bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the isoleucyl‐tRNA synthetase reaction by total rate equations
Authors: AIRAS R. Kalervo
Published: March 2005

Derivation of a steady‐state rate equation for the aminoacyl‐tRNA synthetases is described, and its suitability for the analysis of various details of the reaction is tested. The equation is applied to the magnesium and spermidine dependences of the isoleucyl‐tRNA synthetase reaction. Earlier work [Airas, R. K. (1990) Eur. J. Biochem. 192, 401–409] is expanded by experiments and calculations of the tRNA kinetics. The analysis suggests the following new details in addition to the earlier results: (a) The binding of tRNA to the enzyme (and not only the rate of the aminoacylation reaction) is affected by the presence of the Mg2+ and spermidine in the tRNA molecule. At least two bound Mg2+ or spermidines are required. (b) tRNA and PPi partly inhibit the binding of each other to the enzyme. (c) The transfer reaction is rather slow, and, at least under some conditions, it participates in rate limitation. (d) A Mg2+‐induced reduction in the aminoacylation rate seems to be directed to the dissociation of the aminoacyl‐tRNA from the enzyme. This dissociation rate is enhanced if a Mg2+ is first dissociated from the enzyme or tRNA. An increase in the Mg2+ concentration shifts the rate limitation from the transfer reaction towards dissociation of the product.
Affinity labelling of the active site of brain phosphatidylinositol 4‐kinase with 5′‐fluorosulphonylbenzoyl‐adenosine
Authors: SCHOLZ Glen , BARRITT Greg J. , KWOK Francis
Published: March 2005

5′‐p‐Fluorosulphonylbenzoyl‐adenosine (FSO2BzAdo), an affinity labelling analogue of ATP, was used to label the active site of sheep brain phosphatidylinositol 4‐kinase (PtdIns 4‐kinase). The incubation of PtdIns 4‐kinase with concentrations of FSO2BzAdo as low as 50μ resulted in considerate inactivation of the enzyme. (e.g. 55% less after 60 min with 50μ FSO2BzAdo). The kinetics of inactivation of PtdIns 4‐kinase by FSO2BzAdo suggest a two‐step mechanism, in which a rapid reversible binding of FSO2BzAdo to the enzyme is followed by a covalent sulphonation step. The first‐order rate constant (k2) for the inactivation of PtdIns 4‐kinase was calculated to be 0.063 min−1, and the steady‐state constant of inactivation (ki) to be 200μ. Preincubation of the enzyme with either ATP plus Mg2+, or PtdIns alone, prior to addition of FSO2BzAdo reduced the degree of inactivation of the enzyme; suggesting that FSO2BzAdo binds within the active site PtdIns 4‐kinase. Moreover, since ATP plus Mg2+ provided the greatest protection against inactivation, it is concluded that the main site of labelling of PtdIns 4‐kinase by FSO2BzAdo is within the ATP‐binding site of the enzyme. Results obtained from chemical modification experiments, which employed pyridoxal 5′‐phosphate and tetranitromethane, are consistent with a catalytically‐essential lysine being present within the ATP‐binding site of PtdIns 4‐kinase. Therefore, it is hypothesised that the inactivation of PtdIns 4‐kinase by FSO2BzAdo may be due to the labelling of this lysine residue.
Ganglioside binding proteins of calf brain with ubiquitin‐like N‐terminals
Authors: ZDEBSKA Ewa , ANTONIEWICZ Jolanta , NILSSON Bo , SANDHOFF Konrad , FÜRST Werner , JANIK Przemyslaw , KOŚCIELAK Jerzy
Published: March 2005

Two ganglioside‐associated protein components I and II have been isolated from crude ganglioside preparations of calf brain by DEAE‐Sepadex ion‐exchange chromatography. Both components exhibited binding capacity in aqeous media for gangliosides of the ‘ganglio’ series but not for neutral glycosphingolipids (polyglycosylceramides) and only a low capacity for sialosylparagloboside. Each protein bound individual gangliosides with different efficiency. Upon prolonged incubation of component I with gangliosides, complexes with high (30:1) and low (6:1) glycolipid/protein molar ratios were formed. The latter but not the former complex was able to penetrate Sephadex G‐200 beads. Both components inhibited plating efficiency of cultured mouse N2a neuroblastoma cells. The molecular masses of components I and II were determined by SDS/PAGE to be 11 – 12 kDa and 28 kDa, respectively. Carbohydrates (fucose, mannose, galactose, N‐acetylglucosamine, N‐acetylgalactosamine, and some sialic acid) were found only in component II. When examined by reverse‐phase HPLC each component separated into two major closely migrating peaks which were subsequently examined by Edman degradation. Amino acid sequences of the N‐terminal portions of three of these peaks (one peak from component I and both peaks from component II) showed, as far as the sequences were established, identity with the sequence of ubiquitin. It is hypothesized that the proteins may be instrumental in intracellular trafficking of gangliosides.
Purification and characterization of a pore‐forming protein from the marine sponge Tethya lyncurium
Authors: MANGEL Andreas , LEITÃO José M. , BATEL Renato , ZIMMERMANN Herbert , MÜLLER Werner E. G. , SCHRÖDER Heinz C.
Published: March 2005

