bluuebunny/crossref_metadata_2025 huggingface dataset:
Semisynthetic derivatives of inositol 1,4,5‐trisphosphate substituted at the 1‐phosphate group
Authors: HENNE Volkmar , MAYR Georg W. , GRABOWSKI Beate , KOPPITZ Burckhard , SÖLING Hans‐Dieter
Published: March 2005

Derivatives of inositol 1,4,5‐(tris)phosphate [Ins(1,4,5)P3] substituted at phosphate 1 were compared with respect to their calcium releasing effect in permeabilized guinea pig parotid acinar cells and to their inhibitory action on aldolase A. sn‐Glycero(3)‐1‐phospho‐d‐myo‐inositol‐4,5‐(bis)phosphate, but also glycolaldehyde(2)‐1‐phospho‐d‐myo‐inositol‐4,5‐(bis)phosphate [GcaPIns(4,5)P2] and its derivative N‐octyl‐aminoethanol(1)‐1‐phospho‐d‐myo‐inositol‐4,5‐(bis)phosphate stimulated calcium release and inhibited aldolase A. The relative efficacy of the different derivatives of Ins(1,4,5)P3 was similar for both effects. N‐Hydroxyethyl‐2‐aminoethanol(1)‐1‐phospho‐d‐myo‐inositol‐4,5‐(bis)phosphate [HeAetPIns(4,5)P2], another derivative of GcaPIns(4,5)P2 was considerably less effective on both parameters than the other Ins(1,4,5)P3 derivatives. Although the concentration leading to half‐maximal activation of calcium release varied from 1.7 μM for Ins(1,4,5)P3 to 128 μM for HeAetPIns(4,5)P2, the maximal effect was the same for all derivatives. The results indicate that the 1‐phosphate group of Ins(1,4,5)P3 can be modified without or with only minor loss of biological activity. This may be utilized for future studies aiming at elucidating the putative Ins(1,4,5)P3 binding site.
On the nature of the interaction between 4,4′‐diisothiocyanostilbene 2,2′‐disulfonic acid and microsomal glucose‐6‐phosphatase
Authors: SPETH Maria , SCHULZE Hans‐Ulrich
Published: March 2005

The effect of 4,4′‐diisothiocyanostilbene 2,2′‐disulfonic acid (DIDS) on microsomal glucose 6‐phosphate hydrolysis has been reinvestigated and characterized in order to elucidate the topological and functional properties of the interacting sites of the glucose‐6‐phosphatase. The studies were performed on microsomal membranes, partially purified and reconstituted glucose‐6‐phosphatase preparations and show the following. (a) DIDS inhibits activity of the glucose‐6‐phosphatase of native microsomes as well as the partially purified glucose‐6‐phosphatase. (b) Inhibition is reversed when the microsomes and the partially purified phosphohydrolase, incorporated into asolectin liposomes, are modified with Triton X‐114. (c) Treatment of native microsomes with DIDS and the following purification of glucose‐6‐phosphatase from these labeled membranes leads to an enzyme preparation which is labeled and inhibited by DIDS. (d) Preincubation of native microsomes or partially purified glucose‐6‐phosphatase with a 3000‐fold excess of glucose 6‐phosphate cannot prevent the DIDS‐induced inhibition. (e) Inhibition of glucose‐6‐phosphatase by DIDS is completely prevented when reactive sulfhydryl groups of the phosphohydrolase are blocked by p‐mecuribenzoate. (f) Reactivation of enzyme activity is obtained when DIDS‐labeled microsomes are incubated with 2‐mercaptoethanol or dithiothreitol. Therefore, we conclude that inhibition of microsomal glucose 6‐phosphate hydrolysis by DIDS cannot result from binding of this agent to a putative glucose‐6‐phosphate‐carrier protein. Our results rather suggest that inhibition is caused by chemical modification of sulfhydryl groups of the integral phosphohydrolase accessible to DIDS attack itself. An easy interpretation of these results can be obtained on the basis of a modified conformational model representing the glucose‐6‐phosphatase as an integral channel‐protein located within the hydrophobic interior of the microsomal membrane [Schulze et al. (1986) J. Biol. Chem. 261, 16571–16578].
Polymerization and gelation of fibronogen in D2O
Authors: LARSSON Ulf
Published: March 2005

The solution properties of fibrinogen and the thrombin‐induced activation and gelation of fibrinogen in 95% D2O at pH 7.4 were compared to those in H2O under similar conditions.

The initial release rates of fibrinopeptides A and B in D2O were slightly slower than those in H2O. However, the values of the Michaelis‐Menten parameters Km and V for the release of the two peptides in D2O and H2O in the presence of 0.5 M NaCl were about the same.

From turbidity measurements at 450 nm it is obvious that fibrinogen is soluble in a slightly more narrow range of NaCl concentration and that the fibrin gels have a higher degree of lateral aggregation in D2O than in H2O.

The variation of fibrinogen concentration, thrombin concentration, pH and ionic a strength have a similar dependence on the final gel structure and clotting time in D2O and H2O.

SDS‐gel electrophoresis on fibrin samples, which were cross‐linked by factor XIII, yielded results where the cross‐linking of the γ‐chain appeared to be the same in D2O and H2O. The α‐chain cross‐linking was somewhat faster in D2O than in H2O.

When fibrinogen solutions in 95% D2O were incubated at 20 mM CaCl2, a slow gelation of fibrinogen was observed, which was found to be induced by trace amounts of factor XIII. The final gel turbidity appeared to be about the same for this gelation as for that induced by thrombin.

