bluuebunny/crossref_metadata_2025 huggingface dataset:
Cooperative and Antagonistic Interactions of Peptidyl‐tRNA and Antibiotics with Bacterial Ribosomes
Authors: CONTRERAS Antonio , VAZQUEZ David
Published: March 2005

There is a single‐site interaction of [methylene‐14C]thiamphenicol and [methylene‐14C]chloramphenicol with run‐off ribosomes with dissociation constants Kd= 6.8 μM and Kd= 4.6 μM respectively. Similar affinities for the antibiotics are observed in polysomes totally deprived of nascent peptides, or bearing nascent peptides on the A‐site. However, two types of interaction are observed in endogenous polysomes with some ribosomes bearing nascent peptides on the P‐site and others in the A‐site. The lower‐affinity bindings (dissociation constants Kd= 6.4 μM and Kd= 5.9 μM for thiamphenicol and chloramphenicol respectively) are due to the ribosomes bearing nascent peptides on the A‐site. The higher‐affinity bindings (dissociation constants Kd= 2.3 μM and Kd= 1.5 μM for thiamphenicol and chloramphenicol, respectively) are due to the ribosomes bearing nascent peptides on the P‐site. Therefore binding of nascent peptides to the A‐site does not affect the affinities of thiamphenicol and chloramphenicol for the ribosome. On the other hand interaction of the nascent peptides with the P‐site of the ribosomes increases the affinities of both antibiotics for the ribosome. Thiamphenicol and chloramphenicol are thus good inhibitors of peptide bond formation in ribosomes and polysomes. Their affinities are increased precisely when the peptidyl‐tRNA is placed in the P‐site preceeding the peptide bond formation step, which is specifically blocked by the antibiotics.

There is a single‐site interaction per ribosome for [35S]thiostrepton, which does not appear to be affected by the attachment to the ribosomes of mRNA, tRNA and nascent peptides either to the A or the P‐site.

[N‐methyl‐14C]Lincomycin, [N‐methyl‐14C]erythromycin, [G‐3H]streptogramin B and [G‐3H]‐streptogramin A bind to run‐off ribosomes and polysomes totally free from nascent peptides. However, these antibiotics do not interact with ribosomes bearing nascent peptides either in the A or the P‐site and therefore are not active on preformed polysomes. Thus lincomycin and streptogramin A only interact with free ribosomes and 50‐S subunits and block the early rounds of peptide bond formation prior to polysome formation. Erythromycin and streptogramin B do not inhibit either initiation or the first round of peptide bond formation. However, erythromycin and streptogramin B, prebound to the ribosome, block peptide elongation probably by steric hindrance with the growing oligopeptide chain when this reaches a certain critical length.
Subunit Structure of the Methionine‐Repressible Aspartokinase II–Homoserine Dehydrogenase II from Escherichia coli K12
Authors: DAUTRY‐VARSAT Alice , SIBILLI‐WEILL Lise , COHEN Georges N.
Published: March 2005

The quaternary structure of Escherichia coli K 12 aspartokinase II–homoserine dehydrogenase II has been examined. This multifunctional protein has a molecular weight Mr= 176000. It is composed of two subunits having the same molecular weight and the same charge.

The results obtained from the examination of tryptic maps, the number and amino acid composition of cysteine‐containing peptides and the uniqueness of the N‐terminal sequence, strongly suggest that the 2 subunits are identical. The properties of aspartokinase II–homoserine dehydrogenase II can be compared to those of the much better known protein aspartokinase I–homoserine dehydrogenase I.
Localization of Glycerol‐3‐Phosphate Oxidase in the Mitochondrion and Particulate NAD+‐Linked Glycerol‐3‐Phosphate Dehydrogenase in the Microbodies of the Bloodstream Form of Trypanosoma brucei
Authors: OPPERDOES Fred R. , BORST Piet , BAKKER Suzanne , LEENE Wolter
Published: March 2005

We have studied the intracellular location of glycerol‐3‐phosphate oxidase and NAD+‐linked glycerol‐3‐phosphate dehydrogenase, two enzymes involved in the very active dihydroxyacetone phosphate:glycerol‐3‐phosphate cycle in Trypanosoma brucei. Isopycnic centrifugation of a large‐granule fraction resulted in a 10–20‐fold purification of the oxidase and showed that the enzyme behaved like the mitochondrial ATPase, isocitrate dehydrogenase and the particulate malate dehydrogenase. The activity profiles of these four enzymes in sucrose gradients were identical with maximal activity at a density of 1.17 g/cm3. Electron micrographs of a fraction equilibrating at this density only showed mitochondrial profiles.