A pore‐forming protein was detected and purified for the first time from a marine sponge (Tethya lyncurium). The purified protein has a polypeptide molecular mass of 21 kDa and a pI of 6.4. Tethya pore‐forming protein (also called Tethya hemolysin) rapidly lysed erythrocytes from a variety of organisms. After binding to target membranes, the hemolysin resisted elution with EDTA, salt or solutions of low ionic strength and hence resembled an integral membrane protein. Erythrocytes could be protected from hemolysis induced by Tethya hemolysin by addition of 30 mM dextran 4 (4–6 kDa; equivalent hydrodynamic diffusion radius, 1.75–2.3 nm) to the extracellular medium, but not by addition of uncharged molecules of smaller size [sucrose, raffinose and poly(ethylene glycol) 1550; equivalent hydrodynamic diffusion radii, 0.46, 0.57 and 1.2 nm, respectively]. This result indicates that hemolysin is able to form stable transmembrane pores with an effective diameter of about 2–3 nm. Treatment of osmotically protected erythrocytes with Tethya hemolysin caused a rapid efflux of intracellular K+ and ATP, and a rapid influx of extracellularly added Ca2+ and sucrose. In negative‐staining electron microscopy, target erythrocyte membranes exposed to purified Tethya hemolysin displayed ultrastructural lesions but without visible pores.
Regulation of vimentin expression in cultured epithelial cells
Authors: PIEPER Frank R. , VAN DE KLUNDERT Francy A. , RAATS Jos M. , HENDERIK Jos B. , SCHAART Gert , RAMAEKERS Frans C. , BLOEMENDAL Hans
Published: March 2005

Most cell types start expressing vimentin when brought into tissue culture. Using both vimentin‐expressing (HeLa) and vimentin‐negative (MCF‐7) epithelial cell lines, we have identified the cis‐regulatory DNA elements involved in this process. Sequences located 1.1–0.6 kb upstream of the vimentin transcription‐initiation site strongly enhance expression in HeLa cells, but are silenced in MCF‐7 cells. Other regulatory elements in the vimentin promoter (an enhancer 3.2.–2.6 kb upstream and a minimal promoter region including the CAAT‐box) are potentially active in both cell types, but are silenced by the 0.5‐kb fragment in MCF‐7 cells. Deletion of this fragment restores transcriptional activity of a transfected vimentin promoter. Our data indicate that a double AP 1/jun‐binding site present in the 0.5‐kb fragment mediates the induction of vimentin expression in cultured epithelial cells, while silencing sequences located within the same fragment are responsible for the absence of vimentin expression in MCF‐7 cells.

In contrast to MCF‐7 cells, a transfected vimentin promoter and gene are transcriptionally active in the vimentin‐negative epithelial cell line T24. Transfection studies show that type‐III‐intermediate‐filament expression is not impaired at any level in these cells.

Upon transfection and expression of a desmin construct in T24 cells not only desmin, but also vimentin was detected. Both proteins assembled into intermediate filaments. This induction of vimentin expression appeared to be regulated at the post‐transcriptional level.
Regulatory phosphorylation of phosphoenolpyruvate carboxylase in protoplasts from Sorghum mesophyll cells and the role of pH and Ca2+ as possible components of the light‐transduction pathway
Authors: PIERRE Jean Noël , PACQUIT Valérie , VIDAL Jean , GADAL Pierre
Published: March 2005

The light‐dependent phosphorylation of the photosynthetic phosphoenolpyruvate carboxylase (PyrPC) was shown to occur in protoplasts from Sorghum mesophyll cells. It was accompanied by an increase in PyrPC protein‐serine‐kinase activity and conferred the target‐specific functional properties, i.e. an increase in Vmax and apparent Ki for L‐malate, as previously found with the whole leaf. The light‐dependent regulatory phosphorylation of PyrPC was (a) specifically promoted by the weak bases NH4Cl and methylamine (agents which increase cytosolic pH), but not by KNO3, (b) inhibited by the cytosolic protein‐synthesis inhibitor, cycloheximide, thus confirming that protein turnover is a component of the signal‐transduction cascade, as reported in [4], (c) found to moderately decrease in the presence of EGTA and to be strongly depressed when the Ca2+‐selective ionophore A23187 was added to the incubation medium together with EGTA. Addition of Ca2+, but not of Mg2+, to the Ca2+‐depleted protoplasts partially, but significantly, relieved the inhibition. Calcium deprivation apparently affected the in‐situ light‐activation of the PyrPC protein kinase. These data indicated that both Ca2+ and an increase in cytosolic pH are required for the induction of PyrPC protein kinase activity/PyrPC phosphorylation in illuminated protoplasts from Sorghum mesophyll cells.
Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli
Authors: SCHOEDON Gabriele , REDWEIK Udo , FRANK Gerhard , COTTON Richard G. H. , BLAU Nenad
Published: March 2005

The kinetic and regulatory properties of GTP cyclohydrolase I were investigated using an improved enzyme assay and direct determination of the product, dihydroneopterin triphosphate. The enzyme was purified from Escherichia coli to absolute homogeneity as demonstrated by N‐terminal sequencing of up to 50 amino acid residues. A 30‐residue internal fragment showed 42% similarity with rat liver GTP cyclohydrolase I. The enzyme did not obey Michaelis‐Menten kinetics or show a sigmoid reaction curve. The substrate saturation kinetics were found to be slow with low response to minor changes in GTP concentrations. GTP cyclohydrolase I has a relatively high apparent Km. The values are slightly different for enzyme purified by GTP‐agarose (100 μM) and UTP‐agarose (110 μM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP‐cyclohydrolase‐I activity was modulated in Vmax by K+, divalent cations, UTP and tetrahydrobiopterin. Divalent cations, such as Mg2+, had an activating effect with an optimum at 8 mM Mg2+. A different catalytic function and formation of a new, unidentified product by GTP cyclohydrolase I was observed in the presence of Ca2+. In the presence of 1 mM EDTA and Mg2+, GTP‐cyclohydrolase‐I activity was strongly inhibited by chelate complexes. UTP proved not to be a competitive inhibitor, but a positive modulator. The inhibition by chelate complexes was totally abolished by UTP.

