bluuebunny/crossref_metadata_2025_split huggingface dataset:
On the Role of Ribosylthymine in Prokaryotic tRNA Function
Authors: KERSTEN Helga , ALBANI Martin , MÄNNLEIN Elisabeth , PRAISLER Rosemarie , WURMBACH Peter , NIERHAUS Knud H. 
Published: March 2005

tRNAPhe and tRNALys were isolated from an Escherichia coli K12 mutant deficient in ribosylthymine (rT) and from the wild‐type strain. The sequence G‐rT‐Ψ‐C which is common to loop IV of practically all tRNAs used in the elongation cycle of protein synthesis reads G‐U‐Ψ‐C in tile tRNAs of the mutant strain. The purified tRNAs were compared in various steps of protein biosynthesis.The poly(U)‐dependent poly(Phe) synthesis performed with purified Phe‐tRNAPhe and purified elongation factors showed no dependence on the presence or absence of ribosylthymine in the respective tRNAs. In contrast, the corresponding poly(A)‐dependent poly(Lys) synthesis was markedly increased when Lys‐tRNALys lacking rT was used. The analysis of individual functional steps of the poly(A)‐dependent elongation cycle demonstrated that the absence of rT reduced the binding to the A‐site and improved the translocation reaction, whereas the formation of the ternary complex EF‐Tu · GTP · aa‐tRNA as well as both tRNA binding to the P‐site and the peptidyltransferase reaction remained unaffected. The presence of U in place of rT in tRNA increases the misincorporation of leucine in an optimized poly(U)/poly(Phe) system from about 3 in 10000 to 3 in 1000. Our results are in agreement with the view that rT is involved in tRNA binding to the A‐site in contrast to the P‐site, and suggest that the presence of rT in tRNA improves the fidelity of the decoding process at the A‐site of the ribosome.
The Complete Amino‐Acid Sequence of Hen Ovalbumin
Authors: NISBET Andrew D. , SAUNDRY Richard H. , MOIR Arthur J. G. , FOTHERGILL Linda A. , FOTHERGILL John E. 
Published: March 2005

The complete amino acid sequence of hen ovalbumin, comprising 385 residues, has been determined. The sequence was deduced from the 17 cyanogen bromide fragments and from peptides derived by digestion with a number of proteolytic enzymes. The molecular weight of the polypeptide chain of ovalbumin is 42699.Ovalbumin has four sites of postsynthetic modification; in addition to the acetylated N terminus, the carbohydrate moiety is located at Asn‐292, and the two phosphorylated serines are at residues 68 and 344. The ‘signal sequence’ of ovalbumin is between residues 234 and 252. The heptapeptide released during the conversion of ovalbumin to plakalbumin by subtilisin digestion corresponds to residues 346–352. The hen ovalbumin polymorphism characterised by an Asn–>Asp replacement results from a mutation at residue 311.The amino acid sequence of ovalbumin deduced from these amino acid sequence studies is in complete agreement with the sequence of mRNA determined by McReynolds et al. [Nature (Lond.) 273, 723–728 (1978)].
Processing of Naked 45‐S Ribosomal RNA Precursor in vitro by an RNA‐Associated Endoribonuclease
Authors: DENOYA Claudio , COSTA GIOMI Patricia , SCODELLER Eduardo A. , VÁSQUEZ César , LA TORRE José L. 
Published: March 2005

