bluuebunny/crossref_metadata_2025 huggingface dataset:
Allosteric character of the inhibition of rat‐muscle hexokinase B by glucose 6‐phosphate
Authors: GREGORIOU Mary , TRAYER Ian P. , CORNISH‐BOWDEN Athel
Published: March 2005

We previously provided evidence from isotope‐exchange measurements under non‐equilibrium conditions that hexokinase B from rat muscle follows a compulsory‐order mechanism with glucose binding before MgATP, and with both glucose 6‐phosphate and MgATP capable of binding allosterically [Gregoriou, M., Trayer, I. P. & Cornish‐Bowden, A. (1983) Eur. J. Biochem. 134, 283–288]. We have now re‐examined this work in the light of recent criticisms [Ganson, N. J. & Fromm, H. J. (1985) J. Biol. Chem. 260, 12099–12105]. There is no difficulty in obtaining valid estimates of initial rates of isotope exchange when the equilibrium constant is unfavourable, if one uses highly radioactive reactants and low enzyme concentrations, as we did in the experiments we reported previously. However, our earlier suggestion that MgADP can be released within the inhibitory pathway, which was made for the sake of consistency with the catalytic pathway rather than because of any compelling experimental evidence, must be revised to avoid predicting that the rate must be zero in the absence of MgADP. Although our mechanism admits the possibility of substrate inhibition by MgATP, calculations show that there is no need for this to be observable under ordinary conditions. Indeed, with plausible values assumed for the kinetic constants one can calculate theroretical behaviour according to our model that closely resembles the experimental inhibition experiments that have been claimed as evidence against it.
Circular dichroism studies on chromatin models
Authors: PANOU‐POMONIS Eugenia , SAKARELLOS Constantinos , SAKARELLOS‐DAITSIOTIS Maria
Published: March 2005

The sequential polypeptides (L‐Arg‐Xaa‐Gly)n where Xaa represents amino acid residues Ala, Val and Leu, were employed as models of arginine‐rich histones, in studying their interactions with nucleic acids. These polypeptide‐DNA complexes were prepared using gradient dialysis and their conformational properties were investigated by circular dichroism spectroscopy. It was found that poly(L‐Arg‐L‐Val‐Gly) caused pronounced structural changes in the DNA molecule (conformational transition from B to the more compact and asymmetric C form) as a function of ionic strength and polypeptide: DNA ratio. In contrast the DNA interaction with poly (L‐Arg‐L‐Ala‐Gly) and poly (L‐Arg‐L‐Leu‐Gly) increased in the order of Ala → Leu, but with slight structural changes in the DNA secondary conformation. Thus, the importance of the composition, amino acid sequence and conformation of the polypeptides which bind to DNA was demonstrated. The significance of the hydrophobic forces besides the arginine‐phosphate charge interaction, which modulate the nature of the polypeptide‐DNA complexes and their condensation into higher‐ordered tertiary structures, as found in chromatin, was also confirmed.
Binding of 2′(3′)‐O‐(2,4,6‐trinitrophenyl)‐adenosine 5′‐diphosphate opens the pathway for protons through the chloroplast ATPase complex
Authors: WAGNER Richard , PONSE Gudrun , STROTMANN Heinrich
Published: March 2005

The effect of 2′(3′)‐O‐(2,4,6‐trinitrophenyl)‐adenosine 5′‐diphosphate (TNP‐ADP) on photophosphorylation and on the proton conductivity of the thylakoid membrane has been investigated. The results show that TNP‐ADP is a potent competitive inhibitor of photophosphorylation (Ki= 1–2 μM). Moreover, in the absence of ADP and Pi, TNP‐ADP accelerates basal electron transport of chloroplasts. Addition of ADP, which promotes release of the analogue from CF1, completely reverses this effect of TNP‐ADP; likewise Pi alone reverses stimulation of electron transport by TNP‐ADP. Dicyclohexylcarbodiimide treatment, which is known to close CF0 to H+, completely abolishes the effect of TNP‐ADP.

The measurements of the alkalization of the medium and the acidification of the thylakoid lumen following single turnover flashes showed that binding of TNP‐ADP to CF1 increased membrane permeability for H+. Further results suggest that binding of TNP‐ADP to the catalytic site of CF1 opens the CF0‐CF1 complex for H+. Since ADP, as well as Pi alone, reverses the effect, it is concluded that TNP‐ADP induces a conformation of the CF0‐CF1 complex similar to the one triggered by simultaneous binding of ADP plus Pi. This may be achieved by interaction of the TNP residue with the Pi binding site. Thus it seems that the status of the catalytic site(s) in CF1 can be transmitted to the CF0 part to control proton flux through the ATPase complex in an economically reasonable way.
Properties of different Ca2+ pools in permeabilized rat thymocytes
Authors: GUKOVSKAYA Anna S. , ZINCHENKO Valery P. , PETRUNYAKA Victor V. , KHODOROV Boris I. , EVTODIENKO Yury V.
Published: March 2005

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen‐induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin‐permeabilized and intact rat thymocytes using a Ca2+‐selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin‐2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non‐mitochondrial. Ca2+ uptake by the non‐mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 μM. The non‐mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 μM. At free Ca2+ concentration close to the cytoplasmic (0.1 μM) the main calcium pool in permeabilized thymocytes is localized in the non‐mitochondrial compartment.

Ca2+ accumulated in the non‐mitochondrial pool can be released by inositol 1,4,5‐triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP‐dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non‐mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3‐induced Ca2+ mobilization.

Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin‐2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non‐mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+‐containing an Ca2+‐free medium. This indicates a mitogen‐induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.
In‐vivo‐modified gonococcal plasmid pJD1
Authors: KORCH Christopher , HAGBLOM Per
Published: March 2005

The 4207‐bp cryptic plasmid (pJD1) of Neisseria gonorrhoeae has 5‐methylcytosine bases present at several positions in the DNA sequence. Fortuitously, these modified bases lie in the recognition sequences of many restriction enzymes. This feature makes the cryptic plasmid a model system for assaying the effect of these modified cytosines on the activities of the following restriction endonucleases and their isoschizomers: R ·AvaII, R ·BamHI, R ·BglI, R ·Fnu4HI, R ·HaeII, R ·HaeIII, R ·HpaI, R ·HpaII, R ·KpnI, R ·MspI, R ·NaeI, R ·NarI, R ·NciI, R ·NgoI, R ·NgoII, and R ·Sau96I. Of particular interest was the finding that methylation of one of the external cytosines of the palindrome 5′‐CCGG‐3′ prevented its cleavage by R ·MspI, but not by R ·HpaII as had been suggested by Walder et al. [J. Biol. Chem. (1983) 258, 1235–1241].
Kinetics of enzymatic synthesis of peptides in aqueous/organic biphasic systems
Authors: NAKANISHI Kazuhiro , MATSUNO Ryuichi
Published: March 2005

We studied kinetics of thermolysin‐catalyzed peptide synthesis in an aqueous/organic biphasic system theoretically and experimentally. As a model reaction producing a condensation product having no dissociating groups, we used the synthesis of N‐(benzyloxycarbonyl)‐l‐phenylalanine methyl ester (Z‐Phe2 OMe) from N‐(benzyloxycarbonyl)‐l‐phenylalanine (Z‐Phe) and l‐phenylalanine methyl ester (PheOMe). Usually, ethyl acetate was used as the organic solvent. First we studied the kinetics of the synthesis of Z‐Phe2OMe in a buffer solution saturated with ethyl acetate. Then, factors that may affect the kinetics in the biphasic system were examined. The course of Z‐Phe2OMe synthesis in the biphasic system was explained by the rate equations obtained, using the partitions of substrate and product and non‐enzymatic decomposition of PheOMe. In the biphasic reaction syste, the rate of synthesis was lower for a wide range of pH due to the unfavorable partition of PheOMe in the aqueous phase, but yields were higher than in the buffer solution. The effects of the organic solvents on the rate of synthesis could also be explained by variations in the partition coefficient of PheOMe. Finally, we gave a way to predict the aqueous‐phase pH change caused by partitioning of the substrate. The significance of the pH change was shown in connection with the reaction using the immobilized enzyme in an organic solvent.
Ferredoxin reductase levels in the ovaries of pigs and superovulated rats during follicular cell growth and luteinization
Authors: TUCKEY Robert C. , STEVENSON Patricia M.
Published: March 2005

The concentration of ferredoxin reductase, a component of the mitochondrial steroidogenic electron transport chain, was measured in the ovaries of pigs and superovulated rats by a protein blotting procedure using polyclonal antibodies to the purified protein.

The concentration of ferredoxin reductase in porcine granulosa cells doubled during growth of follicles from small (1 – 2mm diameter) to large (6 – 12 mm diameter). The concentration doubled again during the period of luteinization. This is in contrast to the rate of cholesterol side‐chain cleavage, which showed little change during follicular growth but increased by more than tenfold during luteinization. A similar large increase in cholesterol side‐chain cleavage occurs during the period of luteinization in the ovaries of superovulated rats, but as for the pig, only a small increase in ferredoxin reductase was observed.

A threefold increase in the yield of mitochondrial protein from tissue homogenates was found between the granulosa cells of small – medium follicles and the cells of the corpora luteum. The increase in ferredoxin reductase during follicular development and luteinization, therefore, correlates well with the increase in mitochondria in the cells, but does not correlate with the dramatic increase in cholesterol side‐chain cleavage activity which occurs during luteinization. Based on these results, it is unlikely that the level of ferredoxin reductase limits the expression of the full steroidogenic activity of the granulosa cells of the ovary.
Characterization of the elongation factors from calf brain
Authors: CRECHET Jean‐Bernard , CANCEILL Danielle , BOCCHINI Vincenzo , PARMEGGIANI Andrea
Published: March 2005

This work describes a method for the purification of the elongation factors (EF) from calf brain. The elongation factor responsible for the binding of aminoacyl‐tRNA to the ribosome is found in this organ as a light form (EF‐1α) and as a component of heavy, polydispersed aggregates (EF‐1H). EF‐1β, the factor enhancing the EF‐1α GDP/GTP exchange, is part of EF‐1H and of smaller aggregates. The fraction of EF‐1α and EF‐1β not associated with EF‐1H, and EF‐2 have been purified to homogeneity after several chromatographic steps. EF‐1H consists of many proteins; among them, EF‐1α, EF‐1β and an EF‐1γ‐like protein represent three of the major components. This conclusively shows that EF‐1H from calf brain is not a polydispersed aggregate of only EF‐1α. EF‐1β has also been purified to homogeneity from EF‐1H. The property of EF‐1β to aggregate with other proteins suggests that this factor plays an important role in the organization of EF‐1H. The relative molecular mass of the purified factors have been determined as: EF‐1α, 50000; EF‐1β, 30000; the EF‐1γ‐like component, 49000; EF‐2, 85000. Some cross‐reactivity with the antibodies against the prokaryotic counterparts has been shown for EF‐1α, EF‐1β and EF‐2 by functional and immuno‐precipitation methods, suggesting the existence of structural homologies.
Amino‐acid sequence data of β‐tubulin from Physarum polycephalum myxamoebae
Authors: SINGHOFER‐WOWRA Monika , CLAYTON Lesley , DAWSON Peter , GULL Keith , LITTLE Melvyn
Published: March 2005

