bluuebunny/crossref_metadata_2025 huggingface dataset:
Affinity Labeling of Arginyl Residues at the Catalytic Region of Estradiol 17β‐Dehydrogenase from Human Placenta by 16‐Oxoestrone
Authors: INANO Hiroshi , TAMAOKI Bun‐ichi
Published: July 2010

16‐Oxoestrone inhibited competitively the activity of estradiol 17β‐dehydrogenase from human placenta against estradiol in phosphate buffer (pH 7.2), suggesting reversible binding of 16‐oxoestrone to the substrate‐binding site. 16‐Oxoestrone irreversible inactivated the estradiol 17β‐dehydrogenase in borate buffer (pH 8.5) in a time‐dependent manner, following pseudo‐first‐order kinetics. The rate constant (k3) obtained for the inactivation by 16‐oxoestrone was 8.3 × 10−4 s−1. The rate of inactivation was significantly decreased by addition of estrone, estradiol, estriol, NAD(H) and NADP+. Also, the rate was reduced markedly by 2′AMP, 5′ATP and 2′,5′ADP, but not by NMN(H) and 3‐pyridinealdehyde adeninediphospho nucleotide. The inactivation by 16‐oxoestrone was neither prevented by sodium azide nor influenced by light. From these data, 16‐oxoestrone, an χ‐dicarbonyl steroid, was suggested to inactive estradiol 17β‐dehydrogenase by modification of arginyl residues located around the substrate‐binding site of the enzyme. Biphasic inactivation of the enzyme by 16‐oxoestrone was observed with an increase of modified arginyl residues. The first phase of the inactivation was regarded as an affinity labeling of the arginyl residues at or near the substrate‐binding site of the enzyme. Stoichiometry of the inactivation indicated that two arginyl residues were essential for maintenance of the enzyme activity. The second phase was considered as chemical modification of the arginyl residues outside of the catalytic region of the enzyme.
Carbamoylphosphate Synthetase from Pseudomonas aeruginosa
Authors: ABDELAL Ahmed T. , BUSSEY Lee , VICKERS Leland
Published: July 2010

Carbamoylphosphate synthetase from Pseudomonas aeruginosa is subject to repression by pyrimidines and significant derepression by limitation of arginine or pyrimidines. Carbamoylphosphate synthetase was purified to homogeneity from a derepressed strain of P. aeruginosa. The molecular weight of the enzyme was estimated to be 168000 by sucrose gradient ultracentrifugation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme is composed of two non‐identical subunits with molecular weights of 122000 and 44000. Cross‐linking the enzyme prior to electrophoresis yielded an additional band corresponding to a molecular weight of 165000, showing that the enzyme is composed of one of each subunit. The enzyme utilized either glutamine (Km 0.15 mM) or NH3 (Km 17 mM) and requires free Mg2+ for maximal activity with the optimal level between 4 mM and 10 mM. Hill plots of MgATP saturation data yielded coefficients of 1.2 and 1.4 at pH 8.0 and 8.5, respectively. A Hill equation was derived on the assumptions that MgATP binds at the same time to two distinct sub‐sites as was shown to be the case for carbamoylphosphate synthetase from Escherichia coli and that these sub‐sites are strictly non‐interacting. The resulting theoretical Hill coefficients correspond very closely to the experimental coefficients. Carbamoylphosphate synthetase activity was subject to activation by ornithine and N‐acetylornithine and feedback inhibition by UMP. Carbamoylphosphate synthetase from P. aeruginosa does not associate under all conditions examined, establishing that self‐association does not play a role in regulation of enzyme activity as suggested by other workers for the enzyme from E. coli.
Excimer Formation of ATPase from Sarcoplasmic Reticulum Labeled with N‐(3‐Pyrene)maleinimide
Authors: LÜDI Hans , HASSELBACH Wilhelm
Published: July 2010

Sarcoplasmic reticulum ATPase from fast skeletal muscle was labeled in native vesicles with N‐(3‐pyrene) maleinimide. At labeling ratios larger than 1 mol pyrenemaleinimide/2.5 mol ATPase significant amounts of excimers are detected. Excimer concentration decreases at low, non‐solubilizing amounts of detergents (0.2 mg · mg protein−1) and completely disappears after solubilization of the membranes. These results exclude that excimers are formed due to ‘double‐labeling’ of one ATPase molecule. It is concluded that the ATPase exists as an oligomer within the membrane of native vesicles.
The Inhibition Pattern of Antibiotics on the Extent and Accuracy of tRNA Binding to the Ribosome, and Their Effect on the Subsequent Steps in Chain Elongation
Authors: WURMBACH Peter , NIERHAUS Knud H.
Published: July 2010

Ac[14C]Phe‐tRNA (cognate) and Ac[3H]Leu‐tRNA (miscognate) were bound as competing peptidyl‐tRNA analogues to tightly coupled ribosomes programmed with poly(U). The test system allows the determination of the accuracy of tRNA selection at the A and P site, as well as the measurement of the peptidyltransferase and translocation reactions in the presence of various antibiotics. The following results were obtained.

Cognate tRNA could be bound to either the A or P site with nearly 10% specificity. In contrast, miscognate tRNA could be exclusively bound to the P site but poorly to the A site.

The accuracy of tRNA selection (extent of binding) was sixfold higher at the A site than at the P site.

The extent of A site binding for both cognate and miscognate tRNA was severely inhibited by viomycin, tetracycline and neomycin, and to a lesser extent by streptomycin. No inhibition of binding at the P site was found with all antibiotics tested. This finding, and that of the preceding point, is taken as evidence that acetylated aminoacyl‐tRNA (an analogue for a peptidyl‐tRNA) has direct access to the P site and does not reach this site via a transient A site binding.

In addition to the known effects of streptomycin and neomycin on tRNA selection at the A site, viomycin was found to enhance drastically the error fraction at this site.

