bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analysis of the relA Gene Product of Escherichia coli
Authors: PEDERSEN Finn Skou , KJELDGAARD Niels Ole 
Published: March 2005

The relA gene product, ATP:GTP 3′‐pyrophosphotransferase (stringent factor) has been isolated in homogeneous form from an Escherichia coli strain polyploid for this gene at a yield of 1 mg/100 g cells and at a specific activity in a ribosome‐activated assay at 37 °C of 120 μmol guanosine pentaphosphate formed min−1 mg protein−1. The specific activity in a methanol‐activated assay at 25°C was found to be 4 μmol guanosine pentaphosphate formed min−1 mg protein−1. These values are about 100 times higher than reported by others.Our further studies of this enzyme led to the following results.Antibodies raised against this enzyme inhibit the ribosome‐activated synthesis of guanosine tetraphosphate and pentaphosphate but have no effect on the much slower synthesis, detected in the absence of ribosomes.The amount of stringent factor in the relA+ strain CP78 is estimated to about 1 copy per 200 ribosomes.The amount of antibody‐binding material in CP79 (relA) is at least 5 times lower.
Specific Casein Phosphorylation by a Casein Kinase from Lactating Bovine Mammary Gland
Authors: MACKINLAY Antony G. , WEST David W. , MANSON William 
Published: March 2005

A preparation which contains protein kinase activity capable of rephosphorylating dephosphorylated αs1 and β‐caseins has been prepared from lactating bovine mammary gland. This activity is localised in the Golgi fraction and in this respect is similar to that obtained previously from rat tissue. It differs from the rat preparation in being unable to rephosphorylate dephosphorylated ϰ‐casein.
Progressive dephosphorylation of αs1 and β‐caseins increases the rate of their rephosphorylation in the presence both of Ca2+ and of Mg2+. For β‐casein significant non‐specific phosphorylation occurs in the presence of Mg2+ while in the presence of Ca2+ the level of phosphorylation is very low.
Comparison of the products of the action of this kinase on partially dephosphorylated αs1 and β‐caseins indicates that seryl residues grouped with pre‐existing phosphoseryl residues are phosphorylated preferentially in the presence of Mg2+, while the phosphorylation of isolated single seryl residues is promoted by Ca2+.
Digests of αs1‐casein, prepared by treatment with cyanogen bromide, have been fractionated and the three major peptides, separated. After dephosphorylation two of these, which between them contain all of the phosphate of αs1‐casein, are rephosphorylated at sites occupied in the native protein by phosphoseryl residues, i.e., at positions 46, 48, 75 and 115 of the αs1‐casein molecule. Evidence is presented which shows that this specificity of action also operates when intact αs1‐casein is used as phosphate acceptor. It is concluded that the enzyme responsible for specific phosphorylation acts through recognition of an amino acid sequence and does not require the provision of a specific structural conformation in the protein substrate.
The Action of Lysozyme on Peptidoglycan with N‐Unsubstituted Glucosamine Residues
Authors: AMANO Ken‐ichi , HAYASHI Hideyuki , ARAKI Yoshio , ITO Eiji 
Published: March 2005