The differences in solubility for fibrinogen, kinetics for the enzyme reaction and optical properties for the fibrin gels in D2O and H2O may be explained by differences in electrostatic interactions, hydrogen bonding and hydration of fibrinogen in these two media.
Methanogenesis and ATP synthesis in methanogenic bacteria at low electrochemical proton potentials
Authors: KAESLER Bruno , SCHÖNHEIT Peter
Published: March 2005

The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (ΔμH+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 μmol/mg protein). In the absence of extracellular K+ the cells formed methane at a rate of 4 μmol min−1 (mg protein)−1, the intracellular ATP content was 20 nmol/mg protein and the ΔμH+ was 200 mV (inside negative). When K+ was added to the suspensions the measured ΔμH+ decreased to the value calculated from the [K+]in/[K+]out ratio. Using this method of ΔμH+ adjustment, it was found that lowering ΔμH+ from 200 mV ([K+]in/[K+]out= 1000) to 100 mV ([K+]in/[K+]out= 40) had no effect on the rate of methane formation and on the intracellular ATP content. At ΔμH+ values below 100 mV ([K+]in/[K+]out < 40) both the rate of methanogenesis and the ATP content decreased. Methanogenesis completely ceased and the ATP content was 2 nmol/mg when ΔμH+ was adjusted to values lower 50 mV ([K+]in/[K+]out < 7). The data show that methanogenesis from H2 and CO2 and ATP synthesis in M. thermoautotrophicum are possible at relatively low electrochemical proton potentials. Similar results were obtained with Methanosarcina barkeri.

Protonophoric uncouplers like 3,5,3′,4′‐tetrachlorosalicylanilide (TCS) or 3,5‐di‐tert‐butyl‐4‐hydroxy‐benzylidenemalononitrile (SF 6847) were found not to dissipate ΔμH+ below 100 mV in M. thermoautotrophicum even when used at high concentrations (400 nmol/mg protein). This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.
Study of the coinduction by fatty acids of catalase A and acyl‐CoA oxidase in standard and mutant Saccharomyces cerevisiae strains
Authors: SKONECZNY Marek , CHELSTOWSKA Anna , RYTKA Joanna
Published: March 2005

Evidence is presented that Saccharomyces cerevisiae can metabolize fatty acids via the inducible peroxisomal β‐oxidation pathway even when these acids are not the sole carbon source.

The fatty acids of chain length of C10–C18 induce acyl‐CoA oxidase simultaneously with catalase A but have no effect on catalase T and acyl‐CoA dehydrogenase.

The coinduction of both acyl‐CoA oxidase and catalase A is recorded in strains with both active catalase A and T or displaying only catalase A activity.

In mutants lacking catalase A, the induction of acyl‐CoA oxidase is observed without a concomitant increase in catalase activity.

After centrifugation in a linear Ficoll gradient of the particulate fraction from the cells grown on ethanol and oleate the activity of acyl‐CoA oxidase cosediments with catalase A.

The relationship of catalase A to acyl‐CoA oxidase is discussed.
Crystal structure of human dihydrofolate reductase complexed with folate
Authors: OEFNER Christian , D'ARCY Allan , WINKLER Fritz K.
Published: March 2005

The crystal structure of recombinant human dihydrofolate reductase with folate bound in the active site has been determined and the structural model refined at 0.2‐nm resolution. Preliminary studies of the binding of the inhibitors methotrexate and trimethoprim to the human apoenzyme have been performed at 0.35‐nm resolution. The conformations of the chemically very similar ligands folate and methotrexate, one a substrate the other a potent inhibitor, differ substantially in that their pteridine rings are in inverse orientations relative to their p‐aminobenzoyl‐l‐glutamate moieties. Methotrexate binding is similar to that previously observed in two bacterial enzymes but is quite different from that observed in the enzyme from a mouse lymphoma cell line [Stammers et al. (1987) FEBS Lett. 218, 178–1841. The geometry of the polypeptide chain around the folate binding site in the human enzyme is not consistent with conclusions previously drawn with regard to the species selectivity of the inhibitor trimethoprim [Matthews et al. (1985) J. Biol. Chem. 260, 392–399].
The isolation and characterisation of human atrial natriuretic factor produced as a fusion protein in Escherichia coli
Authors: KNOTT Jacqueline A. , SULLIVAN Carole A. , WESTON Anthony
Published: March 2005

Human atrial natriuretic factor [ANF(1–28)] has been isolated from a fusion protein produced in Escherichia coli. ANF(1–28) was linked to a naturally occurring E. coli protein, chloramphenicol acetyltransferase, via unique cleavage sequences susceptible to either human thrombin digestion, or the chemical action of 2‐(2‐nitrophenylsulphenyl)‐3‐methyl‐3′‐bromoindolenine (BNPS‐skatole). The linker sequences were Gly‐Val‐Arg‐Gly‐Pro‐Arg and Trp respectively. The liberated ANF was purified by reversed‐phase HPLC. Optimised cleavage conditions released 5–10% (by mass) of the maximal yield of ANF(1–28) from the fusion protein with the thrombin‐susceptible linker, whilst a 2–5% (by mass) yield was observed from the fusion protein with the tryptophan linker after BNPS‐skatole treatment. The purified cleavage products were biologically active and shown to comprise intact ANF(1–28). Fast‐atom‐bombardment mass spectrometry confirmed [MH]4 of 3079 m/z, consistent with ANF(1–28).
Ricin and α‐sarcin alter the conformation of 60S ribosomal subunits at neighboring but different sites
Authors: TERAO Kazuo , UCHIUMI Toshio , ENDO Yacta , OGATA Kikuo
Published: March 2005

The effects of ricin and α‐sarcin separately or in combination on the conformation of rat liver ribosomes were investigated by measuring the relative accessibility of individual ribosomal proteins to N‐ethylmaleimide after 80s ribosomes were treated with these toxins.

