojirimycin results from a specific inhibition of the α‐1,6‐glucosidase activity of the debranching enzyme
Authors: BOLLEN Mathieu , STALMANS Willy 
Published: March 2005

The glucose analogue 1‐deoxynojirimycin (dNOJ) and some of its N‐substituted derivatives have recently been described as potent inhibitors of the hepatic glycogenolysis induced by glucagon, Ca2+ ionophores or anoxia. The inhibition increased with time, in spite of a persistently high level of phosphorylase a [Bollen, M., Vandebroeck, A. & Stalmans, W. (1988) Biochem. Pharmacol. 37, 905–909].dNOJ equilibrates within 1 min across the plasma membrane of hepatocytes. It is not phosphorylated or oxidized in the cell. The observation that dNOJ did not affect gluconeogenesis excludes the possibility that glucose‐6‐phosphatase is the target for the inhibition of glucose production from glycogen. Neither were the catalytic activities of phosphoglucomutase and phosphorylase a affected by the compound.dNOJ and two N‐substituted derivatives inhibited instantaneously and completely the α‐1,6‐glucosidase activity of the debranching enzyme, with I50 values in the μmolar range. In contrast, the glucanotransferase activity of the latter enzyme was not inhibited by the compounds at 0.2 mM.The effect of dNOJ was further studied in an in vitro model system of glycogenolysis. The results were compatible with a block of glycogenolysis at the time when phosphorylase has removed the available glucosyl residues from the outer chains of the glycogen particles. This mechanism appears to account for the lag in the response of glycogenolysis to dNOJ.
The influence of substoichiometric concentrations of myosin subfragment 1 on the state of aggregation of actin under depolymerizing conditions
Authors: GRAZI Enrico , MAGRI Ermes , RIZZIERI Luciana 
Published: March 2005

In 3 mM KCl, 2 mM Tris/HCl pH 7.5, 22°C, 0.38 μ myosin subfragment 1 delays the depolymerization of F‐actin (7.2 μ measured as monomer). The depolymerization proceeds rapidly for a few minutes and then slows down suddenly when the ratio between the monomers in the actin filaments and myosin subfragment 1 reaches the value of 11. At this time myosin subfragment 1 is substantially all bound to the actin polymers which form an irregular and discontinuous network of filaments running in doublets and in triplets, perhaps cross‐linked by myosin subfragment 1. Depolymerization proceeds then for several hours, apparently ending up with the formation of the 1:1 actin‐S1 heteropolymer. The ratio between the monomers in the actin filaments and myosin subfragment 1 at the end of the rapid depolymerization proceeds is different for different protein preparations and may be as low as 5.5.In 2 mM Tris/HCl pH 7.5, 25°C, 1 μ myosin subfragment 1 is able to induce the formation of undecorated actin filaments from 12 μ ATP–G‐actin. These filaments probably originate by redistribution of myosin subfragment 1 between the newly formed 1/1 actin‐S1 heteropolymer and G‐actin in the medium, a process which allows the transient formation of undecorated actin filaments.
A novel l‐glutamate oxidase from Streptomyces endus
Authors: BÖHMER Annette , MÜLLER Anita , PASSARGE Margit , LIEBS Peter , HONECK Horst , MÜLLER Hans‐Georg 
Published: March 2005

A new flavoenzyme using molecular oxygen to oxidize l‐glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2‐oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including d‐glutamic acid, l‐glutamine and l‐aspartic acid, only l‐glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273, 355 and 457 nm and the isoelectric point is at pH 6.2.The Km value for l‐glutamic acid in air‐saturated phosphate pH 7.0 was estimated to be 1.1 mM, the Km for oxygen was calculated to be 1.86 mM at saturating concentration of l‐glutamic acid. The enzymic reaction is inhibited by Ag+ and Hg2+ ions. The enzyme described here distinctly differs from two microbial l‐glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure.
Insulin‐induced dephosphorylation of hormone‐sensitive lipase
Authors: STRÅLFORS Peter , HONNOR Rupert C. 
Published: March 2005