The peptidyltransferase is inhibited by the drugs in the following order: chloramphenicol > viomycin, neomycin > streptomycin. Equivalent results were found with cognate and miscognate tRNA. The translocation reaction was severely inhibited by viomycin and neomycin and to a lesser extent by streptomycin.

A striking correspondence was found between the error‐inducing capacity (distortion of codon‐anticodon interaction at the A site) and the inhibition of the translocation reaction by various drugs. This finding suggests that codon‐anticodon interaction at the ribosomal A site is an important if not essential prerequisite for the translocation reaction.
The Complete Amino Acid Sequence of the Zn2+‐Containing d‐Alanyl‐d‐Alanine‐Cleaving Carboxypeptidase of Streptomyces albus G
Authors: JORIS Bernard , VAN BEEUMEN Jozef , CASAGRANDE Fabiana , GERDAY Charles , FRÈRE Jean‐Marie , GHUYSEN Jean‐Marie
Published: July 2010

The 22076‐Mr Zn2+ ‐containing d‐alanyl‐d‐alanine‐cleaving carboxypeptidase of Streptomyces abuls G effectively catalyses the transfer of the Nα, Nɛ‐diacetyl‐l‐lysyl‐d‐alanyl electrophilic group of the standard tripeptide substrate Nα, Nɛ‐diacetyl‐l‐lysyl‐d‐alanyl‐d‐alanine to water. It also performs a weak β‐lactamase activity, hydrolysing penicillin into penicilloate at a very low rate. This protein consists of 212 amino acid residues in a single polypeptide chain. The N terminus is partially blocked as a result of the cyclization of the dipeptide Asn‐Gly into anhydroaspartylglycine imide. The protein has been fragmented by cyanogen bromide into five fragments whose sequences have been determined via appropriate subcleavages with various proteases. The ordering of the cyanogen bromide peptide fragments has been carried out (a) by submitting the S‐carboxymethylated protein to complete tryptic digestion and labelling the methionine‐containing peptides thus obtained with iodo[14C]‐acetamide, and (b) by submitting to limited tryptic digestion the S‐[2‐(4′‐pyridyl)ethyl]‐cysteine protein whose amino groups have been blocked by reaction with exo‐cis‐3,6‐endoxo‐Δ4‐tetrahydrophthalic anhydride prior to digestion. The protein contains six cysteine residues in the form of three disulfide bridges. No homology is found by comparing this peptidase with other Zn2+‐containing enzymes (carboxypeptidase A, thermolysin, carbonic anhydrase B and alcohol dehydrogenase) and several completely or partially sequenced, serine‐containing d‐alanyl‐d‐alanine‐cleaving peptidases and Zn2+/serine‐containing β‐lactamases.
Studies on Energy Supply for Genetic Processes
Authors: KALASAUSKAITÉ Elena V. , KADIŠAITÉ Diana L. , DAUGELAVIČIUS Rimas J. , GRINIUS Leonas L. , JASAITIS Antanas A.
Published: July 2010

In this study the hypothesis considering the requirement for an electrochemical proton gradient in the injection of phage T4 DNA into Escherichia coli cell has been verified experimentally. The phage caused a reversible depolarization of cell membrane, while phage ‘ghosts’ induced an irreversible depolarization. The phage infection was strictly dependent on E. coli membrane potential value when phage/cell ratio was 5 and higher. When the ratio was close to 1, the decrease in the membrane potential up to – 100 mV caused practically no effect on the phage infection. The infection inhibition was observed when the membrane potential was lowered below this ‘threshold’ value. On the other hand, the decrease in the membrane potential caused no effect on the phage infection under conditions promoting a concomitant increase in the value of the transmembranous pH gradient. The phage DNA transfer through the membrane of ATPase‐deficient cells was reversibly inhibited by switching off the respiratory chain – the sole generator of a protonmotive force in these mutant cells. The membrane should be kept in the energized state during the phage DNA entrance into the cell. Adsorption of the phage on E. coli was followed by the reversible release of the respiratory control. Thus the results presented here indicate the requirement of the electrochemical proton gradient across the plasma membrane for injection of phage T4 DNA into E. coli. They support the concept postulating an expenditure of host cell metabolic energy for phage T4 DNA transfer through the membrane.
Differential Effect of Tumor Promoters on Phorbol‐Ester‐Receptor Binding and Membrane Fluorescence Anisotropy in C3H 10 T 1/2 Cells
Authors: TRAN Phuong Lan , CASTAGNA Monique , SALA Maria , VASSENT Gerard , HOROWITZ Ann D. , SCHACHTER David , WEINSTEIN I. Bernard
Published: July 2010

The presence of high‐affinity membrane‐associated receptor sites for phorbol ester tumor promoters has been demonstrated with [3H]phorbol dibutyrate ([3H]phorbol Bt2) in the mouse embryo fibroblast cell line C3H 10 T1/2. Binding of [3H]phorbol Bt2 to intact cells occurs rapidly at 37°C and is also rapidly reversed upon addition of excess unlabeled phorbol Bt2. The dissociation can be resolved into two phases at 23°C. Scatchard analysis reveals a biphasic curve which is consistent with two classes of binding sites: a high‐affinity class with a dissociation constant of 16 nM and a total concentration of binding sites of 1.9 pmol/106 cells, and a low‐affinity class with a dissociation constant of 1000 nM and a total concentration of binding sites of 29.4 pmol/106 cells. Unlabeled phorbol Bt2, 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (Myrphobol Ac), and the indole alkaloid tumor promoter teleocidin are potent inhibitors of [3H]phorbol Bt2 binding, producing 50% inhibition at 98 nM, 38 nM and 23nM, respectively. On the other hand, two compounds that lack tumor‐promoting activity, 4α‐phorbol‐12,13‐didecanoate (4α‐phorbol Dec2) and 4‐O‐methyl‐Myrphorbol Ac, only slightly inhibited phorbol Bt2 binding. The effects of these compounds on the physical state of the membrane of intact C3H 10T 1/2 cells was also assessed using two different fluorescence lipophilic probes. Phorbol Bt2. Myrphorbol Ac and teleocidin, but not 4α‐phorbol Dec2, induce significant decreases in the fluorescence anisotropy parameter obtained with both of the probes. However, the dose response curves for these effects are quite different from those obtained when the same compounds were tested for inhibition of [3H]phorbol Bt2 binding. This discrepancy suggests that although C3H 10T 1/2 cells contain an abundance of highaffinity receptor sites for phorbol ester and related tumor promoters, some of the membrane effects of these compounds may be mediated by direct binding of the tumor promoter to lipid domains in cell membrane.
Structure of mouse DNA (cytosine‐5‐)‐methyltransferase
Authors: SPIESS Eberhard , TOMASSETTI Antonella , HERNAIZ‐DRIEVER Pablo , PFEIFER Gerd P.
Published: July 2010

