bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction of actinogelin with actin
Authors: OHTAKI Tetsuya , TSUKITA Sachiko , MIMURA Naotoshi , TSUKITA Shoichiro , ASANO Akira 
Published: March 2005

Nucleation activity of actin polymerization of actinogelin, a calcium‐ sensitive F‐actin cross‐linking protein from rat liver, was measured by a fluorescence enhancement method using pyrenyl‐actin and by high shear viscometry. No stimulation of nucleation by the addition of actinogelin was observed under several ionic conditions using the fluorescent method. Similar results were also obtained by viscometry. Therefore, it can be concluded that actinogelin has no nucleation activity for actin polymerization.By electron microscopy, it was found that actinogelin molecule has a dumbbell shape, binds to side of F‐actin through its end(s), and cross‐links actin filaments by binding with its two ends. It was also found that meshwork formation occurred in low Ca2+ conditions from F‐actin and actinogelin. Under non‐gelling high Ca2+ conditions, binding of actinogelin along the side of F‐actin with its one end was still detected in accordance with the binding assay using ultracentrifugation and protein determination.Under low Ca2+ conditions, the critical gelling concentration of actinogelin measured by low shear viscometry at 20°C was 6 μg/ml for 250 μg/ml of actin. Comparing this value with those of the other actin cross‐linking proteins, it was found that actinogelin was one of proteins with the highest gelation activity. On the other hand, gelation activity of actinogelin in high Ca2+ conditions was one order of magnitude lower; than 50 μg/ml of the protein was required for gelation. At 37°C, gelation activity of actinogelin at low Ca2+ concentration was decreased to about a quarter of that at 20°C, but this was still higher than that of gizzard α‐actinin at 20°C. Thus, role of actinogelin as an efficient and Ca2+ ‐regulated cross‐linker of microfilaments was substantiated.
An assessment of the importance of intralysosomal and of α‐amylolytic glycogenolysis in the liver of normal rats and of rats with a glycogen‐storage disease
Authors: VANDEBROECK Alfons , BOLLEN Mathieu , DE WULF Henri , STALMANS Willy 
Published: March 2005

Mechanisms of glycogenolysis have been investigated in a comparative study with Wistar rats and gsd rats, which maintain a high glycogen concentration in the liver as a result of a genetic deficiency of phosphorylase kinase.In Wistar hepatocytes the rate of glycogenolysis, as modulated by glucagon and by glucose, was proportional to the concentration of phosphorylase a. In suspensions of gsd hepatocytes the rate of glycogenolysis was far too high as compared with the low level of phosphorylase a; in addition, only a minor fraction of the glycogen lost was recovered as glucose and lactate, owing to the accumulation of oligosaccharides. When the gsd hepatocytes were incubated in the presence of an inhibitor of α‐amylase (BAY e 4609) glycogenolysis and the formation of oligosaccharides virtually ceased; the production of glucose plus lactate; already modest in the absence of BAY e 4609, was further decreased by 40%, owing to the suppression of a pathway for glucose production by the successive actions of α‐amylase and α‐glucosidase.Evidence was obtained that gsd hepatocytes are more fragile, and that amylolytic of glycogen occurred in damaged cells and/or in the extracellular medium. This may even occur in vivo, since quick‐frozen liver samples from anesthetized gsd rats contained severalfold higher concentrations of oligosaccharides than did similar samples from Wistar rats. However, administration of a hepatotoxic agent (CCl4) caused hepatic glycogen depletion in Wistar rats, but not in gsd rats.The administration of phloridzin and of vinblastine, which have been proposed to induce glycogenolysis in the lysosomal system, did not decrease the hepatic glycogen level in gsd rats.Taken together, the data indicate that only the phosphorolytic degradaion of glycogen is metabolically important, and that α‐amylolysis is an indication of an increased fragility of gsd hepatocytes, which becomes prominent when these cells are incubated in vitro.
Biosynthesis of wall tiechoic acids in Staphylococcus aureus H, Micrococcus varians and Bacillus subtilis W23
Authors: HARRINGTON Charles R. , BADDILEY James 
Published: March 2005