A peptidoglycan preparation N‐acetylated at about 30% of glucosamine residues was obtained by the treatment of the lysozyme‐resistant cell wall peptidoglycan of Bacillus cereus with acetic anhydride at pH 7. Fractionation of dialyzable material resulting from lysozyme digestion of the glycan component of this peptidoglycan preparation yielded five oligosaccharides designated as S1 to S5 besides the disaccharide GlcNAc‐MurAc.
Oligosaccharide S3, which accounted for about 30% of the disaccharide units recovered as disaccharides and oligosaccharides, was identified as GlcN‐MurAc‐GlcNAc‐MurAc. Oligosaccharide S1, accounting for about 20% of the disaccharide units recovered, was characterized as GlcN‐MurAc‐GlcN‐MurAc‐GlcNAc‐MurAc, while oligosaccharide S2, present in a smaller amount, as GlcNAc‐MurAc‐GlcN‐MurAc‐GlcNAc‐MurAc. Oligosaccharides S4, and S5, present in small amounts, were identified as GlcNAc‐MurAc‐GlcNAc‐MurAc and MurAc‐GlcNAc‐MurAc, respectively.
Oliogsaccharides S1, S3 and S5 proved to be completely insusceptible to lysozyme, whereas S2 was digested by lysozyme to produce GlcNAc‐MurAc and S3. S1 was found to act as a more potent inhibitor than S3 in lysozyme‐catalyzed digestion of polysaccharides.
The results obtained show that the lysozyme‐catalyzed hydrolysis of peptidoglycan oligosaccharides has an obligatory requirement for the N‐acetyl group on the glucosamine residue located in subsite C in the enzyme‐substrate complex.
Effects of Fatty Acid Unsaturation on Chylomicron Metabolism in Normal and Hepatectomized Rats
Authors: FLORÉN Claes‐Henrik , NILSSON Ake 
Published: March 2005

1. Hepatectomized rats were injected intravenously with doubly labelled ([14C]linoleic acid and [3H]palmitic acid) thoracic duct lymphs from rats fed cream, triolein or corn oil. The disappearance of the radioactive fatty acids of different molecular triacylglycerol species and of phospholipids from plasma was studied.2. 73–93% of the injected triacylglycerols had been cleared from plasma within 15 min. At all stages of lipolysis the 3H/14C ratio of the plasma triacylglycerol was the same as in the injected material. If the cream chyle had been cooled to 4 °C before use there was, however, an enrichment of [3H]palmitic acid and of fully saturated triacylglycerols in the remnant particles formed.3. Only 38–50% of the radioactive chyle phosphatidylcholine was eliminated from plasma in 30 min. At this time most of the remaining phosphatidylcholine was, however, in other lipo‐protein classes than the chylomicron remnants.4. Also in intact rats data were obtained, indicating that the major portion of chylomicron phospholipids is transferred to other serum lipoproteins by exchange or net movement rather than being hydrolysed in the 1‐position by lipoprotein lipase or taken up intact by the liver.5. More of both the labelled fatty acids appeared in liver triacylglycerols in experiments with cream chyle than in experiments with corn oil chyle. Data were obtained suggesting that this may be due to a higher uptake of intact triacylglycerol as remnant particles.6. When linoleic acid is fed as a tracer dose in cream, a high proportion (16–36%) is incorporated into chyle phospholipids.
Studies on the Role and Mode of Operation of the Very‐Lysine‐Rich Histone H1 in Eukaryote Chromatin
Authors: HARTMAN Peter G. , CHAPMAN George E. , MOSS Thomas , BRADBURY E. Morton 
Published: March 2005

Limited digestion with trypsin of both calf thymus H1 histone and the fragment 1‐120 of the H1 molecule has resulted in the isolation of the fragment 35‐120. This fragment assumes a globular structure under physiological conditions of pH and ionic strength. The variable N‐terminal portion of the molecule, up to residue 34, is not required for the formation of the H1 globular structure.Proton nuclear magnetic resonance (NMR) and ultracentrifugation studies show that the H1 histone molecule consists of three distinct structural domains under structuring conditions: a random coil ‘nose’ consisting of 35 to 40 residues from the N‐terminal end; a globular ‘head’ involving the next approximately 80 residues; and a random‐coil ‘tail’ of the remainder of the molecule.
Structure de la bacillomycine L, antibiotique de Bacillus subtils
Authors: BESSON Françoise , PEYPOUX Françoise , MICHEL Georges , DELCAMBE Lucien 
Published: March 2005