DNA (cytosine‐5‐)‐methyltransferase was purified as a single polypeptide (190 kDa by SDS‐PAGE) from mouse P815 mastocytoma cells. This enzyme transfers methyl groups to unmethylated as well as to hemimethylated DNA sites with a strong preference for the hemimethylated substrate. A structural analysis of the isolated enzyme by electron microscopical techniques was undertaken. On the basis of the results obtained, we propose a model for the enzyme structure. This model describes the enzyme as a hemi‐elliptical globular structure with dimensions of 5.4–6.7 nm for the height h and 10.3–10.8 nm for the diameter d, respectively; this globular structure bears a small appendix at the flat side. A molecular mass of 235–250 kDa is calculated from the measured dimensions. Limited trypsin digestion of the enzyme led to a 160‐kDa fragment which preserved the gross morphology of the original material. The possible structure function relationships are discussed.
Kinetics of calcium binding to calbindin mutants
Authors: FORSÉN Sture , LINSE Sara , THULIN Eva , LINDEGÅRD Boel , MARTIN Stephen R. , BAYLEY Peter M. , BRODIN Peter , GRUNDSTRÖM Thomas
Published: July 2010

The kinetics of calcium dissociation from wild‐type bovine calbindin D9k (the smallest protein known with a pair of EF‐hand calcium‐binding sites) and five mutants with single amino‐acid substitutions and/or deletions has been studied by stopped‐flow fluorescence methods, using the calcium chelator Quin 2. The modifications are confined to the N‐terminal half of the molecule, at or near the first calcium‐binding site (I). Substitutions and deletions of amino acids in the calcium‐binding loop of site I primarily affect the rate of Ca2+ dissociation from this site with only minor effects on the dynamic properties of the C‐terminal calcium‐binding site (II). This finding corroborates and extends previous kinetic results obtained from 43Ca‐NMR studies on the same set of mutants. By contrast, removal of the hydrogen bond between Tyr‐13 and Glu‐35, an interaction linking the two α‐helices flanking site I, through replacement of Tyr‐13 with Phe, has no observable effect on the rate of Ca2+ dissociation from the protein.

Comparison of this kinetic data with binding‐constant data, previously obtained in our laboratories, shows that the decrease in Ca2+‐affinity of site I, observed in most mutants, is predominantly due to an increased off‐rate from this site. At low ionic strengths the second‐order rate constants for Ca2+‐binding to both Ca2+ sites of calbindin D9k are calculated to be of the order of 109 M−1 s−1 for all proteins studied. At higher ionic strengths (0.1 M KCl) the rates of Ca2+ dissociation from both sites are increased by a factor of three or more, suggesting a transition state which is ionic in nature.
Mutants with base changes at the 3′‐end of the 16S RNA from Escherichia coli
Authors: ROTTMANN Norbert , KLEUVERS Barbara , ATMADJA Johannes , WAGNER Rolf
Published: July 2010

The functionally important 3′ domain of the ribosomal 16S RNA was altered by in vitro DNA manipulations of a plasmid‐encoded 16S RNA gene. By in vitro DNA manipulations two double mutants were constructed in which C1399 was converted to A and G1401 was changed to either U or C and a single point mutant was made wherein G1416 was changed to U.

Only one of the mutated rRNA genes could be cloned in a plasmid under the control of the natural rrnB promoters (U1416) whereas all three mutations were cloned in a plasmid under the control of the λ PL promoter. In a strain coding for the temperature‐sensitive λ repressor cI857 the mutant RNAs could be expressed conditionally.

We could show that all three mutant rRNAs were efficiently incorporated into 30S ribosomes. However, all three mutants inhibited the formation of stable 70S particles to various degrees.

The amounts of mutated rRNAs were quantified by primer extension analysis which enabled us to assess the proportion of the mutated ribosomes which are actively engaged in in vivo protein biosynthesis. While ribosomes carrying the U1416 mutation in the 16S RNA were active in vivo a strong selection against ribosomes with the A1399/U1401 mutation in the 16S RNA from the polysome fraction is apparent. Ribosomes with 16S RNA bearing the A1399/C1401 mutation did not show a measurable protein biosynthesis activity in vivo. The growth rate of cells harbouring the different mutations reflected the in vivo translation capacities of the mutant ribosomes. The results underline the importance of the highly conserved nucleotides in the 3′ domain of the 16S RNA for ribosomal function.
Viscosity‐dependent energy barriers and equilibrium conformational fluctuations in oxygen recombination with hemerythrin
Authors: LAVALETTE Daniel , TETREAU Catherine
Published: July 2010

The recombination kinetics of photo‐dissociated oxyhemerythrin (Sipunculus nudus) have been investigated between 298 K and 90 K. Fast geminate recombinations compete with oxygen escape into the solvent, from which a subsequent slower bimolecular rebinding takes place. In phosphate buffer (pH 7.7) at 278 K, the fast and slow processes are exponential and have comparable amplitudes. Whereas the oxygen escape rate rapidly decreases upon increasing the viscosity, the inward rate from the solvent is found to be independent of viscosity, up to about 50 cP (50 mPa · s). The data suggest that a Brownian‐motion‐driven displacement of one or several side‐chain residues is implied in oxygen escape from within the protein and also that hemerythrin undergoes a conformational change in the deoxy state.

