bluuebunny/crossref_metadata_2025 huggingface dataset:
Speicifc response of serine protease mRNA to a protein‐free diet in the rat pancreas
Authors: DAKKA Nadia , WICKER Catherine , PUIGSERVER Antoine
Published: March 2005

The time course of response of pancreatic chymotrypsin, trypsin and elastase to a protein‐free carbohydraterich diet was studied in Wistar rats. The levels of serine proteases in pancreatic tissue were markedly decreased for the first two days of diet consumption and further increased up to day 13 without reahing, however, the corresponding levels in control rats. This biphasic pattern of respnse may be due to some changes in the secretory process and/or the synthesis of enzymes in the acinar cell. The relative rate of synthesis of pancreatic enzymes was found to vary linearly with time and tobe regulated in inverse proportion to nutritional substrates. Although the animals were totally deprived of dietary proteins, synthesis of acidic isoenzymic forms of serine protease was nevertheless increased. The specific response of these proteases to a protein‐free diet was mediated, at least partly, by changes in the levels of the corresponding specific mRNAs. As early as day 2 after diet consumption, the level of chymotrypsin mRNA was markedly enhanced in sharp contrast with that of amylase. The difference was further increased up to the fifth day of dietary manipulation. It is suggested that changes in cytoplasmic concentrations of chymotrypsin mRNA most probably resulted from transcriptional control of the corresponding gene although a possible increase in mRNA stability could not be ruled out.
Fluorinated analogues of spermidine as substrates of spermine synthase
Authors: BAILLON Jean G. , MAMONT Pierre S. , WAGNER Joseph , GERHART Fritz , LUX Philippe
Published: March 2005

A series of mono‐and geminal difluorinated analogues of spermidine (4‐azaoctane‐1, 8‐diamine) have been tested as potential substrates of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase (EC 2.5.1.22).

Substitution of the hydrogen atoms of the methylene group at position 7 by one or two fluorine atoms decreases 8‐fold and 160‐fold the apparent Km values for the HTC cell enzyme respectively. Similarly, the Km values of 7‐monofluoro and 7, 7‐difluorospermidine for the pure bovine enzyme are reduced 8‐fold and 100‐fold respectively, incomparison with spermidine. Di‐, but not monofluoro substitution, in the 6‐position causes a 5‐fold reduction in the affinity for the HTC cell enzyme. Gem‐fluorine substitution in the 2‐position a abolishes substrate capability.

In addition to their high affinity for spermine synthase, 7‐monoflurospermidine and 7, 7‐difluorospermidine cause substrate inhibition. This phenomenom, which is more pronounced in the case of the difluorinated analogues is pH‐dependent.

These enzymatic findings are discussed with regard to the protonation sites of the spermidine analogues, determined by potentiometric titration, which vary as a function of the number and position of the fluorine substituents relative to the basic amino groups.
Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase
Authors: BALNY Claude , HOOPER Alan B.
Published: March 2005

Hydroxylamine oxidoreductase (HAO) of the ammonia‐oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O · HNO2+ 2e−+ 2 H+. The heme‐like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first‐order rate constants k1, and k2,. CO binds to ferrous P460 with apparent first‐order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from‐15°C to 20.7°C and at hydrostatic pressures ranging over 0.1‐80 MPa. From the measurements, thermodynamic parameters ΔV‡ (activation volume), ΔG‡, ΔH‡ and ‡‡S‡, have been calculated.

CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on ΔV# (‐30 ml Δ mol−1), ΔG‡, ΔH‡ or S‡ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between In k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation.

Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene gh col resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from −3.6 ml ± mol−1 to 57 ml · mol−1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0°C and, in water or ethylene glycol, the linear dependence of In k1 on P was displaced downwards as the temperature changed from 3.5°C to ‐15°C.

Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was ‐30 ml & mol−1 in ethylene glycol. The activation volume increased with increasing temperature. The Arrhenius plot had a downward inflection at about 0°C yet the linear dependence of In k2 on P had no downward displacement in this temperature range. At high pressure and low temperatures the rate of heme reduction increased with decreasing temperature. In contrast to the reaction of CO with the substrate site, the results suggested that changes in enzyme conformation imposed by changes in solvent, temperature or pressure affected the rates of intramolecular electron transfer from the substrate site to c hemes.
Expression in non‐lymphoid cells of mouse recombinant immunoglobulindirected against the tumour marker human placental alkaline phosphatase
Authors: DE WAELE Peter , FEYS Véronique , VAN DE VOORDE André , MOLEMANS Francis , FIERS Walter
Published: March 2005

From a mouse hybridoma cell line secreting a monoclonal antibody directed against the tumour marker human placental alkaline phosphatase, mRNA coding for the H and L chains of this antibody was isolated and cloned as cDNA. Sequence analysis of the H and L chain cDNAs confirmed the IgG2b,K; subtype previously established. Recloning the H and L chain cDNA information into SV40‐based vectors enabled us to obtain expression of functional immunoglobulin upon cotransfection into COS or CHO dhfr cells. This illustrates that non‐lymphoid cells also have the capacity to assemble active immunoglobulins.
Osmotic pressure measurements, of the ligand‐linked dissociation equilibrium of human oxyhemoglobin
Authors: SCHÖNERT Hansjürgen , STOLL Bernhard
Published: March 2005

The tetramer/dimer equilibrium of human oxyhemoglobin has been measured by an osmometric method as a function of pH, chloride and 2,3‐bisphosphoglycerate concentrations at 21°C. The binding constants of chloride and 2,3‐bisphosphoglycerate to the tetramer and the dimer were thus evaluated at various pH values. 2,3‐Bisphosphoglycerate is bound to the dimer more strongly than to the tetramer. The dimer has a second binding site for 2,3‐bisphosphoglycerate. The data for 2,3‐bisphosphoglycerate binding were corroborated by direct binding measurements with the ultrafiltration method.
Histidyls in lobster arginine kinase
Authors: ROUX‐FROMY Madeleine , PHO Dang Ba , KASSAB Rhida
Published: March 2005

The role of histidyls in lobster arginine kinase (EC 2.7.3.3) has been studied by 1H‐NMR spectroscopy of the enzyme and its complexes with substrates or their analogues and 31P‐NMR spectroscopy of complexes with ADP.