A processing endoribonuclease was isolated from the cytoplasm of chick embryos. The enzyme was easily obtained using an RNA extraction procedure based on a mild deproteinization with Sarkosyl and cold phenol/ chloroform. This technique assured the recovery of several proteins and the endoribonuclease in association with the RNA.It was demonstrated that this endoribonuclease was capable of promoting, in vitro, a precise processing of naked 45‐S ribosomal RNA precursor to molecules resembling the intermediates as well as the 28‐S and 18‐S cytoplasmic RNAs found in vivo. The presence of magnesium ions was required for the correct processing function of the enzyme. In addition, under the same conditions, the mature ribosomal RNA substrates were degraded at a slower rate by this RNA‐associated RNase.It was possible to fractionate the enzymatic preparation into two different populations by means of a sucrose gradient: one associated and the other partially free of an RNA component. The effect of the intrinsic RNA associated with the endoribonuclease on the enzymatic activity was tested by analyzing both the enzymatic populations and the total enzymatic preparation treated with pronase or with immobilized pancreatic RNase. In all cases in which the RNA component was present, the enzyme showed processing activity. On the other hand, when the RNA component was absent or at least partially degraded the enzyme proved to be more active in processing precursor molecules and in promoting extensive degradation of mature RNA species.Although the presence of RNA in association with the enzyme was demonstrated, its role in the regulation of the enzymatic activity is yet not clear.
A Reinvestigation of the Phosphorylation of Rabbit Skeletal‐Muscle Glycogen Synthase by Cyclic‐AMP‐Dependent Protein Kinase
Authors: EMBI Noor , PARKER Peter J. , COHEN Philip 
Published: March 2005

Glycogen synthase preparations which were free of endogenous phosphorylase kinase were phosphorylated to different extents with cyclic‐AMP‐dependent protein kinase and the phosphopeptides obtained by tryptic digestion were analysed. Limited tryptic digestion of the native enzyme quantitatively released two phosphopeptides (site 1 a and site 1 b) which were soluble in trichloroacetic acid. The trichloroacetic‐arid pellet contained a distinct phosphorylation site, which was shown to be identical to the serine residue which is phosphorylated by phosphorylase kinase (site 2).Sites 1 a, 1 b and 2 accounted for virtually all of the phosphorylation of glycogen synthase up to 2.5 molecules phosphate incorporated per subunit. Site 1 a was initially phosphorylated 7–10‐fold faster than site 2, and 15–20‐fold faster than site 1 b. The phosphorylation of site 1 a became much slower after about 0.5 molecules of phosphate had been incorporated per subunit.Cyclic‐GMP‐dependent protein kinase also phosphorylated glycogen synthase at sites 1 a, 1 b and 2. The order of phosphorylation was the same as that observed with cyclic‐AMP‐dependent protein kinase (site 1 a > site 2 > site 1 b).All three sites phosphorylated by cyclic‐AMP‐dependent protein kinase could be dephosphorylated by protein phosphatase 1. Site 2 was initially dephosphorylated 5–10‐fold faster than site la and more than 100‐fold faster than site 1 b. The MgATP‐dependent protein phosphatase dephosphorylated site 2, site 1a and site 1 b to a very similar manner to protein phosphatase 1.The different rates of phosphorylation and dephosphorylation of the three sites allowed the demonstration to be made that the activity of glycogen synthase depended on the state of phosphorylation of site 2 and site 1 a. The phosphorylation of site 1 b did not influence the activity under the conditions used.Incubation of glycogen synthase with high levels of phosphorylase kinase did not lead to the phosphorylation of either site 1 a or 1 b, or of the three sites phosphorylated by glycogen synthase kinase 3 (sites 3a, 3b and 3c). Similarly no phosphorylation of sites 3a, 3b or 3c by cyclic‐AMP‐dependent protein kinase was detected. It is concluded that site 2 is the only site at which overlapping substrate specificity occurs between these three glycogen synthase kinases.
Evidence for New Forms of Cardiac Myosin Heavy Chains in Mechanical Heart Overloading and in Ageing
Authors: KLOTZ Catherine , SWYNGHEDAUW Bernard , MENDES Hélène , MAROTTE Franiçoise , LEGER Jean J. 
Published: March 2005

