bluuebunny/crossref_metadata_2025_split huggingface dataset:
Peptide‐Bond Formation on the Ribosome
Authors: Eckermann David J. , Greenwell Philip , Symons Robert H. 
Published: March 2005

The acceptor substrate activity of peptidyl transferase of both Escherichia coli and rat liver ribosomes has been investigated using 3′‐N‐aminoacyl analogues of puromycin and 5′‐O‐phosphoryl and 5′‐O‐nucleotidyl derivatives of 3′‐N‐glycylpuromycin aminonucleoside. Two simple assay systems were used with washed ribosomes; the fragment reaction with CpApCpCpA‐(Ac‐L‐[3H]Leu) as donor substrate and the reaction with N‐acetyl‐L‐[3H]phenylalanyl‐tRNA as donor substrate in the presence of poly(U). The results can be summarized as follows.The conditions of the fragment reaction impose severe restrictions on the structural requirements for acceptor substrate activity, especially for E. coli ribosomes. Of the 21 puromycin analogues tested, only 3′‐N‐L‐phenylalanyl‐puromycin aminonucleoside and its 5′‐O‐cytidylyl derivative gave high (>50% of that of puromycin) activity with E. coli ribosomes while all other analogues gave essentially negligible (<5% of that of puromycin) activity. However, with rat liver ribosomes, 3′‐N‐L‐phenylalanyl‐puromycin aminonucleoside gave high activity while all other analogues containing a single benzene ring gave low (5‐24% of that of puromycin) to moderate (25–50% of that of puromycin) activity. These observations plus the low activity of 5′‐O‐cytidylyl (3′‐N‐L‐phenylalanyl)‐puromycin aminonucleoside with rat liver ribosomes represent the most marked differences found between the two types of ribosomes.In the N‐acetyl‐L‐[3H]phenylalanyl‐tRNA assay system with E. coli ribosomes, there was low to moderate activity of a number of the more hydrophobic aminoacyl analogues of puromycin which had negligible activity in the fragment reaction. With rat liver ribosomes, there was a general enhancement of activity of aminoacyl analogues above that seen in the fragment reaction. With the phosphoryl and nucleotidyl analogues, however, only 5′‐O‐cytidylyl(3′‐N‐L‐phenylalanyl)‐puromycin aminonucleoside showed high activity with both types of ribosomes and the results were essentially similar to those of the fragment reaction. In general, therefore, the structural requirements for activity of aminoacyl analogues, but not of nucleotidyl analogues, tended to be more stringent in the fragment reaction as compared with the N‐acetyl‐L‐[3H]phenyl‐alanyl‐tRNA assay. It is suggested that the methanol present in the fragment reaction may cause this by disruption of hydrophobic binding at the puromycin binding site.
Covalent Structure of Calf‐Thymus ALK‐Histone
Authors: Sautielère Pierre , Tyrou Danièle , Laine Bernard , Mizon Jacques , Ruffin Pierre , Biserte Gérard 
Published: March 2005