The precursors for linkage unit (LU) synthesis in Staphylococcus aureus H were UDP‐GIcNAc, UDP‐N‐acetylmannosamine (ManNAc) and CDP‐glycerol and synthesis was stimulated by ATP. Moraprenol‐PP‐GlcNAc,‐ManNAc‐(glycerol phosphate)1–3 was formed from chemically synthesised moraprenol‐PP‐GlcNAc, UDP‐ManNAc and CDP‐glycerol in the presence of Triton X‐100. LU intermediates formed under both conditions served as acceptors for ribitol phosphate residues, from CDP‐ribitol, which comprise the main chain. The intial transfer of GlcNAc‐1‐phosphate from UDP‐GlcNAc was very sensitive to tunicamycin whereas the subsequent transfer of ManNAc from UDP‐ManNAc was not.Poly(GlcNAc‐1‐phosphate) and LU synthesis in Micrococcus varians, with endogenous lipid acceptor, UDP‐GlcNAc and CDP‐glycerol, was stimulated by UDP‐ManNAc. Synthesis of LU on exogenous moraprenol‐PP‐GlcNAc, with Triton X‐100, was dependent on UDP‐ManNAc and CDP‐glycerol and the intermediates formed served as substrates for polymer synthesis. Membranes from Bacillus subtilis W23 had much lower levels of LU synthesis, but UDP‐ManNAc was again required for optimal synthesis in the presence of UDP‐GlcNAc and CDP‐glycerol. Conditions for LU synthesis on exogenous moraprenol‐PP‐GlcNAc were not found in this organism.LU synthesis on endogenous acceptor in the absence of UDP‐ManNAc was explained by contamination of membranes with UDP‐GlcNAc 2‐epimerase. Under appropriate conditions, low levels of this enzyme were sufficient to convert UDP‐GlcNAc into a mixture of UDP‐Glc‐NAc and UDP‐ManNAc and account for LU synthesis. The results indicate the formation of prenol‐PP‐GlcNAc‐ManNac‐(glycerol phosphate)1–3 which is involved in the synthesis of wall teichoic acids in S. aureus H, M. varians and B. subtilis W23 and their attachment to peptidoglycan.
Rhodamine 6G inhibits the matrix‐catalyzed processing of precursors of rat‐liver mitochondrial proteins
Authors: KUZELA Stefan , JOSTE Vigg , NELSON B. Dean 
Published: March 2005

Several inner membrane proteins from rat liver mitochondria have been translated for the first time in rabbit reticulocyte lysates. These include the Rieske iron‐sulfur protein, cytochrome c1 and core protein I of the cytochrome bc1 complex, the α and β subunits of F1 ATPase, and subunit IV of cytochrome oxidase. All were translated from free polysomes as larger‐molecular‐mass precursors, and were processed to their mature forms by isolated liver mitochondria or by the isolated mitochondrial matrix fraction. In vitro processing, catalyzed by the isolated matrix fraction, is inhibited by rhodamine 6G. The latter is a fluorescent probe, which accumulates specifically in mitochondria of whole cells and which is used extensively to visualize mitochondrial morphology. The concentration of rhodamine 6G required for inhibition in vitro is similar to that of o‐phenanthroline. Rhodamine 6G inhibits matrix‐catalyzed processing of all precursors tested, indicating that the mechanism of inhibition is common for a variety of functionally unrelated precursors. The novel action of rhodamine 6G reported here can form the basis for its inhibition of precursor processing in intact hepatoma cells [Kolarov, J. & Nelson, B. D. (1984) Eur. J. Biochem. 144, 387–392].
Nucleotide sequence and transcription of the fbc operon from Rhodopseudomonas sphaeroides
Authors: GABELLINI Nadia , SEBALD Walter 
Published: March 2005