The Structure of Bacillomycin L, an Antibiotic from Bacillus subtilisThe structure of bacillomycin L, an antifungal agent isolated from the culture medium of a strain of Bacillus subtilis, has been investigated. The peptide moiety contains one mole each of D‐aspartic acid, L‐aspartic acid, L‐glutamine, L‐serine, D‐serine, L‐threonine, and D‐tyrosine. The lipid moiety is a mixture of 3‐amino‐12‐methyltridecanoic acid (46%), 3‐amino‐12‐methyltetrade‐canoic acid (38%), 3‐amino‐14‐methylpentadecanoic acid (11%), and two minor homologues.The peptide sequence and the cyclic structure were determined by structural analysis of the peptides obtained by mild acid hydrolysis.The molecular weight was determined by the thermoosmotic method; this showed that bacillomycin L has a monomeric structure which is given in Formula 1.
Purification and Comparative Study of Adenine and Guanine Phosphoribosyltransferases from Schizosaccharomyces pombe
Authors: NAGY Maria , RIBET Anne‐Marie 
Published: March 2005

1. Adenine and guanine phosphoribosyltransferases from the yeast Schizosaccharomyces pombe were purified respectively 1200‐fold and 1000‐fold from the crude extracts giving specific activities respectively of 18 and 5.7 μmol purine nucleotide monophosphates mg protein−1 min−1. The purified enzymes remain stable for at least a month when conserved at −20°C in the presence of 6 mM 5‐phosphoribosyl‐1‐pyrophosphate (P‐Rib‐P2) and 2 mM MgCl2.2. Both phosphoribosyltransferases have multiple, apparently buffer‐independent, pH optima; both have an absolute requirement for Mg2+, but, contrary to other organisms studied, the effects of inhibitory concentrations of Mg2+ are non‐competitive towards P‐Rib‐P2 binding.3. The hyperbolic P‐Rib‐P2versus rate curve of guanine phosphoribosyltransferase (but not that of adenine phosphoribosyltransferase) becomes sigmoidal in the presence of fixed concentrations of the reaction product (GMP).4. Guanine phosphoribosyltransferase is ten times more sensitive to inhibition by reaction products in comparison to adenine phosphoribosyltransferase. The inhibition of the first enzyme by other purine and pyrimidine nucleotides appears much more specific relative to the last enzyme.5. Both activities are separated in two active forms by filtration through a controlled‐pore glass column CPG‐10. The estimation of molecular weights of these forms gives 48000 and 42000 for guanine phosphoribosyltransferase and 50000 and 44000 for adenine phosphoribosyltransferase. In the presence of 5 mM MgCl2 the last enzyme, but not the first, dissociates into a single form of apparent molecular weight 30000.6. Electrophoresis on polyacrylamide gel of crude as well as of purified extracts reveals two active bands of adenine phosphoribosyltransferase but only a single active band of guanine phosphoribosyltransferase.
Kinetics of Sheep‐Liver Cytoplasmic Aldehyde Dehydrogenase
Authors: MacGIBBON Alastair K. H. , BLACKWELL Leonard F. , BUCKLEY Paul D. 
Published: March 2005

1. Sheep liver cytoplasmic aldehyde dehydrogenase showed little pH dependence of V or kcat. Some buffer anion effects were noted.2. The oxidation of aldehydes at pH 7.6 was quantitative but irreversible. The initial velocity data indicated a sequential mechanism for the addition of substrates. Inhibition by NADH and the product analogue 2‐bromo‐2‐phenylacetic acid, together with the known tight binding of NADH to the free enzyme, indicated an ordered mechanism with NAD+ as leading substrate.3. Values for the rate of binding and dissociation of NAD+ were obtained from the steady‐state data. The values obtained were virtually identical with those which could be calculated from the data for the horse liver cytoplasmic enzyme. Close similarities are in general apparent for the horse and sheep liver cytoplasmic enzymes and with other tissue aldehyde dehydrogenases.4. Apparent substrate activation was observed with high concentrations of both acetaldehyde and propionaldehyde, a limiting value of 0.25 s−1 being obtained for kcat. No isotope effect was observed on V using [1‐2H]propionaldehyde as substrate suggesting that NADH release might be rate‐limiting in the steady‐state.5. The implications of the non‐linear steady‐state behaviour are discussed.
Frequency Distribution of Messenger Sequences within Polysomal mRNA and Nuclear RNA from Rat Liver
Authors: SIPPEL Albrecht E. , HYNES Nancy , GRONER Bernd , SCHÜTZ Günther 
Published: March 2005