Highly purified calf thymus ALK‐histone (histone rich in alanine, lecine and lysine) was isolated from F2a2 histone fraction by ion‐exchange chromatography on Biorex 70 followed by gel filtration chromatography on Sephadex G‐100. Peptides obtained by enzymatic hydrolyses (trypsin, chymotrypsin, thermolysin) of native or maleylated protein were fractionated by ion‐exchange chromatography on Chromobeads P.Thermolysin peptides which account for 127 of the 129 residues of the protein provide overlaps of tryptic and chymotryptic peptides and led us to establish the complete amino‐acid sequence of ALK‐histone as follows: Ser1Gly‐Arg‐Gly‐Lys‐Gln‐Gly‐Gly‐Lys‐Ala‐10Arg‐Ala‐Lys‐Ala‐ Lys‐Thr‐Arg‐Ser‐Ser‐Arg20‐Ala‐Gly‐Leu‐Gln‐Phe‐Pro‐Val‐Gly‐Arg‐Val30‐His‐Arg‐Leu‐Leu‐Arg‐Lys‐Gly‐Asn‐Tyr‐Ala40‐Glu‐Arg‐Val‐Gly‐Ala‐Gly‐Ala‐Pro‐Val‐Tyr50‐Leu‐Ala‐Ala‐Val‐Leu‐Glu‐Tyr‐Leu‐Thr‐Al60‐Glu‐Ile‐Leu‐Glu‐Leu‐Ala‐Gly‐Asn‐Ala‐Ala70‐Arg‐Asp‐Asn‐Lys‐Lys‐Thr‐Arg‐Ile‐Ile‐Pro80‐Arg‐His‐Leu‐Gln‐Leu‐Ala‐Ile‐Arg‐Asn‐Asp90‐Glu‐Glu‐Leu‐Asn‐Lys‐Leu‐Leu‐Gly‐Lys‐Val100‐Thr‐Ile‐Ala‐Gln‐Gly‐Gly‐Val‐Leu‐Pro‐Asn110‐Ile‐Gln‐Ala‐Val‐Leu‐Leu‐Pro‐Lys‐Lys‐Thr120Glu‐Ser‐His‐His‐Lys‐Ala‐Lys‐Gly‐Lys129.The amino end is acetylated. The sequence of ALK‐histone is characterized by two hydro‐phobic regions, the first from Val‐43 to Ala‐70 and the second from Val‐100 to Pro‐117 and presents many repetitive or analogous sequences.The NH2‐terminal sequence of ALK‐histone is highly basic and shows a remarkable analogy with that of GRK‐histone.A cluster of five basic residues His‐His‐Lys‐Ala‐Lys‐Gly‐Lys is present at the COOH‐terminal end.
Thyrotropin‐Binding Properties of Isolated Thyroid Cells and Their Purified Plasma Membranes
Authors: Verrier Bernard , Fayet Guy , Lissitzky Serge 
Published: March 2005

Plasma membranes have been prepared from porcine thyroid cells stimulated by dibutyryl adenosine 3′:5′‐monophosphate in culture. These cells show the histiotypic organization in follicles and the metabolic properties of gland follicular cells. The plasma‐membrane fractions have a density in sucrose of 1.18 g/ml (about 80%) and 1.16 g/ml (about 20%). The yield is 2 mg per 6 × 108 cells. Electron microscopy showed vesicular structures and sheets of membranes often joined by membrane junctions. Both showed the triple layer characteristic of the unit membrane ultrastructure. Less than 3% of contamination by microsomes was derived from the assay of NADPH‐cytochrome c reductase and NADH oxidase. No contamination by mitochondria was indicated from succinate dehydrogenase activity measurements. NaF‐and thyrotropin‐stimulated adenylate cyclase, 5′‐nucleotidase and alkaline phosphatase are concentrated in the plasma‐membrane preparations.Specific binding of 125I‐labeled thyrotropin of high specific radioactivity (2.5 Ci/μmol) and preserved biological activity was compared to that in intact cells and to the plasma membranes from which they derived. The rate constants of the thyrotropin association with cell or membrane (0.56 and 0.11 nM−1 min−1, respectively) and dissociation (0.36 and 0.25 min−1, respectively) were measured independently at 35°C (cells) and 27°C (membranes). The association constants derived from these rate constants (1.5 and 0.4 nM−1 for cells and membranes) are very similar to that (1.9 and 0.6 nM−1) determined independently from equilibrium data. Therefore plasma membranes purified from cultured thyroid cells represent an excellent material to study the molecular events linked to receptor activation.The relation between thyrotropin concentration and adenylate cyclase activation of membranes was studied in time conditions of equilibrium binding of thyrotropin. The semilogarithmic plot of thyrotropin concentration correlates sigmoidally with the percentage of adenylate cyclase activation. Half‐maximum stimulation is obtained for a thyrotropin concentration of 1.8 nM which correlates remarkably with the dissociation constant of 1.78 nM determined for the binding of the hormone to membranes.
The Denaturation of Lactoferrin and Transferrin by Urea
Authors: Teuwissen Bruno , Schanck Karin , Masson Pierre L. , Osinski Pierre A. , Heremans Joseph F. 
Published: March 2005