Heavy chains of myosin rods and subfragment 1 were isolated from normal hearts and from mechanically overloaded hearts of young and older rats. These myosin heavy‐chain fragments were cleaved by cyanogen bromide or partially proteolysed by pronase and by chymotrypsin after denaturation with sodium dodecyl sulfate. The peptides, analyzed by electrophoresis on a one‐dimensional polyacrylamide slab gel, varied depending on the origin of the cardiac myosin heavy chains. Some bands present in the peptide patterns of the normal heart of young rats were missing from the pattern of greatly hypertrophied hearts and vice versa. We conclude that mechanical overloading of the heart stimulates the synthesis of cardiac myosin ‘isozyme’ with a heavy‐chain primary structure which is differen: from that observed in the normal heart of young rat.The patterns from myosin heavy‐chain peptides from the hearts of older rats were different from those for peptides from young rat hearts; these results also indicate the presence of a new myosin heavy, chain specific to ageing. No difference was detected between the peptide patterns of heavy chains isolated from hypertrophied hearts of young and older rats, and those isolated from normal hearts of older rats.
Stabilisierung von Enzymen and Antiseren durch Schweres Wasser
Authors: LEMM Ursula , WENZEL Martin 
Published: March 2005

Stabilisation of Enzymes and Antisera by Heavy Water The purpose of our investigations was to study the protective effect of 2H2O on the denaturation of biochemically active proteins, e.g. the enzymes lactate dehydrogenase, hexokinase and creatine kinase and the antibodies against the steroid hormones estrone and estradiol.The proteins were dissolved in buffer prepared either with water or heavy water. These protein solutions were exposed to heat or extremes of pH. After this the solutions were well diluted with buffer of normal water, so that the contents of heavy water was not more then 6%. The biochemical activity, i.e. enzyme‐activity or binding capacity was then measured under standard conditions.When lactate dehydrogenase was heated in H2O its activity decreased by about 80%, whereas the activity of the enzyme incubated in 2H2O was only slightly reduced. The lesser inactivation in 2H2O is not the consequence of the generally smaller dissociation of 2H2O, because the protective effect of 2H2O was evident at pH 7.5 and pH 8.2. With the enzyme hexokinase it could be shown, that the protective effect of albumin against denaturation is even more pronounced in 2H2O. Not only proteins which are dissolved in 2H2O are protected against inactivation by heat, but also lyophilized enzymes e.g. creatine kinase are stabilized if the hydrogen bonds are exchanged by deuterium.Besides these enzymes we tested the antibodies of the steroid hormones estrone and estradiol. A protective effect of 2H2O against heat inactivation was shown with estradiol‐antibody. The prolongation of half‐life after heating in 2H2O instead of H2O was three times. Estrone‐antibody was less sensitive against extremes of pH, if it was kept in 2H2O. 2H2O had an effect in alkaline as well as in acid ranges.There is a sharp increase in protection at low 2H2O‐concentrations, at higher concentrations of 2H2O its protective effect increases more slowly. From a practical point of view that means: to obtain a reasonable protective effect against denaturation a 2H2O‐concentration of 15–30% is sufficient.
Equilibrium Aspects of the Binding of myo‐Inositol Hexakisphosphate to Human Hemoglobin as Studied by 31P NMR and pH‐Stat Techniques
Authors: ZUIDERWEG Erik R. P. , HAMERS Laurentius F. , de BRUIN Simon H. , HILBERS Cornelis W. 
Published: March 2005