Tetrahydrobiopterin showed an inhibitory effect, with 50% inhibition at 100 μM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobiopterin. Using monoclonal antibody 1F11 (related to the GTP‐binding site), and monoclonal antibody NS7 (mimicking tetrahydrobiopterin), different binding sites were demonstrated for GTP and tetrahydrobiopterin on each enzyme subunit. Western‐blot competition analysis revealed a UTP‐binding site different from the binding sites of GTP and tetrahydrobiopterin. Based on the kinetic behaviour and the kind of modulations observed we defined GTP cyclohydrolase I as an M‐class allosteric enzyme.
Effects of chemical modification of Anabaena flavodoxin and ferredoxin‐NADP+ reductase on the kinetics of interprotein electron transfer reactions
Authors: MEDINA Milagros , GOMEZ‐MORENO Carlos , TOLLIN Gordon
Published: March 2005

The influence of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin‐NADP+ reductase (FNR), and of carboxyl groups (using glycine ethl ester) in flavodoxin (Fld), on the kinetics of electron transfer between FNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC 7119. Reduction by laser‐generated 5‐deazariboflavin semiquinone of the FAD moiety of phenylglyoxal‐modified FNR occurred with a second‐order rate constant 2.5‐fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred (reduced Fd) by FNRox (oxidized FNR) were dramatically decreased (∼ 100‐fold) when phenylglyoxal‐modified FNR was used. In contrast to the reaction involving the native proteins, no ionic strength effects on Kobs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal‐modified FNRsq (FNR semiquinone) was used to reduce Fldox (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first‐order rate constant for Fldsq (Fld semiquinone) formation via intracomplex electron transfer from FNRsq was approximately 12‐fold smaller than that obtained for the native FNR (600 s−1 vs 7000 s−1). Again, ionic strength effects were diminished. The glycine‐ethyl‐ester‐modified Fld yielded a limiting first‐order rate constant for intracomplex electron transfer from FNRso to Fldox which was approximately 7‐fold smaller (1000 s−1) than that: obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved.
Evidence for two histidine ligands at the diiron site of methane monooxygenase
Authors: Drummond D. , SMITH S. , DALTON Howard
Published: March 2005

Circular dichroism spectroscopy has shown the hydroxylase component of methane monooxygenase to have a high helical content. The apoprotein has the same secondary structure as the holoenzyme. Chemical modification shows 12 histidines to be reactive with diethylpyrocarbonate in the holoenzyme, whereas 14 are reactive in the apoenzyme. Two histidine residues are implicated as iron ligands. Further chemical modification results suggest a cysteine residue is in close proximity to the diiron centre.
A mutant of human insulin‐like growth factor II (IGF II) with the processing sites of proinsulin
Authors: ZARN Jürg A. , LÜTHI Christine , GIGER Roman J. , SIGRIST Adrian , HUMBEL René E.
Published: March 2005

A mutant of human insulin‐like growth factor II (IGF II) was constructed by site‐directed mutagenesis: the nucleotides coding for Ser33 and Ser39 were changed to yield Arg and Lys, respectively, thus creating two pairs of basic residues, Arg‐Arg and Lys‐Arg, as flanking sequences of the remaining C domain. [Arg33, Lys39]IGF II was expressed in NIH‐3T3 cells as a processed two‐chain peptide with a deletion of amino acid residues 37–40 and crosslinked by three disulfide bonds. This des(37–40)[Arg33]IGF II showed 3.6‐fold and 7.4‐fold reduced affinities to the type 1 and type 2 IGF receptor overexpressing cells, respectively, whereas the thymidine incorporation potency was the same as that of wild‐type IGF II. We speculate that the discrepancy between the reduced binding to the type 1 IGF receptor and the full thymidine incorporation potency is due to the 6.1‐fold reduced affinity of the expressed mutant to the co‐expressed IGF binding protein 3 (IGFBP‐3). The results suggest that des(37–40)[Arg33]IGF II assumes a conformation very similar to IGF II, and that the entire length of the C domain is not essential for biological activity.
Cloning and expression of a heat‐inducible hsp70 gene in two species of Hydra which differ in their stress response
Authors: GELLNER Klaus , PRAETZEL Gabriele , BOSCH Thomas C. G.
Published: March 2005

A heat‐inducible, intron‐containing member of the hsp70 gene family has been isolated and characterized in Hydra magnipapillata and Hydra oligactis, two species previously shown [Bosch, T. C. G., Krylow, S. M., Bode, H. R. & Steele, R. E. (1988) Proc. Natl Acad. Sci. USA 85, 7927–7931] to differ in their stress response. The gene, hsp70.1, encodes a 654‐amino‐acid protein of predicted molecular mass 70 kDa with 78% amino acid identity to Xenopus HSP70. Northern‐blot analysis revealed that polyps of H. oligactis accumulate significantly less hsp70.1 mRNA after heat shock than polyps of H. magnipapillata. In nuclear run‐off experiments, we found that transcriptional induction of hsp70.1 expression in response to stress is similar in both species. Thus, the previously reported inability of H. oligactis to synthesize heat‐shock proteins in response to stress is at least in part due to reduced stability of hsp70.1 mRNA during heat shock.
A novel calcium‐binding protein from Euglena gracilis
Authors: GUMPEL Nicola J. , SMITH Alison G.
Published: March 2005

