bluuebunny/crossref_metadata_2025_split huggingface dataset:
Translation of Vitellogenin mRNA in the Presence of 7‐Methylguanosine 5′‐Triphosphate
Authors: WILLEMS Margherita , WIERINGA Bé , MULDER Janny , AB Geert , GRUBER Max 
Published: March 2005

Messenger RNAs for total estrogenized chicken liver proteins and for two of their major constituents (vitellogenin and serum albumin), globin mRNA and Mengo virion RNA were translated in the wheat germ system and in the mRNA‐dependent reticulocyte lysate in the presence of 7‐methylguanosine 5′‐triphosphate. Protein synthesis inhibition was studied as a function of the concentration of cap analog, mRNA and translational factors. The following observations were made. (a) Synthesis directed by total poly(A)‐containing RNAs, purified vitellogenin and serum albumin mRNAs could be completely inhibited by cap analogs; messenger RNA in poysomal RNA preparations was less efficiently inhibited than purified mRNAs. (b) Monitoring immunologically translation of the population of liver messengers, vitellogenin mRNA was found to be more inhibited than serum albumin mRNA. (c) Cap analogs inhibited translation of the capped globin mRNA competitively in the wheat germ system. (d) By increasing the concentration of translational factors inhibition could be reversed. (e) Cap analogs did not inhibit translation of the uncapped Mengo virion RNA in the reticulocyte lysate but did do so, partially and not competitively, in the wheat germ system. (f) By increasing the concentration of wheat germ extract the inhibitable portion of mengo‐directed synthesis increased.These results support speculation that cap analogs compete with mRNAs on the basis of affinity for initiation complex formation. Implications regarding the behaviour of the intact reticulocyte lysate and of uncapped viral RNAs in presence of cap analog are discussed.
Poly(adenylic acid)‐Containing RNA of Euglena gracilis during Chloroplast Development
Authors: VERDIER Gérard 
Published: March 2005

During light‐induced chloroplast development of dark‐grown Euglena gracilis, changes in the polyadenylated RNA distribution have been studied by hybridization of complementary DNA (cDNA) to poly(A)‐containing RNA from different preparations: total cell polyribosomes of dark‐grown cultures illuminated for 0, 1, 6, 24 h, and isolated chloroplasts extracted after 24 h of illumination.The hybridization kinetics revealed three or four frequency classes in the different poly(A)‐containing RNA populations. The high complexity of the poly(A)‐containing RNA sequences synthesized after only 1 h of illumination suggested a transcription mainly from nuclear genome origin. The disribution of poly(A)‐containing RNA was strongly modified after 24 h of illumination and a new class appeared corresponding to abundant sequences. At this stage of the greening, 73% of the poly(A)‐containing RNA sequences were common to the poly(A)‐containing RNA from‐grown cultures; so the sequences synthesized after illumination represented qualitatively a large diversity, but quantitatively, a low proportion of the poly(A)‐containing RNA population.The majority of poly(A)‐containing RNA sequences obtained from isolated chloroplasts was also present in total cell polyadenylated RNA population after 24 h of greening. However. a class corresponding to the most abundant RNA sequences represented 50% of poly(A)‐containing RNA from isolated chloroplasts and only 11% of total cell poly(A)‐containing RNA preparation. The estimated complexities, either for this specific class or for the total polyadenylated RNA population from isolated chloroplasts, suggested the presence of RNA sequences from nuclear origin at the level of chloroplast preparations.
The Dicyclohexylcarbodiimide‐Binding Protein of the Mitochondrial ATPase Complex from Neurospora crassa and Saccharomyces cerevisiae
Authors: SEBALD Walter , GRAF Thomas , LUKINS Harold Bruce 
Published: March 2005

Incubation of mitochondria from Neurospora crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low‐molecular‐weight polypeptide. This dicyclohexylcarbodiimide‐binding protein is identical with the smallest subunit (Mr8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein.The dicyclohexylcarbodiimide‐binding protein is extracted from whole mitochondria with neutral chloroform/methanol both in the free and in the inhibitor‐modified form. In Neurospora and yeast, this extraction is highly selective and the protein is obtained in homogeneous form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide label is enriched in the purified protein up to 50‐fold compared to whole mitochondria.Based on the amino acid analysis, the dicyclohexylcarbodiimide‐binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N‐terminal amino acid is tyrosine in Neurospora and formylmethionine in yeast.
Limited Proteolysis of the Pyruvate Dehydrogenase Multienzyme Complex of Escherichia coli
Authors: HALE Geoffrey , PERHAM Richard N. 
Published: March 2005