The fbc operon from Rhodopseudomonas sphaeroides encodes the three redox carriers of the ubiquinol‐cytochrome‐c reductase (b/c1 complex): FeS protein, cytochrome b and cytochrome c1 [Gabellini, N. et al. (1985) EMBO J. 2, 549‐553]. The nucleotide sequence of 3874 bp of cloned R. sphaeroides chromosomal DNA, including the three structural genes fbcF, fbcB and fbcC has been determined. The reading frames of the fbc genes could be identified readily since the encoded amino acid sequences are highly homologous with the sequences of the corresponding mitochondrial polypeptides.Initiation and termination points for transcription have been investigated by S1 nuclease protection analysis. The transcription of the fbc operon starts approximately 240 base pairs upstream from the start codon of the fbcF gene and terminates 120 base pairs downstream from the stop codon of the fbcC gene. Nucleotide sequences resembling recognition signals for the binding and release of the RNA polymerase were identified.The N‐terminal amino acid sequence of the mature cytochrome c1 was obtained by automated Edman degradation of the isolated subunit, confirming the fbcC reading frame and indicating that the bacterial pre‐apocytochrome c1 has a transient leader sequence including 21 residues. The N‐terminal sequence of one hydrophilic peptide of the FeS protein has been also obtained confirming the fbcF reading frame.The deduced amino acid sequences are discussed in relation to the known primary structures of the homologous proteins from mitochondria and chloroplasts. The primary structures of the polypeptides are evaluated with respect to (a) their topology in the membrane, (b) their biogenesis, (c) the structure of the catalytic sites and (d) subunit interactions.
Characterization of three different flavodoxins from Azotobacter vinelandii
Authors: KLUGKIST Jan , VOORBERG Jan , HAAKER Huub , VEEGER Cees 
Published: March 2005

The flavodoxins from Azotobacter vinelandii cells grown N2‐fixing and from cells grown NH4OAc have been purified and characterized. The purified flavodoxins from these cells are a mixture of three different flavodoxins (Fld I, II, III) with different primary structures. The three proteins were separated by fast protein liquid chromatography; Fld I eluted at 0.38 M K Cl, Fld II at 0.43 M K Cl and Fld III at 0.45 M K Cl. The most striking difference between the three flavodoxins was the midpoint potential (pH 7.0, 25°C) of the semiquinone/hydroquinone couple, which was −320 mV for Fld I and −500 mV for the other two flavodoxins (Fld II and Fld III).All three flavodoxins were present in cells grown on NH4OAc. In cells grown on N2 as N source only Fld I and Fld II were found. The concentration of Fld II was 10‐fold higher in N2‐fixing cells than in cells grown on NH4OAc. Evidence has been obtained that Fld II is involved in electron transport to nitrogenase.As will be discussed, our observation that preparations of Azotobacter flavodoxin are heterogeneous, has consequences for the published data.
Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii
Authors: KLUGKIST Jan , HAAKER Huub , VEEGER Cees 
Published: March 2005

The involvement of the cytoplasmic membrane in electron transport to nitrogenase has been studied. Evidence shows that nitrogenase activity in Azotobacter vinelandii is coupled to the flux of electrons through the respiratory chain.To obtain information about proteins involved, the changes occurring in A. vinelandii cells transferred to nitrogen‐free medium after growth on NH4Cl (derepression of nitrogenase activity) were studied. Synthesis of the nitrogenase polypeptides was detectable 5 min after transfer to nitrogen‐free medium. No nitrogenase activity could be detected until t= 20 min, whereupon a linear increase of nitrogenase activity with time was observed. Synthesis of nitrogenase was accompanied by synthesis of flavodoxin II and two membrane‐bound polypeptides of Mr 29000 and 30000. Analysis with respect to changes in membrane‐bound NAD(P)H dehydrogenase activities revealed the induction of an NADPH dehydrogenase activity, which was not detectable in membranes isolated from cells grown in the presence of NH4OAc. This induced activity was associated with the appearance of a polypeptide of Mr 29000 in the NADPH dehydrogenase complex.
Formation of an aspartyl phosphate intermediate in the reactions of nucleoside phosphotransferase from carrots
Authors: STELTE Beatrix , WITZEL Herbert 
Published: March 2005