At higher viscosities and lower temperature only the geminate phase is observed and the kinetics progressively depart from an exponential. Below about 130 K, the kinetics resemble those reported in the literature for heme proteins. They are consistent with a temperature‐independent non‐equilibrium frozen distribution of conformational substates. However, between 190 K and 130 K, the profile of the kinetics is invariant on a log/log plot and the results simply differ by a translation along the log t axis. It is shown that this property is expected only for a temperature‐dependent distribution of substates in a Boltzmann equilibrium. From room temperature, where rebinding is exponential, down to the ‘freezing’ temperature, the geminate recombinations display a variety of kinetic laws. It can be shown, however, that for a broad class of substate distributions, the initial slope of the kinetic plot follows an Arrhenius relationship. The activation energy is equal to that of the exponential rate constant measured at high temperature. This result establishes the conditions under which protein data obtained from low‐temperature kinetics can be extrapolated to physiological temperature.
Microheterogeneity and structures of neutral glycans present in quail ovomucoid
Authors: ALONSO Josefa María , BOULENGUER Patrick , WIERUSZESKI Jean‐Michel , LEROY Yves , MONTREUIL Jean , FOURNET Bernard
Published: July 2010

Anionic chromatography of oligosaccharide‐alditols obtained by alkaline hydrolysis of quail ovomucoid glycopeptides resulted in the isolation of a major neutral fraction (83%) which was separated into 14 fractions by high‐performance liquid chromatography on an alkylamine silica column. The structures of nine oligosaccharide‐alditols were determined. By the identification of mannobiosido‐, and mannotriosido‐di‐N‐acetylchitobiose mixed with N‐acetyl‐lactosaminic and oligomannosidic‐types oligosaccharides, we demonstrate the high microheterogeneity of the glycans presents in the neutral fraction of this glycoprotein and suggest that ovomucoid glycosylation uses several pathways.
Signal‐Transduction Therapy
Authors: Levitzki Alexander
Published: July 2010

In the past decade it has become apparent that many diseases result from aberrations in signaling pathways. These include proliferative diseases such as cancers, atherosclerosis and psoriasis and inflammatory conditions such as sepsis, rheumatoid arthritis and tissue rejection. These findings refocused the research of the medical community to seek new modalities for disease management which essentially consist of designing drugs which intercept cell signaling. In this review, the emerging success in using tyrosine kinase blockers and other signal interceptors, such as protein kinase C blockers, Ras blockers, Ca2+signaling inhibitors and estrogen antagonists which inhibit growth of cancer cellsin vitroandin vivo, will be discussed. These signal interceptors, especially tyrosine‐kinase blockers, are also able to block inflammatory responses and the proliferation of vascular smooth muscle cells and psoriatic keratinocytes. The utility of signal interceptors in analyzing signal‐transduction pathways is also discussed.
A Super‐Family of Medium‐Chain Dehydrogenases/Reductases (MDR)
Authors: Persson Bengt , Zigler J. Samuel , Jörnvall Hans
Published: July 2010

The protein super‐family of medium‐chain alcohol dehydrogenases (and glutathione‐dependent formaldehyde dehydrogenase), polyol dehydrogenases, threonine dehydrogenase, archaeon glucose dehydrogenase, and eye lens reductase‐active ζ‐crystallins also includes Escherichia coli quinone oxidoreductase, Torpedo VAT‐1 protein, and enoyl reductases of mammalian fatty acid and yeast erythronolide synthases. In addition, two proteins with hitherto unknown function are shown to belong to this super‐family of medium‐chain dehydrogenases and reductases (MDR). Alignment of ζ‐crystallins/quinone oxidoreductases/VAT‐1 reveals 38 strictly conserved residues, of which approximately half are glycine residues, including those at several space‐restricted turn positions and critical coenzyme‐binding positions in the alcohol dehydrogenases. This indicates a conserved three‐dimensional structure at the corresponding parts of these distantly related proteins and a conserved binding of a coenzyme in the two proteins with hitherto unknown function, thus ascribing a likely oxidoreductase function to these proteins. When all forms are aligned, including enoyl reductases, a ζ‐crystallin homologue from Leishmania and the two proteins with hitherto unknown function, only three residues are strictly conserved among the 106 proteins characterised within the super‐family, and significantly these residues are all glycines, corresponding to Gly66, Gly86 and Gly201 of mammalian class I alcohol dehydrogenase. Notably, these residues are located in different domains. Hence, a distant origin and divergent functions, but related forms and interactions, appear to apply to the entire chains of the many prokarotic and eukaryotic members. Additionally, in the ζ‐crystallins/quinone oxidoreductases, a highly conserved tyrosine residue is found. This residue, in the three‐dimensional structure of the homologous alcohol dehydrogenase, is positioned at the sub‐unit cleft that contains the active site and could therefore be involved in catalysis. If so, this residue and its role may resemble the pattern of a conserved tyrosine residue in the different family of short‐chain dehydrogenases/reductases (SDR).
Location of the Two Genes Encoding Glutaconate Coenzyme A‐Transferase at the Beginning of the Hydroxyglutarate Operon in Acidaminococcus fermentans
Authors: Mack Matthias , Bendrat Klaus , Zelder Oskar , Eckel Elke , Linder Dietmar , Buckel Wolfgang
Published: July 2010