Five histidyls were detected by 1H‐NMR in native enzyme (His 1 to His 5). Three of them appeared possibly to be implicated in catalysis: His 3, whose pH/titration was affected by arginine binding, and His 1 and 4, show from paramagnetic relaxation by Mn2+ to be close (≤ 1.2 and ≤ 1.27 nm respectively) to the metal cofactor. His 4 was broadened beyond detection in the presence of any adenine nucleotide. In the enzyme reversibly inactivated by histidine ethoxyformylation, the modified histidyl was His 1. In the transition state analogue complex (in which NO‐3 mimics the transferred phosphoryl), Hill plots of histidyl pH/titration curves showed that His 1 and His 3 were both interacting with the same set of three titratable groups and hence spatially close. 31P‐NMR demonstrated that ADP binding in this complex was unaffected by the chemical modification of His 1.

It is concluded that His‐ethoxyformyl‐enzyme is inactive because ethoxyformyl‐His 1 is unable to titrate. This is consistent with His 1 acting as the acid‐base catalyst. However our results, which do not indicate any catalytic role of His 3, exclude any H‐bonding of His 1 on either substrate. Involvement is needed of at least one other titratable residue for the proton evolved in the catalysis to exchange directly with the guanidino substrate.
ATP‐induced activation of the aminoacylation of tRNA by the isoleucyl‐tRNA synthetase from Escherichia coli
Authors: AIRAS R. Kalervo
Published: March 2005

The rate of aminoacylation of tRNA catalyzed by the isoleucyl‐tRNA synthetase form Escherichia coli has been measured. A steady‐state kinetic analysis of the rate as a function of the concentration of ATP gave nonlinear Hanes plots. ATP behaves as an activator of the reaction. The activation is observed at a low magnesium ion concentration and in the presence of spermidine. The presence of inorganic pyrophosphate or AMP enhances the activation. The results are consistent with a mechanism in which the binding of a second molecule of ATP increases the rate of dissociation of Ile‐tRNA from the enyzme.
Assignment of individual heme EPR signalsof Desulfovibrio baculatus (strain 9974) tetraheme cytochrome c3
Authors: MOURA Isabel , TEIXEIRA Miguel , HUYNH Boi H. , LeGALL Jean , MOURA José J. G.
Published: March 2005

An EPR redox titration was performed on the tetraheme cytochrome c3 isolated from Desulfovibrio baculatus (strain 9974), a sulfate‐reducer. Using spectral differences at different poised redox states of the protein, it was possible to individualize the EPR g‐values of each of the four hemes and also to determine the mid‐point redox potentials of each individual heme: heme 4 (−70 mV) at gmax= 2.93, gmed= 2.26 and gmin= 1.51; heme 3 (− 280 mV) at gmax= 3.41; heme 2 (−300 mV) at gmax= 3.05, gmed= 2.24 and gmin= 1.34; and heme 1 (−355 mV) at gmx= 3.18. A previously described multi‐redox equilibria model used for the interpretation of NMR data of D. gigas cytochrome c3 [Santos, H., Moura, J. J. G., Moura, I., LeGall, J. & Xavier, A. V. (1984) Eur. J. Biochem. 141, 283‐296] is discussed in terms of the EPR results.
Activation of phosphofructokinase 2 by insulin in cultured hepatocytes without accompanying changes of effector levels or cAMP‐stimulated protein kinase activity ratios
Authors: MÜLLER Andreas , UNTHAN‐FECHNER Kirsten , PROBST Irmelin
Published: March 2005

Activation of glycolysis by insulin in cultured adult rat hepatocytes is accompanied by an activation of phosphofructokinase 2 (PFK 2). PFK 2 activation might be caused by insulin‐dependent changes of (a) metabolite levels, (b) basal and (c) Br8cAMP‐stimulated cAMP‐dependent protein kinase activity; this problem was investigated.

Cells cultured with 0.1 nM insulin for 48 h exhibited a low glycolytic rate and low fructose 2,6‐bisphosphate [Fru(2,6)P2] levels. Addition of insulin increased Fru(2,6)P2 and Fru(l,6)P2 levels sequentially which points to PFK 2 as first target enzyme of insulin action.

Concentrations of Glc6P, Fru6P, phosphoenolpyruvate, glycerol 3‐phosphate and citrate, which modulate PFK 2/fructose 2,6‐bisphosphatase 2 activity, were not altered by insulin.

Activation of PFK 2 by insulin occurred without changes in the levels of total and protein‐bound cAMP. Bound cAMP amounted to about 14% of total cAMP.

Insulin neither decreased the basal dissociation state of the cAMP‐dependent protein kinase nor lowered the sensitivity of the kinase towards cAMP in cell extracts.