Starting with 7.7 mg of a β‐tubulin isolated from myxamoebae of the slime mould Physarum polycephalum, 90% of the sequence has been determined by the Edman degradation of peptides generated by cyanogen bromide, trypsin and Staphylococcus aureus protease. Differences to other β‐tubulins are mainly conservative and spread evenly throughout the chain except for a high concentration at the C‐terminus. The Physarumβ‐tubulin shows most homology to Chlamydomonasβ‐tubulin (90.5%) and least homology to yeast β‐tubulin (S. cerevisiae, 73.4%). Two tryptic peptides were isolated in approximately equal quantities which were identical except in one position (S/ALTVPELTQRMFDA) showing that at least two β‐tubulins are present in myxamoebae. However, since this was the only heterogeneity found, these β‐tubulins are probably very similar.
Chemical modification of the brown‐fat‐mitochondrial uncoupling protein with tetranitromethane and N‐ethylmaleimide
Authors: RIAL Eduardo , NICHOLLS David G.
Published: March 2005

Treatment of brown adipose tissue mitochondria with tetranitromethane or N‐ethylaleimide decreases the affinity with which inhibitory nucleotide GDP binds to the tissue‐specific uncoupling protein. Both reagents modify cysteine residues which are ‘accessible’ and ‘buried’ to 5,5′‐dithio‐bis(2‐nitrobenzoic acid) (Nbs2). Modification of the single Nbs2‐accessible residue correlates with the loss of high‐affinity binding sites for GDP. Tetranitromethane does not affect the Cl− or H+ permeability of the protein in the absence of nucleotide, while N‐ethylmaleimide increases both by 70–80%. Bound GDP is a less effective inhibitor of Cl− permeability after N‐ethylmaleimide or tetranitromethane treatment, but retains much of the ability to inhibit H+ permeation.
Analysis of the puromycin reaction
Authors: GEIGENMÜLLER Ute , HAUSNER Thomas‐Peter , NIERHAUS Knud H.
Published: March 2005

The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re‐examine the exclusion principle for peptidyl‐tRNA binding, which states that only one peptidyl‐tRNA (AcPhe‐tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained.

The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe‐tRNA over ribosomes, and in the absence of EF‐G, this criterion is fulfilled at 0°C, where the P‐site‐bound material reacts with puromycin (quantitative reaction after 50 h), while the A‐site‐bound material does not. In contrast, at 37°C the extent of the puromycin reaction can exceed the binding values by 2–4‐fold (‘repetitive reaction’). In the presence of EF‐G a repetitive puromycin reaction is seen even at 0°C, i.e. EF‐G can already promote a translocation reaction at 0°C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0°C, if only catalytic amounts of EF‐G are used.

Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies.

Ribosomes were saturated with AcPhe‐tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P‐site location of the AcPhe‐tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe‐tRNA binding to the P site.

Precharging the P sites of 70S ribosomes with one Ac[14C]Phe‐tRNA molecule per ribosome prevented additional Ac[3H]Phe‐tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[3H]Phe‐tRNA per ribosome.
Tetracycline can inhibit tRNA binding to the ribosomal P site as well as to the A site
Authors: GEIGENMÜLLER Ute , NIERHAUS Knud H.
Published: March 2005

A and P sites of Escherichia coli ribosomes were titrated with AcPhe‐tRNAPhe, in the absence or presence of tetracycline. The P‐site location of the bound AcPhe‐tRNA was assessed by means of a quantitative puromycin reaction. The results demonstrate that, in agreement with the generally held view, tetracycline exclusively inhibits the A‐site binding, if the statistical number of bound acyl‐tRNA molecules per ribosome does not exceed about 0.5. However, above this value the P site becomes sensitive to tetracycline as well. It follows that the tightly coupled 70S ribosomes used in functional studies appear to be functionally heterogeneous, i. e. those P sites which cannot be affected by tetracycline are preferentially occupied by AcPhe‐tRNA, whereas higher concentrations of this tRNA species are required to fill tetracycline‐sensitive P sites. Furthermore, the results imply that under tRNA saturation conditions the tetracycline inhibition cannot be used as an indicator for the site location of bound tRNA.
Properties of the complexes of riboflavin 3′,5′‐bisphosphate and the apoflavodoxins from Megasphaera elsdenii and Desulfovibrio vulgaris
Authors: VERVOORT Jacques , van BERKEL Willem J. H. , MAYHEW Stephen G. , MÜLLER Franz , BACHER Adelbert , NIELSEN Peter , LeGALL Jean
Published: March 2005

Megasphaera elsdenii and Desulfovibrio vulgaris apoflavodoxins have been reconstituted with riboflavin 3′,5′‐bisphosphate. Several biochemical and biophysical properties of the complexes have been investigated and the results are compared with the properties of the native proteins.