The particle‐bound NAD+‐linked glycerol‐3‐phosphate dehydrogenase activity was found to band at a density of 1.23 g/cm3 in sucrose. A fraction equilibrating at this density was highly enriched in microbodies; it contained few mitochondrial profiles and less than 3% of the specific oxidase activity of the mitochondrial fraction. Because of the absence of microbody markers in T. brucei we turned to the insect trypanosome Crithidia luciliae, which contains a high activity of the microbody marker catalase. In C. luciliae part of the catalase activity bands at the same density as the particle‐bound NAD+‐linked glycerol‐3‐phosphate dehydrogenase (Q = 1.26 g/cm3).

From these results we conclude that in T. brucei bloodstream forms glycerol‐3‐phosphate oxidase is located in the mitochondrion and not in the microbodies, as proposed by a number of other investigators, and that the particle‐bound NAD+‐linked glycerol‐3‐phosphate dehydrogenase is located within the microbodies. It is likely that this highly active dehydrogenase plays an essential role in glycolysis.
A Trypsin‐Resistant Fragment from Complexes of Ribosomal Protein S4 with 16‐S RNA of Escherichia coli and from the Uncomplexed Protein
Authors: NEWBERRY Veronica , YAGUCHI Makoto , GARRETT Roger A.
Published: March 2005

A fragment of ribosomal protein S4 was prepared by limited trypsin digestion of a specific complex between protein S4 and 16‐S RNA. It was characterised for amino acid sequence and the N‐terminal 46 amino acids were found to be absent. An intermediate fragment, cut at Arg‐43, was also observed at low trypsin concentrations. Evidence is presented that the protected fragment constitutes the primary RNA‐binding region of the protein. No smaller protein fragments were found that rebound to the RNA. A mechanism for the degradation of the N‐terminal region of the protein is proposed and two probable functions of the excised region are given.

Under milder trypsin digestion conditions than for the complex, the same fragment, cut at Arg‐46, was also prepared from the free protein. This result, together with that from a control experiment, indicates that at least within this local region, the protein conformation is conserved in both the free protein and the protein · RNA complex. This is the first direct evidence for the conservation of conformation in a protein when both complexed and uncomplexed with a ribosomal RNA.
Transcription of Superhelical DNA from Cell Nuclei
Authors: COLMAN Alan , COOK Peter R.
Published: March 2005

Structures resembling nuclei may be released by gently lysing human or frog cells in solutions containing non‐ionic detergents and 1.95 M NaCl. These structures, or nucleoids, sediment in sucrose gradients containing intercalating agents in the manner characteristic of DNA that is intact, supercoiled and circular. They are depleted of nuclear protein and contain no endogenous RNA polymerase activity. We describe conditions for the transcription in vitro of these nucleoids by the RNA polymerase of Escherichia coli. We compared the kinetics of RNA synthesis directed by nucleoids and by DNA prepared using conventional procedures: nucleoids direct RNA synthesis at three to four times the rate of an equivalent weight of pure DNA and under appropriate conditions the DNA of each nucleoid can direct the synthesis of twice its own weight of RNA. Most of the newly‐synthesized RNA remains within the nucleoid. Experiments with the inhibitor, rifampicin, reveal that all RNA synthesis depends upon the initiation of new RNA chains and that nucleoids and pure DNA possess similar numbers of initiation sites. RNA synthesis directed by nucleoids has unusual kinetics: maximal rates are attained only after a lag of about ten minutes. Almost no lag is seen with pure DNA. The lag is due to the slow rate of formation of complexes of polymerase with nucleoid DNA that can initiate. Removal of supercoils by irradiation with γ‐rays or by the addition of the unwinding ligand, ethidium bromide, decreases the numbers of polymerase molecules able to initiate synthesis rapidly and increases the lag before maximal synthetic rates are achieved. Loss of super‐coiling does not alter the maximum synthetic rate. Supercoiling in eukaryotic DNA clearly influences the initiation of RNA synthesis. These results are discussed with reference to the effects of supercoiling on the transcription of prokaryotic templates.
The Major Polypeptides of the Murine‐Mammary‐Tumor Virus Isolated by Plant‐Lectin Affinity Chromatography
Authors: WESTENBRINK Frens , KOORNSTRA Wim , BENTVELZEN Peter
Published: March 2005