By using a double‐labelling technique in which ribosomes were incubated with the toxins and then treated with 3H‐labelled or 14C‐labelled N‐ethylmaleimide, it was found that labelling of protein L14 was specifically reduced by treatment with ricin, and that of proteins L3 and L4 by treatment wit α‐sarcin, suggesting that the toxins alter the conformation of ribosomes in the vicinity of these proteins. When ribosomes were treated with both ricin and α‐sarcin, the extent of labelling of protein L3 was reduced compared to that observed after treatment with α‐sarcin alone.

These results are discussed in relation to previous observations showing that these three proteins are neighbours in the 60S ribosomal subunit and probably play important roles in protein biosynthesis, and in the actions of ricin and α‐sarcin on 28s rRNA.
Identification of a cDNA clone for sorghum leaf malate dehydrogenase (NADP)
Authors: CRÉTIN Claude , LUCHETTA Philippe , JOLY Cécile , MIGINIAC‐MASLOW Myroslawa , DECOTTIGNIES Paulette , JACQUOT Jean‐Pierre , VIDAL Jean , GADAL Pierre
Published: March 2005

The mechanisms underlying the photoregulation of the synthesis of sorghum leaf malate dehydrogenase (NADP) (EC 1.1.1.82) (NADP‐MDH), a key enzyme in C4 photosynthesis, have been investigated. During the greening process a light‐dependent increase in enzyme activity took place, accompanied by de novo synthesis of the protein. In vitro translation experiments showed that this chloroplastic protein is synthesized as a precursor (46 kDa) with a ‘transit peptide’ of about 2.5 kDa. A large increase in NADP‐MDH‐translatable RNAs was also observed during greening. We describe also the construction and characterization of a cDNA clone for NADP‐MDH (pCM18A) in the expression vector λgt11. The use of this homologous probe demonstrated a light‐dependent mRNA accumulation.
A photo‐chemically induced dynamic nuclear polarization NMR study on rabbit and bovine cytochrome b5
Authors: HORI Akira , HAYASHI Fumiaki , KYOGOKU Yoshimasa , AKUTSU Hideo
Published: March 2005

Although it has been indicated that proteins with chromophoric groups are not suitable for photo‐chemically induced dynamic nuclear polarization (photo‐CIDNP) measurements, we have successfully obtained these spectra for a heme protein, cytochrome b5. The characteristics of the spectra differed in some points from those so far reported. The intensities of the signals in the aromatic region were very weak, while those of the β‐methylene protons of one histidine and one tryptophan were extremely strong in comparison with the aromatic protons. It was demonstrated, on the basis of the photo‐CIDNP spectrum, that one of seven histidines, all three tyrosines and a single tryptophan of the rabbit soluble cytochrome b5 are exposed on the surface of the protein. The results of comparison of the photo‐CIDNP spectra for the rabbit soluble and intact, and bovine intact, cytochrome b5 led us to the conclusion that the conformation of the hydrophilic, catalytic part of cytochrome b5 is quite similar among these three proteins. In the presence of Chaps micelles, bovine intact cytochrome b5 was in monomeric form and the histidine signals disappeared from its photo‐CIDNP spectrum. When bovine intact cytochrome b5 was reconstituted into egg yolk phosphatidylcholine liposomes, although separate signals due to the protein part were observed in the normal 1H‐NMR spectrum, no photo‐CIDNP signal could be detected. The normal spectrum suggests that the conformation of the protein embedded in liposomes is similar to that of the oligomeric form without lipids or a detergent.
Control analysis of metabolic networks
Authors: GIERSCH Christoph
Published: March 2005

The summation theorem C1J+…+ CnJ= 1 for flux control coefficients CiJ is shown to be equivalent to the assumption that flux J is a homogeneous function of degree 1 of enzyme concentrations E1,…,En, that is to the assumption J (tE1,…,tEn) =tJ (E1,…,En) for any t≠ 0. Likewise, the summation theorem C1Xj+…+ CnXj= 0 for concentration control coefficients CiXj is equivalent to homogeneity of degree 0 of steady‐state metabolite concentrations Xj, or to Xj (tE1,…,tEn) =Xj (E1,…,En). From this equivalence it is obvious that metabolic control analysis applies only to homogeneous systems.

The summation theorem for flux control coefficients is shown to be equivalent to that for concentration control coefficients, provided all reaction rates vi are homogeneous functions of enzyme concentrations Ei.

The equivalence between homogeneity of flux J and the summation theorem for flux control coefficients is used to analyse branching of fluxes in metabolic pathways in terms of flux control coefficients.
Control analysis of metabolic networks
Authors: GIERSCH Christoph
Published: March 2005

The mathematical background of the connectivity relations of metabolic control theory is analysed. The connectivity relations are shown to reflect general properties of total differentials of reaction rate vi, flux J, and metabolite concentration Xj. Connectivity relations hold for any metabolic network in which all vi are homogeneous functions of enzyme concentration Ei. This notion allows established algebraic methods to be used for the formulation of connectivity relations for metabolic systems in which numerous constraints are imposed on metabolite concentrations. A general procedure to derive connectivity relations for such metabolic systems is given. To encourage a broader audience to apply control theory to physiological systems, an easy‐to‐use graphical procedure is derived for formulating connectivity relations for biochemical systems in which no metabolite is involved in more than one constraint.
Insulin‐like growth factor I receptors on mouse neuroblastoma cells
Authors: OTA Akira , WILSON Gaye Lynn , LEROITH Derek
Published: March 2005

We have characterized receptors for the insulin‐like growth factor (IGF‐I) on the mouse neuroblastoma cell line N18 as well as NG108, the hybrid cell line of N18 and rat glioma (C6). In this cell‐free system, IGF‐I and insulin stimulated the phosphorylation of 95‐kDa and 105‐kDa proteins. Using appropriate antibodies we were able to demonstrate that the IGF‐I receptor β subunit has two subtypes of 95 kDa and 105 kDa. On the other hand, insulin receptor β subunit is a separate single 95‐kDa protein. Enzymatic digestion of IGF‐I receptor β subunit subtypes by glycopeptidase F resulted in similar molecular masses (84 kDa and 86 kDa) on SDS‐PAGE, which suggests that the difference in molecular masses between two subtypes is attributable to the differences in N‐linked complex‐type carbohydrate chains on the extracellular domain of β subunits. This conclusion is further supported by peptides of similar molecular mass following staphylococcal V8 protease digestion.