The dissociation constant of the modified complex of M. elsdenii flavodoxin is increased by a factor of about 23 by comparison with that of the native protein. The rate constant for the formation of the complex of M. elsdenii flavodoxin is about 26 times lower than that for the native protein. The redox potential of the transition between the oxidized and semiquinone state is similar to that of the native protein. On the other hand, the redox potential of the semiquinone‐hydroquinone transition is about 20mV more negative than that of the native protein. Absorbance and circular dichroic spectra of the protein‐bound artificial prosthetic group and the protein‐bound natural prosthetic group are very similar. In both the oxidized and in the fully reduced state only minor differences in interaction between the isoalloxazine ring and the apoprotein for the two flavin derivatives are found by 13C and 15P‐NMR. 31P‐NMR studies show that the 5′‐phosphate group of the two flavin derivatives is bound in the same way and that it is dianionic in the complex. In contrast, the 3′‐phosphate group in riboflavin 3′,5′‐bishosphate is monoanionic or even neutral when bound to the protein. The 3′‐phosphate group is also close to or on the surface of the protein.

Desulfovibrio vulgaris apoflavodoxin has an affinity for riboflavin 3′,5′‐bisphosphate which is 10 times higher as compared to Megasphaera elsdenii apoflavodoxin (Ka= 108 M–1). Also the association rate constant of Desulfovibrio vulgaris apoprotein and riboflavin 3′,5′‐bisphosphate is found to be 10 times faster than for the Megasphaera elsdenii flavodoxin reaction. The dissociation behaviour of native Desulfovibrio vulgaris flavodoxin measured under identical conditions as for the riboflavin 3′,5′‐bisphosphate analog gives a value (Kd∼ 0.2 nM) which is considerably lower than reported earlier [Dubourdieu, M., MacKnight, M. L. & Tollin, G. (1974) Biochem. Biophys. Res. Commun. 60, 649–655].

The results are discussed in the light of the existing crystallographic data of flavodoxins and the recently proposed theory on the regulation of the redox potential in flavoproteins [Moonen, C. T. W., Vervoort, J. & Müller, F. (1984) in Flavins and flavoproteins, pp. 493–496, Walter de Gruyter, Berlin].
Fructose 2,6‐bisphosphate in Dictyostelium discoideum
Authors: ARAGÓN Juan J. , SÁNCHEZ Valentina , BOTO Luis
Published: March 2005

The occurrence of fructose 2,6‐bisphosphate was detected in Dictyostelium discoideum. The levels of this compound were compared with those of cyclic AMP and several glycolytic intermediates during the early stages of development. Removal of the growth medium and resuspension of the organism in the differentiation medium decreased the content of fructose 2,6‐bisphosphate to about 20% within 1 h, remaining low when starvationinduced development was followed for 8 h. The content of cyclic AMP exhibited a transient increase that did not correlate with the change in fructose 2,6‐bisphosphate. If after 1 h of development 2% glucose was added to the differentiation medium, fructose 2,6‐bisphosphate rapidly rose to similar levels to those found in the vegetative state, while the increase in cyclic AMP was prevented. The contents of hexose 6‐phosphates, fructose 1;6‐bisphosphate and triose phosphates changed in a way that was parallel to that of fructose 2,6‐bisphosphate, and addition of sugar resulted in a large increase in the levels of these metabolites. The content of fructose 2,6‐bisphosphate was not significantly modified by the addition of the 8‐bromo or dibutyryl derivatives of cyclic AMP to the differentiation medium. These results provide evidence that the changes in fructose 2,6‐bisphosphate levels in D. discoideum development are not related to a cyclic‐AMP‐dependent mechanism but to the availability of substrate. Fructose 2,6‐bisphosphate was found to inhibit fructose‐1,6‐bisphosphatase activity of this organism at nanomolar concentrations, while it does not affect the activity of phosphofructokinase in the micromolar range. The possible physiological implications of these phenomena are discussed.
Binding mode of nucleosome‐assembly protein (AP‐I) and histones
Authors: ISHIMI Yukio , KOJIMA Masatoyo , YAMADA Masa‐atsu , HANAOKA Fumio
Published: March 2005

Studies were made on the binding mode of the nucleosome‐assembly protein AP‐I with histones H2A + H2B and/or H3 + H4. Histones H2A + H2B bound with AP‐I to form a 7‐S complex which contained twice as much AP‐I (by weight) as histones. Histone H3 + H4 formed an 8‐S complex with AP‐I. The 7‐S and 8‐S complexes did not form a new complex when mixed, but significant amounts of two histone pairs were assembled into a 12‐S complex on mixing the (H2A + H2B)–AP‐I complex (7‐S) with free H3 + H4. In contrast, when the (H3 + H4)–AP‐I complex (8‐S) was incubated with free H2A + H2B, almost no assembly occurred, but the 7‐S complex of H2A + H2B was newly formed. Binding studies by enzyme‐linked immunosorbent assay showed that AP‐I bound with H2A + H2B faster than with H3 + H4.

From these results, it is suggested that AP‐I has a higher binding affinity for histone H2A + H2B than for H3 + H4, and that the 7‐S complex is an intermediate in formation of the 12‐S octamer complex (H2A + H2B + H3 + H4)2
Characterisation of the metal‐ion–GDP complex at the active sites of transforming and nontransforming p21 proteins by observation of the 17O‐Mn superhyperfine coupling and by kinetic methods
Authors: FEUERSTEIN Jürgen , KALBITZER Hans Robert , JOHN Jacob , GOODY Roger S. , WITTINGHOFER Alfred
Published: March 2005