The effect of insulin on the state of phosphorylation of hormone‐sensitive lipase, cellular cAMP‐dependent protein kinase activity and lipolysis was investigated in isolated adipocytes. Increased phosphorylation of hormone‐sensitive lipase in response to isoproterenol stimulation was closely paralleled by increased lipolysis. Maximal phosphorylation and lipolysis was obtained when the cAMP‐dependent protein kinase activity ration was ≥ 0.1, and this corresponded to a 50% increase in the state of phosphorylation of hormone‐sensitive lipase.Insulin (1 nM) reduced cAMP‐dependent protein kinase activity and also reduced lipolysis with both cAMP‐dependent and cAMP‐independent antilipolytic effects up to an activity ratio of ∼ 0.4, above which the antilipolytic effect was lost. Insulin caused a decrease in the state of phosphorylation of hormone‐sensitive lipase at all levels of cAMP‐dependent protein kinase activity. Under basal conditions, with cAMP‐dependent protein kinase activity at a minimum, this reflected a dephosphorylation of the basal phosphorylation site of hormone‐sensitive lipase in a manner not mediated by cAMP. When the cAMP‐dependent protein kinase was stimulated to phosphorylate the regulatory phosphorylation site of hormone‐sensitive lipase, the insulin‐induced dephosphorylation occurred both at the basal and regulatory sites. At low levels of cAMP‐dependent protein kinase activity ratios (0.05–0.1), dephosphorylation of the regulatory site correlated with reduced cAMP‐dependent protein kinase activity, but not at higher activity ratios (> 0.1).Stimulation of cells with isoproterenol produced a transient (1–5 min) peak of cAMP‐dependent protein kinase activity and of phosphorylation of hormone‐sensitive lipase. The state of phosphorylation also showed a transient peak when the protein kinase was maximally and constantly activated. In the presence of raised levels of cellular cAMP, insulin (1 nM) caused a rapid (t1/2∼ 1 min) dephosphorylation of hormone‐sensitive lipase. In unstimulated cells the reduction in phosphorylation caused by insulin was distinctly slower (t1/2∼ 5 min).These findings are interpreted to suggest that insulin affects the state of phosphorylation of hormone‐sensitive lipase and lipolysis through a cAMP‐dependent pathway, involving reduction of cAMP, and through a cAMP‐independent pathway, involving activation of a protein phosphatase activity that dephosphorylates both the regulatory and basal phosphorylation sites of hormone‐sensitive lipase.
Stimulation of insulin of glycolysis in cultured hepatocytes is attenuated by extracellular ATP and puromycin through purine‐dependent inhibition of phosphofructokinase 2 activation
Authors: PROBST Irmelin , QUENTMEIER Armin , SCHWEICKHARDT Christa , UNTHAN‐FECHNER Kirsten 
Published: March 2005

Activation of glycolysis by insulin in cultured rat hepatocytes is preceeded by an activation of phosphofructokinase 2 (PFK 2) and subsequent rise of the fructase 2,6‐bisphosphate [Fru(2,6)P2] level. Extracellular addition of ATP or puromycin prevented the hormonal effect on glycolysis. The mechanism through which the purines abolished glycolytic stimulation was investigated.

50 μM ATP completely prevented the 3–5‐fold insulin‐dependent increase of glycolysis, irrespective of whether the cells initially possessed a low or a high Fru(2,6)P2 content 50 μM puromycin prevented the stimulation of glycolysis by insulin only in cells whose initial Fru(2,6)P2 levels were low and had to be increased by insulin prior to the increase in glycolysis. It did not antagonize the action of insulin in cells with initial high Fru(2,6)P2 content.
ATP exerted effects on its own; it decreased initially high Fru(2,6)P2 levels by 95% within 10 min and decreased the basal glycolytic rate by 60%. Half‐maximal effects on the Fru(2,6)P2 level were obtained with about 25 μM ATP or 15 μM adenosine 5′[β,γ‐methylene]triphosphate. ADP and adenosine‐5‐(γ‐thio]triphosphate were as effective as ATP, whereas 100 μM adenosine 5′[α,β‐methylene]triphosphate elicited no effect. Puromycin neither decreased high Fru(2,6)P2 levels nor inhibited basal glycolysis.
Extracellular ATP (100 μM) led to inhibition of the active form of PFK 2. Intracellular levels of Glc6P, citrate. ATP, ADP and AMP were increased by extracellular ATP, the phosphoenolpyruvate content was decreased, Fru6P and glycerol 3‐phosphate levels stayed constant. Puromycin did not inhibit PFK 2.
Both puromycin and ATP prevented the insulin‐dependent rise of the Fru(2,6)P2 level, they abolished the activation of PFK 2 by the hormone. Puromycin did not block the accumulation of Fru(2,6)P2 provoked by glucose addition; ATP also antagonized the glucose‐dependent increase.
100 μM ATP elevated the cAMP‐dependent protein kinase activity ratio from 0.1 to 0.38 and increased the level of inositol trisphosphate by 16‐fold within 5 min, whereas puromycin was without effect on either level.

It is concluded that the two purines block the insulin effect on glycolysis by preventing the hormone increasing the Fru(2,6)P2 level. The mode of action, however, seems to be different: ATP antagonizes insulin action in that it leads to increased inhibition of PFK 2 whereas puromycin prevents the activation of PFK 2 by insulin.