The interaction of myo‐inositol hexakisphosphate (P6 ‐insitol) with human hemoglobin has been studied as a function of pH using pH‐stat techniques and 31P NMR. With the pH‐stat method the following data were obtained: the association constants for the P6 and P6 complexes at alkaline and acid pH respectively and the proton absorption curves associated with the protein/phosphate interaction for both complexes from pH 5.5 to pH 9. From these data the affinities of P6 towards deoxyhemoglobin (Hb) and carboxyhemoglobjn (HbCO) have been dalculated as a function of pH. The shape of the proton absorption curves was found to be strongly dependent on the ligation state of the hemoglobin molecule.The pH dependence of the 31P NMR spectra of P6 bound to Hb or HbCO provides a monitor for the proton‐binding behaviour of the phosphate groups of P6‐instol when present in the central cavity of the protein. It appears that this behaviour is only slightly dependent on the ligation state of the hemoglobin molecule. The NMR spectral data were interpreted in terms of a model which takes into account the electrostatic interaction between the phosphate groups within the P6‐instol molecule as well as the electrostatic interaction between the phosphate groups and positively charged groups on the protein.To account for the discrepancy between the pH‐stat and 31 P NMR results, i. e. a strong dependence of the proton‐absorption curves and a weak dependence of the proton‐binding behaviour of P6‐instol on the ligation state of the protein respectively, it is proposed that a conformational change takes place in HbCO upon P6‐instol binding.
31P NMR Study of the Kinetics of Binding of myo‐Inositol Hexakisphosphate to Human Hemoglobin
Authors: ZUIDERWEG Erik R. P. , HAMERS Laurentius F. , ROLLEMA Harry S. , de BRUIN Simon H. , HIBERS Corneils W. 
Published: March 2005

The association and dissociation kinetics of the complexes of myo‐inositol hexakisphosphate (P6‐instol) with deoxyhemoglobin (Hb) and carboxyhemoglobin (HbCO) have been investigated by 31P NMR between pH 6.8 and pH 5.5. These complexes represent high‐affinity systems with binding constants varying between 105 M−1 and 2 × 109 M−1. 31P NMR spectra of P6‐instol were recorded in the presence of hemoglobin as a function of the P6‐inositol/hemoglobin molar ratio. It appeared that the exchange of the polyphosphate molecule between the solution and the central cavity binding site is fast on the P6‐instol NMR time scale. This observation cannot be reconciled with a single‐step binding mechanism of P6‐insitol to hemogloin.Analysis of the spectra revealed the occurrence of additional binding of P6‐insitol to both Hb and HbCO. This binding was also observed in pH‐stat experiments performed at low ionic strength. 31P NMR exoeriments carried out with hemoglobin of which the α‐chain N termini were carbamylated, strongly suggest that these termini constitute the additional binding site for P6‐insitolA model is proposed which accounts for the enhancement of exchange kinetics in these high‐affinity systems. In this model a rapid migration is assumed for P6‐insitol between the central cavity binding site and an entry/leaving site on the hemoglobin molecule. Based on this model 31P NMR linewidths and chemical shift patterns for this three‐site exchange problem were calculated.
Insulin Antagonism of Glucocorticoid Induction of Tyrosine Aminotransferase in Cultured Foetal Hepatocytes
Authors: HO Kevin K. W. , CAKE Max H. , YEOH George C. T. , OLIVER Ivan T. 
Published: March 2005

Whereas dexamethasone is unable to induce the premature formation of hepatic tyrosine aminotransferase when administered to foetal rats in utero, the steroid can induce the enzyme in foetal rat liver if the liver is first removed from the environment in ulero and grown in culture. Dexamethasone produced a significant induction of the enzyme at a concentration of 0.1 nM in cultured foetal hepatocytes, but for optimal induction the cells were exposed to 10 nM for 15 h. Growing the hepatocytes in the presence of physiological concentrations of insulin had no effect on the enzyme activity in control cells. However, the induction of the enzyme by dexamethasone was markedly diminished in the presence of insulin. This effect of insulin is both time‐dependent and dose‐dependent with significant inhibition being obtained with 1 nM insulin. Growing foetal hepatocytes in the presence of insulin has no effect on either the cellular level of glucocorticoid receptor or on the ability of dexamethasone receptor complexes to undergo nuclear translocation suggesting that insulin inhibits some event subsequent to translocation.The results are discussed in relation to the postnatal appearance of tyrosine aminotransferase and suggest that the marked decline in the plasma concentration of insulin, that is known to occur at birth, is a major contri butor to the postnatal induction of the enzyme.
Tertiary Structure of tRNAs in Solution Monitored by Phosphodiester Modification with Ethylnitrosourea
Authors: VLASSOV Valentin V. , GIEGÉ Richard , EBEL Jean‐Pierre 
Published: March 2005