Addition of the phosphodiesterase‐resistant Br8cAMP to the cultures increased cAMP levels 3–4‐fold, elevated the protein kinase activity ratio from 0.14 to 0.6 and decreased the Fru(2,6)P2 level and the rate of glycolysis. When Br8cAMP and insulin were given together, insulin was capable of counteracting Br8cAMP in that it activated glycolysis and PFK 2 and elevated the level; however, it did not decrease the elevated protein kinase activity ratio.

It is concluded that insulin presumably does not activate PFK 2 through changes in cAMP and effector levels or through inhibition of cAMP‐dependent protein kinase dissociation. The data support the hypothesis that insulin may act via activation of PFK 2 phosphatase.
The enzymatic activity of proteinase K is controlled by calcium
Authors: BAJORATH Jürgen , HINRICHS Winfried , SAENGER Wolfram
Published: March 2005

The fungal proteinase K (EC 3.4.21.14) is a very potent unusually stable member of the subtilisin family. Its X‐ray structure determined at 0.15‐nm resolution shows two bound Ca2+ ions. Cal isin near‐ideal pentagonal bipyramidal configuration with Asp200 carboxylate and Prol75 peptide C=O in an apical, and Vai177 peptide C=O and four water molecules in an equatorial position, whereas Ca2 displays incomplete octahedral coordination with the carboxylate of Asp260, the peptide C=O of Vail6 and two water molecules. Scatchard analysis of the titration of Ca2+‐free proteinase K with Ca2+ yields a single dissociation constant (7.6±2.5) × 10 −8 M associated with the tightly bound Cal whereas Ca2 is so weakly bound that it cannot be titrated. If proteinase K is depleted of Ca2+ by treatment with EDTA, followed by gel filtration, its enzymatic activity drops within 6 h to 20% of its original value, without autolysis. Addition of excess Ca2+ immediately raises the residual activity to 28%, but full activity is not achieved. Removal of Ca2+ triggers a conformational change of the substrate recognition site because there is a direct connection, via secondary structure hydrogen bonds, between the Cal binding site and the substrate‐recognition site. This is indicated further by circular dichroism and fluorescence‐spectroscopic data, and by reversed‐phase FPLC, carried out in the presence and absence of Ca2+, but the overall structure of the enzyme is not affected. Depletion of Ca2+ also influences binding of longer peptide inhibitors of the chloromethane type, it increases the rate of autolysis after about 48 h, it reduces the thermal stability (measured by activity tests from 65 °C to 46 °C), and it enhances the deactivation by 8 M urea which inactivates to only 65%, whereas sodium dodecyl sulfate totally inactivates at a concentration of 12.5%.
Chemical modification of tyrosine‐38 in P‐Hydroxybenzoate hydroxylase from Pseudomonas fluorescens by 5′‐P‐fluorosulfonylbenzoyladenosine: A probe for the elucidation of the NADPH binding site?
Authors: Van BERKEL Willem J. , MÜLLER Franz , JEKEL Peter A. , WEIJER Wicher J. , SCHREUDER Herman A. , WIERENGA Rik K.
Published: March 2005

P‐Hydroxybenzoate Hydorxylase from Pseudomonas Fluorescens was covalently modified by the nucleotide analog 5′‐(P‐fluorosulfonylbenzoyl)‐adenosine in the presence of 20% dimethylsulfoxide, The inactivation reaction is pH‐dependent and does not obey pseudo‐first‐order kinetics, due to spontaneous hydrolysis of the reagent. The kinetic data further indicate that a weak, reversible enzyme‐inhibitor complex is an intermediate in the inactivation reaction and that only one amino acid residue is responsible for the loss of activity

The inactivation is strongly inhibited by NADPH and 2′, 5′ADP. Steady‐state kinetics and 2′,5′ADP bioaffinity chromatography of the modified enxzyme suggest that the enssential residue is not directly involved in NADPH binding.

Sequence studies show that Tyr‐38 is the main residue protected form modicfication in the presence of NADPH. From crystallorgraphic studies it is known that the hydroxyl group of Tyr‐38 is 1.84 nm away form the active site. Model‐Building studies using computer graphics show that this distance can be accommodated when FSO2BzAdo binds in an extended comformation with the sulfonylbenzoyl protion in an orientaiton different from the nicotinamide ring of NADPH.
A new siliconized‐glass fiber as support for protein‐chemical analysis of electroblotted proteins.
Authors: ECKERSKORN Christoph , MEWES Werner , GORETZKI Hans , LOTTSPEICH Friedrich
Published: March 2005

A new hydrophobic glass‐fiber support is presented, which is well suited to the electrophoretic transfer of proteins from polyacrylamide gels and subsequent protein‐chemical analysis. Modified glass‐fiber sheets are easily prepared by chemical reaction of the surface with poly(methyl‐3,3,3‐trifluoropropylsiloxane) in trifluoroacetic acid. The modification is stable during electroblotting, amino acid sequence analysis and hydrolysis. The siliconized glass fiber exhibits a high protein‐binding capacity, allows the application of well‐established staining procedures, and does not interfere with the analytical methods of modern protein chemistry at the low picomole level. Samples separated by electrophoresis and immobilized on hydrophobic supports fail to exhibit any detectable contamination in amino acid sequence analysis hence allowing the high performance of the available protein‐chemical methods to be exploited.
Heterogeneous distribution of calmodulin‐and cAMP‐dependent regulation of Ca2+ uptake in cardiac sarcoplasmic reticulum subfracitons
Authors: GASSER Jürg , PAGANETTI Paolo , CARAFOLI Ernesto , CHIESI Michele
Published: March 2005

