bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intracellular transport of the major glycoprotein of zymogen granule membranes in the rat pancreas
Authors: HAVINGA Johan R. , STROUS Ger J. , POORT Cees 
Published: March 2005

The intracellular transport and destination of the major glycoprotein associated with zymogen granule membranes in the pancreas (GP‐2) was established. In suspensions of isolated acinar cells from rat pancreas, pulse‐chase experiments were performed. The incorporation of the first newly synthesized GP‐2 molecules into zymogen granule membranes occurred at about 60 min after beginning of the pulse. We demonstrated by using two different methods that newly made GP‐2 reaches the cell surface within the same time span. After 6–8 h chase considerable more newly synthesized GP‐2 has reached the cell surface than would be expected on account of secreted newly synthesized zymogens. These observations strongly suggest that at least part of the GP‐2 molecules bypass the mature zymogen granule compartment on their way to the plasma membrane.GP‐2 is the only protein that appears in discernable quantity in the plasma membrane during 1–4h after a pulse label. Nevertheless GP‐2 comprises only a small percentage of externally 125I‐iodinated plasma membrane proteins. We conclude that GP‐2 has a high turnover rate at the plasma membrane level.Treatment of the acinar cells with the N‐gylcosylation inhibitor tunicamycin does not block the intracellular transport of GP‐2.
Isolation and characterization of a somatomedin‐binding protein from mid‐term human amniotic fluid
Authors: PÓVOA Guilherme , ENBERG Gösta , JÖRNVALL Hans , HALL Kerstin 
Published: March 2005

Human amniotic fluid is rich in a binding protein for somatomedins. This binding protein competes with human placenta membranes for labelled somatomedin A. Consequently, the placenta radioreceptorassay for somatomedin can be used for detection of the binding protein. The protein was isolated from human amniotic fluid by a three‐step procedure: First, stepwise ammonium sulphate precipitation; second, hydrophobic chromatography (phenyl‐Sepharose); and third, anion‐exchange chromatography (fast protein liquid chromatography). The total recovery of binding protein calculated with the placenta radioreceptorassay was 50%.Polyacrylamide gel electrophoresis under native and denaturating conditions of the isolated protein disclosed a single band. The relative molecular mass was 35000, determined by exclusion chromatography, and 32000 under denaturating conditions in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The isoelectric point was 4.3 according to chromatofocusing and the amino acid composition also disclosed a high content of acidic/amidated residues. The N‐terminal amino acid sequence was Ala‐Pro‐Trp‐Gln‐Cys‐Ala‐Pro‐Cys‐Ser‐Ala.
Histidine residues in elongation factor EF‐Tu from Escherichia coli protected by aminoacyl‐tRNA against photo‐oxidation
Authors: JONÁK Jiří , PETERSEN Torben E. , MELOUN Bedřich , RYCHLÍK Ivan 
Published: March 2005

Complexes of Escherichia coli elongation factor EF‐Tu with GTP or GTP and aminoacyl‐tRNA were photo‐oxidized by irradiation with visible light in the presence of rose bengal dye. EF‐Tu was isolated, digested with trypsin, the resulting tryptic peptides were separated by high‐performance liquid chromatography (HPLC), and the position of most of the peptides on the chromatogram was determined. Irradiation of complexes resulted in the inactivation of the factor (as tested by its capacity to interact with aminoacyl‐tRNA) and was accompanied by the loss of its histidine residues (as revealed by amino acid analysis) and by the decrease in the amount of some tryptic peptides (as detected by HPLC).Aminoacyl‐tRNA, bound to EF‐Tu during the irradiation, protected the protein from inactivation, from the loss of histidine residues and some of its peptides from photo‐oxidative degradation. Comparison of quantities of individual tryptic peptides recovered from the irradiated EF‐Tu GTP aminoacyl‐tRNA complex with those from the irradiated EF‐Tu GTP complex revealed that histidine‐containing peptides T12 and T15 as well as methionine‐containing peptide T14 were in the ternary complex markedly protected against the photo‐oxidative degradation. This finding suggests that their histidines, i.e. His‐66 and His‐118 respectively and at least one of the methionines (Met‐91, 98 or 112) present in peptide T14 are located near to or at the binding site of EF‐Tu for aminoacyl‐tRNA and could be involved in the interaction between aminoacyl‐tRNA and the factor.
Computer simulation of purine metabolism
Authors: FRANCO Rafael , CANELA Enrique I. 
Published: March 2005

