bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of mRNA from KB‐Cells by Affinity Chromatography on Polyuridylic Acid Covalently Linked to Sepharose
Authors: Lindberg Uno , Persson Torgny 
Published: March 2005

Messenger RNA from polysomes of KB‐cells was isolated by affinity chromatography on columns of polyuridylic acid covalently linked to Sepharose. The mRNA molecules were retained by the resin apparently via their poly(A) segments by base pairing to the poly(U). Ribosomal RNA and transfer RNA were not retained by the columns and were thus removed from the mRNA. The mRNA was recovered to an extent of 90% and apparently in intact form.This method allows studies of mRNA resulting from unabated synthesis and was used here in studies of the size distribution of different classes of cytoplasmic poly(A)‐containing RNA. The presence of poly(A)‐containing RNA in the non‐polysomal fractions of the cytoplasm was demonstrated. This putative mRNA was shown to constitute about 30% of the total cytoplasmic poly(A)‐containing RNA. Also by using the poly (U)‐Sepharose technique it was demonstrated that actinomycin D at low concentration, 0.04 μg/ml, supresses the appearance of mRNA on polysomes to an extent of 50%. This low concentration of the drug was previously though to effect only ribosomal RNA synthesis.
La glycéraldehyde‐3‐phosphate déshydrogénase du latex d'Hevea brasiliensis
Authors: Jacob Jean‐Louis , d'Auzac Jean 
Published: March 2005

The cytoplasmic serum of Hevea brasiliensis latex contains two glyceraldehyde‐3‐phosphate dehydrogenases. These enzymes have been isolated and purified. One of them has an absolute specificity for NAD (EC 1.2.1.12); the other utilizes mainly NADP and reacts only slowly with NAD; it is similar to the non‐phosphorylating enzyme discovered by Arnon (EC 1.2.1.9). The NAD‐dependent enzyme has a molecular weight which is analogous to that of the other species of glyceraldehyde‐3‐phosphate dehydrogenases. Its optimum is around 8 and its Km‐values for the substrate, NAD and Pi respectively are: 0.1 mM, 0.12 mM and 2.6 mM.The NADP‐dependent enzyme is probably composed of 4 subunits and its molecular weight is estimated to be about 200000. Its optimum pH is around 8.6 and it catalyses a reaction that is irreversible. Its Km‐values for the substrate, NADP and NAD respectively are: 1.2 mM, 10 μM and 12 mM.Influence of some effectors like NADH, NADPH and adenosine phosphates on the enzymatic reaction has been studied. Only NADH has an influence. It inhibits competitively the reaction and the Ki value of the order 13 μM. It appears on the other hand, taking into account the influence of cysteine, that the NADP‐dependent enzyme is more resistant to xodative agents than the NAD‐dependent enzyme. NADP‐dependent enzyme is also inhibited by PO42‐, Mg2+, Ca2+, 3‐phosphoglycerate, glyceraldehyde iodoacetamide and citrate.
Comparison of the Conformation of Mammalian Globins in Solution
Authors: Vodrážka Zdenék , Hrkal Zbyněk , Kodíček Milan B. , Jandová Danuše 
Published: March 2005

By studying the oligomeric state, chiroptical properties and the reactivity of sulfhydryl and phenolic groups, we compared the properties of human, horse, bovine, canine, rabbit and rat globins in solution.The results indicate that, in spite of the differences in the amino acid sequence, all the mammalian globins studied exhibit analogical properties suggesting similar conformation.
Synthesis and Properties of Poly(2‐Methyladenylic Acid)
Authors: Ikehara Morio , Hattori Masao , Fukui Toshikazu 
Published: March 2005

Chemically synthesized 2‐methyladenosine 5′‐diphosphate (m2ADP) was polymerized to yield poly(2‐methyladenylic acid), poly(m2A), using Escherichia coli polynucleotide phosphorylase. In neutral solution poly(m2A) exists with a significant stacking of bases as studied by ultraviolet, circular dichroism and thermal melting methods. In a solution of pH 5.0 poly(m2A) formed a double‐stranded protonated structure, which had melting temperatures, Tm of 90°C at 0.15 M salt concentration. Upon mixing with poly(U), poly(m2A) formed a 1:1 complex with Tmof 21°C at 0.15 M salt concentration.From this and previous findings, i.e. a bulky group at position 2 of the purine distorted the C═O group of the pyrimidine residues at position 2 and inhibited the Watson‐Crick‐type hydrogen bonding, a double‐stranded complex of Hoogsteen‐type for poly(m2A) · poly(U) was predicted.
The Mechanism of Reaction of Methionyl‐tRNA Synthetase from Escherichia coli
Authors: Blanquet Sylvain , Fayat Guy , Waller Jean‐Pierre , Iwatsubo Motohiro 
Published: March 2005