The alkylation by ethylnitrosourea of phosphodiester bonds in yeast tRNAPhe, tRNAVal and in Escherichia coli tRNAGlu, tRNAMetf, tRNAmetm and tRNAPhe was investigated under various conditions.In unfolded tRNAs the reactivities of phosphates in various positions toward the reagent were similar. In the folded tRNAs remarkable differences in reactivities of phosphates located in various positions of the molecules were observed. In yeast and E. coli tRNAPhe, reactivities of phosphates in positions 9, 10, 11, 19, 49, 58, 59 and 60 were found to be strongly decreased. Some decrease in reactivity was observed for phosphates 23 and 24. Spermine and ethidium bromide did not influence the pattern of phosphate alkylation in the TΨC arm of yeast tRNAPhe. Our solution results fit with the crystal structure of tRNAPhe with respect to the potential availability of the phosphates in this tRNA to solvent as shown by others.Judging from the pattern of phosphate reactivities, the structure of E. coli tRNAPhe is very similar to that of yeast tRNAPhe. Upon thermal denaturation of the yeast tRNAPhe, the reactivity of the low‐reactive phosphates increased, demonstrating a cooperative melting curve. A comparison of the pattern of phosphate alkylation in several tRNAs, essentially in their TΨC arms, revealed a striking similarity, suggesting that the folding of these tRNAs is essentially similar.
Proteolytic Digestion Studies of Chromatin Core‐Histone Structure
Authors: BÖHM Lothar , BRIAND Gilbert , SAUTIÈRE Pierre , CRANE‐ROBINSON Colyn 
Published: March 2005

Trypsin digestion of chromatin results in a well‐defined set of limit peptides (P1‐P5) derived from the four core histones. Those from histones H3 and H4 have been identified. P1 is H3 residues 27‐129; P4 is H4 residues 18‐102 and P5 is H4 residues 20‐102.The N‐terminal sequences removed correlate well with the regions that undergo post‐synthetic acetylation and which show the greatest degree of sequence conservation. Autolytic digestion of chromatin releases a peptide (P1′) from H3 representing residues 21‐135. The implications of protease digestion of the higher order structure of chromatin are discussed.
A Reinvestigation of the Purity, Isoelectric Points and Some Kinetic Properties of the Aldehyde Dehydrogenases from Sheep Liver
Authors: AGNEW Kim E. M. , BENNETT Adrian F. , CROW Kathryn E. , GREENWAY Robert M. , BLACKWELL Leonard F. , BUCKLEY Paul D. 
Published: March 2005

Cytoplasmic aldehyde dehydrogenase was shown to be free of contamination by the mitochondrial enzyme by isoelectric focusing.
Both enzymes showed multiple banding in activity stains. The cytoplasmic enzyme gave two very close bands pl = 5.22 ± 0.03 whereas the mitochondrial enzyme showed seven bands, a pair at pl = 5.22 and five further bands of pl 5.48 ± 0.09, 5.56 ± 0.07, 5.56 ± 0.06, 5.70 ± 0.03 and 5.76 ± 0.02. Possible origins of the isoenzymes are discussed.
Disulfiram in a fourfold excess reduced the activity of the cytoplasmic enzyme to 9% of the initial value. The residual activity represents the activity of the disulfiram‐modified enzyme and is not due to mitochondrial contamination. This casts doubt on the role of an essential thiol group.
The mitochondrial enzyme shows a low amplitude (22%) burst in the production of 4‐nitrophenoxide ion during the hydrolysis of 4‐nitrophenyl acetate at pH 7.6. The burst rate constant was 7.3 ± 1 s−1 and the steady‐state rate constant was 0.2 s−1, values similar to those previously reported for the cytoplasmic enzyme.
The mitochondrial enzyme shows a burst in the release of protons during the oxidation of propionaldehyde at pH 7.6. The burst rate constant was 6 s−1 and the amplitude was equal to half the formal enzyme concentration. The significance of these results for the steady‐state mechanism is discussed.
Antibodies to the F1‐ATPase of Rhodospirillum rubrum and Its Purified Native β‐Subunit:
Authors: PHILOSOPH Sonia , GROMET‐ELHANAN Zippora 
Published: March 2005