Glutaconate coenzyme A‐transferase (Gct) from Acidaminococcus fermentans consists of two subunits (GctA, 35725 Da and GctB, 29168 Da). The N‐termini sequences of both subunits were determined. DNA sequencing of a subgenomic fragment of A. fermentans revealed that the genes encoding glutaconate CoA‐transferase (gctAB) are located upstream of a gene cluster formed by gcdA, hgdC, hgdA and hgdB in this order. Further upstream of gctA, a DNA sequence was detected showing significant similarities to σ70‐type promoters from Escherichia coli. Primer‐extension analysis revealed that this specific DNA sequence was indeed the location of transcription initiation in A. fermentans. The entire gene cluster, 7.3 kb in length, comprising gctAB, gcdA and hgdCAB, has tentatively been named the hydroxyglutarate operon, since the enzymes encoded by these genes are involved in the conversion of (R)‐2‐hydroxyglutarate to crotonyl‐CoA in the pathway of glutamate fermentation by A. fermentuns. The genes gctAB were expressed together in E. coli. Cell‐free extracts of a transformant E. coli strain contained glutaconate CoA‐transferase at a specific activity of up to 30 U/mg protein. The recombinant enzyme was purified to homogeneity with a specific activity of 130 U/mg protein by ammonium sulfate fractionation and crystallisation. The amino acid residue directly involved in catalysis was tentatively identified as E54 of the small subunit of the enzyme (GctB).
Posttranslational Processing of Human α2‐HS Glycoprotein (Human Fetuin)
Authors: Jahnen‐Dechent Willi , Trindl Andreas , Godovac‐Zimmermann Jasminka , Müller‐Esterl Werner
Published: July 2010

α2‐HS glycoprotein (α2‐HS) is a major protein occuring in human blood and calciferous tissues. Due to extensive sequence identity, α2‐HS has been grouped with the fetuins, a family of proteins that occur in fetal plasma in high concentrations. Native α2‐HS undergoes a series of posttranslational modifications including proteolytic processing, multiple N‐glycosylations and O‐glycosylations, and sulfation of the carbohydrate side chains. Various two‐chain forms of α2‐HS have been prepared from human plasma, however, the single‐chain precursor has not yet been isolated. Here, we have studied the biosynthesis of α2‐HS by a human hepatoma cell line, HepG2. We demonstrate that a single‐chain form and the two‐chain form of α2‐HS are secreted by this cell line. The α2‐HS forms are further modified by phosporylation on multiple serine residues. Mapping studies indicate that the connecting peptide region releasable from the heavy chain of α2‐HS contains at least one such Phosphorylation site. Our results identify proteolytic trimming and/or phosphorylation as modifications possibly regulating the biological effects of α2‐HS and the homologous fetuins.
A Kinetic Study on Pantetheinase Inhibition by Disulfides
Authors: Pitari Giuseppina , Maurizi Giovanni , Ascenzi Paolo , Ricci Giorgio , Duprè Silvestro
Published: July 2010

The mammalian enzyme pantetheinase, which hydrolyzes pantetheine to pantothenic acid and cysteamine, is inhibited by many thiol reagents and activated by thiols. Two thiol groups of different reactivity and accessibility are involved in the catalytic process [Ricci, G., Nardini, M., Chiaraluce, R., Duprè, S. & Cavallini, D. (1986) Biochim. Biophys. Acta 870, 82–91]. The inhibition kinetics by some natural and synthetic disulfides [pantethine, cystamine, 5,5′‐dithiobis(2‐nitrobenzoic acid), 4,4′‐dithiodipyridine and oxidized mercaptoethanol] has been studied by two experimental approaches, either by monitoring activity after incubation of the enzyme with the inhibitor or by determining the progress curves in the presence of substrate and inhibitor. Data reported here indicate that pantetheinase reacts irreversibly with various disulfides in a time‐dependent manner with the formation of a mixed disulfide apparently preceeded by a conformational change, giving a modified E* form with new kinetic parameters. This modified form may be further competitively inhibited by disulfides interacting with the enzyme at the active site.
Differential Utilization of Alternate Initiation Sites in a Plant Defense Gene Responding to Environmental Stimuli
Authors: Woo Lee Shin , Heinz Ruth , Robb Jane , Nazar Ross N.
Published: July 2010

The upstream regulatory region of an efficiently expressed phenylalanine ammonia‐lyase (PAL5) gene from tomato (Lycopersicon esculentum) was characterized by DNA sequencing and S1‐nuclease‐protection studies. Two transcription initiation sites giving rise to a long and a short transcript were identified, each corresponding to a different putative TATA sequence element with multiple conserved upstream regulatory motifs. When tomato plants were challenged with three different environmental stimuli, wounding, light and Verticillium infection, this gene was observed to utilize the alternate initiation sites differentially, in a tissue‐dependent and stimulus‐dependent manner. In general, basal levels of the shorter transcript were very low but preferentially and strongly stimulated by the environmental factors. In contrast, the longer transcript was less affected, essentially representing a constitutive expression of this gene. The results suggest that the multiple regulatory sites in the PAL5 gene promoter provide for both an essential constitutive expression of phenylalanine ammonia‐lyase as well as an appropriate response to different environmental stresses.
The Solution Conformation of a Trisdecanucleotide Containing the Consensus Binding Site of the dnaA Initiation Protein
Authors: Gray Barry N. , Owen Elisabeth A. , Keniry Max A.
Published: July 2010

The solution structure of a trisdecanucleotide, d(CCTGTGGATAACA) · d(TGTTATCCACAGG) containing the consensus binding site of the dnaA initiation protein has been determined by two‐dimensional NMR techniques and restrained molecular dynamics calculations. Interproton distances were obtained by an iterative complete relaxation matrix algorithm, MARDIGRAS. During molecular dynamics runs, the backbone was restricted with the assistance of experimentally derived distance constraints. A family of refined structures with small pairwise root‐mean‐square deviation values (≈0.08 nm) was obtained. All but one of the pyrimidines were found to adopt the C1′‐exo conformation while the purines were found to adopt the C2′‐endo or C1′‐exo conformation. The six‐membered rings of the purines were found to stack over the six‐membered rings of the pyrimidines while there is virtually no overlap of the pyrimidines over the purines. 5′‐purine‐purine‐3′ and 5′‐pyrimidine‐pyrimidine‐3′ stacking resembles the observed stacking of these bases in other NMR and X‐ray structures of oligonucleotides. The final refined structure exhibited a small curvature and was slightly longer than canonical B‐DNA. The variation of twist angle, proposed as a recognition element for proteins, exhibited symmetry about the centre of the consensus binding site.
Mutational Studies on the α‐Sarcin Loop of Escherichia coli 23S Ribosomal RNA
Authors: Marchant Alan , Hartley Martin R.
Published: July 2010