The interactions of methionyl‐tRNA synthetase, a tetrameric enzyme, with various ligands involved in the methionine‐activation reaction (methionine and analogues, ATP, AMP, PPi, methioninyl adenylate and methionyl adenylate) were examined by equilibrium dialysis, absorbance difference spectroscopy and fluorescence measurements at equilibrium and in a stoppedflow apparatus.Two binding sites for methionine and four for ATP were found by equilibrium dialysis. The number of binding sites and the association constants of these ligands for the enzyme were not affected by the presence of Mg2+ or EDTA. From ligand‐induced fluorescence and absorbance variations, two binding sites were measured for methioninyl adenylate, a structural analogue of methionyl adenylate.The interaction of methionyl‐tRNA synthetase with most of the ligands investigated gave rise to characteristic fluorescence variations of large amplitude. Thus, saturating concentrations of methioninyl adenylate and methionine enhanced the fluorescence emission by 40% and 26%, respectively, while interaction with chemically synthesized methionyl adenylate resulted in 27% quenching. ATP, AMP or PPi, alone or in combinations, did not affect the emission spectrum. However, when ATP was added to enzyme saturated with methionine, the fluorescence level shifted from the initial value of 126% to 82%. Similary, additon of PPi to enzyme saturated with ATP and methionine raised the level of fluorescence from 82% to 112%. In none of the cases investigated did addition of Mg2+ or EDTA affect the final level of ligand‐induced fluorescence modifications. Titration experiments were conducted to measure the association constants of the ligands for the enzyme.The rates of formation of methionyl adenylate and of reversal of this reaction by PPi were determined by stopped‐flow measurements. Comparison of reaction rates in the presence of Mg2+ or EDTA reveals a dramatic enhancement of the rates of both reactions in the presence of Mg2+.
A Myoglobin‐Type Haemoglobin of Chironomus thummi thummi
Authors: Wollmer Axel , Buse Gerhard , Sick Hinrich , Gersonde Klaus 
Published: March 2005

A monomeric haemoglobin of the insect Chironomus thummi thummi is described which does not exhibit a Bohr effect. The thermodynamic parameters ΔG, ΔH, and ΔS are given for the oxygen association. There is a difference of 4 cal per degree in the ΔS values for the acid and alkaline pH. The absence of a Bohr effect at physiological temperatures is explained by the fact that there is a point of intersection at 25 °C in the −ΔG versus T plot for acid and alkaline pH. This point is designated the “isoaffinity temperature”. In the case of other monomeric Chironomus haemoglobins a Bohr effect is observable because their isoaffinity temperatures are remote from the physiological temperature range (haemoglobin III = 184 °C, haemoglobin IV = 377 °C).No conformation isomers can be distinguished by circular dichroism and absorption measurements at 25 °C; isosbestic triangles are not encountered in this case.Electron spin resonance spectroscopy, however, carried out at 77 K, reveals the existence of at least two conformation isomers distinguished by specific high‐ and low‐spin signals. In case of high‐spin H2O‐Hb(III) the acid conformation is characterized by a rhombic distortion of the intramolecular electric field and strong interaction of the d‐electrons of the iron with the two nuclei of the bound imidazole. Transition to the alkaline conformation is followed by a change to axial symmetry and weak interaction of the d‐electrons with the imidazole.In the case of OH‐Hb(III) two sets of low‐spin signals describe two conformations with different rhombic symmetry.By comparing the positions of histidine residues in haemoglobins I, III and IV possible Bohr proton‐binding sites are discussed.
The Membrane Structure Studied with Cationic Dyes
Authors: Dell' Antone Paolo , Colonna Raffaele , Azzone Giovanni Felice 
Published: March 2005