Solubilized polypeptides of the murine mammary tumor virus (MuMT virus) were chromatographed on a column of immobilised concanavalin A. The unbound viral material was rechromatographed on phosphocellulose, resulting in the isolation of the major internal proteins with a molecular weight of 28000 (p28) and 12000 (p12) respectively. The adsorbed glycopolypeptides after elution with methyl α‐d‐mannopyranoside were subjected to gel filtration. The major glycoprotein with a molecular weight of 52000 (gp52) was obtained in an almost pure form. However, a considerable part of gp52 elutes together with a glycoprotein with a molecular weight of 36000 (gp36), suggesting that in addition to the free form of gp52 a complex exists of gp52 plus gp36.
Diacylglycerol Acyltransferase from Rat Liver Microsomes
Authors: HOSAKA Kohei , SCHIELE Ulrich , NUMA Shosaku
Published: March 2005

Diacylglycerol acyltransferase was resolved from rat liver microsomes and separated from glycerolphosphate acyltransferase and 1‐acylglycerolphosphate acyltransferase. The separation was achieved by sucrose‐density‐gradient centrifugation of the enzyme preparation which was obtained by molecular‐sieve chromatography of microsomes solubilized with a nonionic detergent, Triton X‐100.

Although diacylglycerol acyltransferase and 1‐acylglycerolphosphorylcholine acyltransferase were not separated from each other, the two acyltransferases were distinguishable with respect to heat stability and sensitivity to sulfhydryl‐binding reagents.

Studies with the diacylglycerol acyltransferase preparation obtained have shown that this enzyme possesses a broad acyl‐donor specificity, utilizing saturated, monoenoic, dienoic and tetraenoic fatty acyl‐CoA thioesters efficiently.

A simplified assay method for diacylglycerol acyltransferase is described.
On the Structure of Flavin‐Oxygen Intermediates Involved in Enzymatic Reactions
Authors: GHISLA Sandro , ENTSCH Barrie , MASSEY Vincent , HUSEIN Mazhar
Published: March 2005

During the catalytic reactions of flavoprotein hydroxylases and bacterial luciferase, flavin peroxides are formed as intermediates [see Massey, V. and Hemmerich, P. (1976) in The Enzymes, 3rd edn (P. Boyer, ed.) pp. 421–505, Academic Press, New York]. These intermediates have been postulated to be C(4a) derivatives of the flavin coenzyme. To test this hypothesis, modified flavin coenzymes carrying an oxygen substituent at position C (4a) of the isoalloxazine ring were synthesized. They are tightly bound by the apoenzymes of d‐amino acid oxidase, p‐hydroxybenzoate hydroxylase and lactate oxidase; the resulting complexes show spectral properties closely similar to those of the transient oxygen adducts of the hydroxylases. The optical spectra of the lumiflavin model compounds were found to be highly dependent on the solvent environment and nature of the substituents. Under appropriate conditions they simulate satisfactorily the spectra of the transient enzymatic oxygen adducts. The results support the proposal that the primary oxygen adducts formed with these flavoproteins on reaction of the reduced enzymes with oxygen are flavin C(4a) peroxides.
Investigation into the Occurrence and Structure of Lectin Receptors on Human and Bovine Erythrocyte, Milk‐Fat Globule and Lymphocyte Plasma‐Membrane Glycoproteins
Authors: NEWMAN Roland. A. , UHLENBRUCK Gerhard. G.
Published: March 2005

The reduced disaccharide β‐d‐galactosyl(1‐3)‐N‐acetyl‐d‐galactosaminitol was released, after alkaline borohydride and neuraminidase treatment, from human and bovine erythrocyte membrane and fat globule membrane glycoproteins as well as from human and bovine lymphocyte plasma membranes. This disaccharide was found to be substituted to different extents by sialic acid in the various fractions. β‐d‐Galactosyl(1‐3)‐d‐acetyl‐d‐galactosamine is the immunodominant disaccharide of the Thomsen‐Friedenreich antigen (TF) and was detected serologically using specific lectins and antibody‐containing sera.

Bovine fat globule membrane glycoproteins were the only source of Tn antigen. The Tn antigen was detected by anti‐Tn‐containing sera and by the lectins from Salvia sclarea and Helix pomatia as well as gas chromatographic detection of N‐acetylgalactosaminitol after alkaline borohydride treatment of the native glycoproteins. Human milk fat globule membrane glycoproteins showed the presence of blood group A substance by reaction with rabbit anti‐A serum.
The Organisation of Genes for Transfer RNA and Ribosomal RNA in Amoebae and Plasmodia of Physarum polycephalum
Authors: HALL Len , BRAUN Richard
Published: March 2005

Using hybridisation techniques nuclei from both amoebae and plasmodia of Physarum polycephalum were found to contain 275 genes each coding for 5.8‐S, 19‐S and 26‐S rRNA, 685 genes for 5‐S rRNA and 1050 genes for tRNA.