The activity of the Ca2+‐pumping ATPase of cardiac sarcoplasmic reticulum is controlled by the phosphorylation level of the intrinsic membrane protein phospholamban. Phospholamban monomers contain two distinct pphosphorylation sites for either the cAMP‐dependent or the calmodulin‐dependent kinase. The two kinases, however, preferentially phosphorylate different populations of phospholamban molecules and double phosphorylation of the same subunit by their concerted action is a phenomenon that occurs only under particular experimental conditions. This study investigates the phosphorylation pattern of phospholamban in various subfractions derived from dog crdiac sarcoplasmic reticulum. The results show that the endogenous calmodulin‐dependent kinase preferentially phosphorylates the phospholamban population found in association with the cisternal compartments of the reticulum. The differential phosphorylation occurs despite the presence of sufficient amouns of the kinase in all sarcoplasmic reticulum subfractions. On the other hand, phospholamban molecules localized onthe longitudinal system are preferential substrates for the cAMP‐dependent kinase. Possibly, the different lipid and/or protein microenvironment of phospholamban in the various sarcoplasmic reticulum domains is responsible for the apparent heterogeeity of phosphorylation. The present findings are compatible with the concept of additive and independent action of the cAMP‐dependent and calmodulin‐dependent kinases on cardiac sarcoplasmic reticulum. The imply, however, that different regions of the sarcoplasmic reticulum network are controlled by the two regulatory mechanisms.
Long‐term ingestion of ammonium increases acetylglutamate and urea levels without affecting the amount of carbamoyl‐phosphate synthase
Authors: FELIPO Vicente , MIÑANA María‐Dolores , GRISOLÍA Santiago
Published: March 2005

Rats were fed the following diets: standard (20% protein), high‐protein (80%), protein‐free, standard plus ammonium and protein‐free plus ammonium for six weeks. The standard plus ammonium diet was prepared to contain ammonia equivalent to that supplied by the high‐protein diet. Addition of ammonium acetate 20% by mass) to the 20% protein or protein‐free diets results in 2.3‐ and 10‐fold increases of urea excretion respectively, without increase of carbamoyl‐phosphate synthase.

Supplementation of the standard diet with ammonium increases the mitochondrial content of acetylglutamate from 830 to 1590 pmol/mg protein, and of the protein‐free diet from 130 to 1040 pmol/mg. However, ingestion of ammonium did not increase the activity of acetylglutamate synthase. Therefore the efflux of acetylglutamate from mitochondria was determined. After 30 min at 37°C liver mitochondria from rats on standard diet released 61% of the initial acetylglutamate while mitochondria from animals on standard plus ammonium diet released only 20%. These results indicate that ingestion of ammonium increases the content of acetylglutamate in rat liver by decreasing its efflux from mitochondria. This effect is similar to that produced in mice by a high protein diet [Morita et al. (1982) J. Biochem. (Tokyo) 91, 563‐569]. However while the high‐protein diet increases carbamoyl‐phosphate synthase content, the ammonium diet does not.
DNA‐binding properties of gene‐5 protein encoded by bacteriophage M 13
Authors: BULSINK Henk , HARMSEN Ben J. M. , HILBERS Cornelis W.
Published: March 2005

The irreversible dissociation kinetics of complexes of M13‐encoded gene‐5 protein with the polynucleotides poly(dA) and M13 DNA was studied by means of stopped‐flow experiments. A linear decay was found for all gene‐5‐protein · poly(dA) complexes and for the gene‐5‐protein · M13 DNA complexes for which the DNA lattice was completely saturated at the beginning of the dissociation experiments. Only at the end of the dissociation curve was a deviation from linearity observed. A single‐exponential decay was found for the dissociation of gene‐5‐protein · M13 DNA complexes when the DNA was not completely saturated initially. These results could be interpreted by assuming that dissociation of bound protein is only possible from isolated binding sites, while during the dissociation, rearrangement of bound protein clusters takes place continuously, including the formation of newly isolated bound protein. This redistribution results from a translocation of the protein along the lattice, which, for the poly(dA) complex, is fast with respect to the dissociation step, but which is slow for the M13 DNA complex. During this process the equilibrium cluster distribution predicted by the theory of McGhee and Von Hippel [1] is not maintained.

The binding of gene‐5 protein to poly(dA) or poly(dT) does not result in a broadening of the nucleotide resonances in the NMR spectra of these polynucleotides, as had been observed for E. coli DNA‐binding protein and interpreted as an indication for a high rate of translocation of the protein on the polynucleotide [2]. The absence of line broadening for gene‐5‐protein · polynucleotide complexes is caused by the high binding cooperativity. As a consequence the majority of the protein molecules are bound in a cluster which makes the concentration of isolated bound protein very low. This results in a decrease of the signal/noise ratio at higher degrees of binding, but does not lead to line broadening while fast translocation still occurs.
The organization of the human‐plasminogen‐activator‐inhibitor‐1 gene
Authors: STRANDBERG Leif , LAWRENCE Daniel , NY Tor
Published: March 2005