Three effects concerning the organic cations, the aggregation, the metachromasy and the pKα shift, have been studied in comparison at high dye concentrations, with natural and synthetic polyanions and with energized submitochondrial particles.The metachromatic effect has been obtained by increase of the free cationic dye concentration or by interaction of the dye with natural, synthetic polyanions and energized submitochondrial particles. The metachromatic effect is dependent on the pH of the medium, the presence of ionized acidic groups, and the ionic strength of the medium.The polyanion‐induced metachromasy requires the ionization of the acidic groups of the polyelectrolyte. The metachromasy with neutral red requires an acidic pH if induced by increase of the dye concentration or by chondroitin sulphuric acid, whereas it takes place also at alkaline pH if induced by polystyren sulphonic acid or by energized particles. It is inhibited by increase of ionic strength in the case of agar and chondroitin sulphuric acid, but not in the case of the energized particles or polystyrene sulphonic acid.Interaction of neutral red with polystyrene sulphonic acid or energized particles results also in a large apparent pKα shift, which represents the mechanism for obtaining the metachromatic effects at alkaline pH. The apparent pKα, as measured from the extinction of the alkaline band is: 6.0 at 400 μM neutral red, 6.7 at 20 μM neutral red, 7.0 with chondroitin sulphuric acid, 7.1 with the deenergized particles, 8.0 with polystyrene sulphonic acid and 8.1 with energized particles. From thermodynamic considerations it is suggested that the pKα shift requires a decrease of the activity coefficient of the dye following the formation of ion pairs with anionic groups of the membrane. The pKα shift may be taken as a tool for discriminating between a dyeinduced and a membrane‐induced metachromatic effect.A model is proposed for the energized membrane, based upon electrostatic and hydrophobic interactions of the cationic dyes with a layer of oriented nucleophilic sites in an environment of intermediate polarity.
The Iodination of Chymotrypsinogen and Chymotrypsin in Crystal and Solution
Authors: Wright H. Tonie , Weber Bruce H. 
Published: March 2005

Single crystals of active and phenylmethanesulfonyl‐treated γ‐chymotrypsin and γ‐chymotrypsin were iodinated. The sites of iodination and their degree of occupancy were determined crystallographically. It was found that the pattern of iodination depends upon the form of chymotrypsin being iodinated, the pH, and whether the enzyme has been irreversibly inhibited. The effect upon γ‐chymotrypsin catalytic activity of the iodination of specific residues was observed toward acetyl tyrosine ethyl ester.Chymotrypsinogen was iodinated in solution under slightly varying conditions; it was impossible to iodinate chymotrypsinogen crystals in either native or glutaraldehyde crosslinked form. The iodochymotrypsinogens were activated by trypsin under slow and rapid conditions and the catalytic properties of the resulting chymotrypsins examined. The activity of the iodochymotrypsins toward acetyl tyrosine ethyl ester depends upon both the conditions of iodination and those of activation.The crystallographic results are compared with those obtained previously for γ‐ and α‐chymotrypsins and possible explanations for observed differences are advanced. We propose from the kinetic data that the slow interconversion of α‐ and γ‐chymotrypsins is controlled by the ionization of the tyrosine‐146 sidechain.
Complexes of Aminoacyl‐tRNA Synthetases with tRNAs as Studied by Partial Nuclease Digestion
Authors: Hörz Wolfram , Zachau Hans G. 
Published: March 2005

Complexes of tRNAPhe and tRNASer with their cognate aminoacyl tRNA synthetases have been exposed to a number of nucleases under conditions of partial digestion in order to gain information on the topography of the complexes. tRNAPhe was found to be strongly protected by yeast phenylalanyl tRNA synthetase at both nuclease‐sensitive sites, the dihydrouridine loop and the anticodon. tRNASer, on the other hand, was selectively shielded by yeast seryl tRNA synthetase at one of the two nuclease targets, the G‐C‐C‐A terminus, whereas splitting at the other target, the anticodon, was not affected at all by the synthetase.There was complete specificity of shielding: tRNAs were only protected by their respective synthetases. The complexes were sufficiently stable to allow an analysis of protection not only at pH 5.5 but also at pH 7.0. Phenylalanyl tRNA synthetase protected one mole of tRNAPhe, seryl tRNA synthetase two moles of tRNASer against nuclease attack. Denatured tRNAPhe did not bind to phenylalanyl tRNA synthetase.Two effects of ligands on complex stability have been observed. The analog phenylalaninol‐AMP increased the protection by phenylalanyl tRNA synthetase of tRNAPhe but not of Phe‐tRNAPhe; ATP abolished the shielding of the G‐C‐C‐A terminus of tRNASer by seryl tRNA synthetase.A number of tRNAPhe and tRNASer fragments was studied. It was concluded that the terminal 7 nucleotides in tRNAPhe are not required for complex formation. As expected, combinations of homologous tRNA half molecules resembled the intact tRNAs in the degree of protection. When the half molecules were tested separately or in heterologous combinations, phenylalanyl tRNA synthetase afforded some protection for the pG‐half molecule of tRNAPhe and seryl tRNA synthetase for the C‐C‐A‐half molecule of tRNASer.
Isolation of a Glucose‐Binding Lipoprotein from Yeast Plasma Membrane
Authors: Hoeák Jaroslav , KOTYK Arnost 
Published: March 2005