Antibodies prepared against the Rhodospirillum rubrum F1‐ATPase (RrF1) and its purified, native β‐subunit, exhibited cross‐respectively with the following soluble preparations of R. rubrum ATPase: RrF0 . F1, RrF1 and the β‐subunit. Anti‐RrF1, but not anti‐β‐antibodies, also formed precipitin lines with soluble β‐less RrF1, indicating that antigenic determinants of both the β‐subunit and the other four RrF1‐subunits are expressed in the whole RrF1 molecule. Both antibodies agglutinated the R. rubrum chromatophores, suggesting that the β‐subunit is located on the external part of RrF1.
Both antibodies inhibited ATP synthesis and hydrolysis activities of R. rubrum chromatophores, as well as all the soluble ATPase reactions. Similar concentrations of each antibody were required for 50% inhibition of all these reactions, but anti‐RrF1 was always somewhat more effective than anti‐β. These data indicate that the β‐subunit is involved in the catalytic site of the RrF1‐enzyme.
The antibodies prepared against R. rubrum F1‐ATPase and its β‐subunit could bind the soluble chloroplast F1‐ATPase (CF1) and inhibited ATP‐linked reactions carried out by chloroplasts and by soluble CF1. In these reactions, unlike in the R. rubrum ones, anti‐β was a more potent inhibitor than the anti‐RrF1 antibody. The cross‐reaction obtained between the antibodies raised against R. rubrum F1 and its β‐subunit and the chloroplast CF1 indicates the presence of similar antigenic determinants in the photosynthetic prokaryotic and cukaryotic F1‐ATPases, which have been conserved during evolution.
Enzymic Synthesis of Lignin Precursors Comparison of Cinnamoyl‐CoA Reductase and Cinnamyl Alcohol: NADP+ Dehydrogenase from Spruce (Picea abies L.) and Soybean (Glycine max L.)
Authors: LÜDERITZ Thomas , GRISEBACH Hans 
Published: March 2005

Cambial sap of Spruce (Picea abies) proved to be a good source for isolation of cinnamoyl‐CoA reductase and cinnamyl alcohol: NADP+ dehydrogenase. Apparently homogenous enzymes were obtained by a multistep procedure including dye‐ligand chromatography and for the reductase also affinity chromatography on (coenzyme A)‐agarose. An improved purification procedure for the reductase from soybean cell cultures is also reported.Molecular weights and subunit composition of reductase and dehydrogenase from spruce are very similar to those of the corresponding enzymes from soybean.Reduction of feruloyl‐CoA to coniferaldehyde catalysed by the reductase is a freely reversible reaction with an equilibrium constant of 5.6 × 10−4M at pH 6.25. A strong dependence of the Michaelis constants on the type of buffer was found. For reductase the Km‐value of feruloyl‐CoA in phosphate buffer (5.2 μM) is about 14‐times smaller than in citrate buffer (73 μM).Pronounced differences in substrate specificities between the enzymes from spruce and soybean were found, which reflect the different lignin composition of gymnosperms and dicotyledenous angiosperms. From the kinetic constants of the enzymes it can be concluded that under physiological conditions feruloyl‐CoA is the preferred substrate for the reductase from both sources whereas sinapoyl‐CoA is a substrate only for the soybean reductase and sinapyldehyde a substrate only for the soybean dehydrogenase.4‐Coumaroyl‐CoA is a poor substrate for the reductase from both spruce and soybean. This result is consistent with the low content of 4‐coumaryl alcohol units in gymnosperm and angiosperm lignin.