Plasminogen activator inhibitor 1 (PAI‐1) is a member of the serine protease inhibitor super family (SERPINS) which is thought to play an integral role in the control of plasminogen activation. PAI‐1 inhibits both tissue‐type plasminogen activator and urokinase‐type plasminogen activator and may therefore be implicated in the control of various physiological processes. We have isolated the PAI‐1 gene including its 5′‐flanking sequence. The gene was characterized by restriction enzyme analysis, Southern blotting and DNA sequencing of all the coding parts as well as the 5′‐flanking region. The PAI‐1 gene contains nine exons and eight introns distributed over approximately 12.3 kb of DNA. All exon/intron boundaries agree with the ‘GT‐AG’ rule. To characterize the presumptive promoter region, 800 bp of the 5′‐flanking region was sequenced and potential binding sites for transacting transcriptional factors were localized. The transcription initiation site was identified by S1 protection experiments and is located 25 base pairs downstream of a TATA consensus sequence. By aligning the gene structure of PAI‐1 and four other SERPINS and extrapolating a general tertiary structure to these SERPINS, we find that most introns map between subdomain structures of the proteins. Evidence is presented supporting an intron loss model for the evolution of the SERPIN family.
Inositolphospholipid‐accelerated activation of prekallikrein by activated factor XII and its inhibition by β2‐glycoprotein I
Authors: SCHOUSBOE Inger
Published: March 2005

Inositolphospholipid‐accelerated activation of prekallikrein by α‐factor XIIa was determined by measuring the appearance of kallikrein amidolytic activity towards the chromogenic substrate, D‐prolyl‐phenylalanyl‐arginyl p‐nitroanilide (S‐2302). The activation reaction did not exhibit normal Michaelis‐Menten kinetics. The Hill coefficient was found to be 1.6 indicating that the activation followed an allosteric reaction mechanism. The temperature dependence of the reaction showed a thermal transition at 30°C, which in addition to the allosteric reaction mechanism is indicative of a conformational change of prekallikrein following binding to the inositolphospholipid. The reaction exhibited pH optimum at pH 7.2 and ionic strength optimum at 50 mM NaCl. At optimal conditions the apparent KA value and the kcat/KA value for factor XIIa on prekallikrein were calculated to be 73 nM and 9.3×106 s−1 M−1, respectively. Kinetic constants could not be calculated at salt concentrations higher than the optimal concentrations, as Lineweaver‐Burk plots were curvilinear in agreement with the Hill coefficient greater than unity. The activation was inhibited competitively by β2‐glycoprotein I with a Ki value of 77 nM as determined by the Dixon plot.
Interaction of the β‐adrenergic receptor with Gs following delipidation
Authors: BEN‐ARIE Nissim , GILEADI Carina , SCHRAMM Michael
Published: March 2005

Preparations of β‐adrenergic receptor and Gs from turkey erythrocytes were delipidated by previously developed procedures. Three synthetic phospholipids, dioleoylglycerophosphoethanolamine, dioleoylglycerophosphocholine and dioleoylglycerophosphoserine plus an unphosphorylated lipid, were all required to restore receptor‐mediated activation of Gs by GTP[γS]. The same lipids were necessary for the reconstitution of the isoproterenol‐enhanced GTPase. The requirement for the unphosphorylated lipid could be fulfilled by 1‐monooleoyl glycerol, α‐tocopherol or oleic acid.
Primary structures and catalytic properties of isoenzymes encoded by the two 4‐coumarate: CoA ligase genes in parsley
Authors: LOZOYA Edmundo , HOFFMANN Heidi , DOUGLAS Carl , SCHULZ Wolfgang , SCHEEL Dierk , HAHLBROCK Klaus
Published: March 2005

We have determined the primary structures of two 4‐coumarate: CoA ligase (4CL) isoenzymes in parsley (Petroselinum crispum) by sequencing near full‐length cDNAs corresponding to the two 4CL genes, Pc4CL‐1 and Pc4CL‐2, present in this plant. Comparison of the cDNA and genomic nucleotide sequences showed that each 4CL gene is organized in five exons separated by introns of varying lengths. The positions of introns are the same in both genes and 97‐99% of the corresponding nucleotide sequences are identical. The two isoenzymes, which are nearly identical in their primary structures, were separated by ion‐exchange chromatography, and were found to be indistinguishable with regard to substrate specificity. Assignment to Pc4CL‐1 and Pc4CL‐2 was achieved by comparison with catalytically active 4CL proteins, isolated from Escherichia coli cells which had been transformed with plasmids harboring the corresponding cDNAs.
Preparation of chemically ‘mutated’ aprotinin homologues by semisynthesis
Authors: BECKMANN Jürgen , MEHLICH Armin , SCHRÖDER Werner , WENZEL Herbert R. , TSCHESCHE Harald
Published: March 2005

The semisynthesis of homologues of aprotinin (BPTI) is described. The P1 amino acid residue of these homologues was substituted by other amino acids using peptide synthetic methods. The reactive‐site‐modified inhibitor (with the Lys15−‐Ala16 peptide bond hydrolyzed) was used as starting material. All carboxyl groups of the modified inhibitor were esterified with methanol, then the Lys15 methyl ester group was hydrolyzed selectively. Afterwards, Lys15 itself was split off. A new amino acid residue was incorporated by using water‐soluble carbodiimide combined with an acylation catalyst. tert‐Butyl‐ester‐protected amino acids were used for reinsertion. The method was tested by re‐insertion of Lys15 to reconstitute the original inhibitor. Thirteen BPTI homologues with coded (Lys, Glu, Gly, Ala, Val, Ile, Leu) or uncoded amino acids (Abu, Ape, alle, Ahx, tLeu, Neo) in position 15 were synthesized and the specificity of the inhibitors investigated. Amongst these, [Val15]BPTI was shown to be an excellent inhibitor for human polymorphonuclear leukocyte elastase having a complex dissociation constant of 0.11 nM. This inhibitor showed no detectable affinity to bovine pancreatic trypsin.
Characterization of the nucleoside‐binding site inside the Tsx channel ofEscherichia coliouter membrane Reconstitution experiments with lipid bilayer membranes
Authors: BENZ Roland , SCHMID Angela , MAIER Christl , BREMER Erhard
Published: March 2005