Kinetic studies on the interaction of three Ha‐ras‐encoded p21 proteins with GDP and MgGDP have yielded values for the association (106–107 M−1 s−1) and dissociation (10−3–10−5s−1) rate constants at 0°C. Dramatic differences in the rate constants were not observed for the three proteins. Under non‐physiological conditions (absence of Mg2+), the rate constant for GDP release was an order of magnitude faster for the viral protein p21v than for the cellular form p21c or the T24 mutant p21t but this was reduced to a factor of about 3 in the presence of Mg2+. In all cases, there was an increase of about one order of magnitude in the rate of GDP release on removing magnesium. The binding affinities ranged from 5.7×1010 M−1 for p21c to 1.3×1011 M−1 for p21v. Electron paramagnetic resonance (EPR) measurements on Mn2+ bound together with stereospecifically 17O‐labelled GDP showed direct coordination of a β‐phosphate oxygen to the metal ion with a superhyperfine coupling constant of 0.16–0.22mT, but no interaction with the α‐phosphate oxygens at the active site of all three proteins. The association constant of Mn(II) to p21 proteins in the absence of nucleotide was estimated to be > 105 M−1. In agreement with the EPR results, experiments on the metal ion dependence of the binding of thiophosphate analogs of GDP provided further evidence for the absence of direct coordination of the metal ion to the α‐phosphate group. These results have been used to construct a model for the interactions of Mg · GDP with the active site of p21 proteins.
Tissue‐specific expression of porphobilinogen deaminase
Authors: GRANDCHAMP Bernard , DE VERNEUIL Hubert , BEAUMONT Carole , CHRETIEN Stany , WALTER Olivier , NORDMANN Yves
Published: March 2005

Porphobilinogen deaminase (hydroxymethylbilane synthase; EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway, catalyzes the stepwise condensation of four porphobilinogen units to yield hydroxymethylbilan, which is in turn converted to uroporphyrinogen III by cosynthetase.

We compared the apparent molecular mass of porphobilinogen deaminase from erythropoietic and from non‐erythropoietic cells by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immune‐blotting. The results indicate that two isoforms of porphobilinogen deaminase can be distinguished and differ by 2000 Da.

Analysis of cell‐free translation products directed by mRNAs from human erythropoietic spleen and from human liver demonstrates that the two isoforms of porphobilinogen deaminase are encoded by distinct messenger RNAs.

We cloned and sequenced cDNAs complementary to the non‐erythropoietic form of porphobilinogen deaminase encoding RNA. Comparison of these sequences to that of human erythropoietic mRNA [Raich et al. (1986) Nucleic Acids Res. 14, 5955–5968] revealed that the two mRNA species differ by their 5′ extremity. From the mRNA sequences we could deduce that an additional peptide of 17 amino acid residues at the NH2 terminus of the non‐erythropoietic isoform of porphobilinogen deaminase accounts for its higher molecular mass.

RNase mapping experiments demonstrate that the two porphobilinogen deaminase mRNAs are distributed according to a strict tissue‐specificity, the erythropoietic form being restricted to erythropoietic cells.

We propose that a single porphobilinogen deaminase gene is transcribed from two different promoters, yielding the two forms of porphobilinogendeaminase mRNAs. Our present finding may have some relevance for further understanding the porphobilinogen deaminase deficiency in certain cases of acute intermittent porphyria with an enzymatic defect restricted in non‐erythropoietic cells.
Tyrosination of microtubules and non‐assembled tubulin in brain slices
Authors: BELTRAMO Dante M. , ARCE Carlos A. , BARRA Héctor S.
Published: March 2005

Brain slices were used to examine comparatively the incorporation of [14C]tyrosine into the C terminus of α‐tubulin of the microtubule and non‐assembled tubulin pools. We found that the incorporation of [14C]tyrosine from 5 min up to 60 min of incubation was higher in microtubules than in non‐assembled tubulin. The possibility that this result was due to the activity of tubulin carboxypeptidase or tubulin:tyrosine ligase during the in vitro isolation of tubulin was discarded.

We also found that tubulin:tyrosine ligase was mainly associated with microtubules when brain slices were homogenized under microtubule‐preserving conditions. Conversely the enzyme behaved as a soluble entity when homogenization was performed under conditions that do not preserve microtubules. In addition, soluble tubulin:tyrosine ligase did not become sedimentable when in vitro conditions were changed to induce the formation of microtubules.

The results presented in this work indicate the possibility that, in vivo, microtubules and not tubulin dimers are the major substrate for tubulin:tyrosine ligase. This is in contrast with previous findings from in vitro experiments, which showed a preference of the ligase for non‐assembled tubulin.
Biochemical analysis of synoviocytes from normal and arthritic rats
Authors: BERNABEU Carmelo , PÉREZ‐MACEDA Blanca , LÓPEZ‐BOTE Juan Pedro , MARQUET Alberto , LARRAGA Vicente
Published: March 2005

Adjuvant‐induced polyarthritis in rats is a common model system used for the study of the synovitis that occurs in rheumatoid arthritis. Synoviocytes A, the major cell type covering the internal surface of the joint, could be involved in the pathogenesis of rheumatoid arthritis because of their increased proliferation and the intraarticular manifestations of the disease. So far only a few molecular studies have been reported on synoviocytes upon arthritis induction. We report here changes in polypeptides, between control and arthritic synoviocytes, by using two different radiolabeling methods and two‐dimensional gel electrophoresis analysis. Major differences were found using metabolic labeling on regions of tropomyosins, cyclin, tubulins and vimentin. In addition, external surface labeling of the cells with lactoperoxidase showed clear differences between control and arthritic synoviocytes in the region of 77 – 100‐kDa proteins. Some of these differences can be reproduced by certain macrophage activators such as phorbol myristate acetate and lipopolysaccharide acting on synoviocytes in vitro and in vivo respectively. These results exclude the possibility that the changes observed were due to a possible infiltration of other cell types in the arthritic synovia and strongly support the existence of an activated state of synoviocytes associated with arthritis induction.
A peptidase activity exhibited by human serum pseudocholinesterase
Authors: BOOPATHY Rathanam , BALASUBRAMANIAN Aiylam S.
Published: March 2005