Lactoferrin and transferrin were submitted in their metal‐free forms as well as in their ironsaturated forms to various concentrations of urea, in the presence or absence of 2‐mercaptoethanol. The progressive unfolding of the proteins was followed by their optical activity in the visible region.The change in the three‐dimensional structure of lactoferrin when binding iron was found to be more profound than in transferrin whose stability is more dependent on disulphide bridges. In the absence of 2‐mercaptoethanol, the concentrations of urea which caused 50% denaturation at pH 7.4 were found to be respectively 4 M for apolactoferrin, 4.9 M for apotransferrin, 5.5 M for Fe·transferrin and 8 M for Fe·lactoferrin. In the presence of 2‐mercaptoethanol, these values became respectively 2 M for apotransferrin, 2.5 M for apolactoferrin, 3.9 M for Fe·transferrin and 6.7 M for Fe·lactoferrin.The loss of iron‐binding ability, estimated from absorbance at 460 nm, was found to follow strictly the evolution of the [m']546 parameter.
The Reaction of the Iron‐Sulfur Protein Hydrogenase with Carbon Monoxide
Authors: Thauer Rudolf K. , Käfer Barbara , Jungermann Kurt , Zähringer Marianne 
Published: March 2005

The iron‐sulfur protein hydroenase from Clostridium pasteurianum was shown to be specifically inhibited by carbon monoxide. Cyanide (10 mM), azide (10 mM), and fluoride (10 mM) were without effect.The inhibition of hydrogenase by carbon monoxide was studied under pre‐steady‐state and under steady‐state conditions. The data obtained from both studies indicate a 1:1 stoichiometry of the reversible reaction between an independent active site of hydrogenase and carbon monoxide. The second‐order rate constant of the formation (k+1) and the first‐order rate constant of the dissociation (k−1) of the hydrogenase · carbon‐monoxide complex at pH values between 6.5 and 8.5 and at 25°C were found to be 5.7 s−1mM−1 and 0.013 s−1, respectively. The equilibrium constant K was calculated from pre‐steady‐state data to be 440 mM−1 and from steady‐state data to be 420 mM−1.The carbon monoxide inhibition of hydrogenase could be reversed bylight. The light sensitivity indicates that in the active site iron is being attacked by carbon monoxide.
Structural Aspects of Sheep‐Heart Phosphofructokinase
Authors: Brennan Stephen O. , Davis Paul F. , Midwinter Graeme G. 
Published: March 2005

The sedimentation pattern of the purified enzyme has been ivestigated over a protein concentration range of 0.7 to 14.5 mg cm−3. Two distict 7‐S and 30‐S boundaries were observed at all concentrations. A minor amount of 19‐S material was also present. The 30‐S boundary was asymmetric, and its concentration dependence was characteristic of a polymerising system in rapid reversible equilibrium.The dissolved crystallin enzyme, under similar conditions, sedimented as single asymmetric 30‐S peak. X‐ray crystallography indicated unit cell dimensions of 60×25×22 nm, implying a protein weight per asymmetric unit of about 106 daltons.Dodecylsulphate‐gel electrophoresis indicated a protomer molecular weight of 80000 to 85000, which was consistent with a corrected sedimentation coefficient of 3.8 S and a molecular weight of 90000 for maleyl‐phosphofructokinase, and with a corrected sedimentation coefficient of 3.9 S for the urea‐dissociated enzyme.When maleylation was carried out on carboxymethylphosphofructokinase in 7.5 M urea, the enzyme was further dissociated to a subunit with molecular weight 40000. Peptide mapping of tryptic peptides, in which peptides containing arginine, histidine, tryptophan and tyrosine were located, was consistent with an 85000‐Mr form composed of two identical subunits.
Metabolism of Isolated Kidney Tubules
Authors: Guder Walter G. , Wieland Otto H. 
Published: March 2005