Analysis of IGF‐I receptor β subunit subtypes in these cells may provide insights into the mechanism of action of IGF‐I on neural tissues.
Involvement of Ni protein in the functional coupling of the atrial natriuretic factor (ANF) receptor to adenylate cyclase in rat lung plasma membranes
Authors: RESINK Thérèse J. , PANCHENKO Michail P. , TKACHUK Vsevolod A. , BÜHLER Fritz R.
Published: March 2005

In the presence of 1 μM atrial natriuretic factor (ANF) and low (0.1 mM) Mg2+ concentrations, the initial rate of binding of [3H]guanosine 5′‐[β,γ‐imido)triphosphate ([3H]p[NH]ppG) to rat lung plasma membranes was increased twofold to threefold. ANF‐dependent stimulation of the initial rate of [3H]p[NH]ppG binding was reduced at high (5 mM) Mg2+ concentrations. Preincubation of membranes with p[NH]ppG (5 min at 37°C) eliminated the ANF‐dependent effect on [3H]p[NH]ppG binding whereas ANF‐dependent [3H]p[NH]ppG binding was unaffected by similar pretreatment with guanosine 5′‐[β‐thio]diphosphate (GDP[βS]). An increase in ANF concentration from 10 pM to 1 μM caused a 40% decrease in forskolin‐stimulated or isoproterenol‐stimulated adenylate cyclase activities (IC50 5 nM) in rat lung plasma membranes. GTP (100 μM) was obligatory for the ANF‐dependent inhibition of adenylate cyclase, which could be completely overcome by the presence of 100 μM GDP[βS] or the addition of 10 mM Mn2+. Reduction of Na2+ concentration from 120 mM to 20 mM had the same effect. Pertussis toxin eliminated ANF‐dependent inhibition of adenylate cyclase by catalyzing ADP‐ribosylation of membrane‐bound Ni protein (41‐kDa α subunit of the inhibitory guanyl‐nucleotide‐binding protein of adenylate cyclase). The data support the notion that one of the ANF receptors in rat lung plasma membranes is negatively coupled to a hormone‐sensitive adenylate cyclase complex via the GTP‐binding Ni protein.
Biosynthesis of vitamin B‐12 in anaerobic bacteria
Authors: VOGT Joachim R. A. , LAMM‐KOLONKO Lydia , RENZ Paul
Published: March 2005

In anaerobic microorganisms the origin of C atoms 2 and 4–7 of the 5,6‐dimethylbenzimidazole moiety of vitamin B‐12 is still unknown. In order to tackle this problem we added several 14C‐labeled putative precursors to Eubacterium limosum fermentations. The degradation of the isolated vitamin B‐12 revealed that only d‐erythrose, 14C‐labeled in different positions, was efficiently incorporated into the 5,6‐dimethylbenzimidazole part.

The 5,6‐dimethylbenzimidazole obtained from an experiment with d‐[U‐4C]erythrose was further degraded. It was found that C‐2 was unlabeled, whereas half of the label was located in C‐5 plus C‐6, and the other half in C‐4 plus C‐7.

These results demonstrate that in E. limosumd‐erythrose is a precursor of C‐atoms 4, 5, 6 and 7 of the 5,6‐dimethylbenzimidazole part of vitamin B‐12.
Lipid–protein interaction
Authors: GOÑI Felix M. , CÓZAR Manuel , ALONSO Alicia , DURRANI Aziz A. , GARCÍA‐SEGURA Luis M. , LEE David C. , MONREAL Jaime , CHAPMAN Dennis
Published: March 2005

Bovine myelin proteolipid apoprotein (PLA), obtained in high yield and purity by a novel ultrafiltration procedure, has been used to study the perturbations produced by this protein on phosphatidylcholine bilayers, using infrared spectroscopy, nuclear magnetic resonance and fluorescence polarisation. PLA interacts with phospholipids in a similar manner to other intrinsic proteins. For bilayers in the fluid state, the fatty‐acyl chain static order, as measured by deuterium NMR, is slightly increased in the presence of the protein, except at very high PLA concentrations. Phosphorus NMR reveals some perturbation of the phospholipid polar group by PLA, but to a smaller degree than occurs with other intrinsic proteins. An increase in static order above tc (the onset temperature for gel‐to‐fluid transition) is also detected by infrared spectroscopy. Studies using steady‐state polarisation of diphenylhexatriene fluorescence indicate that the microviscosity of the bilayer increases as a function of the protein mole fraction. From these data an estimation of the average number of lipids perturbed per protein monomer has been made, and a figure of 37 phospholipid molecules determined. The data are compatible with a picture of a hydrophobic polypeptide, perturbing the phospholipids close to it, but allowing rapid (> 104 s−1) exchange with all the lipid molecules in the system.
Degradation of the human erythrocyte membrane band 3 studied with the monoclonal antibody directed against an epitope on the cytoplasmic fragment of band 3
Authors: CZERWIŃSKI Marcin , WAŚNIOWSKA Kazimiera , STEUDEN Iga , DUK Maria , WIȨDŁHA Antoni , LISOWSKA Elwira
Published: March 2005