Incubation of the pyruvate dehydrogenase multienzyme complex of Escherichia coli with trypsin at pH 7.0 leads to limited proteolysis of the constituent polypeptide chains and, more slowly, to loss of enzymic activity. The core component, lipoate acetyltransferase, is the most susceptible to tryptic cleavage, whereas the lipoamide dehydrogenase component is apparently unaffected. The susceptiable peptide bonds in lipoate acetyltransferase involve lysine residues, probably on exposed loops of polypeptide chain.
No protection against the action of trypsin is afforded to the individual enzymes by their self‐assembly into the complex, suggesting that no gross conformational changes in the subunits take place on assembly.
The principal large fragments of lipoate acetyltransferase chains produced by limited tryptic digestion of native complex have molecular weights (estimated by sodium dodecylsulphate/polyacrylamide gel electrophoresis) of about 39000 and 36000. The fragment of molecular weight 36000 appears to be the ultimate main product. This fragment, presumably a folding domain of some kind, may be represented twice in the lipoate acetyltransferase chain (assumed molecular weight 83000).
The major product of tryptic digestion of the native complex remains of high molecular weight. Similarly, the proteolysed lipoate acetyltransferase/lipoamide dehydrogenase subcomplex retains its ability to form a complex of high molecular weight with pyruvate decarboxylase, the third component enzyme. The large folding domains of the lipoate acetyltransferase chains appear to be of major importance in the maintenance of subunit interactions in the enzyme complex.
These results with trypsin provide a satisfying rationale for the degradation of the native complex by endogenous proteinases encountered in earlier work.
Proteolysis of Human Ceruloplasmin
Authors: MOSHKOV Kirill A. , LAKATOS Susan , HAJDU János , ZÁVODSZKY Péter , NEIFAKH Solomon A. 
Published: March 2005

Highly purified human ceruloplasmin was isolated from fresh donor blood in the presence of inhibitors of proteolysis and from stored retroplacental blood serum both with and without inhibitors of proteolysis. According to the data of electrophoresis. ultracentrifuge sedimentation velocity and sedimentation equilibrium, all the ceruloplasmin samples were homogeneous, their molecular weight being 130 000.
The dissociation of the samples treated by dodecylsulphate, guanidine · HCl, and urea was studied by means of quantitative analytica and preparative electrophoresis, and sedimentation equilibrium. The dissociation patterns depended on whether inhibitors were used in the isolation procedure. Polypeptides with molecular weights of 130 000, 112 000 and 16 000 (minor component) were obtained, if phenylmethylsulfonyl fluoride and/or 6‐aminohexanoic acid were used; if these compounds were not used, the dissociation yielded additional polypeptides of molecular weights of 100 000 (minor component), 64 000 and 48 000. Under proteolysis‐favouring conditions the relative amount of these polypeptides increased. Prolonged storage of samples under sterile conditions without inhibitors of proteolysis resulted in a decrease of the relative amount of polypeptides with molecular weights of 130 000, 112 000, 100 000, 64 000 and 48 000, contrary to that of the 16 000‐Mr polypeptide, which increased. At the same time new polypeptides appeared with molecular weights of 42 000 and 21 500–23 000.
Spontaneous specific fragmentation of the ceruloplasmin molecule is due to trace amounts of proteases, which seem to originate from blood plasma.
Limited tryptic hydrolysis of the ceruloplasmin globule resulted in the appearance of polypeptides with the same molecular weights which were observed in spontaneous fragmentation.
A conclusion is drawn that the ceruloplasmin molecule in vivo is a single polypeptide chain with at least five bonds which in vitro are the points of specific proteolytic fragmentation, yielding six principal fragments.
The Association of the Serotonin‐Sensitive Aryl Acylamidase with Acetylcholinesterase in the Monkey Brain
Authors: OOMMEN Anna , BALASUBRAMANIAN Aiylam S. 
Published: March 2005