We have isolated a novel cDNA from Euglena gracilis that encodes a protein composed of 24.9% aspartate with an estimated pI of 3.56, and a deduced molecular mass of 73 542 Da. The first 20 or so amino acids are hydrophobic and resemble a signal sequence. The rest of the polypeptide is composed of a 23‐amino‐acid repeat. There are 30 repeats, of which 23 are full length. Part of the consensus sequence derived from the repeats has some similarity to the loop of the EF‐hand type calcium‐binding motif. Evidence is presented that a fusion protein of this novel protein with β‐galactosidase can bind calcium. Northern blotting indicates a single transcript of 2.3 kb (the same size as the cDNA). In‐vitro translation of the cDNA gives a protein that migrates on SDS/PAGE with an apparent molecular mass of 120–125 kDa. The protein is processed into a smaller, protease‐protected form (110–120 kDa) when translated in the presence of canine pancreatic microsomal vesicles. This suggests that the protein is targeted across the endoplasmic reticulum membrane in vivo, and is the first report of a signal sequence from E. gracilis. We propose that the cDNA obtained encodes a novel calcium‐binding protein that is either secreted or resident in the endomembrane system of E. gracilis, and call it the acidic‐repeat protein.
Mechanism of the degradation of non‐enzymatically glycated proteins under physiological conditions
Authors: SMITH Patricia R. , THORNALLEY Paul J.
Published: March 2005

The degradation of fructosamines, formed from the non‐enzymic glycation of proteins under physiological conditions, to advanced glycation end products was investigated by studying the model peptide fructosamine Nε‐(1‐deoxy‐d‐fructos‐1‐yl)hippuryl‐lysine (DHL). At pH 7.4 and 37°C in aerobic phosphate buffer, DHL degraded to form Nε‐carboxymethyl‐hippuryl‐lysine, and hippuryllysine over a 29‐day incubation period. The expected Nε‐(3‐lactato)hippuryl‐lysine and ‘hippuryl‐lysylpyrraline’ derivatives were not found. Superoxide radicals and hydrogen peroxide were formed during the degradation of DHL but were also both consumed during the degradation reaction. Reversal of the Amadori rearrangement was not a major fate of the fructosamine.

The formation of Nε‐carboxymethyl‐hippuryl‐lysine was decreased by desferrioxamine, catalase, superoxide dismutase, catalase with superoxide dismutase, anaerobic conditions and aminoguanidine. The formation of hippuryl‐lysine was decreased by desferrioxamine, catalase and catalase with superoxide dismutase, but was increased by the addition of aminoguanidine. Nε‐Carboxymethyl‐serine and unmodified lysine residues are major peptide‐based end products in the degradation of lysyl‐fructosamine under physiological conditions. Oxygen, redox‐active metal ions, catalase, superoxide dismutase and the pharmacological agent aminoguanidine are expected to be influential on the rate and fate of fructosamine degradation.
Inhibition of cytochrome‐P450 reductase by polyols has an electrostatic nature
Authors: VOZNESENSKY Andrei I. , SCHENKMAN John B.
Published: March 2005

The present study was undertaken to examine the nature of the inhibitory action of glycerol on the liver microsomal monooxygenase system. In agreement with earlier observations, glycerol inhibited benzphtamine N‐demethylation by liver microsomes of the phenobarbital‐treated rabbit. The presence of glycerol in the medium did not affect binding of the substrate to cytochrome P450. Another polyol, ethylene glycol, was equally efficient in inhibiting benzphetamine N‐demethylation. Both also inhibited reduction of rabbit cytochrome P450 LM2, cytochrome c and potassium ferricyanide by NADPH‐cytochrome‐P450 reductase in microsomes. Recently, we showed that the stimulation of electron transfer by increased ionic strength is due to neutralization of electrostatic interaction between NADPH ‐ cytochrome‐P450 reductase and its charged redox partners [Voznesesky, A. I. & Schenkman, J. B. (1992) J. Biol. Chem. 267, 14669–14676]. Polyols have an opposite effect to that of salt on ionic properties of a solution. They decrease the dielectric constant, thereby promoting electrostatic interactions between proteins. Addition of polyols decreased the conductivity of the medium. When rates of electron transfer to charged acceptors, cytochrome P450, cytochrome c and potassium ferricyanide, at various salt and polyol concentrations, relative to activities in 200 mM sodium phosphate, were plotted as a function of the conductivity the data for each acceptor fit on the same line. In contrast, neither alteration of ionic stregth nor polyol addition affected the rate of electron transfer from NADPH ‐ cytochrome‐P450 reductase to an uncharged acceptor 1,4‐benzoquinone. The data obtained is consistent with our earlier suggestion that charge repulsion limits redox interactions between rabbit cytochrome P450 LM2 and its reductase at low ionic strength, and suggest that the observed action of polyols is the result of enhancement of electrostatic interactions that inhibits electron transfer between NADPH ‐ cytochrome‐P450 reductase and its charged redox partners. In congruence with the hypothesis, the Km of rabbit cytochrome P450.LM2 for NADPH ‐ cytochrome‐P450 reductase was increased almost one order of magnitude by elevating the glycerol content from 5% to 25% (by vol.) without a change in Vmax.
Studies on the Aminopeptidase Activity of Rat Cathepsin H
Authors: ROTHE Markus , DODT Johannes
Published: March 2005