The mouse hybridoma monoclonal antibody BIII.136 of the IgG2a class is specific for human erythrocyte band‐3 protein. It was shown by means of immunoblotting and immunoprecipitation assays that the antibody recognized an epitope located in the cytoplasmic pole of the band‐3 molecule within approximately 20 kDa from the N‐terminal end. The N‐terminal fragments of band‐3 protein, migrating in SDS/polyacrylamide gel electrophoresis in the 60‐kDa, 40‐kDa and 20‐kDa regions, were detected with the antibody in untreated red‐cell membranes as products of autolysis of band‐3 protein. A correlation was found between the amount of these fragments and erythrocyte age, which suggests that partial degradation of band 3 proceeds in vivo during senescence of erythrocytes. The further degradation of band‐3 protein in vitro was not observed in intact erythrocytes stored at 4°C, but progressed distinctly after hemolysis of red cells, during washing and storing the membranes.
Guinea‐pig liver leukotriene A4 hydrolase
Authors: HAEGGSTRÖM Jesper , BERGMAN Tomas , JÖRNVALL Hans , RÅDMARK Olof
Published: March 2005

Leukotriene A4 hydrolase from perfused guinea‐pig liver was purified 1200‐fold to near homogeneity with a yield of about 20%. Apparent values of Km and Vmax at 37°C (27 μM and 68 μmol × mg−1× min−1), turnover number, and activation energy for the conversion of leukotriene A4 into leukotriene B4 were estimated from kinetic data obtained at ‐10°C, 0°C and +10°C (Arrhenius plots). Physical properties including Mr (67000–71000), pH optimum, isoelectric point and Stokes' radius were determined. The amino acid composition and N‐terminal amino acid sequence were established after carboxymethylation of the enzyme. Unlike liver cytosolic epoxide hydrolase, the purified enzyme did not catalyze the conversion of leukotriene A4 into (5S, 6R)‐5, 6‐dihydroxy‐7,9‐trans‐11,14‐cis‐icosatetraenoic acid.
Rat hepatoma (HTC) cell 6‐phosphofructo‐2‐kinase differs from that in liver and can be separated from fructose‐2,6‐bisphosphatase
Authors: LOISEAU Anne M. , RIDER Mark H. , FORET Dominique , ROUSSEAU Guy G. , HUE Louis
Published: March 2005

6‐Phosphofructo‐2‐kinase was purified from rat liver and hepatoma (HTC) cells. The HTC cell enzyme had kinetic properties different from those of the liver enzyme (more sensitive to inhibition by citrate and not inhibited by sn‐glycerol 3‐phosphate) and was not a substrate of the cyclic‐AMP‐dependent protein kinase. Unlike the liver enzyme, which is bifunctional and phosphorylated by fructose 2,6‐[2‐32P]bisphosphate, the HTC cell enzyme contained no detectable fructose‐2,6‐bisphosphatase activity and phosphorylation by fructose 2,6‐[2‐32P]‐bisphosphate could not be detected. HTC cell fructose‐2,6‐bisphosphatase could be separated from 6‐phospho‐fructo‐2‐kinase activity by purification. Antibodies raised against liver 6‐phosphofructo‐2‐kinase did not precipitate HTC cell fructose‐2,6‐bisphosphatase whose kinetic properties were completely different from those of the liver enzyme.
A protein covalently bound to ColEl DNA
Authors: DRYGIN Yuri F. , ZUKLYS Kazys L. , TERSKICH Alexey V. , BOGDANOV Alexey A.
Published: March 2005

ColE1 DNA was isolated from Escherichia coli as a relaxation complex of supercoiled DNA and proteins. Treatment of the complex with either protein‐denaturing agents (SDS, phenol etc.) or proteolytic enzymes converted the supercoiled DNA to an open‐circular form (relaxation). The relaxation complex was separately labelled in vivo with [3H]Leu or [14C]Leu, [35S]Met or (32P)phosphate and extensively purified. Complete hydrolysis of the relaxed complex with DNase I and P1 nuclease produced a 36‐kDa protein which, we believe, is covalently bound to ColEl DNA. On the other hand, the relaxed complex was treated with tosylphenylalanylchloromethane‐treated‐trypsin and the DNA‐peptide(s) produced was (were) isolated and digested with the nucleases as above. The resulting nucleotidylpeptide(s) was (were) isolated by DEAE‐Sephadex chromatography. The only 5′‐dCMP was released from the nucleotidylpeptide(s) by snake venom phosphodiesterase treatment. O‐Phosphoserine was found in acid hydrolysates of the DNA‐peptide(s). We suggest that in the relaxation event the 36‐kDa protein becomes covalently linked to ColEl DNA via a phosphodiester bond between dC and the serine residue.
Importance of polysomal mRNA‐associated polypeptides for protein synthesis initiation in yeast
Authors: HERRERA Flor , TRIANA Ledia , BOSCH Irene
Published: March 2005

The polysomal mRNA from the cell‐free system of the yeast Saccharomyces cerevisiae, in the absence of exogenous energy, binds to the 40s ribosomal subunit thus forming a 48 s preinitiation complex which, with energy added, is converted into 80s initiation complex. By using ribosomes with a high affinity to polysomal mRNA (pmRNA) from an edeine‐resistant mutant of S. cerevisiae in place of wild‐type ribosomes, increased quantities of the 48s preinitiation complex are obtained.