Pyruvate dehydrogenase activity was measured in extracts of isolated kidney cortex tubules, prepared by collagenase treatment. The measured activities were comparable to those described previously for whole kidney homogenates.Incubation of tubules in vitro in the absence of exogenous substrates led to a steady increase in enzyme activity over 30 min. This increase was not significantly changed by the addition of several gluconeogenic substrates including lactate, glutamine, glutamate, glycerol, fructose and malate. 1 mM oleate, 5 mM acetate and 20 mM 2‐oxoglutarate however, prevented this increase completely. In contrast, glucose and dihydroxyacetone accelerated the activation of pyruvate dehydrogenase during incubation in vitro. 20 mM pyruvate very rapidly activated pyruvate dehydrogenase with a following decrease below the activity of control experiments without substrate. 1 mM oleate and 20 mM 2‐oxoglutarate counteracted the increase in pyruvate dehydrogenase activity caused by glucose and dihydroxyacetone and had no effect in the presence of 20 mM pyruvate.Determination of the inactive part of pyruvate dehydrogenase after addition of purified pyruvate dehydrogenase phosphatase revealed that all changes observed were probably caused by enzymatic interconversion of pyruvate dehydrogenase.The results are compared with previously published observations on the regulation of pyruvate dehydrogenase in vivo and confirm the role of fatty acids in the regulation of kidney cortex pyruvate metabolism.
Translational Control of Antibody Synthesis
Authors: Stevens Ronald H. 
Published: March 2005

Mouse plasma cytoma cells (5563) contain 2.5 times more L‐chain mRNA than H‐chain mRNA. The ratio of L‐ to H‐chain protein produced however appears related to the growth rate of the cells. During exponential growth, up to 2.5 times more L‐chain than H‐chain protein is produced, but as cells approach stationary growth phase, L‐ and H‐chain protein approach equal synthesis. The apparent decrease in L‐chain synthesis upon shifting from exponential to stationary growth appears not to be due to a relative reduction in peptide elongation but rather reflects differences in ribosome loading. During exponential growth, L‐chain polyribosomes contain 6–8 ribosomes/L‐chain mRNA whereas in stationary growth the figure drops to less than 3–4 ribosomes/L‐chain mRNA. A similar shift of polyribosome size is noted with total cellular polyribosome.Polyribosomes engaged in H‐chain synthesis remain fully loaded with ribosomes during conditions of L‐chain polyribosome size reduction. The size of polyribosomes synthesizing H‐chain protein can be reduced however under conditions of an increase in the intracellular level of H2L2.
Δ4‐3β‐Hydroxysteroid Dehydrogenase Activity in Rat Liver
Authors: Lax E. Rodney , Schriefers Herbert 
Published: March 2005

A simple optical method for the assay of Δ4‐3β‐hydroxysteroid dehydrogenase activity in cell‐free preparations of rat liver was designed. This test is based on the fact that under the chosen conditions no reduction of the Δ4‐bond occurs. Thus the Δ4‐3β‐hydroxysteroid dehydrogenase activity can be directly measured by the production of Δ4‐3β‐ketosteroids, the sum of which in turn may be quantitated by their isonicotnic acid hydrazone formation.Δ4‐3β‐Hydroxysteroid dehydrogenases are localized in both cytoplasmic and microsomal fractions, exhibiting higher activity with NAD than with NADP. Microsomal enzyme activities show a significant sex difference which is more pronounced with NADP as coenzyme than with NAD (male: female activity ratios 5.8 with NADP, 1.9 with NAD). No sex differences were observed in the cytoplasmic enzyme activity. These results indicate the existence of two NAD‐dependent enzyme activities of which the microsomal activity shows sexual differences. On the basis of the male:female activity ratios a further separate NADP‐dependent microsomal enzyme activity can also be distinguished.
Structure of the Porcine Vasoactive Intestinal Octacosapeptide
Authors: Mutt Viktor , Said Sami I. 
Published: March 2005