Three synthetic substrates H‐Arg‐NH‐Mec, Bz‐Arg‐NH‐Mec and H‐Cit‐NH‐Mec (Bz, benzoyl; NH‐Mec, 4‐methylcoumaryl‐7‐amide; Cit, citrulline) were used to characterize specificity requirements for the P1‐S1 interaction of cathepsin H from rat liver. From rapid equilibrium kinetic studies it was shown that Km, kcat and the specificity constants kcat/Km are quite similar for substrates with a free α‐amino group. In contrast, a 25‐fold decrease of kcat/Km was observed for the N‐terminal‐blocked substrate Bz‐Arg‐NH‐Mec. The activation energies for H‐Arg‐NH‐Mec and Bz‐Arg‐NH‐Mec were determined to be 37 kJ/mol and 55 kJ/mol, respectively, and the incremental binding energy ΔΔGb of the charged α‐amino group was estimated to ‐8.1 kJ/mol at pH 6.8. The shown preference of cathepsin H for the unblocked substrates H‐Arg‐NH‐Mec and H‐Cit‐NH‐Mec was further investigated by inspection of the pH dependence of kcat/Km. The curves of the two substrates with a charged α‐amino group showed identical bell‐shaped profiles which both exhibit pKa1 and pKa2 values of 5.5 and 7.4, respectively, at 30°C. The residue with a pKa1 of 5.5 in the acid limb of the activity profile of H‐Arg‐NH‐Mec was identified by its ionization enthalpy ΔHion= 21 kJ/mol as a β‐carboxylate or γ‐carboxylate of the enzyme, whereas the residue with a pKa2 of 7.4 was assigned to the free α‐amino group of the substrate with a ΔHion of 59 kJ/mol. Bz‐Arg‐NH‐Mec showed a different pH‐activity profile with a pKa1 of 5.4 and a pKa2 of 6.6 at 30°C. Cathepsin H exhibits no preference for a basic P1 side chain as has been shown by the similar kinetics of H‐Arg‐NH‐Mec and the uncharged, isosteric substrate H‐Cit‐NH‐Mec. In summary, specific interactions of an aniouic cathepsin H active site residue with the charged α‐amino group of substrates caused transition state stabilization which proves the enzyme to act preferentially as an aminopeptidasc.
Purine–cytosine permease of Saccharomyces cerevisiae
Authors: BRÈTHES Daniel , NAPIAS Christian , TORCHUT Elisabeth , CHEVALLIER Jean
Published: March 2005

The cloned FCY2 gene (strain pAB4) of the purine‐cytosine permease (PCP) of Saccharomyces cerevisiae and the cloned allele fcy2‐21 (strain pAB25) introduced into an S. cerevisiae strain carrying a chromosomal deletion at the FCY2 locus [Weber, E., Rodriguez, C., Chevallier, M. R. & Jund, R. (1990) Mol. Microbiol. 4, 585–596] were studied.

The influence of external pH (varying over 3.5–6) has been analysed on the uptake of adenine, hypoxanthine and cytosine (Ktapp' apparent Michaelis constant and Vm) and on the binding constants of these three solutes (Kdapp' apparent half‐saturation constant and Bmax' total binding sites) determined on plasma membranes.

For pAB4, the variations of Ktapp and Vm were the same for the three bases, i.e. an increase in Ktapp when the pH increased and a maximum Vm around pH 5. For pAB25, Ktapp values varied in the same way and were significantly higher for the three bases than those found in pAB4. There was almost no variation of Vm for adenine, and there was a continuous decrease when the pH incréased in the Vm of hypoxanthine and cytosine.

Equilibrium binding measurements were performed for the three bases with plasma membrane isolated from pAB4 and pAB25. One single class of binding sites was detected. For pAB4, the affinity increased when the pH decreased for the three bases. The affinity of PCP for adenine was always greater than for cytosine or hypoxanthine. For pAB25, the same phenomenom was observed. However, the curves showing the variation of Kdapp as a function of pH were shifted towards more acidic pH values. A model was used to fit the experimental binding data obtained with hypoxanthine for the calculation of the dissociation constants of its binding to PCP and to determine the ionization constants of an amino acid involved in ligand binding. For pAB4, at acid pH, the dissociation constant was 1.7 ± 0.4 μM. An amino acid displaying a pK of 3.8 was determined; this value was shifted to pK 4.8 when hypoxanthine was bound. For pAB25, the main effects of the mutation were a large decrease in the affinity of PCP for hypoxanthine (Kd of 14.4 ± 4.3 μM) and a shift in the pK of the amino acid towards a more acidic pH (about 2.9). The pK of this group remained similar to the value obtained with pAB4 when hypoxanthine was bound. From these data, it is proposed that the binding of hypoxanthine and H+ is a random process.
Crystallographic studies of the binding of protonated and unprotonated inhibitors to carbonic anhydrase using hydrogen sulphide and nitrate anions
Authors: MANGANI Stefano , HÅKANSSON Kjell
Published: March 2005

The structures of human carbonic‐anhydrase‐II complexes with the anionic inhibitors hydrogen sulphide (HS−) and nitrate (NO3−) have been determined by X‐ray diffraction at 0.19‐nm resolution from crystals soaked at pH 7.8 and 6.0, respectively. The modes of binding of these two anions differ markedly from each other. The strong inhibitor HS− replaces the native zinc‐bound water/hydroxide (Wat263) leaving the tetrahedral metal geometry unaltered and acts as a hydrogen‐bonding donor towards Thr199γ. The weak NO3− inhibitor does not displace Wat263 from the metal coordination but occupies a fifth binding site changing the zinc coordination polyhedron into a slightly distorted trigonal bipyramid. The interaction of NO3− with the metal is weak; the nearest of its oxygen atoms being at a distance of 0.28 nm from the zinc ion. The binding of nitrate to the enzyme is completed by a hydrogen bond to the metal coordinated Wat263 and a second one to a water molecule of the active‐site cavity. The structures of the two complexes help to rationalize the binding of anionic inhibitors to carbonic anhydrase and the binding mode displayed by NO3− may be relevant to the catalytic mechanism.
Sequence‐specific 1H‐NMR assignment and determination of the secondary structure of bovine heart fatty‐acid‐binding protein
Authors: LÜCKE Christian , LASSEN Dirck , KREIENKAMP Hans‐Jürgen , SPENER Friedrich , RÜTERJANS Heinz
Published: March 2005