The serotonin‐sensitive aryl acylamidase was partially purified from monkey brain. The aryl acylamidase activity was inhibited by serotonin (Ki= 0.425 mM) and tryptamine (Ki= 3.6 mM) but not by a number of other amines. It was also inhibited by acetylcholine (Ki= 2 mM) and its analogues and homologues. The relationship of aryl acylamidase to acetylcholinesterase was examined. The ratios of specific activities of aryl acylamidase and acetylcholinesterase in the different steps of purification were approximately constant and the percentage recoveries of both enzyme activities were comparable. Elution profiles of both enzyme activities from concanavalin‐A‐Sepharose, Sephadex G‐200 and DEAE‐Sephadex A‐25 columns were similar. Both enzyme activities migrated in a similar fashion on gel electrophoresis in different percentage gels for different time intervals. Both enzymes showed similar distribution in the various anatomical regions and in the different subcellular fractions of monkey brain.Eserine and neostigmine, potent competitive inhibitors of acetylcholinesterase also potently inhibited aryl acylamidase in a non‐competitive manner. Inhibition of both enzymes was 100% at 10 μM of both the inhibitors. Tetraisopropylpyrophosphoramide, a selective inhibitor of pseudo‐cholinesterase, did not inhibit either the brain acetylcholinesterase or aryl acylamidase at 10μM. Serotonin inhibited acetylcholinesterase only at concentrations above 10 mM. Dixon plots of one inhibitor in the presence of additional inhibitors indicated that serotonin, butyrylcholine, acetylcholine, neostigmine and eserine (all non‐competitive inhibitors of aryl acylamidase) acted at the same site as inhibitors of aryl acylamidase.The serotonin‐insensitive aryl acylamidase of monkey liver was also insensitive to acetylcholine and eserine and was not found associated with acetylcholinesterase [A. Oommen and A. S. Balasubramanian (1978) Biochem. Pharmacol. 27, 891–895]. Erythrocyte membrane known to contain true cholinesterase had aryl acylamidase activity sensitive to serotonin, acetylcholine and eserine. All these considerations suggest that the serotonin‐sensitive aryl acylamidase activity is a property of true cholinesterase. Although some of the experimental results would suggest that in the monkey brain the two activities may be associated with the same protein with two different active sites further experiments are needed to confirm this.
Yeast Argininosuccinate Synthetase
Authors: HILGER François , SIMON Jean‐Paul , STALON Victor 
Published: March 2005

Yeast argininosuccinate synthetase has been purified to homogeneity. The enzyme was found to have a molecular weight of 228000 as determined by gel sieving. It is composed of identical subunits of Mr 49000 as shown by gel electrophoresis.The quaternary structure as determined by cross‐linking of the subunits with glutaraldehyde, followed by gel electrophoresis with dodecylsulfate, is tetrameric.The saturation functions by citrulline and aspartate are hyperbolic; with MgATP as the variable substrate a sigmoid character, dependent on the concentration of citrulline, aspartate, argininosuccinate and arginine, was observed. The positive cooperativity is reduced by increasing concentrations of citrulline and asparate; it is increased by argininosuccinate and arginine.Kinetic analysis provided evidence for a random addition of substrates. Initial velocity studies as well as product and dead‐end inhibition studies comply with a rapid‐equilibrium random model, except for the interconversion of the central quaternary complexes; the different kinetic constants have been established on this basis.Yeast arginonosuccinate synthetase has a double metabolic function: anabolic in the biosynthesis of arginine, catabolic as the first enzyme of citrulline utilization as nitrogen source. The kinetic properties of the enzyme point to a physiologically well‐adjusted activity for both roles and to an economic and efficient utilization of ATP.
Quaternary Structure of Higher Plant Glyceraldehyde‐3‐Phosphate Dehydrogenases
Authors: CERFF Rüdiger 
Published: March 2005