The nucleoside phosphotransferase from carrots forms N‐phosphorylhydroxylamine when substrates are hydrolysed in the presence of hydroxylamine. Denaturation of the enzyme after short incubation with the substrates leads to a protein, in which, after reduction with [3H]NaCNBH3 and completer hydrolysis with 6 M HCl, labelled homoserine can be detected.The first experiment provides evidence for an activated phosphorylenzyme, the second experiment shows that the intermediate is an acyl phosphate formed by a nucleophilic attack of an aspartate β‐carboxylate group.
Topology of the binding site of blood‐clotting factors in model membranes
Authors: PRIGENT‐DACHARY Jeanne , FAUCON Jean‐Francois , BOISSEAU Michel‐René , DUFOURCQ Jean 
Published: March 2005

Factors II, X and IX are blood‐clotting proteins which bind to phospholipid interfaces in the presence of Ca2+ to activate coagulation. The topology of their binding site on the membrane was investigated in two ways.First, the transition temperature changes of equimolar mixtures of dipalmitoylglycerophosphocholine/phosphatidylserine and dimyristoylglycerophosphocholine/dipalmitoylglycerophosphoserine were examined by the fluorescence polarization of 1,6‐diphenylhexatriene. Results show that Ca2+ triggers a shift of about 3–4°C and that blood‐clotting factors further increase this shift by about 1.5 °C. This suggests that in the gel phase, Ca2+ induces some aggregation of the phosphatidylserine molecules which is reinforced by blood proteins.Second, isothermal energy transfer experiments were performed with natural lipids in their fluid phase. The tryptophan residues of the factors were the energy donors, and pyrene covalently bound to a fatty acid chain of either phosphatidylcholine or phosphatidic acid was the energy acceptor. These pyrene‐phospholipids probe either the neutral or the acidic component of phospholipid mixtures.It is concluded that the binding sites of the factors are constituted by both types of lipids and that their composition depends on the membrane. Factor II exhibits some specificity for acidic phospholipids and seems to be surrounded by non‐interacting zwitterionic lipids. Factor IX appears to be surrounded by statistically the same amount of charged and zwitterionic lipids.We also demonstrate that binding can also occur without Ca2+. This Ca2+‐independent binding probably involves electrostatic and hydrophobic forces but its physiological significance remains to be elucidated.
The primary structure of the α subunit of human elongation factor 1
Authors: BRANDS Jolanda H. G. M. , MAASSEN J. Antonie , VAN HEMERT Formijn J. , AMONS Reinout , MÖLLER Wim 
Published: March 2005

The primary structure of the α subunit of elongation factor 1 (EF‐ 1α) from human MOLT 4 cells was determined by cDNA sequencing. The data show that the conservation of the amino acid sequence is more than 80% when compared with yeast and Artemia EF‐1α. An inventory of amino acid sequences around the guanine‐nucleotide‐binding site in elongation factor Tu from Escherichia coli and homologous amino acid sequences in G proteins, initiation and elongation factors and proteins from the RAS family shows two regions containing conserved seqence elements.Region I has the sequence apolar‐Xaa‐Xaa‐Xaa‐Gly‐Xaa‐Xaa‐Yaa‐Xaa‐Gly‐Lys‐Thr(Ser)‐Xaa‐Xaa‐Xaa‐Xaa‐X‐apolar. Except for RAS proteins, Yaa is always an acidic amino acid residue. Region II is characterized by the invariant sequence apolar‐apolar‐Xaa‐Xaa‐Asn‐Lys‐Xaa‐Asp. In order to facilitate sequence comparison we have used a graphic display, which is based on the hydrophilicity values of individual amino acids in a sequence.
L16, a bifunctional ribosomal protein and the enhancing effect of L6 and L11
Authors: BAXTER Ross Macpherson , ZAHID Nasir 
Published: March 2005