Baker's yeast membranes were solubilized with Tween 80, the material was chromatographed on Sephadex G‐150 and hydroxyapatite and finally treated with ammonium sulphate and in an isopycnic gradient of sucrose, to yield a lipoprotein complex which appears homogeneous in disc electrophoresis. The complex binds d‐glucose, d‐xylose, d‐arabinose, l‐sorbose, but not d‐galactose and l‐arabinose, showing thus the specificity of the typical glucose carrier from baker's yeast. The binding is suppressed by dodecylsulphate and by treatment at 90°C, but not by 2,4‐dinitrophenol or N‐ethylmaleimide.The molecular weight of the protein (per binding site) appears to be about 5000, that of the lipoprotein (from analysis of P and N) about 35000. The lipid part contains phosphatidylcholine, phosphatidylethanolamine and probably lysophosphatidylcholine. There are a minimum of 3 × 106 glucose‐binding sites per cell.
The Presence of an Exonuclease in Highly Purified DNA Polymerase from Bakers' Yeast
Authors: Helfman William B. 
Published: March 2005

Two DNA polymerase activities from bakers' yeast can be separated by DEAE‐cellulose chromatography, one of which can be dissociated from its DNAase activity by subsequent chromatography on phosphocellulose. Both of the DEAE‐cellulose polymerases prefer nicked double‐stranded DNA and utilize single‐stranded DNA efficiently only at high ratios of enzyme to template. DNA polymerase activity is completely inhibited by p‐hydroxymercuribenzoate, suggesting that cysteine is essential for activity.The DNAase has been shown to be coincident with polymerase activity during chromatography. This enzyme hydrolyzes single‐stranded DNA preferentially, releasing deoxymononucleotides as the sole product. The rate of hydrolysis is unaffected by the presence of a 2′,3′‐dideoxy terminus on poly(dT).
Steroid‐Binding Properties of a Plasma‐Membrane Fraction from Rat Liver
Authors: Blyth Carol A. , CLARK Rosemary P. , Freedman Robert B. , HAMMOND Jane , James Derrick W. , RABIN Brian R. , Ridge David , VINTER Jill , Williams David 
Published: March 2005

The binding curves of oestradiol and testosterone to plasma membranes from male or female rat livers show no specific sites of high affinity.
Plasma membranes from male and female rat livers show no disulphide interchange activity. It is concluded that no steroid‐promoted association of ribosome with plasma membranes occurs.
Microsomes prepared from a homogenate from which plasma membranes have already been isolated, show a slightly higher concentration of specific high‐affinity steroid‐binding sites than do standard preparations of microsomes. It is concluded that standard microsomal preparations are contaminated only to a slight extent by fragments derived from the plasma membrane.
The Primary Structure of the Major Parvalbumin from Hake Muscle
Authors: Capony Jean‐Paul , PECHÈRE Jean‐François , Rydèn Lars , DEMAILLE Jacques 
Published: March 2005

Work on the amino acid sequence of the major parvalbumin from hake muscle has been pursued with the aim of obtaining large fragments and overlapping peptides permitting the tryptic peptides sequenced previously to be ordered and the structure of the N‐terminal portion of the molecule to be established.Cyanogen‐bromide cleavage at the single methionine and tryptic digestion limited to the single arginine after maleylation were found useful in this respect and, with the help of the previous data, allowed the last 44 residues of the molecule to be placed unambiguously.Additional information was obtained from a chymotryptic digest of the protein which resulted in almost complete elucidation of the sequence of the first 24 residues and valuable knowledge on two other fragments, 12 and 29‐residues long, respectively.Application of both limited and extensive peptic‐digestion, the first one supplemented with the diagonal technique for cysteine peptides, permitted the remaining gaps to be filled, yielding an unique sequence of 108 residues now in full agreement with the amino acid composition of the protein.The location of the three amide groups of this parvalbumin is described and completes the description of its primary structure.
Carbamoylation of Proteins Following Administration to Rats of Carbamoyl Phosphate and Cyanate and Effects on Memory
Authors: Crist Robert D. , Grisolia Santiago , Bettis Carol J. , Grisolia James 
Published: March 2005