DNA complementary to polysomal poly(A)‐containing mRNA (cDNA) of male rat liver was used to study the diversity of messenger sequences in the nucleus and in polysomes.1. Hybridization of cDNA against an excess of its own polysomal mRNA template revealed that about 10000 different mRNA species are expressed in the liver tissue. They are distributed in a wide frequency range derived from approximately 0.5% of the total genome.2. Hybridization of the cDNA against total nuclear RNA shows that messenger sequences comprise less than 1% of the mass of total nuclear RNA. Messenger sequences have a different frequency distribution in nucleus and cytoplasm.3. In hybridizations using cDNA, which had been fractionated into sequences representing abundant and scarce polysomal mRNA molecules, it was found that although abundant cytoplasmic messenger sequences are also abundant in the nucleus, they exist in a significantly lower frequency range in the nuclear compartment.
Detergent Influence on Rat‐Liver Galactosyltransferase Activities towards Different Acceptors
Authors: BRETZ Rolf , STÄUBLI Willy 
Published: March 2005

1. Galactosyltransferase activities in postnuclear supernatants and Golgi fractions from rat liver were assayed with two improved and simplified methods, using high‐ and low‐molecular‐weight acceptors. Transfer to N‐acetylglucosamine was measured after the separation of the reaction product N‐acetyllactosamine from all other radioactive molecules (including galactose) on an ion‐exchange column partially converted to the borate form. To determine the transfer of galactose to a glycoprotein acceptor we used ovomucoid, which accepts galactose without any previous chemical or enzymic modification.2. Both enzymic activities were enriched 60–80‐fold (compared with the post‐nuclear supernatant) in Golgi fractions, which were isolated on two subsequent sucrose gradients and identified morphologically by their high contents of stacked Golgi elements. The two activities could not be resolved by isolation of the Golgi fractions or by detergent solubilization. Each acceptor inhibited the galactose transfer to the other one (up to 95%), presumably because both compete for the same enzyme.3. The transferase activities were enhanced by the nonionic detergent Triton X‐100. The degree of activation depended directly on the amount of Triton bound to the membrane, i.e. the Triton/phospholipid ratio and not the w/v concentration of the detergent in the assay medium. This relationship persisted, regardless of the purity of the Golgi preparation: Half‐maximal activation occurred at the same Triton/phospholipid ratio in postnuclear supernatants as well as in isolated Golgi fractions. The activation could not be explained by complete solubilization, because 50% of the fully activated enzyme could still be sedimented (1 h, 100000 ×g).4. Galactose transfer to the high‐molecular‐weight acceptor required a higher Triton/phospholipid ratio for half‐maximal activation than did the transfer to the monosaccharide N‐acetylglucosamine (1 mg/mg compared with 0.5 mg/mg). The degree of activation maximally achieved was much higher with the protein acceptor (400%) than with the sugar (150%). Because both activities are probably due to the same enzyme, it is suggested that these differences in activation reflect properties of the membrane rather than the enzyme, e.g. the presence of a tight diffusion barrier for ovomucoid and the breakdown of this barrier by the detergent.
A Kinetic Study of the pH‐Dependent Properties of the Ferric Undecapeptide of Cytochrome c (Microperoxidase)
Authors: WILSON Michael T. , RANSON Richard J. , MASIAKOWSKI Piotr , CZARNECKA Eva , BRUNORI Maurizio 
Published: March 2005