A computer model of purine metabolism, including catabolism, salvage pathways and interconversion among nucleotides, is given. Steady‐state rate equations corresponding to metabolic enzymes are written based on information from the literature about their kinetic behaviour. Numerical integration of this set of equations is performed employing selected parameters taken from the literature. After stabilization of purine compound concentrations is reached, simulation of enzyme deficit and enzyme overproduction is carried out. The latter is calculated by varying specified maximum velocities in the numerical integration. A pattern of intermediate metabolite concentrations is found. These results form a basis for the comparison of normal patterns or patterns reflecting the effects of inborn errors of metabolism.The aim of this paper is to demonstrate the usefulness of this computer simulation method in complex metabolism pathways.
Neuronal nuclei and glial nuclei from mammalian cerebral cortex
Authors: PEARSON Edwin C. , BATES David L. , PROSPERO Terence D. , THOMAS Jean O. 
Published: March 2005

We have characterised the histone and DNA contents of neuronal and glial nuclei from ox cerebral cortex which have, respectively, repeat lengths of 162 base pairs and 201 base pairs. Although the neuronal population cannot be obtained completely free of glial nuclear contamination, the degree of contamination is easily determined by counting, and has been allowed for in all the methods used here.

By diphenylamine assay and flow cytofluorometry we find that the DNA contents of both nuclear types are essentially equal, and equivalent to the diploid value, contrary to some reports.
By quantification of the core histones in known numbers of nuclei with respect to an added external standard, we have shown that the ratio of core histone octamers in the two nuclear types, neuronal and glial, is the inverse of the ratio of repeat lengths. Thus the same proportion of DNA is associated with core histone octamers in the two nuclear types, most simply all of the DNA.
By complete radiolabelling of the lysine side chains of the histones with methyl [1‐3H]acetimidate we have determined the stoichiometry of H1 relative to the core histones. Neuronal nuclei‐have a low H1 content of 0.45 molecule H1/nucleosome on average; glial nuclei have the ‘normal’ 1 H1 molecule/nucleosome. In neuronal nuclei about half of the nucleosomes therefore probably lack H1.Whether there is any relation between the low H1 content and the short DNA repeat length of neuronal nuclei, on the one hand, and their high transcriptional capacity (at least when assayed in vitro), on the other, remains to be established.
Import and processing of cytochrome b‐c1 complex subunits in isolated hepatoma ascites cells
Authors: KOLAROV Jordan , NELSON B. Dean 
Published: March 2005

The import and processing of cytochrome c1 and the iron sulfur protein of the cytochrome b‐c1 complex were studied in Zajdela hepatoma ascites cells. Both peptides were synthesized as larger percursor molecules which were approximately 2–3kDa and 5–6kDa larger than the mature forms of apocytochrome c1 and apo‐iron sulfur protein, respectively. Comparison of these precursors to those reported for functionally homologous peptides in yeast and Neurospora indicate significant size changes have occurred in mammals.Rhodamine 6G, a specific vital stain for mitochondria, is a potent inhibitor of precursor processing in isolated hepatoma cells. Both precursor to cytochrome c1 and precursor to FeS accumulate in the soluble and paniculate fractions obtained by digitonin treatment of tumor cells treated with Rhodamine 6G. Appearance of the mature peptides was abolished. The precursors are unstable, however, and disappear from the cytosolic and membrane fractions during a 10 min chase. Comparison of the effects of Rhodamine 6G and carbonylcyanide m‐chlorophenylhydrazone on precursor processing shows that: (a) Rhodamine 6G is a more effective inhibitor of processing, (b) it has less of an inhibitory effect on cellular protein synthesis, and (c) it inhibits processing under conditions in which it appears to have little influence on coupled B. D. Nelson, Biokemiska Institutionen, Stockholms Universitet, Arrheniuslaboratoriet, Bergiusvägen 65, Frescati, S‐106‐91 Stockholm, Sweden respiration in whole cells. The data suggest that the most likely mode of action of Rhodamine 6G is on the matrix processing step.
α‐2→4‐Interlinked 3‐deoxy‐d‐manno‐octulosonic acid disaccharide
Authors: BRADE Helmut , RIETSCHEL Ernst‐Theodor 
Published: March 2005