The nearly complete sequence‐specific 1H resonance assignment of the pI = 4.9 isoform of cytosolic 15‐kDa fatty‐acid‐binding protein from bovine heart (H‐FABPc) by homonuclear two‐dimensional NMR spectroscopy is presented. Regular secondary structure elements were identified from NOE spectra and the sequence locations of slowly exchanging backbone amide protons. The molecular structure of the protein was found to consist mainly of ten antiparallel β‐strands and two short α‐helices. The data presented here for the first time for a hydrophobic molecule transporter of the fatty‐acid‐binding protein type is the basis for a complete tertiary structure determination currently in progress.
Conformation/activity studies of rationally designed potent anti‐adhesive RGD peptides
Authors: GURRATH Marion , MÜLLER Gerhard , KESSLER Horst , AUMAILLEY Monique , TIMPL Rupert
Published: March 2005

The Arg‐Gly‐Asp (RGD) sequence is a universal cell‐recognition site of various extracellular proteins that interact with integrin cell‐surface receptors. In order to design low‐molecular‐mass RGD protein antagonists, the determination of the biologically active conformation is a prerequisite. We present a method that yields detailed insight into the steric factors which govern the binding of the ligands to their receptors by systematically scanning the conformational space accessible for the tripeptide sequence RGD. The investigation is based on the conformationally controlled design of homodetic cyclic oligopeptides and their structural determination, coupled with biological assays. For this purpose, a whole set of cyclic pentapeptides and hexapeptides has been synthesized and their three‐dimensional structures in solution analyzed by modern two‐dimensional NMR techniques in combination with restrained and free molecular dynamics simulations. Their biological activity was compared with that of linear GRGDS in inhibition assays of tumor cell adhesion to laminin P1 and vitronectin substrates. An up to 100‐fold, and in part selective, increase in activity was observed for two cyclic pentapeptides. Most other peptides showed a decreased activity which, however, was useful to correlate activity with rather small variations in conformation.

Detailed comparative studies of the systematically designed conformations and the corresponding anti‐adhesive activities offer an access to lead structures for a rational indirect drug design of peptide and peptidomimetic pharmaceuticals with strong interfering activity for integrin‐mediated cell–cell and cell–matrix interactions.
Molecular analysis of the protein‐protein interaction between the E9 immunity protein and colicin E9
Authors: WALLIS Russell , MOORE Geoffrey R. , KLEANTHOUS Colin , JAMES Richard
Published: March 2005

The specificity‐determining region of the colicin E9 immunity protein (Im9) for its interaction with its cognate E colicin has been localized to residues 16–43 of the 86‐amino‐acid protein by the use of gene fusions. A comparison of the alignment of residues in this region of the Im2, Im8 and Im9 proteins have identified nine candidate specificity‐determining residues. Using site‐directed mutagenesis, we have changed each of these residues in the Im9 protein to the residue found in the same position in the Im8 protein. The immunity phenotype conferred by the mutant immunity protein was then tested. Of the nine residues, only one (Val34 to Asp) showed any evidence of conferring immunity to colicin E8. Changing other residues in the specificity‐determining region to the equivalent Im8 residue did not affect the phenotype conferred by the mutant protein, with the exception of the change of Val37 to Glu, which resulted in low‐level E8 immunity. While the substitutions at positions 34 and 37 of the Im9 protein introduced immunity towards ColE8, they did not diminish the immunity towards ColE9, suggesting that the two immunity proteins may have a common specificity framework which can be modified by single mutations. In addition, we have used chemical modification of the unique cysteine residue of Im9 (Cys23) in order to probe further this specificity‐determining region. Cys23 in the purified Im9 protein is accessible to modification with the thiol‐specific reagent 5,5′‐dithiobis(2‐nitrobenzoic acid) and the stoichiometry of labelling is close to 1:1. This residue, however, cannot be labelled by 5,5′‐dithiobis(2‐nitrobenzoic acid) when the Im9 protein is complexed to colicin E9. This result is consistent with the Cys23 residue being buried in the complex. However, when the purified Im9 protein modified at Cys23 with a variety of reagents was used in DNase inhibition assays with colicin E9, the modified Im9 proteins still possessed anti‐DNase activity but only up to a certain derivative molecular mass. These results are discussed in terms of the proximity of Cys23 to the specificity‐determining region.
Expression and secretion of wheat germ agglutinin by Saccharomyces cerevisiae
Authors: NAGAHORA Hitoshi , ISHIKAWA Keiichiro , NIWA Yoshio , MURAKI Michiro , JIGAMI Yoshifumi
Published: March 2005