The α‐sarcin loop, located in domain VI of Escherichia coli 23S rRNA, is a universally conserved sequence involved in the binding of elongation factors to the ribosome and is the site of action of ribosome‐inactivating proteins. Six mutations were created in this loop with the aim of establishing whether the mutant 23S rRNA could be assembled into functional ribosomes. In order to distinguish between plasmid‐derived (mutant) and chromosome‐derived (wild‐type) 23S rRNAs, an oligonucleotide tag sequence was introduced into the plasmid‐borne 23S rRNA gene. The tag sequence had no apparent effect on ribosome assembly or function. Two of the bases mutated (at positions A2660 and G2661) have been implicated in the binding of both elongation factor Tu and elongation factor G to the ribosome [Moazed, D., Robertson, J. M. & Noller, H. F. (1988) Nature 334, 362–364]. A further two bases (at positions C2658 and G2663) have been proposed to form a Watson‐Crick base pair involved in the formation of a tetraloop structure required for ribosome function [Szewczak, A. A., Moore, P. B., Chan, Y.‐L. & Wool, I. G. (1993) Proc. Natl Acad. Sci USA 90, 9581–9585]. It is inferred that the identity of the bases at positions 2658 and 2663 are of critical importance for ribosome structure and function, and that this function cannot be restored by a second mutation which potentially restores a Watson‐Crick base pair, but with reversed position. Of five single mutants (each mutant containing one of the mutations C2658G, A2660G, G2661A, G2663C and G2664C) and one double mutant (containing both mutations C2658G and G2663C) only the two mutants with the single mutations G2661A and G2664C were incorporated into ribosomes at a level comparable to that of 23S rRNA expressed from a wild‐type plasmid. However, the G2664C mutation resulted in a decrease in growth rate and a gradual loss of viability. rRNAs containing the G2663C single mutation and the C2658G and G2663C double mutation showed reduced incorporation into 50S subunits and these did not enter into ribosome couples.
A Common System Controls the Induction of Very Different Genes
Authors: Datz Martina , Joris Bernard , Azab Essam A. M. , Galleni Moreno , Van Beeumen Jozef , Frère Jean‐Marie , Martin Hans H.
Published: July 2010

Among the Enterobacteriaceae, Proteus vulgaris is exceptional in the inducible production of a 29‐kDa β‐lactamase (cefuroximase) with an unusually high activity towards the β‐lactamase‐stable oximino‐cephalosporins (e.g. cefuroxime and cefotaxime). Sequencing of the corresponding gene, cumA, showed that the derived CumA β‐lactamase belonged to the molecular class A. The structural gene was under the direct control of gene cumR, which was transcribed backwards and whose initiation codon was 165 bp away from that of the β‐lactamase gene. This resembled the arrangement of structural and regulator genes ampC and ampR of the 39‐kDa molecular‐class‐C β‐lactamase AmpC present in many enterobacteria. Moreover, cloned genes ampD and ampG for negative modulation and signal transduction of AmpC β‐lactamase induction, respectively, were also able to restore constitutively CumA overproducing and non‐inducible P. vulgaris mutants to the inducible, wild‐type phenotype. The results indicate that controls of the induction phenomena are equivalent for the CumA and AmpC β‐lactamase. Very different structural genes can thus be under the control of identical systems.
Characteristics of the Paramagnetic 1H‐NMR Spectra of the Ferricytochrome c‐551 Family
Authors: Timkovich Russell , Cai Mengli , Zhang Bali , Arciero David M. , Hooper Alan B.
Published: July 2010

Heme proton resonances have been assigned for ferricytochromes c‐551 isolated from four distinct species of bacteria. While the available structure information indicates that the four cytochromes have very similar conformations in solution, including the chirality of the methionine ligand sulfur bond, the chemical shifts of the paramagnetically shifted resonances are surprisingly different, more so than has been previously reported for a homologous series of ferricytochromes. The resonances are contrasted in terms of chemical shift and the temperature dependence of the shift, which gives rise to a very strong anti‐Curie effect for some specific protons. Non‐methyl heme resonances do display an approximately conserved set of chemical shifts, but the heme methyl groups demonstrate a wide range of values. The 121 heme methyl group is always the highest frequency heme methyl, but the relative positions of the other methyl groups may change. The 71 heme methyl group always displayed strong anti‐Curie behavior, while the 121 methyl group displayed normal Curie behavior. The behavior of the other methyl groups was variable. Possible reasons for the range of observations will be discussed. In spite of their NMR differences, all the ferricytochromes c‐551 demonstrated comparable electron‐transfer rates to a membrane‐bound cytochrome reductase system.
Purification and Characterization of the 30S Ribosomal Proteins from the Bacterium Thermus thermophilus
Authors: Tsiboli Paraskevi , Herfurth Elke , Choli Theodora
Published: July 2010