Reconstitution of purified Tsx protein fromEscherichia coliinto lipid bilayer membranes showed that Tsx formed small ion‐permeable channels with a single‐channel conductance of 10 pS in 1 M KCI. The dependence of conductance versus salt concentration was linear, suggesting that Tsx has no binding site for ions. Conductance was inhibited by the addition of 20 mM adenosine. Titration of the Tsx‐mediated membrane conductance with different solutes including free bases, nucleosides, and deoxynucleosides suggested that the channel contains a binding site for nucleosides but not for sugars or amino acids, and binding increased in the following order: free base, nucleoside, and deoxynucleoside. Among the five nucleosides the stability constant for the binding increased in the order of cytidine, guanosine, uridine, adenosine, and thymidine. Control experiments revealed that the binding of the nucleosides is independent of ion concentration in the aqueous phase, i.e. there was no competition between nucleosides and ions for the binding site inside the channel. The binding of the solutes to the channel interior can be explained by a one‐site two‐barrier model for the Tsx channel. The advantage of a binding site inside a specific porin for the permeation of solutes is discussed with respect to the properties of a general diffusion pore.
Tuna pepsinogens and pepsins
Authors: TANJI Masao , KAGEYAMA Takashi , TAKAHASHI Kenji
Published: March 2005

Three pepsinogens (pepsinogens 1, 2, and 3) were purified from the gastric mucosa of the North Pacific bluefin tuna (Thunnus thynuus orientalis). Their molecular masses were determined to be 40.4 kDa, 37.8 kDa, and 40.1 kDa, respectively, by SDS/polyacrylamide gel electrophoresis. They contained relatively large numbers of basic residues when compared with mammalian pepsinogens. Upon activation at pH 2.0, pepsinogens 1 and 2 were converted to the corresponding pepsins, in a stepwise manner through intermediate forms, whereas pepsinogen 3 was converted to pepsin 3 directly. The optimal pH of each pepsin for hemoglobin digestion was around 2.5. N‐acetyl‐L‐phenylalanyl‐L‐diiodotyrosine was scarcely hydrolyzed be each pepsin. Pepstatin, diazoacetyl‐DL‐norleucine methyl ester in the presence of Cu2+, 1,2‐epoxy‐3‐(p‐nitrophenoxy)propane and p‐bromophenacyl bromide inhibited each pepsin, although the extent of inhibition by each reagent differed significantly among the three pepsins.

The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences of the NH2‐terminal regions of pepsins. Similarities in the activation segment region among the three tuna pepsinogens were rather low, ranging over 28–56%. A phylogenetic tree for 16 aspartic proteinase zymogens including the three tuna pepsinogens was constructed based on the amino acid sequences of their activation segments. The tree indicates that each tuna pepsinogen diverged from a common ancestor of pepsinogens A and C and prochymosin in the early period of pepsinogen evolution.
Alterations in the extracellular domain of M13 procoat protein make its membrane insertion dependent on secA and secY
Authors: KUHN Andreas
Published: March 2005

The products of genes secA and secY (SecA and SecY) are putative components of a bacterial protein export machinery and are required for the export of many periplasmic and membrane proteins. Only a few proteins, among them the M13 procoat protein, insert independently of SecA and SecY. To investigate the reason why the procoat protein inserts independently of sec functions, various hybrid proteins were constructed. By in‐frame gene fusions the central procoat region, which translocates across the membrane, was extended in size. Fragments of the ompA gene ranging from 522–294 bp were ligated with the procoat gene. The hybrid proteins were inserted into the membrane and processed normally, but only in the presence of functional SecA and SecY.
Secondary structure determination for α‐neurotoxin from Dendroaspis polylepis polylepis based on sequence‐specific 1H‐nuclear‐magnetic‐resonance assignments
Authors: LABHARDT Alexander M. , HUNZIKER‐KWIK Eng‐Hiang , WÜTHRICH Kurt
Published: March 2005

Sequence‐specific assignments are presented for the polypeptide backbone protons and a majority of the amino‐acid‐side‐chain protons of α‐neurotoxin from Dendroaspis polylepis polylepis, and individual amide proton‐exchange rates with the solvent are reported. The secondary structure and the hydrogen‐bonding patterns in the regular secondary structure elements are deduced from nuclear Overhauser effects and the sequence locations of the slowly exchanging amide protons. The molecule includes a three‐stranded antiparallel β‐sheet, and there are indications that two additional short chain segments are arranged in an antiparallel β‐sheet. These structural elements are similar, but not identical, to either the secondary structure reported for erabutoxin b in single crystals, or the solution structure of cytotoxin CTXIIb from Naja mossambica mossambica.
Increased oxygen affinity with normal heterotropic effects in hemoglobin Loire [α88(F9)Ala→Ser]
Authors: BAKLOUTI Faouzi , BAUDIN‐CHICH Véronique , KISTER Jean , MARDEN Michael , TEYSSIER Georges , POYART Claude , DELAUNAY Jean , WAJCMAN Henri
Published: March 2005