NAD(P)+‐induced changes in the aggregational state of prepurified NADP‐linked glyceraldehyde‐3‐phosphate dehydrogenase (EC 1.2.1.13) were used to isolate the enzyme from Spinacia oleracea, Pisum sativum and Hordeum vulgare. Each of the three plant species contains two separate isoenzymes. Isoenzyme 1 (fast moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 55–70% saturation. It shows two separate subunits in dodecylsulfate gels, which are probably arranged as A2B2 in the native enzyme molecule. Isoenzyme 2 (slow moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 70–95%. It contains a single subunit of the same Mr as subunit A in isoenzyme 1 and is apparently a tetramer (A4). The molecular weights of subunits A/B for spinach, peas and barley were determined as 38000/40000, 38000/42000 and 36000/39000 respectively.
The NAD‐specific glyceraldehyde‐3‐phosphate dehydrogenase (EC 1.2.1.12) was purified from Spinacia oleracea and Pisum sativum by affinity chromatography on blue Sepharose CL‐6B. The enzyme from both plant species is shown to be a tetramer of subunits with Mr 39000.
The present findings contrast with heterogeneous results obtained previously by other authors. These results suggested that there are considerable interspecific differences in the quaternary structure of glyceraldehyde‐3‐phosphate dehydrogenases from higher plants.
Localisation of an Endonuclease Specific for Double‐Stranded RNA within the Nucleolus and Its Implication in Processing Ribosomal Transcripts
Authors: GRUMMT Ingrid , HALL Stanton H. , CROUCH Robert J. 
Published: March 2005

Nucleoli of both chick embryos and mouse Ehrlich ascites cells contain an enzymatic activity that is very similar to RNase DII, an enzyme isolated from total chick embryos for its ability to degrade double‐stranded RNA. The enzyme can be extracted by low salt/EDTA from nucleoli and is associated with pre‐ribosomal 80‐S and 55‐S particles. Under ionic conditions which are inhibitory for the nucleolytic activity the transcript in vitro of nucleoli is not processed and sediments around 45 S. Under salt conditions which are optimal for the nucleolar enzyme the nucleolar transcripts are cleaved to distinct intermediate‐sized moleculues. Addition of the chicken RNase DII or RNase III to the nucleolar transcription system results in a similar shift of the chain length of the RNA molecules.It is concluded that a nucleolar RNase recognizing double‐stranded regions in the pre‐ribosomal RNA is involved in the maturation of ribosomal RNA.
Effects of Phospholipase A2 and Albumin on the Calcium‐Dependent ATPase and the Lipid Composition of Sarcoplasmic Membranes
Authors: SWOBODA Gertrude , FRITZSCHE Joachim , HASSELBACH Wilhelm 
Published: March 2005

The calcium‐dependent ATPase activity of phospholipase‐A2‐digested sarcoplasmic vesicles decreases concomitantly with the contents of residual lysophospholipids and fatty acids when increasing albumin concentrations are applied.
Delipidated albumin preferentially removes unsaturated fatty acids and lysophosphatidylcholine. A complete removal of the phospholipids by albumin does not occur.
The membrane‐bound lysophospholipids were analysed with respect to type of phospholipid, plasmalogen content and fatty acid chains by means of thin‐layer chromatography and gas chromatography.
While the fatty acid composition of the lysophospholipids is independent of the degree of delipidation, the composition of the residual free fatty acids is found to change with the albumin concentration.
Reactivation of the Ca2+‐ATPase by oleate leads to reasonable activities at room temperature as long as a minimum of about 30 lysophospholipid moleucules per ATPase is left. The course of the residual Ca2+‐ATPase activity with the degree of delipidation is related to the presence of unsaturated fatty acids.
No specific role of either sphingomyelin or the plasmalogens has been found.
Dual Localization of Long‐Chain Acyl‐CoA Hydrolase in Rat Liver: One in the Microsomes and One in the Mitochondrial Matrix
Authors: BERGE Rolf Kristian , FARSTAD Mikael 
Published: March 2005

Subcellular fractionation studies of rat liver localized the activity of palmitoyl‐l‐carnitine hydroalse to the microsomal fraction whereas palmitoyl‐CoA hydrolase activity was found both in the microsomal fraction and in mitochondria.An unusual biphasic saturation curve for palmitoyl‐CoA was observed when intact mitochondria were used, possibly reflecting the existence of an intramitochondrial hydrolase activity. Disruption of the mitochondrial structure doubled the palmitoyl‐CoA hydrolysis. Discontinuous sucrose gradient centrifugation and digitonin of rat liver mitochondria demonstrated that a palmitoyl‐CoA hydrolase was associated with the matrix fraction.Pure matrix and microsomal fractions showed that the two hydrolase activities were differently affected by the presence of divalent cations. Both the specific activity and the saturation concentration of palmitoyl‐CoA were higher for the microsomal enzyme than for the matrix‐associated enzyme.
Globin mRNA Precursor
Authors: AGLIANÓ Anna Maria , NACCI Aldo , REYMOND Christophe , APPLEBY David , SPOHR Georges 
Published: March 2005