L16 exhibits both peptide bond and transesterification activities when reconstituted into 2 M LiCl core particles. L6 and L11, when reconstituted in a similar manner in the absence of L16, manifest significant transesterification activity. Both L6 and L11 enhance the transesterification activity of L16; L11 being more active than L6 in this respect. However, both L6 and L11 have minimal effect on peptide bond formation when reconstituted with L16 at concentrations more than 2.5 M equivalents. Both L6 and L11 exhibit a differential effect on transesterification.The affinity‐labelling agents, like PhCH2SO2F, diisopropylfluorophosphate and ethoxyformic anhydride, have been used to explore the role of residues in peptide bond formation and transesterification. It is proposed that the Ser‐Phe combination present in L16, L11 and L6 is involved in transesterification in addition to the single histidine in L16. The single histidine in L16 appears to be important in the catalysis of peptide bond formation and transesterification.
Covalent linking of poly(ethyleneglycol)‐bound NAD with Thermus thermophilus malate dehydrogenase
Authors: EGUCHI Tamotsu , IIZUKA Takashi , KAGOTANI Tadashi , LEE Joung Hee , URABE Itaru , OKADA Hirosuke 
Published: March 2005

Poly(ethyleneglycol)‐bound NAD (PEG‐NAD) was covalently linked to Thermus thermophilus malate dehydrogenase with a bifunctional reagent, 3,3′‐(1,6‐dioxo‐1,6‐hexanediyl)bis‐2‐thiazolidinethione. The covalently linked malate‐dehydrogenase–PEG–NAD complex (MDH‐PEG‐NAD) was purified by DEAE‐Sephadex column chromatography to remove unbound PEG‐NAD, and fractionated by blue‐Sepharose column chromatography into four preparations: MDH‐PEG‐NAD I, MDH‐PEG‐NAD II, MDH‐PEG‐NAD III and MDH‐PEG‐NAD IV. The average numbers of NAD moieties covalently bound per subunit of MDH‐PEG‐NAD I, MDH‐PEG‐NAD II, MDH‐PEG‐NAD III and MDH‐PEG‐NAD IV were 1.2, 1.2, 0.8 and 0.5, respectively, and the values were confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. 60–80% bound NAD moiety of these preparations of MDH‐PEG‐NAD was reduced by the enzyme moiety in the presence of L‐malate, and the specific activity of the enzyme moiety of the preparations was more than 80% that of the native enzyme.MDH‐PEG‐NAD I has the following properties. The Km value for exogeneous NAD is three times that of the native enzyme. The coenzyme activity of its NAD moiety is 20–40% that of native NAD for alcohol and lactate dehydrogenases. The complex catalyzes the oxidation of L‐malate in the presence of the redox system of 5‐ethylphenazinium ethyl sulfate and a tetrazolium salt with a rate constant of 0.11 s−1. The coenzyme moiety of the complex can also be recycled by coupled reactions of the active site of the same complex and alcohol dehydrogenase. These results indicate that MDH‐PEG‐NAD works as an NAD(H)‐regeneration unit for coupled reactions.
Genes coding for the elongation factor EF‐1α in Artemia
Authors: LENSTRA Johannes A. , Van VLIET Anja , ARNBERG Annika C. , Van HEMERT Formijn J. , MÖLLER Wim 
Published: March 2005