The amino acid sequence of the porcine vasoactive intestinal octacosapeptide is His‐Ser‐Asp‐Ala‐Val‐Phe‐Thr‐Asp‐Asn‐Tyr‐Thr‐Arg‐Leu‐Arg‐Lys‐Gln‐Met‐Ala‐Val‐L ys‐Lys‐Tyr‐Leu‐Asn‐Ser‐Ile‐Leu‐Asn‐NH2. Its amino acid residues 1, 2, 6 and 7, counted from the N‐terminus, are identical to those in the corresponding positions in both porcine glucagon and secretin. The residues in positions 3, 12, 13, 14 and 23 are identical to those in secretin, but not in glucagon, and position 10 is occupied by a tyrosyl and position 28 by an asparaginyl residue, like in glucagon but not in secretin. If in addition to identical amino acid residues also chemically similar residues, such as isoleucine in position 26 of the octacosapeptide, as compared to leucine in secretin and glucagon, are taken into consideration then the similarity between these three polypeptides is still more evident.At a more remote level there is some structural resemblance also between these peptides and the other four peptides from the intestinal wall, cholecystokinin‐pancreozymin, motilin, the gastric inhibitory peptide and substance P, the structures of which are known.The elucidation of the structure of the octacosapeptide was facilitated by the finding that pancreatic kallikrein preferentially cleaved only one of the three bonds in its N‐terminal cyanogen bromide heptadecapeptide that are susceptible to cleavage with trypsin.
Isolation and Characterisation of a Mitochondrially Synthesized Precursor Protein of Cytochrome Oxidase
Authors: Werner Sigurd 
Published: March 2005

Cytochrome oxidase was prepared from Neurospora crassa. An individual subunit of the enzyme (subunit 3, apparent molecular weight 21000) was isolated by recycling gel chromatography.Specific antibodies to cytochrome oxidase and to subunit 3 were produced.The antibody to cytochrome oxidase precipitates the enzyme from detergent‐treated mitochondria. The precipitated cytochrome oxidase contains seven polypeptide subunits. The growth labelling kinetics of subunit 3 within the enzyme complex was studied.The antibody to subunit 3 recognizes an antigen in solubilized mitochondria, which is not identical with cytochrome oxidase. This component was isolated from mitochondria by immunological techniques. It was identified as a precursor protein, which has the following characteristics, (a) The protein is translated on mitochondrial ribosomes. (b) The precursor has a half‐life of about 2 min. (c) Precursor protein and subunit 3 display identical apparent molecular weights, (d) It is tightly associated with mitochondrial membranes. (e) The precursor represents about 0.5‰ of the mitochondrial membrane protein.It is concluded that the isolated precursor is a genuine precursor protein of subunit 3 of cytochrome oxidase.
Polysaccharide Biosynthesis in Acetobacter xylinum
Authors: GarcÍA Rodolfo C. , Recondo Eduardo , Dankert Marcelo 
Published: March 2005

The incorporation of radioactivity in vitro from UDP‐[14C]glucose into endogenous acceptors in a crude enzymatic preparation from Acetobacter xylinum is described.A fraction of the total incorporated radioactivity could be extracted with organic solvents, like butanol. The remainder was mostly solubilized by hot alkali, leaving an insoluble fraction.No incorporation was observed in the absence of magnesium ions. Mn2+ could only partially replace Mg2+, and had an inhibitory effect when both ions were present.Analysis of the butanol‐soluble fraction permitted the isolation of at least three components characterized as: lipid‐diphosphate α‐glucose, lipid‐diphosphate α‐cellobiose and lipid‐monophosphate β‐galactose.In the presence of [β‐32P]UDP‐glucose the system incorporated radioactivity only into the diphosphate derivatives, indicating the transfer of a α‐glucose 1‐phosphate group from UDP‐glucose. Experiments with doubly labelled substrate confirmed these results.The role these compounds might play in polysaccharide biosynthesis is discussed.
Isotope Effects and Hydrogen Transfer during Simultaneous Metabolism of Ethanol and Cyclohexanone in Rats
Authors: Cronholm Tomas 
Published: March 2005