After mild acid hydrolysis, a disaccharide of 3‐deoxy‐d‐manno‐octulosonic acid (dOclA) was obtained from Re‐mutant lipopolysaccharide of Salmonella minnesota, Salmonella godesberg, Proteus mirabilis, and Escherichia coli, and from the lipopolysaccharide of an S. minnesota Rb2 mutant. Combined gas‐liquid chromatography/ mass spectrometry of the reduced and permethylated derivatives indicated that the disaccharide is interlinked by a 2	4‐glycosidic bond in all lipopolysaccharides tested. In addition, it was shown by gas‐liquid chromatography of appropriate synthetic standards and a previously characterized α2	4‐linked dOclA disaccharide (derived from lipopolysaccharide of S. godesberg) that the non‐reducing dOclA residue possesses the α configuration. In the case of lipopolysaccharide of S. minnesota Rb2 mutant, this result, together with earlier findings, suggests that it contains a linear dOclA trisaccharide of the sequence dOclA (α2–4)dOclA(α2–4)dOclA. The results show that a dOclA (α2–4)dOclA disaccharide represents a common architectural principle in enterobacterial lipopolysaccharides.
Characterization of the transcription products of glyceraldehyde 3‐phosphate‐dehydrogenase gene in HeLa cells
Authors: DANI Christian , PIECHACZYK Marc , AUDIGIER Yves , EL SABOUTY Sanaa , CATHALA Guy , MARTY Louise , FORT Philippe , BLANCHARD Jean‐Marie , JEANTEUR Philippe 
Published: March 2005

We have partially purified the messenger RNA coding for glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from HeLa cells and obtained a cDNA clone containing part of its sequence. Using this clone to probe electrophoregrams of RNA transferred to nitrocellulose, we have investigated the characteristics of nuclear and cytoplasmic transcripts in these cells.In the cytoplasm, nature GAPDH mRNA was detected in Northern blots as an intense band, apparently unique, of approximately 1400 nucleotides. The half‐life of this mRNA was determined both from the decay kinetics, after a chase with actinomycin D, and from the labeling kinetics during an accumulation experiment. Both kinds of experiments yielded a half‐life value of about 8 h, while the accumulation experiment indicated that steady‐state GAPDH mRNA amounted to about 1.6% of cytoplasmic poly(A)‐rich RNA.Much longer species, likely to be restricted to the nucleus, were also detected in RNA extracted from total cells. At least three discrete species of 1600,4000, 5800 and 6800 bases were observed above a trailing background extending up to about 8000 bases. This value is commensurate with a functional size of the GAPDH transcription unit in the order of 13000 bases, which we determined by measuring the size of the ultraviolet inactivation target. Until direct evidence can be obtained at the genomic level, the present results provide the first clue to the existence of introns, presumably at least four, in a GAPDH gene from a higher eucaryote.
Conformational stability of the myosin rod
Authors: CROSS Robert A. , BARDSLEY Ronald G. , LEDWARD David A. , SMALL J. Victor , SOBIESZEK Apolinary 
Published: March 2005

Chymotryptic cleavage patterns of myosin rods from pig stomach, chicken gizzard, and rabbit skeletal muscle indicate that short (∼45 nm) heavy meromyosin subfragment 2 (SF2) is a consistent product of all three rods, whereas long (∼ 60 nm) SF2 is derived only from skeletal muscle myosin.Differential scanning calorimetry was used to follow the thermally induced melting transition of the rods and certain of their subfragments. In 0.12 M KCl, sodium phosphate buffer, pH 6.2–7.6, the light meromyosin (LMM) and SF2 domains of each rod had essentially identical conformational stabilities. Temperature midpoints for the melting transitions were 54–56°C for the two smooth muscle myosin rods and 50–53°C for the skeletal muscle myosin rod. In 0.6 M KCl buffer, melting transitions for the smooth muscle myosin rods were essentially unchanged, but skeletal muscle myosin rods showed multiphase melting, with major transitions at 43°C and 52°C. The first of these was tentatively attributed to LMM, and the second to SF2. In 0.12 M KCl buffer, the LMM transition was stabilised so that it superimposed on that of SF2. No melting was observed in any of the rods at physiological temperature. These results indicate that, excluding a possible but only narrow hinge region, the entire myosin rod has essentially uniform conformational stability at physiological pH and ionic strength, and thus that the contractile and elastic properties of the cross‐bridge exist in the heavy meromyosin subfragment 1 (SF1) domains of the molecule.
pH‐induced co‐operative effects in hysteretic enzymes
Authors: RICARD Jacques , NOAT Georges , NARI Joannès 
Published: March 2005