Both [14C]carbamoyl phosphate and sodium [14C]cyanate injected in rats were extensively incorporated into blood proteins, red cells and tissues, including brain. The rate of cyanate binding to blood protein plateaus 4 h after intraperitoneal administration of [14C]cyanate. The rapid disappearance of free [14C]cyanate was shown to be largely due to protein binding and the release of 14CO2 was found to decrease sharply at 4 h when cyanate binding is essentially complete.Retention of carbamoylated proteins was measured after injection of [14C]cyanate, and at 110 days, when bound radioactivity in blood protein was negligible, brain protein retained about 50% of the radioactivity found at 6 h.A dose‐related decrease in learning ability was observed when rats injected with 50, 100 and 150 μmol cyanate per day were tested in a Lashley maze No. III filled with water. This retardation persisted when the animals were retested nine weeks after termination of cyanate injections. When retested at eight months, no retardation could be demonstrated. At this time, only 10% modified proteins should remain in the brain according to the data presented on retention of carbamoylated proteins.The application of this data to a biochemical model for learning based on synaptic facilitation by modified proteins is discussed, as well as the implied effect on humans treated for sickle‐cell disease by administration of cyanate or carbamoyl phosphate.
Studies on the Apparent Instability of Neurospora Tryptophan Synthase
Authors: Yu Peter‐Hao , KULA Maria‐Regina , Tsai Hsin 
Published: March 2005

Neurospora tryptophan synthase prepared by a standard procedure was found to be contaminated with endogenous protease(s). Such a protease is responsible for the apparent instability of the tryptophan synthase. The rate of inactivation of tryptophan synthase was found to be directly proportional to the level of proteolytic activity. The protease is synthesized during all phases of cell growth. However, the proteolytic activity is most pronounced in the aged cultures.The protease which has been purified by Sephadex G‐200 column chromatography and poly‐acrylamide‐gel electrophoresis exhibits the properties of an endopeptidase. It is relatively insensitive to p‐mercuribenzoate and is insensitive to EDTA. It is very sensitive, however, to the proteolytic inhibitor phenylmethanesulfonyl fluoride.During the course of this study, a potent protease inhibitor has also been isolated from Neurospora. It is concentrated in a fraction precipitated with ammonium sulfate at 30 to 45% saturation. The isolated inhibitor is heat‐resistant, non‐dialyzable and is excluded by Sephadex G‐25. It strongly inhibits the Neurospora protease but has no effect on trypsin.
Regulation of Yeast Inorganic‐Pyrophosphatase Activity by Divalent Cations
Authors: Baykov Alexander A. , AVAEVA Svetlana M. 
Published: March 2005

The stability constants of the magnesium and calcium complexes of inorganic pyrophosphate and the manganese complex of ATP were measured with a specific ion electrode at pH 5.6 and 25°C. These data were used to calculate the concentrations of free metals, free substrates, and the metal · substrate complex in their mixtures. The effect of these variables on the activity of the inorganic pyrophosphatase from baker's yeast was investigated and it was concluded that the active enzyme · substrate complex has at least three sites specific to Mg2+ or Ca2+ and two sites specific to Mn2+ per protein molecule. Calcium inhibits the enzymatic hydrolysis of inorganic pyrophosphate due to displacement of three magnesium ions from the enzyme complex with the substrate.A simple semi‐automatic analyzer is described which measures inorganic phosphate in solution at concentrations down to 2 μM.
Phoratoxin, a Toxic Protein from the Mistletoe Phoradendron tomentosum subsp. macrophyllum (Loranthaceae)
Authors: Mellstrand S. Tore , SAMUELSSON Gunnar 
Published: March 2005

In a simplified procedure for isolation of phoratoxin, the dried plant material (stems and leaves) was extracted with 2% aqueous acetic acid. The extract was treated with polyamide to remove colored impurities and subjected to gel filtration on Sephadex G‐25. The high‐molecular‐weight fraction was chromatographed on SE‐Sephadex, using a gradient elution technique, whereby pure phoratoxin was obtained.The material was homogeneous by standard criteria of protein purity. The molecular weight, determined in the ultracentrifuge by the sedimentation equilibrium method, is 5000. Phoratoxin has the following amino acid composition (residues/mole): alanine 2, arginine 3, aspartic acid 4, 1/2‐cystine 6, glycine 6, histidine 1, Isoleucine 3, leucine 1, lysine 4, phenylalanine 1, proline 2, serine 5, threonine 5, tryptophan 1, tyrosine 1, valine 1. The molecular weight, calculated from the amino acid composition, is 4884. The material contains no reducing sugars, amino sugars or sialic acids. The LD50, determined by intraperitoneal injection in mice, is 0.57 ± 0.05 mg/kg bodyweight.