The identity of a peptidase activity with human serum pseudocholinesterase (PsChE) purified to apparent homogeneity was demonstrated by (a) co‐elution of both peptidase and PsChE activities from procainamide‐Sepharose and concanavalin‐A–Sepharose affinity chromatographic columns; (b) comigration on polyacryl‐amide gel electrophoresis; (c) co‐elution on Sephadex G‐200 gel filtration and (d) coprecipitation at different dilutions of an antibody raised against purified PsChE. The purified enzyme showed a single protein band on gel electrophoresis under non‐denaturing conditions. SDS gel electrophoresis under reducing conditions, followed by silver staining, also gave a single protein band (Mr∼ 90000). Peptidase activity using different peptides showed the release of C‐terminal amino acids. Blocking the carboxy terminal by an amide or ester group did not prevent the hydrolysis of peptides. There was no evidence for release of N‐terminal amino acids.

Potent anionic or esteratic site inhibitors of PsChE, such as eserine sulphate, neostigmine, procainamide. ethopropazine, imipramine, diisopropylfluorophosphate, tetra‐isopropylpyrophosphoramide and phenyl boronic acid, did not inhibit the peptidase activity. An anionic site inhibitor (neostigmine or eserine) in combination with an esteratic site inhibitor (diisopropylfluorophosphate) also did not inhibit the peptidase. However, the choline esters (acetylcholine, butyrylcholine, propionylcholine, benzoylcholine and succinylcholine) markedly inhibited the peptidase activity in parallel to PsChE. Choline alone or in combination with acetate, butyrate, propionate. benzoate or succinate did not significantly inhibit the peptidase activity. It appeared that inhibitor compounds which bind to both the anionic and esteratic sites simultaneously (like the substrate analogues choline esters) could inhibit the peptidase activity possibly through conformational changes affecting a peptidase domain.
Identification of the C‐terminally α‐amidated amino acid in peptides by high‐performance liquid chromatography
Authors: SCHMIDT Wolfgang E. , CONLON J. Michael , MUTT Viktor , CARLQUIST Mats , GALLWITZ Baptist , CREUTZFELDT Werner
Published: March 2005

A sensitive method for the rapid identification of the C‐terminally amidated amino acid in peptides is described. Peptides containing the α‐amide group at the C‐terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C‐terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid α‐amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high‐performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20—50 pmol. The C‐terminal α‐amides of neurokinin‐A (Met‐NH2), mammalian secretin (Val‐NH2), pancreatic polypeptide (Tyr‐NH2) and peptide HI (Ile‐NH2) are unequivocally determined at a level of 0.5—2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin‐58 was identified in this fraction by detection of phenylthiocarbamoyl‐phenylalaninamide. The method is suitable for the identification of the C‐terminal α‐amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an α‐amidated amino acid at the C‐terminus.
Massive overproduction of dihydrofolate reductase in bacteria as a response to the use of trimethoprim
Authors: FLENSBURG John , SKÖLD Ola
Published: March 2005

Among several observations of greatly increased levels of chromosomal dihydrofolate reductase as a cause of resistance to high concentrations of the antifolate drug trimethoprim, in clinically isolated bacteria, one is described here of a strain of Escherichia coli overproducing dihydrofolate reductase several hundredfold. The chromosomally located resistance gene of this strain was isolated, inserted into a plasmid vector, and analyzed for its nucleotide sequence. The structural gene for the overproduced dihydrofolate reductase was found to be identical to that of E. coli K12, with nine exceptions, of which seven resulted in synonymous codon usage. Two transversions resulted in a substitution of Gly for Trp at amino acid position 30, and of Gln for Glu at position 154. Six of the nine base changes resulted in codons more frequently used. The Gly substitution which leads to a less commonly used codon, was thought to relate to the observed threefold increase in Ki for trimethoprim. Furthermore, a C→T transition was found in the –35 region of the promoter, increasing its homology with the E. coli consensus promoter sequence. In the ribosome‐binding area of the resistant strain, finally, seven base changes were observed, two of which resulted in a five‐base sequence of complementarity with the 3′‐end of ribosomal 16S RNA. The distance between the —10 site of the promoter and the start codon for translation was finally increased one base pair by the insertion of an A at position + 9 in the resistant strain. These genetic changes towards more efficient transcriptional and translational start sequences and towards increased mRNA expressivity are interpreted to reflect an evolutionary adaptation to the presence of antifolates.
Properties, biosynthesis and processing of a sulfur‐rich protein in Brazil nut (Bertholletia excelsa H.B.K.)
Authors: S. M. SUN Samuel , ALTENBACH Susan B. , LEUNG Filomena W.
Published: March 2005