The total protein mixture of the 30s subunit (TP‐30) of the bacterium Thermus thermophilus has been purified using reverse‐phase HPLC and the proteins obtained were identified both by means of two‐dimensional polyacrylamide gel electrophoresis as well as by amino‐terminal amino acid microsequence analysis. The proteins are numbered according to their primary structural similarity with known prokaryotic ribosomal proteins. Eight of them, namely proteins S6, S7, S9, S10, S14, S15, S16 and S17 run at different positions in the two‐dimensional gel electrophoresis system to those suggested [Sedelnikova, S. C., Agalarov, M. B., Garber, M. & Yusupov, M. M. (1987) FEBS Lett. 220, 227–230]. All characterized proteins are homologous to known ribosomal proteins from other species, except for a small basic protein which shows homology only to a ribosomal protein from spinach chloroplasts [Choli, T., Franceschi, F., Yonath, A. & Wittmann‐Liebold, B. (1993) Biol. Chem. Hoppe‐Seyler 374, 377–383; Subramanian, A.‐R. (1984) Trends Biochem. Sci. 9, 491–494].
Insulin‐Like Growth Factor‐II is a Substrate for Dipeptidylpeptidase I (Cathepsin C)
Authors: Kiess Wieland , Terry Cheryl , Burgess Wilson H. , Linder Barbara , Lopaczynski Wlodzimierz , Nissley Peter
Published: July 2010

We observed that the lysosomal enzyme, dipeptidylaminopeptidase I (DAP‐I) caused the release of trichloroacetic‐acid‐soluble radioactivity from rat 125I‐insulin‐like growth factor‐II (IGF‐II). This activity could be blocked by dipeptide inhibitors of DAP‐I, and was enhanced by chloride. Treatment of unlabeled rat IGF‐II with DAP‐I converted approximately 50% of the IGF‐II to a species with a slightly shorter elution time on reverse‐phase HPLC, whereas treatment of human IGF‐II caused complete conversion to the species with the shorter elution time. Rat IGF‐II purified from the rat BRL 3A cell line is a mixture of two molecules beginning with Ala‐Tyr‐Arg‐Pro‐Ser‐ and Tyr‐Arg‐Pro‐Ser‐ [Marquardt, H., Todaro, G. J., Henderson, L. E. & Oroszlan, S. (1981) J. Biol. Chem. 256, 6859–6865] while human IGF‐II begins with Ala‐Tyr‐Arg‐Pro‐Ser‐. Determination of the N‐terminal amino acid sequence of human IGF‐II before and after digestion with DAP‐I showed that DAP‐I cleaved Ala‐Tyr, terminating at Arg‐Pro‐; the rat IGF‐II species beginning with Tyr‐Arg‐Pro‐Ser‐ was resistant to digestion. In order to compare DAP‐I‐treated IGF‐II with native IGF‐II for binding to IGF receptors and IGF‐binding proteins and in a bioassay, rat and human IGF‐II were treated with DAP‐I and the digested and undigested species were isolated by reverse‐phase HPLC. The IGF‐II/mannose 6‐phosphate receptor was purified from rat placental membranes, the IGF‐I receptor was solubilized from human placental membranes and IGF‐binding proteins were partially purified from adult and three‐day‐old rat sera by sequential gel filtration on Sephadex G‐200 (pH 8.0) and Sephadex G‐50 (acid pH). The dose/response curves of the two IGF‐II species were indistinguishable in radioreceptor assays utilizing the IGF‐II/mannose 6‐phosphate receptor and the IGF‐I receptor and in IGF competitive binding assays utilizing partially purified IGF‐binding proteins. The DAP‐I‐digested and native IGF‐II species were also equipotent in stimulating [3H]thymidine incorporation into DNA in the human osteosarcoma cell line, MG‐63. We conclude that DAP‐I cleaves an N‐terminal dipeptide from IGF‐II and that this does not result in a change in the biological activity of the molecule.
Purification and Characterization of Coenzyme F390 Synthetase from Methanobacterium thermoautotrophicum (strain H)
Authors: Vermeij Paul , Detmers Frank J. M. , Broers Frank J. M. , Keltjens Jan T. , Van Der Drift Chris
Published: July 2010

Coenzyme F390 synthetase catalyzes the formation of 8‐hydroxyadenylylated‐coenzyme F420 (coenzyme F390‐A) from coenzyme F420 and ATP in some methanogenic Archaea. The presence of coenzyme F390 was found when these organisms were exposed to oxygen. To get more insight into the defined function of coenzyme F390, the coenzyme F390 synthetase from Methanobacterium thermoautrophicum was purified from a cell‐free extract and its catalytic properties were determined. The synthetase was purified 150‐fold to a specific activity of 0.45 μmol · mim−1· mg protein−1. The enzyme consisted of one polypeptide of approximately 51 kDa. The isolated enzyme showed a tendency to aggregate into dimers and tetramers upon concentration. Co‐elution during purification of GTP‐dependent coenzyme F390 synthetase activity suggested that the synthetase is also capable of 8‐hydroxyguanylylated‐coenzyme F420, (coenzyme F390‐G) formation.

Initial‐velocity measurements of the two‐substrate reaction showed that the enzyme kinetics for the coenzyme F390 synthetase reaction proceeded by a ternary‐complex mechanism. The coenzyme F390 synthetase displayed a Km for coenzyme F420 of 39 μM and a Km for ATP of 1.7 mM.

In contrast to the enzyme in the cell‐free extract, the isolated enzyme was active under aerobic and anaerobic conditions. Treatment with air was not required to obtain the enzyme in an active form. However, 1,5‐dihydro‐coenzyme F420 (coenzyme F420H2) appeared to be a potent competitive inhibitor (Ki 3 μM) with respect to coenzyme F420. The latter findings may explain why the enzyme could only be detected in crude extracts that had been exposed to air, i.e. treatment with air causes the oxidation of reduced coenzyme F420 present in anaerobic extracts. The results of this study are discussed in view of the proposed role for coenzyme F390 in methanogenic metabolism.
The Short and Long Forms of the α Subunit of the Stimulatory Guanine‐Nucleotide‐Binding Protein are Unequally Redistributed During (–)‐Isoproterenol‐Mediated Desensitization of Intact S49 Lymphoma Cells
Authors: Kvapil Petr , Novotny Jiri , Svoboda Petr , Ransnäs Lennart A.
Published: July 2010