The ferric form of the haem undecapeptide, derived from horse cytochrome c by peptic digestion, undergoes at least three pH‐induced transitions with pK values of 3.4, 5.8 and 7.6. Temperature‐jump experiments suggest that the first of these is due to the binding of a deprotonated imidazole group to the ferric iron while the second and third arise from the binding of the two available amino groups present (the α‐NH2 of valine and the ɛ‐NH2 of lysine).Molecular models indicate that steric restraints on the peptide dictate that these amino groups may only coordinate to iron atoms via intermolecular bonds, thus leading to the polymerization of the peptide.Cyanide binding studies are in agreement with these conclusions and also yield a value of 3.6 × 106 M−1 s−1 for the intrinsic combination constant of CN− anion with the haem.A model is proposed which describes the pH‐dependent properties of the ferric undecapeptide.
A Steady‐State‐Kinetic Random Mechanism for Glutathione S‐Transferase A from Rat Liver
Authors: JAKOBSON I. , WARHOLM M. , ASKELÖF P. , MANNERVIK B. 
Published: March 2005

The steady‐state kinetics of glutathione S‐transferase A from rat liver were studied with the substrates glutathione and 3,4‐dichloro‐1‐nitrobenzene varying from 1 μM to 5 mM and from 1 μM to 0.5 mM, respectively. The mathematical description of the experimental data required a rate law which was at least third degree with respect to concentration of each of the substrates. The rate equation of a generalized random sequential mechanism was fitted by a nonlinear regression program to the kinetic‐data set and was found to be superior to other two‐substrate models tested. Product inhibition studies with S‐(2‐chloro‐4‐nitrophenyl)‐glutathione were also in accordance with a random sequential mechanism for the forward reaction. The second product of the reaction, chloride, was a very poor inhibitor. In the regression analysis of the data, the correlation of experimental error with velocity was taken into account by weighting. For this purpose an empirical error function was used.Binding of the product S‐(2‐chloro‐4‐nitrophenyl)‐glutathione was studied by the method of equilibrium partition in an aqueous two‐phase system. The binding could be described by a rectangular hyperbola which showed binding at a ratio of 1.95 molecules of product per molecule of enzyme at saturating product concentration. No cooperativity could be established by the binding studies and it is assumed that each of the two subunits of the enzyme can bind one molecule of S‐(2‐chloro‐4‐nitrophenyl)‐glutathione.The results of the different experiments were all in accordance with the proposition of a steady‐state random sequential model for the mechanism of action of GSH S‐transferase A, provided that kinetically significant enzyme‐product complexes were introduced. In structural and functional terms the model has the following simple features: the two subunits of the enzyme are not assumed to display any cooperativity, and a subunit contains only one catalytically relevant binding site for GSH and GSH derivatives and one for the electrophilic substrate; these sites constitute subsites of the catalytically active center of the enzyme.
Biosynthesis of N‐Glycolylneuraminic Acid in Porcine Submandibular Glands
Authors: BUSCHER Hans‐Peter , CASALS‐STENZEL Jorge , SCHAUER Roland , MESTRES‐VENTURA Pedro 
Published: March 2005