Genes encoding pre‐protein and prepro‐protein of wheat germ agglutinin isolectin 2 (WGA2) were chemically synthesized and expressed in the yeast Saccharomyces cerevisiae under the control of the ENO1 promoter. Yeast harboring either a pre‐WGA2 or a prepro‐WGA2 gene expression plasmid secreted a mature form of WGA2 into the culture medium. The amount of WGA2 secreted by the strain KS58‐2Ddel, which has a ssll mutation causing a supersecretion of human lysozyme [Suzuki, K., Ichikawa, K. & Jigami, Y. (1989) Mol. Gen. Genet. 219, 58–64], was 20‐fold greater than that secreted by the wild‐type strain KK4. The recombinant WGA2 from the cells containing the preproWGA2 gene expression plasmid was purified to homogeneity by a three‐step ion‐exchange chromatography scheme. As in wheat, the N‐terminal signal peptide of recombinant WGA2 purified from yeast culture was processed to form an N‐terminal 5‐oxoprolyl (pyroglutamyl) residue. Likewise, we found that the C‐terminal pro‐region of recombinant WGA2 had also been processed in yeast. Using electrospray ionization mass spectrometry, we found the processed C‐terminus to be heterogeneous in both recombinant WGA2 purified from yeast and in authentic WGA2. The major component of the recombinant WGA2 contained two additional amino acids at its C‐terminus compared to that of authentic WGA2. In spite of this difference in the C‐terminus, the recombinant WGA2 exhibited a sugar binding activity that was indistinguishable from that of authentic WGA2.
Short‐chain and medium‐chain aliphatic‐ester synthesis inSaccharomyces cerevisiae
Authors: MALCORPS Philippe , DUFOUR Jean‐Pierre
Published: March 2005

In the yeastSaccharomyces cerevisiae, the enzymes which catalyse the synthesis of ethyl acetate, ethyln‐hexanoate and isoamyl acetate were partly resolved from a fraction containing slowly sedimenting lipoproteins released during cell disruption with glass beads. Solubilization with detergents and fractionation by affinity chromatography have demonstrated the presence of at least three, and probably four, ester synthases which differ in their catalytic properties.

Isoamyl‐acetate synthase was solubilized and extensively purified to apparent homogeneity by successive chromatographies on various columns. On the basis of its specific activity in cell‐free extracts, the enzyme was purified 19000‐fold with a 5% activity yield. As judged by SDS/PAGE, it consists of a single polypeptide chain with a molecular mass of 57 ± 3 kDa and its apparent pIis 5.5. The enzyme acetylates isoamyl alcohol, ethanol and 12‐DL‐hydroxystearic acid from acetyl‐CoA but is unable to usen‐hexanoyl‐CoA as a cosubstrate. This enzyme, defined as an acetyl‐CoA: O‐alcohol acetyltransferase, could be the product of one of the anaerobically induced genes inS. cerevisiae.
The control of protein phosphatase‐1 by targetting subunits
Authors: ALESSI Dario , MACDOUGALL Lindsay K. , SOLA Maria M. , IKEBE Mitsui , COHEN Philip
Published: March 2005

The major protein phosphatase that dephosphorylates smooth‐muscle myosin was purified from chicken gizzard myofibrils and shown to be composed of three subunits with apparent molecular masses of 130, 37 and 20 kDa, the most likely structure being a heterotrimer. The 37‐kDa component was the catalytic subunit, while the 130‐kDa and 20‐kDa components formed a regulatory complex that enhanced catalytic subunit activity towards heavy meromyosin or the isolated myosin P light chain from smooth muscle and suppressed its activity towards phosphorylase, phosphorylase kinase and glycogen synthase. The catalytic subunit was identified as the β isoform of protein phosphatase‐1 (PP1) and the 130‐kDa subunit as the PP1‐binding component. The distinctive properties of smooth and skeletal muscle myosin phosphatases are explained by interaction of PP1β with different proteins and (in conjunction with earlier analysis of the glycogen‐associated phosphatase) establish that the specificity and subcellular location of PP1 is determined by its interaction with a number of specific targetting subunits.
Fatty‐acid‐binding protein in locust flight muscle
Authors: HAUNERLAND Norbert H. , ANDOLFATTO Peter , CHISHOLM Joan M. , WANG Zhixiang , CHEN Xinmei
Published: March 2005

Fatty‐acid‐binding protein (FABP) from the flight muscle of the locust, Schistocerca gregaria, is similar to mammalian heart FABP in its primary structure and biochemical characteristics. We have studied developmental changes using enzyme‐linked immunosorbent assays, RNA hybridization and electron microscopy of immunogold‐labeled sections. Locust muscle FABP is the most abundant soluble muscle protein in fully developed adult locusts, comprising 18% of the total cytosolic protein. At the beginning of the adult stage, however, no FABP is detectable. Its concentration rises during the following 10 days, after which it reaches its maximal value. FABP mRNA is present shortly after adult ecdysis; its concentration increases for 10 days, before it diminishes and reaches a constant, low level, probably needed to maintain the established FABP level. The protein is abundant in cytosol and nuclei, but virtually absent in mitochondria.
Dominant role of glucagon in the initial induction of phosphoenolpyruvate carboxykinase mRNA in cultured hepatocytes from fetal rats
Authors: PEGORIER Jean‐Paul , SALVADO Josepa , FORESTIER Marguerite , GIRARD Jean
Published: March 2005

The injection of streptozotocin to 18‐day‐old rat fetuses induced, 2 days later, a 50% fall in plasma insulin and a twofold increase in plasma glucagon concentrations and liver cAMP levels. Phosphoenolpyruvate carboxykinasc mRNA that were undetectable in the fetal rat liver, accumulated 48 h after streptozotocin injection, their concentration being 30% of that found in the liver of 1‐day‐old newborn rats in whom liver phosphoenolpyruvate carboxykinase gene expression is maximal. Physiological concentrations of glucagon (0.7 ± 0.2 nM) induced, within 2 h, phosphoenolpyruvate carboxykinase mRNA accumulation in cultured hepatocytes from 20‐day‐old fetuses. The addition of insulin (0.01 – 100 nM) inhibits, by no more than 30%, the glucagon‐induced phosphoenolpyruvate carboxykinase mRNA accumulation. Exposure of fetal hepatocytes to insulin for 24 h did not change the glucagon dose/response curve and did not lead to a more efficient inhibition of the glucagon‐induced phosphoenolpyruvate carboxykinase mRNA accumulation, despite a clear stimulatory effect on the rate of lipogenesis. In contrast, when hepatocytes were cutured in the presence of dexamethasone, the glucagon‐induced phosphoenolpyruvate carboxykinase mRNA accumulation can be totally inhibited by pharmacological concentrations of insulin (10 nM).