The pmRNA is found associated with several polypeptides having molecular masses of 115–98 kDa, 72 kDa, 60 kDa and 51 kDa. These polypeptides, labelled with 125I, interact with 40s and 80s ribosomes and are essential for the formation of the 48s and 80s initiation complexes inasmuch as deproteinized pmRNA alone cannot initiate the process. In addition, other polypeptides present in the cytosol are required to carry out the above‐mentioned steps of protein synthesis.
Ligand‐triggered conformational perturbations elicit changes at the single cysteinyl residue of spinach calmodulin
Authors: YUAN Shixing , HAUG Alfred
Published: March 2005

Following application of stoichiometric amounts of Ca2+ or specific partner peptides to spinach calmodulin, dynamic changes in the nanosecond range could be monitored at a strategically anchored fluorescence or spin probe. For these studies the single cysteinyl residue 26 of spinach calmodulin was labelled with a thiol‐specific proxyl (i.e. 2,2,5,5‐tetramethyl‐1‐pyrrolidinyl‐oxyl) spin probe or with a bimane fluorescence probe. With Ca2+ and a specific ligand (mastoparan) present, fluorescence studies (anisotropy, lifetime) indicated that the rotational motion of the protein complex becomes slower relative to the motion of calmodulin in the absence of the specific ligand. The probe's attachment site 26 appears to reside in a fairly polar microenvironment as reported by a series of proxyl spin probes varying in label length. The rotational correlation time of the shortest spin probe markedly changed upon binding of a specific peptide to a calmodulin region distant from that of the monitoring spin probe. We interpret these observations as indicating that ligand‐triggered conformational perturbations are eliciting specific responses at the cysteinyl residue 26 of spinach calmodulin.
Dissociation of the sodium‐ion‐translocating oxaloacetate decarboxylase of Klebsiella pneumoniae and reconstitution of the active complex from the isolated subunits
Authors: DIMROTH Peter , THOMER Anna
Published: March 2005

Oxaloacetate decarboxylase was reconstituted from the purified α subunit and a Triton X‐100 extract of bacterial membranes devoid of this protein. Upon freezing of oxaloacetate decarboxylase in salt solutions, the enzyme was split into subunits and the catalytic activity was abolished. The catalytically active decarboxylase complex was reconstituted by decreasing the salt concentration of the dissociated sample. The conditions for the inactivation were critical for an optimum recovery of catalytically active enzyme during reconstitution, and modest dissociating conditions generally improved the yield of the reconstitutively active decarboxylase. The dissociated enzyme has been separated by chromatography on avidin‐Sepharose into two fractions: fraction I, that was not retained on the column, consisted of the β+γ subunits, and fraction II consisted of the biotin‐containing α subunit. Oxaloacetate decarboxylase was reconstituted from a mixture of the isolated α and β+γ subunits. The Na+ transport activity was recovered, if a mixture of subunits α and β+γ was incorporated into liposomes, or by a sequential reconstitution, starting with the formation of proteoliposomes with the integral membrane proteins β+γ and completed by an attachment of the peripheral subunit α.
DNA primase activity found in an α‐like DNA polymerase obtained from Halobacterium halobium
Authors: NAKAYAMA Masashi , BEN‐MAHREZ Kamel , KOHIYAMA Masamichi
Published: March 2005

An aphidicolin‐sensitive DNA polymerase was purified from extracts of Halobacterium halobium. The analysis of this a‐like DNA polymerase on polyacrylamide gels under denaturing conditions revealed two peptides with molecular masses of 70 kDa and 60 kDa in equal amounts. Like the DNA polymerase α isolated from eukaryotes, the α‐like DNA polymerase possesses primase activity using UTP and polydeoxyadenylate as template. The primase activity was sensitive to aphidicolin and inhibited by an antiserum against the α‐like DNA polymerase of H. halobium. The primase activity was dependent on the presence of high salt concentrations.
Influence of the bound nucleotide on the molecular dynamics of actin
Authors: HEGYI György , SZILÁGYI László , BELÁGYI József
Published: March 2005

Rotational dynamics of actin spin‐labelled with maleimide probes at the reactive thiol Cys‐374 were studied. Replacement of the bound nucleotide by Br8ATP in G‐actin and Br8ADP in F‐actin causes significant increase of the rotational correlation time of the spin probe, indicating reduced motion in both G and F‐actin. The orientation dependence of the electron paramagnetic resonance spectra in oriented F‐actin filaments revealed an altered molecular order of the probe when the nucleotide was a Br‐substituted one. The bound nucleotide affects the myosin S1 ATPase activation by actin; both Vmax, and Kactin decreased significantly when the bound nucleotide of actin was Br8ADP.
Complex formation of human thrombospondin with osteonectin
Authors: CLEZARDIN Philippe , MALAVAL Luc , EHRENSPERGER Anne‐Sylvie , DELMAS Pierre D. , DECHAVANNE Marc , MCGREGOR John L.
Published: March 2005

Human thrombospondin, a 450‐kDa glycoprotein isolated from platelets and endothelial cells, specifically interacts with osteonectin, a protein of 30 kDa isolated from bovine bones and human platelets. Using ELISA, purified osteonectin binds to solid‐phase‐adsorbed thrombospondin with a dissociation constant (Kd) of 0.7 nM. Binding of thrombospondin to solid‐phase‐adsorbed osteonectin was also observed (Kd= 0.86 nM). The interaction of thrombospondin with solid‐phase‐adsorbed osteonectin was significantly decreased (81% inhibition) when using an excess of fluid‐phase osteonectin. Thrombospondin‐osteonectin complex formation was calcium‐dependent as shown by a 50–80% inhibition in the presence of EDTA. None of the proteins known to interact with thrombospondin (fibrinogen, fibronectin, collagen, plasminogen) had a significant inhibitory effect on thrombospondin‐osteonectin complex formation. This selective interaction was confirmed by affinity chromatography. Iodinated osteonectin, previously incubated with purified thrombospondin, specifically bound to an anti‐thrombospondin monoclonal antibody (P10) linked to protein‐A –Sepharose 4B. Elution of the anti‐thrombospondin antibody from protein A allowed the recovery of the thrombospondin‐osteonectin complex in the eluate, as judged by SDS/polyacrylamide gel electrophoresis and autoradiography. Blotting of purified thrombospondin to osteonectin adsorbed onto nitrocellulose further confirmed complex formation. In addition, when released from thrombin‐stimulated platelets, thrombospondin and osteonectin bound to anti‐thrombospondin IgG‐coated plates indicating that osteonectin was complexed to thrombospondin once the platelet‐release reaction has occurred.
High‐affinity binding of fungal β‐glucan fragments to soybean (Glycine max L.) microsomal fractions and protoplasts
Authors: COSIO Eric G. , PÖPPERL Heike , SCHMIDT Walter E. , EBEL Jürgen
Published: March 2005