A new model which provides an explanation for pH‐induced co‐operativity of hysteretic enzymes is proposed. The essence of the model is that a region, or a domain, of the enzyme undergoes a spontaneous ‘slow’ conformational change which does not affect the geometry of the active site. The region which undergoes this spontaneous conformational transition bears an ionizable group. Kinetic co‐operativity occurs if the pK of this ionizable group changes upon this conformational transition. Thus co‐operativity does not arise from a distortion of the active site.An interesting prediction of the model is that at ‘extreme’ pH values co‐operativity must be suppressed. Although the kinetic equation pertaining to the model is of the 2:2 type, co‐operativity is not maximum or minimum at half‐saturation of the enzyme by the substrate, as occurs with 2:2 binding isotherms. A new index of maximum or minimum kinetic co‐operativity, whether this extreme occurs at half‐saturation or not, has been proposed which allows the change of kinetic co‐operativity to be followed as a function of pH.It is believed that this model will be useful in explaining the behaviour of enzymes attached to biological polyelectrolytes, such as membranes or cell envelopes.
pH‐induced co‐operative effects in hysteretic enzymes
Authors: NARI Joannès , NOAT Georges , RICARD Jacques 
Published: March 2005

A β‐glucosyltransferase, extracted and purified from the cell walls of isolated soybean cells, displays hysteretic behaviour. The enzyme is monomeric and has a negative co‐operative between pH 5.5 and 7.5. Below and above these pH values, the enzyme follows, or approaches, classical Michaelis‐Menten kinetics.The free enzyme and the enzyme‐glucose complex exhibit, upon pH jumps, conformational transitions which may be followed by monitoring the fluorescence of enzyme‐bound toluidinylnaphthalene sulfonate.Taken together these results are consistent with the model of pH‐induced co‐operativity described in the preceding paper in this journal. This special type of co‐operativity relies on a change of the pK value of a strategic ionizable group located outside the active site in a region (or a domain) of the protein which undergoes the conformational transition. The result that at ‘low’ and ‘high’ pH values, the enzyme follows or approaches Michaelis‐Menten kinetics is explained by assuming that the conformational changes do not affect the active site.
Increased ribosomal affinity for mRNA causes resistance to edeine in a mutant of Saccharomyces cerevisiae
Authors: HERRERA Flor , MORENO Nancy , MARTÍNEZ José Antonio 
Published: March 2005

The effect of edeine on the translation of mRNA or poly(U)‐directed polyphenylalanine synthesis has been studied in an edeine‐resistant mutant of Saccharomyces cerevisiae under three different experimental conditions: in the whole lysate system, in a micrococcal‐nuclease‐treated lysate, and in a high‐salt‐treated lysate. The results indicate that translation of messenger is more resistant to edeine in the whole lysate than in the depleted lysates: these observations suggest that resistance to edeine is associated with the presence of endogenous mRNA.It is shown that 40S mutant subunits have a higher affinity for polysomal RNA than 40S wild‐type subunits. Since the mRNA binding is inhibited by 7‐methylguanosine 5′‐monophosphate, the interaction between polysomal RNA and 40S ribosomes is specific for mRNA.The data demonstrate that in each of the depleted lysates, with edeine initially present, the formation of the 80S initiation complex is inhibited. However, edeine inhibition of [3H]methionine binding to 80S ribosomes is overcome completely in the mutant extract by preincubation of this lysate with polysomal RNA.The results indicate that the mutant may carry a specific change in a messenger‐binding factor or in a ribosomal protein thereby permitting an increased stability of the messenger‐ribosome complex which consequently results in an increased resistance of the mutant lysate to edeine.
Developmental regulation of gene expression in wheat embryos
Authors: CUMING Andrew C. 
Published: March 2005

The early‐methionine‐labelled (Em) polypeptide is the most abundant single polypeptide found in the cytosolic fraction (30000 ×g supernatant) of dry wheat embryos. Synthesis of this polypeptide is readily detectable during the earliest stages (0–3 h) of wheat embryo germination, by labelling with [35S]methionine. Thereafter, synthesis of the Empolypeptide declines rapidly.A DNA sequence encoding a portion of this polypeptide has been isolated by the molecular cloning of DNA complementary to the messenger RNA from dry wheat embryos. The identity of this cloned sequence has been confirmed by hybrid‐selected translation of the Emmessenger, the product being analysed by one‐ and two‐dimensional polyacrylamide gel electrophoresis, and by analysis of its partial proteolytic digestion products.Gel blot hybridisation of RNA isolated from embryos at successive stages of germination with this cloned DNA sequence confirms that the decline in the synthesis of the Empolypeptide, detected by labelling in vivo, is a consequence of the degradation of its corresponding messenger RNA.
Effects of the ribosomal subunit association on the chemical modification of the 16S and 23S RNAs from Escherichia coli
Authors: MEIER Norbert , WAGNER Rolf 
Published: March 2005