From these in‐vivo and in‐vitro studies, it is concluded that, under physiological conditions, the postnatal rise in plasma glucagon concentration is more important than the fall in the plasma insulin concentration for the primary induction of liver phosphoenolpyruvate carboxykinase gene expression.
Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli
Authors: FRANKEN Peet A. , VAN DEN BERG Lambertus , HUANG James , GUNYUZLU Paul , LUGTIGHEID Richard B. , VERHEIJ Hubertus M. , DE HAAS Gerad H.
Published: March 2005

Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine‐less mutant has been expressed as a Cro‐LacZ fusion protein in Escherichia coli, from which a pro‐PLA2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HP‐PLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HP‐PLA2 sequence. This protein was expressed in E. coli as a 68‐kDa Cro‐LacZ fusion protein. CNBr cleavage liberated the HP‐PLA2 fragment which was reoxidized in vitro. The [Met8→Leu]HP‐PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8. p‐Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2‐dioctanoyl‐sn‐glycero‐3‐phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2‐dioctanoyl‐sn‐glycero‐3‐phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol > phosphatidylethanolamine ≫ phosphatidylcholine. The enzyme has low activity on monomeric 1,2‐diheptanoyl‐sn‐glycero‐3‐phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP‐PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n‐hexadecylphosphocholine. The HP‐PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2. In mixed micelles of taurodeoxycholate and 1,2‐didodecanoyl‐sn‐glycero‐3‐phosphocholine, the competitive inhibition of HP‐PLA2 by the R and S enantiomers of 2‐tetradecanoylaminohexanol‐1‐phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine ≪ phosphoethanolamine < phosphoglycol. The best inhibitor, (R)‐2‐tetradecanoylaminohexanol‐1‐phosphoglycol, binds 2200 times stronger than the substrate to the HP‐PLA2 active site.
A comparative laser‐flash absorption spectroscopy study of algal plastocyanin and cytochrome c552 photooxidation by photosystem I particles from spinach
Authors: HERVÁS Manuel , DE LA ROSA Miguel A. , TOLLIN Gordon
Published: March 2005

Laser‐flash kinetic absorption spectroscopy has been used to compare the rate constants for electron transfer from reduced plastocyanin and cytochrome c552, obtained from the green alga Monoraphidium braunii, to photooxidized P700 (P700+) in photosystem I (PSI) particles from spinach. Sigmoidal protein concentration dependence for the observed electron‐transfer rate constants are obtained for both proteins. In the absence of added salts, the P700+ reduction rate increases as the pH decreases from approximately 8 to 5.5, then decreases to pH, 3.5, this effect being more pronounced with cytochrome c552 than with plastocyanin. At neutral pH, plastocyanin is a more efficient electron donor to P700+ than cytochrome c552, whereas at pH 5.5, which is closer to physiological conditions, the two redox proteins react with approximately equal rate constants. In the presence of increasing concentrations of added salts, the P700+ reduction rate constants for both proteins increase at pH >5.5, but decrease at pH <4. At neutral pH, the observed rate constants for both algal proteins have a biphasic dependence on sodium chloride concentration, increasing in a parallel manner with increasing salt concentration, reaching a maximum value at 50 mM NaCl. then decreasing. A similar biphasic dependence is obtained with magnesium chloride, but in this case the maximum value is reached at salt concentrations ten times smaller, suggesting a specific role for the divalent cations in the electron‐transfer reaction.
Expression of a pheromone‐binding protein in insect cells using a baculovirus vector
Authors: KRIEGER Jürgen , RAMING Klaus , PRESTWICH Glenn D. , FRITH David , STABEL Silvia , BREER Heinz
Published: March 2005

A cDNA encoding a pheromone‐binding protein from the male silkmoth Antheraea pernyi has been integrated into the genome of the Autographa californica multiple nuclear polyhydrosis virus such that the transcription was under the control of the strong polyhedrin promoter. Recombinant pheromone‐binding protein was expressed in a baculovirus‐infected insect cell line (Sf9) and secreted from the cells into the culture medium. Using a two‐step protocol, recombinant pheromone‐binding protein has been isolated and purified to homogeneity. Pheromone binding of recombinant protein has been demonstrated using a tritiated analog of (E,Z)‐6,11‐hexadecadienyl acetate.
Amphibian lutropin and follitropin from the bullfrog Rana catesbeiana
Authors: HAYASHI Hiroaki , HAYASHI Tomoko , HANAOKA Yoichi
Published: March 2005

The amino acid sequence of the α‐subunit of the gonadotropins, lutropin and follitropin from bullfrog, Rana catesbeiana, has been determined. The α‐subunit was identified in both hormones by the amino acid composition and ovulation activity of lutropin in the Xenopus ovary, by means of reconstituted hormones in various combinations. The amino acid sequences of two identical α‐subunits from lutropin and follitropin were determined or deduced by different strategies. The α‐subunit of those gonadotropins have 97 amino acid residues, the longest among the known α‐subunits of gonadotropins, and one arginine insertion at position 29. Ten cysteine residues and two sugar‐chain binding sites at Asn57 and Asn83 are completely conserved among the species. The molecular mass of this subunit is 11 026 Da not including the sugar chains. The bullfrog α‐subunit has approximately 70% sequence identity with mammalian α‐subunits.