Increased homotropic allosteric effect, while maintaining normal heterotropic effects, was observed in hemoglobin Loire. The oxygen binding curves, at equilibrium, and the kinetic measurements demonstrated that the substitution of α88(F9) Ala for a Ser results in increased oxygen affinity and decreased n50 value. The function of the residues involved in the Bohr effect or in the regulation by 2,3‐bisphosphoglycerate is not altered. The effects of bezafibrate, which binds specifically to the α chains, was similar to that observed in Hb A. The functional properties of Hb Loire may be explained by a slight displacement of some key residues of the C‐terminal region of the α chain destabilizing the T structure.
Phosphoenolpyruvate‐dependent flavinylation of 6‐hydroxy‐D‐nicotine oxidase
Authors: NAGURSKY Heiner , BICHLER Veronika , BRANDSCH Roderich
Published: March 2005

The reaction leading to the flavinylation of apo‐6‐hydroxy‐D‐nicotine oxidase was investigated in cell‐free extracts of Eschericia coli carrying the 6‐hydroxy‐D‐nicotine oxidase (6‐HDNO) gene on the expression plasmid pDB222. It was demonstrated that the reaction required phosphoenolpyruvate (P‐pyruvate) in addition to FAD. When [32P]P‐pyruvate or [14C]P‐pyruvate were used in the reaction with apo‐6‐HDNO, no phosphorylated or pyruvylated apo‐protein could be detected, however. In order to drive the reaction to completion, FAD and P‐pyruvate had to be present simultaneously in the reaction mixture. When apo‐6‐HDNO, highly purified by affinity chromatography, was used in the reaction with P‐pyruvate and FAD, no additional protein fraction was required. A possible reaction scheme for the formation of holoenzyme from 6‐HDNO is discussed.
Soaking in Cs2SO4 reveals a caesium‐aromatic interaction in bovine‐liver rhodanese
Authors: Ulco KOOYSTRA P. J. , KALK Kor H. , HOL Wim G. J.
Published: March 2005

Soaking crystals of rhodanese (thiosulphate: cyanide sulphurtransferase) in 2 M caesium sulphate reveals three caesium binding sites of this enzyme. One of these had been described before as a binding site for sodium ions and is located in a cleft close to the active site. In this site the monovalent cation is coordinated by five oxygen atoms. The first additional binding site seems to be quite special. The caesium ion is bound to the phenyl ring of a tryptophan residue. It is further liganded by two oxygen atoms. The third binding site is a result of crystal packing effects: caesium is liganded by four oxygen atoms, provided by two rhodanese molecules and one sulphate ion.

It is likely that the binding of caesium affects the fluorescence of the tryptophan residue with which it interacts. Such possible effects should also be kept in mind when caesium ions are used as a quencher in fluorescence studies of proteins in general.
Use of the inhibition of enzymatic antioxidant systems in order to evaluate their physiological importance
Authors: MICHIELS Carine , REMACLE José
Published: March 2005

Chemical inhibitors of the different antioxidant enzymes were systematically testet either on purified enzymes of after incubation with human fibroblasts in culture. Inhibition values were obtained for catalase with aminotriazole, for superoxide dismutase with diethyldithiocarbamate, for glutathione peroxidase with mercaptosuccinate, for glutathione reductase with bischloroethylnitrosourea and for glutathione synthesis with buthionine sulfoximine. Viability of cells incubated with these inhibitors was then tested under normal conditions and under high oxygen pressure; the data were correlated with the above‐mentioned inhibitory values. Cell viability was particularly affected when the glutathione‐related enzymes, especially glutathione peroxidase, were inhibited.
Dissociation of outer‐sphere water is rate‐limiting for the binding of ligands in the active site of horse liver alcohol dehydrogenase
Authors: SARTORIUS Christian , ZEPPEZAUER Michael
Published: March 2005

The association of imidazole and auramine O to native horse‐liver alcohol dehydrogenase [Zn(II)LADH] and active‐site specifically cobalt(II)‐substituted horse‐liver alcohol dehydrogenase [Co(II)LADH], respectively, has been investigated. In all cases [except imidazole binding to Zn(II)LADH in the presence of auramine O] the association rates approached an upper limit (kmax). The kmax values were compared for the metal ligands imidazole (monodentate), 1,10‐phenanthroline and 2,2′‐bipyridine (bidentate; see also the preceding paper), and for auramine O which does not coordinate to the catalytic metal ion. Independent of the large differences in their structure and metal‐bonding capability, all these compounds exhibit common, maximum, limiting rate constants of about 60 s−1 and 200 s−1 for Co(II)LADH and Zn(II)LADH, respectively. These results demonstrate that kmax is strongly dependent on the catalytic metal ion but not on the ligand. The absence of spectral changes in the d‐d transitions of the catalytic Co(II) ion upon auramine O binding to Co(II)LADH indicates that the rate‐limiting step is not accompanied by a major conformational change. Finally, it is concluded that reactions in the inner coordination sphere of the catalytic metal ion (i.e. the metal‐bound water molecule) are not responsible for the step characterized by kmax.

We propose the rate‐limiting step to consist of the dissociation of one or several water molecules from the second coordination sphere of the catalytic metal ion in the active site of LADH in its open conformation.
Relationship between the tyrosination state of tubulin and the activities of tubulin: tyrosine ligase and tubulin carboxypeptidase in rat muscle during development
Authors: ALONSO Alejandra del C. , ARCE Carlos A. , BARRA Héctor S.
Published: March 2005

Tubulin can be post‐translationally modified by the incorporation or the release of a tyrosine residue at the COOH‐terminus of the α subunit. The present study demonstrates that rat muscle soluble preparations contain tubulin carboxypeptidase besides tubulin:tyrosine ligase.