70S ribosomes and 30S and 50S ribosomal subunits from Escherichia coli were modified under non‐denaturing conditions with the chemical reagent dimethylsulfate. The ribosomal 23S and 16S RNAs were isolated after the reaction and the last 200 nucleotides from the 3′ ends were analyzed for differences in the chemical modification. A number of accessibility changes could be detected for 23S and 16S RNA when 70S ribosomes as opposed to the isolated subunits were modified.In addition to a number of sites which were protected from modification several guanosines showed enhanced reactivities, indicating conformational changes in the ribosomal RNA structures when 30S and 50S subunits associate to a 70S particle. Most of the accessibility changes can be localized in double‐helical regions within the secondary structures of the two RNAs. The results confirm the importance of the ribosomal RNAs for ribosomal functions and help to define the RNA domains which constitute the subunit interface of E. coli ribosomes.
Dual function of calmodulin (δ) in phosphorylase kinase
Authors: HESSOVÁ Zuzana , VARSÁNYI Magdolna , HEILMEYER Ludwig M. G. 
Published: March 2005

The Ca2+‐independent activity of fast skeletal muscle phosphorylase kinase, A0, can be reversibly stimulated by heparin more than 20‐fold; concomitantly the Ca2+‐dependent A2 activity is abolished completely. Heparin also drastically changes the aggregation state of the enzyme; aggregated species contain significantly less δ and show an about fivefold higher A0 activity than the tetrameric form containing δ stoichiometrically. We interpret this to mean that δ has two functions in the phosphorylase kinase: an inhibitory one with respect to A0 and an activating one with respect to A2. The inhibition of A0 by Ca2+‐free δ is released, i.e. A0 increases when this subunit dissociates from the holoenzyme.The maximally heparin‐stimulated A0 activity, A0,hep, is enriched from a crude extract to the same degree and approximately with the same yield as the major activity, A2. The phosphorylase kinase is not eluted from DEAE‐cellulose as a symmetrical bell‐shaped protein peak. The peak fraction contains the activities A2 and A0,hep, superimposed and yields a nearly homogeneous sedimentation boundary with an s20,w value of 25.5 S. The A0 yields a much broader eluation profile showing a distinct maximum from the A2 activity which contains slower sedimenting species of 12.1 S, some tetrameric enzyme of 22.7 S and higher aggregated material. Over the whole profile the activity ratio A2/A0 decreases about sevenfold whereas the ratio A2/A0,hep is constant on average. This shows that A0 is an intrinsic activity of phosphorylase kinase.The heparin‐activated A0 activity or A0 itself in the presence of the phosphorylase phosphatase inhibitor, fluoride, can trigger a Ca2+‐independent flash activation of phosphorylase in a protein‐glycogen complex. Thus, A0 could be responsible for the conversion of phosphorylase b to a at 20 nM free Ca2+ in resting, hormone‐stimulated, muscle.
Time course of polynucleosome relaxation and ADP‐ribosylation
Authors: NIEDERGANG Claude P. , de MURCIA Gilbert , ITTEL Marie‐Elisabeth , POUYET Jean , MANDEL Paul 
Published: March 2005

Isolated rat pancreatic polynucleosomes were poly(ADP‐ribosylated) with purified calf thymus poly(ADP‐ribose) polymerase. A time course study was performed using an NAD concentration of 200 μM and changes in nucleosomal structure were investigated by means of electron microscopy visualization and sedimentation velocity determinations. In parallel, analyses of histone H1 poly(ADP‐ribosylation) and determinations of DNA polymerase α activity on ADP‐ribosylated polynucleosomes were done at different time intervals.A direct kinetic correlation between ADP‐ribose incorporation, polynucleosome relaxation amd histone H1 hyper‐ADP‐ribosylation was established. In addition, DNA polymerase α activity was highly stimulated on ADP‐ribosylated polynucleosomes as compared to control ones, suggesting increased accessibility of DNA to enzymatic action.Because of the strong evidence implicating histone H1 in the maintenance of higher‐ordered chromatin structures, the present study may provide a−basis for the interpretation of the involvement of the histone H1 ADP‐ribosylation reaction in DNA rearrangements during DNA repair, replication or gene expression.