1. The morphology of porcine submandibular gland tissue is demonstrated by light and electron microscopy.2. Subcellular particles from porcine submandibular glands were prepared by a procedure adapted to the presence of viscous mucus, and were fractionated on continuous and discontinuous sucrose gradients.3. Six particulate fractions were obtained by discontinuous gradient centrifugation. These were identified by electron microscopy, marker enzymes and nucleic acid content and were designated as follows: (1) plasma membranes, (2) smooth membranes and light Golgi membranes, (3) heavy Golgi membranes, (4) membrane mixture and mitochondria, (5) mitochondria and (6) membrane mixture and mucin residues. The bulk of rough membranes and cell nuclei was found in the 3000 ×g sediment before fractionation.4. Sialyltransferase and N‐acetylneuraminate monooxygenase showed highest activities in the Golgi membrane fractions representing 45% and 36%, respectively, of total tissue activity. The specific activities of these membrane‐bound enzymes were respectively 13 and 10 times higher when compared with the homogenate.5. The acylneuraminic acid/protein ratio was lowest in the Golgi membranes when compared with the other particulate fractions and the cytosol glycoprotein. The molar N‐acetylneuraminic acid/N‐glycolylneuraminic acid ratio was highest in the two Golgi membrane fractions (average 45:55) and in the pool of free acylneuraminic acids (41:59), and lowest in the cytosol glycoprotein (10:90).6. Approximately 2.6% of the total acylneuraminic acids occurring in porcine submandibular glands are freely diffusible, 80% of them representing free N‐acetylneuraminic acid and N‐glycolyl‐neuraminic acid in a molar ratio of about 4:6 and 20% representing cytidylyl‐glycosides of N‐acetylneuraminic acid and N‐glycolylneuraminic acid in a ratio of 6:4.7. In radioactive labelling experiments with surviving slices of porcine submandibular glands the highest specific radioactivities were found in the pool of free acylneuraminic acids, followed by N‐acetylneuraminic acid and N‐glycolylneuraminic acid bound to the Golgi membranes. N‐Acetylneuraminic acid and N‐glycolylneuraminic acid of the cytosol glycoproteins have lowest turnover rates.8. Based on these experiments a model is presented describing two pathways of N‐acetylneuraminic acid modification in the course of glycoprotein biosynthesis. At least 40% of N‐acetylneuraminic acid is hydroxylated before its transfer onto Golgi membranes and linkage to growing glycoprotein molecules and about 50% thereafter. This results in a molar N‐acetylneuraminic acid/N‐glycolyl‐neuraminic acid relationship of 1:9 in the secreted glycoprotein. It is assumed that free N‐acetylneuraminic acid is modified by the soluble cytosol monooxygenase whereas the glycoprotein‐bound N‐acetylneuraminic acid is hydroxylated by the corresponding membrane‐bound enzyme.
Transcription Fidelity and Structural Integrity of Isolated Nucleoli
Authors: BEEBEE Trevor J. C. , BUTTERWORTH Peter H. W. 
Published: March 2005

1. RNA was transcribed in vitro using isolated nucleoli, the endogenous form A RNA polymerase and mercurated UTP as one of the nucleoside triphosphate substrates. The products were isolated from endogenous nucleolar RNA sequences by chromatography on sulphydryl‐Sepharose and analysed by hybridisation in vast DNA excess. The results of competition‐hybridisation experiments suggested that a large proportion of the transcript was ribosomal RNA although some sequences had been transcribed from DNA of lower reiteration frequency.2. Analyses of the constituents of nucleoli following isolation by the sonication procedure suggested that the nucleolar DNA, particularly the ribosomal cistrons, are severely degraded. Furthermore, indications were obtained that the transcription complexes were damaged and many of the nascent RNA chains appeared to have been sheared from near the growing points.
α‐D‐Galactosidase from Soybeans Destroying Blood‐Group B Antigens
Authors: HARPAZ Noam , FLOWERS Harold M. , SHARON Nathan 
Published: March 2005

α‐D‐Galactosidase was isolated from untoasted soybean meal and purified to homogeneity by affinity chromatography on N‐ɛ‐aminoacaproyl α‐D‐galactopyranosylamine‐Sepharose. The purified enzyme destroyed the B‐specificity of human ovarian cyst B‐glycoprotein with an accompanying increase in H‐specificity, and converted human type‐B erythrocytes to type O.The enzyme consists primarily of a tetramer, molecular weight 150000 ± 5000 at pH 4.0, and of a monomer, molecular weight 40000 ± 3000 at pH 8.0. Polyacrylamide gel electrophoresis in dodecyl sulfate at pH 7.2 distinguished between two types of monomeric unit of similar molecular weight. N‐terminal alanine was identified as the sole N‐terminal amino acid residue. The enzyme was shown to be devoid of carbohydrate.