We report here that desensitization of the β‐adrenergic receptor‐triggered transmembrane signalling in S49 wild‐type lymphoma cells, induced by (–)‐isoproterenol (1 μM), results in unequal intracellular redistribution of the splicing variants of the α subunit of the stimulatory guanine‐nucleotide‐binding regulatory (Gsαprotein (Gsα‐shortand Gsα‐long) and alters the functional characteristics of the membrane‐associated signal transduction complex. We found that two cellular pools of membranes, light‐density membranes and plasma membranes prepared by sucrose‐density‐gradient centrifugation of cell homogenates differed in their content of Gsαsplicing subforms and, moreover, that prolonged activation of the β‐adrenergic pathway induced intermembrane redistribution of the splicing variants of Gsα. Short (10 min) as well as prolonged (1 h) (–)‐isoproterenol treatment of the cells shifted Gsα‐shortfrom light‐density membranes to plasma membranes and increased the total amount of light‐density membrane‐bound Gsα‐long; in parallel, the maximal (–)‐isoproterenol‐stimulated or AlF−4‐stimulated adenylyl cyclase activities measured in the plasma membrane pools prepared from treated cells decreased. The functional characteristics of the membrane‐bound Gsαpools were examined by a cyc−‐reconstitutive adenylyl cyclase assay where extracts of the plasma membrane and light‐density‐membrane pools, respectively, were mixed with plasma membranes derived from the mutant S49 cell line, cyc−, lacking Gsα. The maximal cyc−‐reconstitutive activities of the extracts prepared from light‐density membranes of short‐term as well as long‐term desensitized cells increased compared to control cells. These findings may indicate differences in the functioning of the splicing variants of Gsα.
Site‐Directed Mutagenesis Clarifies the Substrate Position within the Three‐Dimensional Model of the Active Site of Herpes Simplex Virus Type‐1 Thymidine Kinase
Authors: Michael Martina , Fetzer Jürgen , Folkers Gerd
Published: July 2010

Site‐directed mutagenesis was used to experimentally verify the 3D model of the active site of herpes simplex virus type‐1 thymidine kinase (HSV 1 TK) obtained by homology modelling. For this purpose, D215 and K317 were replaced by R and G, respectively, at homologous positions in the aciclovir‐insensitive bovine herpes virus type‐1 thymidine kinase (BHV 1 TK). Wild‐type and mutated enzymes were expressed in Escherichia coli using a gene fusion vector and purified to homogeneity. While both mutants had the same Km value for thymidine as the recombinant wild‐type enzyme (0.2 μM), Vmax was decreased to 20–25% of the original wild‐type value. The recombinant wild‐type enzyme was inhibited by the substrate analogue aciclovir with a Ki of 146 μM. Both mutants were able to phosphorylate aciclovir to about the same extent as the wild‐type enzyme. These findings suggest that neither D215 nor K317 are directly involved in substrate binding. Therefore, a rearrangement of the 3D model is suggested, concerning the assignment of the substrate‐binding site and co‐substrate‐binding site at the right and left side of the phosphate‐binding loop, respectively.
Evolution of Gene Regulation as Revealed by Differential Regulation of the Chicken Lysozyme Transgene and the Endogenous Mouse Lysozyme Gene in Mouse Macrophages
Authors: Bonifer Constanze , Bosch Franz X. , Faust Nicole , Schuhmann Antje , Sippel Albrecht E.
Published: July 2010

Lysozyme gene expression is a marker for macrophage differentiation in vertebrates. We have previously shown that expression of the complete chicken lysozyme gene domain in macrophages of transgenic mice is directly correlated to the copy number of integrated transgenes. Thus, the chicken lysozyme locus in the mouse acts as an independent regulatory unit irrespective of its random position in the host genome. This finding allowed a comparative analysis of the regulation of the endogenous mouse lysozyme M gene and the chicken lysozyme transgene in the same animal. We demonstrate by transcript analysis of total tissue RNA and by in situ hybridization, that both genes are expressed in macrophages. In macrophages of the same animal the regulation of both genes in response to external signals was distinctly different: the lysozyme transgene responded to various agents influencing macrophage activation, in contrast, mouse lysozyme RNA levels remained unchanged under these conditions. Thus, as in chicken macrophages, the chicken lysozyme expression level in mouse macrophages is coupled to the macrophage activation status, while the mouse lysozyme is not. Our results suggest, that the cis ‐regulatory elements of lysozyme genes have evolved more rapidly than the function and expression of the trans ‐acting factors involved in the regulation of macrophage‐specific gene activation.
Isolation and Characterisation of Subcomplexes of the Mitochondrial NADH: Ubiquinone Oxidoreductase (Complex I)
Authors: Finel Moshe , Majander Anna S. , Tyynelä Jaana , De Jong A. Mariette P. , Albracht Simon P. J. , Wikström Måtin
Published: July 2010

Enzymically active subcomplexes were purified from bovine mitochondrial NADH: ubiquinone oxidoreductase (complex I) by sucrose‐gradient centrifugation in the presence of detergents. These subcomplexes, named Iλ, IS, and IλS, catalyse ferricyanide and ubiquinone‐1 (Q‐1) reduction by NADH at similar rates to complex I, but do not catalyse the reduction of decylubiquinone. In addition, the Q‐1 reductase activity of all the subcomplexes is insensitive to rotenone. Chemical and EPR analyses of the subcomplexes show that FMN and all the Fe‐S clusters of complex I are present, but that the line shape of cluster 2 is modified. The smallest subcomplex, IλS, contains only approximately 13 subunits, as compared to approximately 22 in the previously described sub‐complex Iα [Finel, M., Skehel, J. M., Albracht, S. J. P., Fearnley, I. M. & Walker, J. E. (1992) Biochemistry 31, 11425–11434], but it retains the 75, 51‐, 49‐, 30‐, 24‐, 23‐ (TYKY) and 20‐kDa (PSST) subunits, which are suggested to form a functional core that comprises the EPR‐detectable Fe‐S clusters 1–4, and FMN. The structural and functional implications of such an arrangement are discussed.