An abundant seed protein, which is exceptionally rich in the sulfur‐containing amino acids, methionine (18%) and cysteine (8%), is synthesized in Brazil nut embryos about 9 months after flowering. This sulfur‐rich protein consists of two low‐molecular‐mass polypeptide components, a 9‐kDa polypeptide and a 3‐kDa polypeptide. The two‐subunit polypeptides associate through disulfide linkage(s) to form a 12‐kDa protein molecule. We have demonstrated through in vitro translation studies, using RNA from 9‐month‐old embryos, that the sulfur‐rich protein is synthesized as a larger precursor polypeptide of 18 kDa. In addition, data from in vivo labelling studies of 9‐month‐old Brazil nuts suggest that there are two intermediate precursors of the sulfur‐rich protein, one of 15 kDa and another of 12 kDa. One of these precursors, the 12‐kDa polypeptide, accumulates for a 2‐month period in the developing embryos. From these data we infer that at least three stepwise cleavages are involved in the maturation of the sulfur‐rich protein from its 18‐kDa precursor.
Glyceraldehyde‐phosphate dehydrogenase from Trypanosoma brucei
Authors: MISSET Onno , VAN BEEUMEN Jozef , LAMBEIR Anne‐Marie , VAN DER MEER Rob , OPPERDOES Fred R.
Published: March 2005

Trypanosoma brucei contains two glyceraldehyde‐phosphate (GAPDH; EC 1.2.1.12) isoenzymes; one is located in glycosomes and represents 80% of the total activity, whereas the other is present in the cytosol. The purification of the cytosolic GAPDH, which is identical in both bloodstream‐form and insect‐stage trypanosomes, is described, and the enzyme compared with its glycosomal counterpart. Cytosolic GAPDH is specific for NAD. It is a tetrameric enzyme with subunits of 33.5 kDa, 5 kDa smaller than those of the glycosomal GAPDH. The native enzyme has a pI of 7.9, which is 1.5 pH units less basic than the glycosomal enzyme. Both enzymes display maximal activity at pH 8 but the cytosolic enzyme has a much broader activity profile especially towards lower pH values.

Sequence comparison of the first 85 amino acids reveals that the N‐terminal parts of both isoenzymes differ by 52%. The N terminus of the cytosolic isoenzyme resembles the corresponding N termini of ten other known GAPDH sequences in that they all lack three amino‐acid insertions, which so far only have been found in the glycosomal isoenzyme of T. brucei. This observation explains in part the great difference in subunit size between the two T. brucei isoenzymes and suggests that at least one of these insertions is responsible for import of the glycosomal isoenzyme into the organelle.
Primary structure of the Streptomyces R61 extracellular DD‐peptidase
Authors: DUEZ Colette , PIRON‐FRAIPONT Claudine , JORIS Bernard , DUSART Jean , URDEA Mickey S. , MARTIAL Joseph A. , FRÈRE Jean‐Marie , GHUYSEN Jean‐Marie
Published: March 2005

An 11450‐base DNA fragment containing the gene for the extracellular active‐site serine DD‐peptidase of Streptomyces R61 was cloned in Streptomyces lividans using the high‐copy‐number plasmid pIJ702 as vector. Amplified expression of the excreted enzyme was observed. Producing clones were identified with the help of a specific antiserum directed against the pure DD‐peptidase. The coding sequence of the gene was then located by hybridization with a specific nucleotide probe and sub‐fragments were obtained from which the nucleotide sequence of the structural gene and the putative promoter and terminator regions were determined. The sequence suggests that the gene codes for a 406‐amino‐acid protein precursor. When compared with the excreted, mature DD‐peptidase, this precursor possesses a cleavable 31‐amino‐acid N‐terminal extension which has the characteristics of a signal peptide, and a cleavable 26‐amino‐acid C‐terminal extension. On the basis of the data of Joris et al. (following paper in this journal), the open reading frame coding for the synthesis of the DD‐peptidase was established. Comparison of the primary structure of the Streptomyces R61 DD‐peptidase with those of several active‐site serine β‐lactamases and penicillin‐binding proteins of Escherichia coli shows homology in those sequences that comprise the active‐site serine residue. When the comparison is broadened to the complete amino acid sequences, significant homology is observed only for the pair Streptomyces R61 DD‐peptidase/Escherichia coli ampC β‐lactamase (class C). Since the Streptomyces R61 DD‐peptidase and β‐lactamases of class A have very similar three‐dimensional structures [Kelly et al. (1986) Science (Wash. DC) 231, 1429–1431; Samraoui et al. (1986) Nature (Lond.) 320, 378–380], it is concluded that these tertiary features are probably also shared by the β‐lactamases of class C, i.e. that the Streptomyces R61 DD‐peptidase and the β‐lactamases of classes A and C are related in an evolutionary sense.
Complementation between LLC‐PK1 mutants affected in polypeptide hormone‐receptor function
Authors: JANS David A. , RESINK Thérèse J. , HEMMINGS Brian A.
Published: March 2005

The mutant LLC‐PK1 cell lines FIB6 and FIB5/N4 were examined for responsiveness to the polypeptide hormones calcitonin and vasopressin. Both mutants exhibited little or no activation of adenylate cyclase or cAMP‐dependent protein kinase (cAMP‐PK) in response to calcitonin, but responded to vasopressin. Analysis of calcitonin receptor function demonstrated that both mutants bound less than 9 fmol 125I‐labeled salmon calcitonin/mg cellular protein, which was about 1% of parental activity (642 fmol calcitonin bound/mg). Concomitant with reduced calcitonin binding, both mutants exhibited increased vasopressin binding (greater than 272 fmol [[3H]Arg]vasopressin bound/mg) compared to parental (166 fmol bound/mg). The concentration of vasopressin for half‐maximal stimulation of adenylate cyclase in both mutants was comparable to that for LLC‐PK1 cells (40 pM) and hence the increased binding activity was concluded to be due to increased numbers of functional vasopressin receptors in the mutants.