We have recently reported the existence of binding sites in soybean membranes for a β‐glucan fraction derived from the fungal pathogen Phytophthora megasperma f. sp. glycinea, which may play a role in the elicitor‐mediated phytoalexin response of this plant [Schmidt, W. E. & Ebel, J. (1987) Proc. Natl Acad. Sci. USA 84, 4117–4121]. The specificity of β‐glucan binding to soybean membranes has now been investigated using a variety of competing polyglucans and oligoglucans of fungal origin. P. megaspermaβ‐glucan binding showed high apparent affinity for branched glucans with degrees of polymerization greater than 12. Binding affinity showed good correlation with elicitor activity as measured in a soybean cotyledon bioassay. Modification of the glucans at the reducing end with phenylalkylamine reagents had no effect on binding affinity. This characteristic was used to synthesize an oligoglucosyl tyramine derivative suitable for radioiodination. The 251I‐glucan (15–30 Ci/mmol) provided higher sensitivity and lower detection limits for the binding assays while behaving in a manner identical to the [3H]glucan used previously. More accurate determinations of the Kd value for glucan binding indicated a higher affinity than previously shown (37 nM versus 200 nM). The 251I‐glucan was used to provide the first reported evidence of specific binding of a fungal β‐glucan fraction in vivo to soybean protoplasts. The binding affinity to protoplasts proved identical to that found in microsomal fractions.
Control of the tRNA‐tufB operon in Escherichia coli
Authors: VAN DELFT Joost H. M. , TALENS Anneke , DE JONG Paul J. , SCHMIDT Denise S. , BOSCH Leendert
Published: March 2005

The mechanism underlying feedback inhibition of tufB expression has been studied in vivo by gene‐dosage experiments and by gene and operon fusions involving lacZ. Raising the cellular EF‐Tu content, by introducing a multicopy plasmid encoding EF‐TuA into the cell, repressed the level of EF‐TuB but left the content of tRNAThr3, encoded by the tRNA‐tufB operon, unaffected. This indicates that autoregulation of chromosomal tufB expression does not occur by modulating transcription initiation at the promoter of the tRNA‐tufB operon. This conclusion is further substantiated by experiments with a tRNA':lacZ operon fusion. The molecular ratio of chromosome‐borne tufA and tufB transcripts also remained unaltered under conditions of excess EF‐Tu, though experiments with a tRNA‐tufB':lacz operon fusion showed a decrease of tufB transcripts. Our data further exclude drastic effects of the autogenous repressor on processing of the contranscript of the operon into monocistronic tufB RNA and on alteration of EF‐TUB turnover. Two possible mechanisms remain, which cannot yet be decided between. One is modulation of EF‐Tu by transcription termination either directly or indirectly by affecting antitermination. The second is translational repression. In vitro translation of transcripts derived from SP6 clones did not reveal any feedback inhibition of EF‐TUB synthesis. Surprisingly, addition of EF‐Tu to a coupled transcription/translation systems was found to block transcription initiation at the primary promoter of the tRNA‐tufB operon by over 90%. Although this in vitro effect of EF‐Tu could not be demonstrated in vivo, possibly because of a difference in higher‐order structure between plasmid‐borne and chromosome‐borne DNA, it indicates that under certain conditions EF‐Tu binds very specifically to the tRNA‐tufB operon promoter or its upstream region.
Control of the tRNA‐tufB operon in Escherichia coli
Authors: VAN DELFT Joost H. M. , BOSCH Leendert
Published: March 2005

The expression of tufB, one of the two EF‐Tu‐encoding genes in Escherichia coli, is under autogenous control. Feedback inhibition of tufB expression by plasmid‐borne EF‐Tu has been used to answer the question of whether or not the integrity of the guanine‐nucleotide‐binding domain of EF‐Tu is required for the autoregulatory role of the factor protein. We show that a large deletion of tufB, causing the elimination of an 81‐amino‐acid segment from the plasmid‐borne EF‐Tu, does not abolish tufB repression. We conclude that the autoregulation of the cellular EF‐Tu level is not dependent on an intact guanine‐nucleotide‐binding domain and does not require binding of GTP to EF‐Tu. The repressor activity of the deletion derivative of EF‐Tu can be measured despite a rapid disappearance of the (altered) mutant protein from the soluble cytoplasmic fraction of the cell. Degradation and assembly in larger complexes are responsible for this disappearance.
Mapping of six epitopes on haemocyanin subunit Aa6 by immunoelectron microscopy
Authors: BILLIALD Philippe , LAMY Josette , TAVEAU Jean Christophe , MOTTA Geneviève , LAMY Jean
Published: March 2005