The globin mRNA sequences present in duck erythroblast nuclei appear under non‐denaturing conditions to be associated with heterogeneous nuclear RNA (hnRNA) molecules of various sizes.Under denaturing conditions, however, the bulk of the globin mRNA sequences associated with hnRNA are released as molecules of size close to that of the active globin mRNA.To find out whether hydrogen‐bonded structures occur in situ or arise after RNA extraction. nuclei were treated with aminomethyltrioxalen and exposed to ultraviolet light. This treatment generates covalent links between opposite strands of double‐stranded nucleic acids, which were visualised by electron microscopy. It appears that, after cross‐linking, a fraction of the globin mRNA sequences present in nuclei is associated with high‐molecular‐weight hnRNA molecules by a link which is resistant to stringent denaturing conditions. In particular globin mRNA sequences are found associated with a band of 0.9×106 molecular weight approximately.It is suggested that within the erythroblast nucleus, globin mRNA sequences are associated by hydrogen bonds with RNA of high molecular weight. These structures may represent intermediate steps in globin mRNA processing.
Amino Acid Sequence Studies of Horseradish Peroxidase
Authors: WELINDER Karen Gjesing 
Published: March 2005

Horseradish peroxidase C dominates quantitatively among the isoperoxidases of horseradish root and has an isoelectric point close to 9. It consists of a hemin prosthetic group, 2 Ca2+and 308 amino acid residues, including 4 disulfide bridges, in a single polypeptide chain that carries 8 neutral carbohydrate side‐chains. The molecular weight of the polypeptide chain is 33890. Assuming an average carbohydrate composition of (GlcNAc)2, Man3, Fuc, Xyl for each carbohydrate chain, the molecular weight of native horseradish peroxidaseCis close to 44000.Cyanogen bromide fragments of reduced and carboxymethylated apo‐peroxidase were purified by a combination of gel filtration and isoelectric focusing in urea, and cystine‐containing tryptic fragments of apo‐peroxidase were purified by gel filtration followed by disulfide cleavage and rechromatography at the initial conditions.The present paper discusses (a) isoelectric points and charge distribution within the native protein, the apoprotein and the cyanogen bromide fragments, (b) a buried pyrrolidonecarboxylyl amino terminus, (c) heterogeneity at the carboxyl terminus, and (d) a possible domain structure, likely from partial tryptic digestion.
Purification and Properties of Sheep‐Liver Aldehyde Dehydrogenases
Authors: MACGIBBON Alistair K. H. , MOTION Rosemary L. , CROW Kathryn E. , BUCKLEY Paul D. , BLACKWELL Leonard F. 
Published: March 2005

Sheep liver cytoplasmic aldehyde dehydrogenase was purified to homogeneity to give a sample with a specific activity of 380 nmol NADH min−1 mg−1.
An amino acid analysis of the enzyme gave results similar to those reported for aldehyde dehydrogenases from other sources. The isoelectric point was at pH 5.25 and the enzyme contained no significant amounts of metal ions.
On the binding of NADH to the enzyme there is a shift in absorption maximum of NADH to 344 nm, and a 5.6‐fold enhancement of nucleotide fluorescence. The protein fluorescence (λexcit= 290 nm, λemission= 340 nm) is quenched on the binding of NAD+ and NADH.
The enhancement of nucleotide fluorescence on the binding of NADH has been utilised to determine the dissociation constant for the enzyme · NADH complex (Kd= 1.2 ± 0.2 μM). A Hill plot of the data gave a straight line with a slope of 1.0 ± 0.3 indicating the absence of co‐operative effects.
Ellman's reagent reacted only slowly with the enzyme but in the presence of sodium dodecyl‐sulphate complete reaction occurred within a few minutes to an extent corresponding to 36 thiol groups/enzyme.
Molecular weights were determined for both cytoplasmic and mitochondrial aldehyde dehydrogenases and were 212000 ± 8000 and 205000 respectively. Each enzyme consisted of four subunits with molecular weight of 53000 ± 2000.
Properties of the cytoplasmic and mitochondria1 aldehyde dehydrogenases from sheep liver were compared with other mammalian liver aldehyde dehydrogenases.