Somatic cell hybrids formed between each mutant and LLC‐PK1 cells exhibited normal hormone binding and activation of cAMP‐PK in response to both vasopressin and calcitonin. The mutations affecting receptor function in FIB6 and FIB5/N4 were accordingly concluded to be recessive. Somatic cell hybrids between FIB6 and FIB5/N4 showed no complementation of the mutant phenotype, indicating that both cell lines were affected in the same gene. In contrast, somatic cell hybrids between FIB5/N4 and the ‘receptorless’ mutant M18 (which lacks functional calcitonin and vasopressin receptors) exhibited approximately the same responsiveness to vasopressin and to calcitonin as LLC‐PK1. Complementation between two different mutations affecting polypeptide receptor function was thus observed. The results are discussed in terms of a proposed common pathway for processing of calcitonin and vasopressin receptors.
Prostaglandin E1 binds to Z protein of rat liver
Authors: DUTTA‐ROY Asim K. , GOPALSWAMY Narasimh , TRULZSCH Dietmar V.
Published: March 2005

Z protein or fatty‐acid‐binding protein is abundant in the cytosol of many cell types including liver cells. It is considered to play an important role in intracellular transport and metabolism of long‐chain fatty acids and other organic anions. We studied the role of Z protein in the metabolism of prostaglandin E1 (PGE1). Binding of tritiated prostaglandin E1 to this fatty‐acid‐binding protein (Z protein) purified from rat liver was determined. The binding of [3H]prostaglandin E1 to Z protein is rapid, saturable and reversible. Scatchard analysis of [3H]PGE1 binding to Z protein showed a single class of binding sites with a dissociation constant (Kd) of 37 nM. The binding capacity is 110 nmol/mg Z protein. Optimal [3H]PGE1 binding occurred at pH 7.4. The presence of 3 mM MgCl2 stimulated the prostaglandin E1 binding to Z protein. Competition experiments show that the binding of this autacoid to Z protein is highly specific. It could not be displaced by other prostaglandins (PGA1, PGA2, PGE2, PGB1, PGI2, PGD2, PGF2α, and 6‐ketoPGF1α). Z protein might be involved in the metabolism of prostaglandins in the cytosol.
Interaction of mitogenic bacterial lipoprotein and a synthetic analogue with mouse lymphocytes
Authors: BIESERT Lothar , SCHEUER Werner , BESSLER Wolfgang G.
Published: March 2005

Lipoprotein from the outer membrane of Escherichia coli constitutes a potent mitogen and polyclonal activator for B lymphocytes of different species. The binding of lipoprotein to murine spleen cells was investigated using water‐soluble 125I‐labelled citraconylated lipoprotein from E. coli B/r. Our results indicate that the binding of this B‐cell mitogen to splenocytes is a saturable, time‐ and dose‐dependent, reversible process; about 9.7×108 lipoprotein molecules were bound to each cell. The mechanism of the binding of lipoprotein to lymphocytes was investigated by using the synthetic analogue of its N‐terminal part, S‐[2,3‐bis(palmitoyloxy)‐(2RS)‐propyl]‐N‐palmitoyl‐(R)‐cysteinyl‐(S)‐eryl‐(S)‐seryl‐(S)‐asparaginyl‐(S)‐alanine (tripalmitoyl pentapeptide). This compound had been shown by us previously to be the molecular part of lipoprotein responsible for mitogenicity and exhibited, in all experiments performed, a stimulatory activity towards B lymphocytes comparable, or even superior, to native lipoprotein. Binding proteins for the synthetic N‐terminus were enriched by affinity chromatography, using an affinity column prepared by coupling the mitogenic compound to CPG‐aminopropyl controlled‐pore glass beads by the carbodimide method. [3H]Leucine‐labelled murine spleen cells were solubilized by the nonionic detergent NP40 and applied to the affinity adsorbent. Proteins bound to the column were selectively eluted by a solution of tripalmitoyl pentapeptide, and the fractions were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Our results indicate the presence of a major binding protein of Mr 35000 on mouse primary lymphocytes for the biologically active N‐terminal structure of lipoprotein, which might play a role as membrane receptor in mitogenic B lymphocyte activation.
Molecular characterisation of the DNA ligase gene, CDC17, from the fission yeast Schizosaccharomyces pombe
Authors: BARKER David G. , H. M. WHITE Julia , JOHNSTON Leland H.
Published: March 2005

We have sequenced a 4200‐base‐pair fragment of Schizosaccharomyces pombe DNA which encompasses the entire DNA ligase gene, CDC17. S1 mapping has enabled us to identify two small introns (40 and 62 nucleotides) at the 5′ end of the coding region of the gene and their 3′ internal conserved sequences match the CTRAY consensus found in other S. pombe introns. The major transcription initiation and 3′ polyadenylation sites have been mapped and are preceded by higher eukaryotic‐like TATA and AATAAA sequences respectively. Furthermore, the CDC17 mRNA carries a poly(A) tail whose length (approximately 250 nucleotides) is typical of that found in higher eukaryotic mRNAs, and is in contrast to the much shorter polyadenylated sequences found for the mRNAs of the budding yeast, Saccharomyces cerevisiae. The deduced amino acid sequence of the S. pombe DNA ligase predicts a protein of 86 182 daltons, and an overall 53% homology with the same enzyme from S. cerevisiae. In particular, a stretch of 24 amino acids with 100% sequence homology spans the putative ATP‐binding region which is also conserved in T4 and T7 bacteriophage DNA ligases.