bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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].
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
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.
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.
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.
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.
Urea synthesis from ammonia in periportal and pericentral regions of the liver lobule
Authors: KARI Frank W. , YOSHIHARA Harumasa , THURMAN Ronald G. 
Published: March 2005

Rates of urea synthesis were determined in periportal and pericentral regions of the liver lobule in perfused liver from fed, phenobarbital‐treated rats by measuring the extra O2 consumed upon infusion of NH4Cl with miniature O2 electrodes and from decreases in NADPH fluorescence detected with micro‐light‐guides.
Urea synthesis by the perfused rat liver supplemented with lactate (5 mM), ornithine (2 mM) and methionine sulfoximine (0.15 mM), an inhibitor of glutamine synthetase, was stimulated by stepwise infusion of NH4Cl at doses ranging from 0.24 mM to 3.0 mM. A good correlation (r= 0.92) between decreases in NADPH fluorescence and urea production was observed when the NH4Cl concentration was increased.
Sublobular rates of O2 uptake were determined by placing miniature oxygen electrodes on periportal or pericentral regions of the lobule on the liver surface, stopping the flow and measuring decreases in oxygen tension. From such measurements local rates of O2 uptake were calculated in the presence and absence of NH4Cl and local rates of urea synthesis were calculated from the extra O2 consumed in the presence of NH4Cl and the stoichiometry between O2 uptake and urea formation. Rates of urea synthesis were also estimated from the fractional decrease in NADPH fluorescence, caused by NH Cl infusion in each region, measured with micro‐light‐guides and the rate of urea synthesis by the whole organ.
When perfusion was in the anterograde direction, maximal rates of urea synthesis, calculated from changes in fluorescence, were 177 ± 31 μmol g−1 h−1 and 61 ± 24 μmol g−1 h−1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, however, rates were 76 ± 23 μmol g−1 h−1 in periportal areas and 152 ± 19 μmol g−1 h−1 in pericentral regions.
During perfusion in the anterograde direction, urea synthesis, calculated by changes in O2 uptake, was 307 ± 76 μmol g−1 h−1 and 72 ± 34 μmol g−1 h−1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, urea was synthesized at rates of 54 ± 17 μmol g−1 h−1 and 387 ± 99 μmol g−1 h−1 in periportal and pericentral regions, respectively. Thus, maximal rates of urea synthesis were dependent upon the direction of perfusion. In addition, rates of urea synthesis were elevated dramatically in periportal regions when the flow rate per gram liver was increased (e.g. 307 versus 177 μmol g−1 h−1).
These results demonstrate that periportal regions of the liver lobule have a greater maximal capacity to synthesize urea when flow is in the physiological anterograde direction. In contrast, the primary site of urea synthesis can be shifted rapidly to pericentral regions when the direction of flow is reversed. Thus, maximal rates of urea synthesis in the perfused liver are observed in regions of the liver lobule with the highest O2 tensions.
The oxygenation‐linked binding of neutral red to spiny lobster hemocyanin
Authors: MAKINO Nobuo 
Published: March 2005

The structural change of lobster hemocyanin in cooperative O2 binding was studied by the dye‐binding method. It was found that neutral red shows an O2‐linked binding to hemocyanin with a higher affinity for the oxy form. The number of the dye‐binding sites was estimated to be three in the hexameric molecule of oxyhemocyanin.The course of the structural change in the partially oxygenated hemocyanin was examined using the absorbance change of the bound dye as a measure. It was found that the fractional change in the dye binding was considerably greater than the degree of O2 saturation of hemocyanin.The three‐state allosteric model, which was proposed for explanation of the O2 binding properties of lobster hemocyanin [N. Makino (1986) Eur. J. Biochem. 154, 49–55], was also consistent with the effects of the dye on the O2 binding to the native hemocyanin. On the basis of this model, the dye binding to partially oxygenated hemocyanin could be connected with the populations of the affinity states. It was inferred that the binding of neutral red reflects the quaternary structure of the protein.In contrast, O2 binding to the stripped (EDTA‐treated) hemocyanin showed a considerable decrease in the cooperativity in the presence of the dye. The O2‐binding isotherms could not be explained by the three‐state model. It is suggested that the subunit interaction is partially blocked by the dye in the absence of divalent cations.
Studies on the functional topography of Escherichia coli RNA polymerase
Authors: GRACHEV Mikhail A. , KOLOCHEVA Tatyana I. , LUKHTANOV Evgeny A. , MUSTAEV Arkady A. 
Published: March 2005

RNA polymerase was treated in the presence of promoter‐containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate. This treatment was followed by addition of a pyrimidine [α‐32P]NTP. Due to ‘catalytic competence’ of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active‐center‐catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula ‐pNpN (where = radioactive phosphate) appeared covalently attached to the enzyme. Such affinity labelling was super‐selective because affinity reagent residues bound outside the active center were not elongated and thus remained non‐radioactive.Labelling took place only when the combination of the reagent and [α‐32P]NTP corresponded to the sequence of nucleotides of the promoter. With reagents having short ‘arms’, only the β subunit was labelled; the targets were His and/or Lys residues. With reagents having longer ‘arms’, the σ subunit was also labelled.
Synthesis of β‐glucans in Prototheca zopfii
Authors: RIVAS Liliana A. , LEZICA Rafael PONT 
Published: March 2005

Membrane preparations from the non‐photosynthetic alga Prototheca zopfii incorporate glucose from UDP‐[3H]glucose into the trichloroacetic‐acid‐insoluble fraction and the polysaccharides insoluble in hot alkali. Time course and pulse‐chase experiments indicate that the acid‐insoluble fraction was a precursor of the alkali‐insoluble fraction. Isolation of 3H‐labeled membrane or soluble fraction showed that only membrane fractions were able to transfer radioactivity into polysaccharides. Treatment of glucosylated membranes with trypsin or cellulase only partially affect their transfer ability, indicating that the precursor was internalized in vesicles.Analysis of the in vitro synthesized polysaccharides by enzymatic and acid hydrolysis showed that glucose and cellobiose were present as radioactive sugars. Permethylation of the polysaccharide indicates that 80% of the glucose was β‐1,4‐bonded with 20% in β‐1,3‐linkagés. This polysaccharide was found to be identical with the cell‐wall β‐glucan obtained in vivo [Rivas, L. A. & Pont Lezica, R. (1978) Planta (Berl.) 165, 348–353].