Monoclonal antibodies directed against the haemocyanin of the scorpion Androctonus australis were raised in order to map antigenic determinants (epitopes). The method of mapping employed in this study is molecular immunoelectron microscopy. It consists of a direct electron microscopic observation of antigenic molecules labelled with monoclonal antibodies. The epitopes are then localized in a small region of the external surface of the antigenic molecule whose architecture and quaternary structure are well known. Six monoclonal antibodies have been selected and epitopes have been circumscribed within a small area of one subunit among the 24 subunits composing the whole antigenic molecule.
The enantiomeric error frequency of aspartate aminotransferase
Authors: KOCHHAR Sunil , CHRISTEN Philipp
Published: March 2005

The enantiomeric error frequency of aspartate aminotransferase (mitochondrial isoenzyme from chicken) was assessed by adding the enzyme in high concentration (0.89 mM) to a mixture of l‐glutamate and 2‐oxoglutarate (12 and 1.2 mM, respectively, at pH 7.5 and 25° C). The substrates continuously undergo the transamination cycle under these conditions. Thereby, L‐glutamate is progressively racemized, a 1:1 ratio of two enantiomers being reached within 240 h. The enantiomeric error frequency, i.e. the ratio of the rate of D‐glutamate production and the rate of the transamination reaction with glutamate and 2‐oxoglutarate as substrates, is 1.5 × 10−7. d‐Glutamate is also converted to a 1:1 racemic mixture. The racemizing activity of a mixture of free pyridoxal 5′‐phosphdte and pyridoxamine 5′‐phosphate is about two orders of magnitude lower than that of aspartate aminotransferase. The error frequency of the enzyme in the case of the C4 substrate pair aspartate and oxalacetate is 3.4 × 10−8, i.e. 4 times lower than that with the C5 substrate pair.
Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k
Authors: WENDT Boel , HOFMANN Theo , MARTIN Stephen R. , BAYLEY Peter , BRODIN Peter , GRUNDSTRÖM Thomas , THULIN Eva , LINSE Sara , FORSÉN Sture
Published: March 2005

The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF‐hand calcium‐binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ ‐binding sites. The Ca2+‐loaded forms of the wild‐type and mutant calbindins are too stable to be significantly denatured by heating at 90° C or by adding 8 M urea. For the Ca2+ ‐free (apo) forms thermal unfolding appears to be only half complete at 90° C, while denaturation is complete in 7–8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu‐17 → Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state.

According to X‐ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr‐13) is hydrogen‐bonded to the carboxyl group of Glu‐35, thus linking the two α helices flanking the N‐terminal Ca2+ site. The pKof ionization of the Tyr‐13 hydroxyl group was over 13 for calcium forms of the wild‐type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms.
Only one out of the three strong ribosomal binding sites of the early region of bacteriophage T7 exhibits high translational efficiency in fragments of about 30 base pairs
Authors: FATSCHER Hans Peter , GEISEN Rolf M. , FUCHS Eckart
Published: March 2005

Within the early region of bacteriophage T7 three genes, 0.3, 1 and 1.3, are most efficiently expressed. They belong to the strongest initiation signals of Escherichia coli. In the T7 wild‐type situation the proteins are produced with a molar ratio of gene 1:1.3:0.3 protein = 1:3.9:9.7. DNA fragments of about 30 base pairs comprising the ribosomal binding sites (RBS) of these genes were synthesized and cloned into two derivatives of the pDS1 vector just upstream of the mouse dihydrofolate reductase gene. Although all tested RBS fragments contained an initiation triplet, a Shine‐Dalgarno sequence and some nucleotides upstream and downstream of this region, only the gene 1.3 RBS fragment showed high efficiency whereas those of genes 0.3 and 1 were at the border of significance. The amount of synthesized mRNA was about the same for all three constructs. A major influence of vector‐derived sequences on the RBS activity could be ruled out. The high translational activity of the short 1.3 gene RBS seems to be largely due to its primary structure. The other two RBSs studied require much longer sequences for high activity.
An EPR study of the rotational dynamics of actins from striated and smooth muscle and their complexes with heavy meromyosin
Authors: MOSSAKOWSKA Malgorzata , BELÁGYI Jozef , STRZELECKA‐GOLASZEWSKA Hanna
Published: March 2005

The rotational motions of the actin from rabbit skeletal muscle and from chicken gizzard smooth muscle were measured by conventional and saturation transfer electron paramagnetic resonance (EPR) spectroscopy using maleimide spin‐label rigidly bound at Cys‐374. The conventional EPR spectra indicate a slight difference in the polarity of the environment of the label and in the rotational mobility of the monomeric gizzard actin compared to its skeletal muscle counterpart. These differences disappear upon polymerization. The EPR spectra of the two actins in their F form and in their complexes with heavy meromyosin (HMM) did not reveal any difference in the rotational dynamic properties that might be correlated with the known differences in the activation of myosin ATPase activity by smooth and skeletal muscle actin.

Our results agree with earlier EPR studies on skeletal muscle actin in showing that polymerization stops the nanosecond rotational motion of actin monomers and that F‐actin undergoes rotational motion having an effective correlation time of the order of 0.1 ms. However, our measurements show that complete elimination of the nanosecond motions requires prolonged incubation of F‐actin, suggesting that the slow formation of interfilamental cross‐links in concentrated F‐actin solutions contributes to this process.

We have also used the EPR spectroscopy to study the interaction between HMM and actin in the F and G form. Our results show that in the absence of salt one HMM molecule can cooperatively interact with eight monomers to produce a polymer which closely resembles F‐actin in its rotational mobility but differs from the complex of F‐actin with HMM. The results indicate that salt is necessary for further slowing down, in a cooperative manner, the sub‐millisecond internal motion in actin polymer and for a non‐cooperative change in the intramonomer conformation around Cys‐374 on the binding of HMM.