The elongation factor EF‐1α is one of the most abundant proteins in eukaryotic cells, where it catalyzes the binding of aminoacyl‐tRNA to ribosomes. The genes coding for this protein in the brine shrimp Artemia were analyzed by gene cloning, electron microscopy and chromosomal blot hybridization. There are only a few (about four) copies of one type of gene per haploid genome. These genes contain five exons divided over 104 base pairs. Local rearrangements give rise to a number of gene variants. Cross‐hybridizations of Artemia cDNA probes with yeast and Drosophila DNA revealed two different yeast EF‐1α genes and one or two different Drosophila genes, respectively.Nucleotide sequencing revealed signals for synthesis and processing of EF‐1α transcripts as well as the exact location of exons. One interruption in the coding sequence corresponds closely to a splice junction in the gene coding for the homologous chloroplast protein EF‐Tu from Euglena gracilis, presumably of prokaryotic origin. The first exon in the chloroplast gene codes for the region of EF‐Tu that is homologous to regions of the elongation factor EF‐G and of the initiation factor IF2, respectively.
Mechanism of the control of dioxygen binding in a dimeric cobalt‐substituted insect hemoglobin
Authors: YU Nai‐Teng , MACKIN THOMPSON Helen , ZEPKE Diethardt , GERSONDE Klaus 
Published: March 2005

Resonance Raman spectroscopy has been used to investigate the allosteric control mechanism for O2 binding in a cobalt‐substituted dimeric insect hemoglobin (CTT II), which exhibits a large Bohr effect due to a pH‐induced transition between two ligand affinity states. Substitution of cobalt for iron in CTT II does not modify the Bohr effect, but permits the resonance enhancement (hence the detection) of Raman lines corresponding to the vibrations of the axial ligand‐cobalt bonds.Using 16O2/18O2 isotope substitution the O‐O and Co‐O2 stretching and the Co‐O‐O bending mode have been assigned to the two affinity states of this hemoglobin: v (O‐O) changes from 1152 cm −1 (pH 5.5; t conformation) to about 1125 cm−1 (pH 9.5, r conformation), v(Co‐O2) from 512 cm−1 (pH 5.5) to 537 cm−1 (pH 9.5) and δ (Co‐O‐O) from 378 cm−1 (pH 5.5) to 390 cm−1 (pH 9.5). The Co‐Nɛ(His) stretching mode has also been detected changing from 313 cm−1 (pH 5.5) to 307 cm−1 (pH 9.5). For the first time, reciprocal behaviour between the Co‐Nɛ and Co‐O2 bonds and between the Co‐O2 and the O‐O bonds in an allosteric hemoglobin are demonstrated. Furthermore, the pH sensitivity of a vinyl bending mode in the range of 411–415 cm−1 has been investigated and shown also to reflect the t⇌r conformation transition.
Control of urea production, glutamine release and ammonia uptake in the perfused rat liver by the sympathetic innervation
Authors: BALLÉ Christoph , JUNGERMANN Kurt 
Published: March 2005

The nervous control of hepatic urea and glutamine release and of ammonia uptake was studied in the rat liver perfused in situ.
Electrical stimulation of the nerve bundles around the hepatic artery and the portal vein resulted in a reduction of urea release, of glutamine output and of ammonia uptake. At the same time, as observed before [Hartmann et al. (1982) Eur. J. Biochem. 123, 521–526], nerve stimulation led to a decrease of portal flow as well as to an increase of glucose release and a shift of lactate uptake to output.
Noradrenaline infusion mimicked the nerve‐dependent metabolic and hemodynamic changes in a first approximation only at the highly unphysiological concentration of 0.1 μM. It was without effect at 0.01 μM, which might be reached in the sinusoids as a result of overflow from the vasculature.
In the presence of sodium nitroprusside nerve stimulation no longer reduced urea output, glutamine release and ammonia uptake or portal flow, yet it still increased glucose and lactate release.
Phentolamine clearly reduced the alterations after nervous stimulation of urea output, ammonia uptake and portal flow, while propranolol was essentially not effective. The nerve‐stimulation‐dependent reduction of glutamine release was almost abolished in the presence of phentolamine and lowered to 50% by propranolol.
Glucagon stimulated urea output but had no influence on glutamine release, ammonia uptake and portal flow. Nerve stimulation antagonized the glucagon‐stimulated urea release.
The present results suggest that in the perfused liver α‐sympathetic hepatic nerves regulate urea release, glutamine output and ammonia uptake predominantly by an indirect mechanism via hemodynamic alterations, but glucose release by a direct mechanism also in the absence of circulatory changes.