The state of tyrosination of tubulin and the activities of both the ligase and the carboxypeptidase were examined in rat muscle during development. The proportion of tyrosinated tubulin with respect to tyrosinable tubulin (tyrosinated plus detyrosinated tubulin) decreased from 83% (new‐born rats) to 28% (adult rats) with the corresponding increase in detyrosinated tubulin. The activities of the enzymes decreased continuously and in a near parallel fashion during development. These results indicate that the changes in the tyrosination state of tubulin can not be explained merely by changes in the enzyme activities.

We also compared the ability of rat muscle and brain [14C]tyrosinated tubulin to act as substrate of the carboxypeptidase. Muscle tubulin was found to be a less efficient substrate than brain tubulin.
Monoclonal antibodies neutralizing mammalian DNA topoisomerase I activity
Authors: ODDOU Pascale , SCHMIDT Ute , KNIPPERS Rolf , RICHTER Arndt
Published: March 2005

We have isolated three different monoclonal antibodies specific for mammalian type‐I DNA topoisomerase. The antibodies react with three closely adjacent epitopes located in a central section of the enzyme (between amino acid residues 344 and 483). Two of the antibodies inhibit an early step of the nicking/closing pathway. We provide evidence showing that the antibodies do not block the association of the enzyme with DNA. The antibodies are useful for immunocytochemical investigation and for further exploration of the biochemical function of mammalian type‐I DNA topoisomerase.
Two‐dimensional 1H‐NMR investigation of ribonuclease T1
Authors: HOFFMANN Eberhard , RÜTERJANS Heinz
Published: March 2005

Ribonuclease T1 was studied by two‐dimensional 1H‐NMR spectroscopy. Resonance assignments were obtained for the backbone protons of 95 amino acid residues and most of its side‐chain protons using sequence‐specific assignment procedures. The secondary structure elements of ribonuclease T1 were identified by an investigation of medium‐ and long‐range nuclear Overhauser effects between the backbone and Cβ protons. A low‐resolution three‐dimensional structure of ribonuclease T1 was deduced from qualitative interpretation of long‐range nuclear Overhauser effects.
Structural and functional characterization of mutants of recombinant single‐chain urokinase‐type plasminogen activator obtained by site‐specific mutagenesis of Lys158, Ile159 and Ile160
Authors: LIJNEN H. Roger , NELLES Luc , VAN HOEF Berthe , DEMARSIN Eddy , COLLEN Désiré
Published: March 2005

Single‐chain urokinase‐type plasminogen activator (scu‐PA) is converted to urokinase by hydrolysis of the Lys158‐Ile159 peptide bond. Site‐directed mutagenesis of Lys158 to Gly or Glu yields plasmin‐resistant mutants with a 10–20‐fold reduced catalytic efficiency for the activation of plasminogen [Nelles et al. (1987) J. Biol. Chem. 262, 5682–5689]. In the present study, we have further evaluated the enzymatic properties of derivatives of recombinant scu‐PA (rscu‐PA), produced by site‐directed mutagenesis of Lys158, Ile159 or Ile160, in order to obtain additional information on the structure/function relations underlying the enzymatic properties of the single‐and two‐chain u‐PA moieties.

[Arg158]rscu‐PA (rscu‐PA with Lys158 substituted with Arg) appeared to be indistinguishable from wild‐type rscu‐PA with respect to plasminogen‐activating potential (catalytic efficiency k2/Km=0.21 mM−1 s−1 versus 0.64 mM−1 s−1), conversion to active two‐chain urokinase by plasmin (k2/Km=0.13 μM−1 s−1 versus 0.28 μM−1 s−1), as well as its specific activity (48000 IU/mg as compared to 60000 IU/mg) and its fibrinolytic potential in a plasma medium (50% lysis in 2 h with 2.8 μg/ml versus 2.1 μg/ml). [Pro159]rscu‐PA (Ile159 substituted with Pro) and [Gly159]rscu‐PA (Ile159 converted to Gly) are virtually inactive towards plasminogen (k2/Km less than 0.004 mM−1 s−1). They are however converted to inactive two‐chain derivatives by plasmin following cleavage of the Arg156‐Phe157 peptide bond in [Pro159]rscu‐PA and of the Lys158‐Gly159 peptide bond in [Gly159]rscu‐PA. [Gly158,Lys160]rscu‐PA (with Lys158 converted to Gly and Ile160 to Lys) has a low catalytic efficiency towards plasminogen both as a single‐chain form (k2/Km=0.012 mM−1 s−1) and as the two‐chain derivative (k2/Km=0.13 mM−1 s−1) generated by cleavage of both the Arg156‐Phe157 and/or the Lys160‐Gly161 peptide bonds by plasmin.

These findings suggest that the enzymatic properties of rscu‐PA are critically dependent on the amino acids in position 158 (requirement for Arg or Lys) and position 159 (requirement for Ile). Conversion of the basic amino acid in position 158 results in a 10–20‐fold reduction of the catalytic efficiency of the single‐chain molecule but yields a fully active two‐chain derivative. The presence of Ile in position 159 is not only a primary determinant for the activity of the two‐chain derivative, but also of the single‐chain precursor. Cleavage of the Arg156‐Phe157 or the Lys160‐Gly161 peptide bonds by plasmin yields inactive two‐chain derivatives.