The metabolism of ethanol in vivo was studied by measurements of tritium isotope effects and extent of hydrogen transfer from ethanol to a reducible substrate of liver alcohol dehydrogenase. The specific radioactivity of ethanol in bile increased by a factor of two in 6–8 h following administration of [1‐3H]ethanol, 11 mmol/kg. This indicates a tritium isotope effect of about 1.6 on the association of [1‐3H]ethanol to the alcohol dehydrogenase‐NAD complex or on the rate of conversion of the ternary complex.Cyclohexanone was used as the reducible substrate of liver alcohol dehydrogenase. The major metabolite in bile was cyclohexyl glucuronide. During the simultaneous metabolism of [1,1 ‐2H2]‐ethanol with a deuterium excess of 95 atoms % the cyclohexanol formed had a deuterium excess of about 65 atoms %. This dilution cannot be explained by the isotope effect and is most likely due to the reversible dissociation of the enzyme‐NADH complex resulting in exchange with a less‐labelled free‐NADH pool.Ethanol metabolism increased the rate of cyclohexanone elimination, probably by increasing the concentration of alcohol dehydrogenase‐NADH complex. A large dose of cyclohexanone decreased the ethanol‐elimination rate, possible due to competitive inhibition.
Anion‐Exclusion and Anion‐Exchange Chromatography of Nucleotides
Authors: Singhal Ram P. 
Published: March 2005

Anion‐exclusion chromatography, previously shown to resolve nucleosides, has been applied to separate nucleotides on “cation‐exchange” columns at acid pH values. The study of pH and ionic effects indicates that these separations occur by anion repulsion, not by cation exchange. Cation exchange, previously held reponsible for these separations, is inoperative since nucleotides at acid pH values bear net negative charges, as do the fixed anions of the resin. The separations can be explained by anion‐exclusion chromatography) which involves two factors: repulsion between identically charged solute and resin ions, and partition between aqueous eluant and organic resin matrix. The results indicate that separations by anion exclusion are as good as or better than those by anion‐exchange chromatography.Anion‐exchange chromatography has also been reinvestigated to develop systems that can be used to separate the four major nucleotides in single eluants and to separate most nucleosides, nucleotides and other RNA hydrolysis products in one analysis.The ion‐exclusion separations are compared with those of anion‐exchange chromatography. The advantages and disadvantages of both methods are discussed. A typical example of the analysis of a nucleoside‐nucleotide mixture derived from the total hydrolysis of tRNAs for the assay of the base comparison and for the analysis of terminal end groups is described.
NADPH‐Dependent Formation of Thymidine diphosphodihydrostreptose from Thymidine diphospho‐d‐glucose in a Cell‐Free System from Streptomyces griseus and Its Correlation with Streptomycin Biosynthesis
Authors: Ortmann Rainer , Matern Ulrich , Grisebach Hans , Stadler Peter , Sinnnwell Volker , Paulsen Hans 
Published: March 2005

When thymidine diphospho‐d‐[U‐14C]glucose was incubated with a cell‐free extract from a streptomycin‐producing strain of Streptomyces griseus in the presence of NADPH, Radioactive dTDP‐Dihydrosteptose Was formed in addition to radioactive dTDP‐4‐keto‐6‐deoxyglucose and dTDP‐rhamnose. Dihydrostreptose was identified by chromatographic, gas chromatographic and electrophoretic comparison with synthetic dihydrostreptose characterized by its nuclear magnetic resonance spectrum.
dTDP‐dihydrostreptose is very labile and decomposes to dihydrostreptose monophosphate and further to free dihydrostreptose.
dTDP‐dihydrostreptose and dTDP‐rhamnose were also obtained when dTDP‐4‐keto 6‐deoxyglucose was the substrate of the reaction. Dihydrostreptose synthesis could be inhibited completely by 1 mM p‐chloromercuribenzoate, whereas rhamnose formation was not inhibited.
In the course of fermentation the activities of arginine: x‐amidinotransferase, an enzyme involved in streptomycin biosynthesis, and of the enzymatic activity for dTDP‐dihydrostreptose formation ran parallel and reached a maximum before onset of streptomycin formation.
A good correlation between the activity of “dTDP‐dihydrostreptose synthase”, arginine : x‐amidinotransferase activity and streptomycin production was found in several strains of S. griseus. No dihydrostreptose formation could be detected in a hydroxystreptomycin‐producing strain of S. griseocarneus.
Streptomycin but not dihydrostreptomycin could be detected in the fermentation medium of the S. griseus strain used for preparation of the cell‐free extract. The role of dTDP‐dihydrostreptose in streptomycin biosynthesis is at present unknown.