Hybridisation of these RNA species to both amoebal and plasmodial DNA fractionated on CsCl gradients reveal that the 5.8‐S, 19‐S and 26‐S rRNA genes are located at a satellite position (Q= 1.714 g/cm3) with respect to the main band of DNA, whereas 4‐S and 5‐S RNA genes are located exclusively in the main band of DNA (Q= 1.702 g/cm3).

This result was confirmed by demonstrating that only the 5.8‐S, 19‐S, and 26‐S rRNA species hybridise to purified plasmodial ribosomal DNA.

The 19‐S and 26‐S rRNA genes of amoebae are located on extrachromosomal DNA molecules of a discrete size (Mr= 38 × 106) with identical properties to plasmodial ribosomal DNA.
The Use of Formaldehyde in RNA‐Protein Cross‐linking Studies with Ribosomal Subunits from Escherichia coli
Authors: MÖLLER Klaus , RINKE Jutta , ROSS Alexander , BUDDLE Gerard , BRIMACOMBE Richard
Published: March 2005

Radioactive subunits from Escherichia coli ribosomes were treated with formaldehyde, with a view to inducing a controlled cross‐linking reaction between protein and ribosomal RNA. Under the conditions described, about 10–15% of the total protein remained bound to the RNA in the presence of dodecyl sulphate, and little or no irreversible protein‐protein cross‐linking was observed. The RNA‐protein complexes are of a rather unstable nature, and the reaction is spontaneously reversible.

The proteins involved in the cross‐linked complexes were examined by polyacrylamide gel electrophoresis, after removal of unbound protein and nuclease treatment to digest the RNA‐protein complex. This showed that the individual ribosomal proteins were cross‐linked to varying degrees, and most of the proteins were involved in the reaction at least to some extent. A distinct group of proteins from each subunit were, however, reproducibly cross‐linked in high yield.

The formaldehyde‐treated subunits were subjected to a controlled hydrolysis with ribonuclease T1, and RNA fragments of known nucleotide sequence containing the cross‐linked proteins were isolated. Despite the reversibility of the cross‐linking reaction, sufficient cross‐linked protein survived the experiment to enable an analysis to be made of proteins associated with the various RNA fragments.

In the 50‐S subunit, proteins L27 and L32 were found to be cross‐linked to the 18‐S RNA fragment which arises from the 3′ end of 23‐S RNA. L2, L6 and L16 were also found in this fragment in small amounts. In contrast, L13 appeared to be linked to both the 18‐S RNA and the 13‐S RNA fragment from the 5′ end of 23‐S RNA. In the 30‐S subunit, proteins S3, S5, S9 (11), S13, and to some extent S4, S7, S12 and S18, were found associated with an RNA region comprising approximately 900 nucleotides at the 5′ end of 16‐S RNA. S4, S9 (11), S13, and to a lesser extent S7 and S14, were found in an RNA region comprising about 450 nucleotides near the 3′ end of 16‐S RNA, but lacking the 3′‐terminal 150 nucleotides. Protein S21 was cross‐linked to 16‐S RNA, but was not found in either of the two RNA regions above.
Characterisation of RNA Fragments Obtained by Mild Nuclease Digestion of 30‐S Ribosomal Subunits from Escherichia coli
Authors: RINKE Jutta , ROSS Alexander , BRIMACOMBE Richard
Published: March 2005

When Escherichia coli 30‐S ribosomal subunits are hydrolysed under mild conditions, two ribonucleoprotein fragments of unequal size are produced. Knowledge of the RNA sequences contained in these hydrolysis products was required for the experiments described in the preceding paper, and the RNA sub‐fragments have therefore been examined by oligonucleotide analysis. Two well‐defined small fragments of free RNA, produced concomitantly with the ribonucleoprotein fragments, were also analysed.

The larger ribonucleoprotein fragment, containing predominantly proteins S4, S5, S8, S15, S16 (17) and S20, contains a complex mixture of RNA sub‐fragments varying from about 100 to 800 nucleotides in length. All these fragments arose from the 5′‐terminal 900 nucleotides of 16‐S RNA, corresponding to the well‐known 12‐S fragment. No long‐range interactions could be detected within this RNA region in these experiments.

The RNA from the smaller ribonucleoprotein fragment (containing proteins S7, S9, S10, S14 and S19) has been described in detail previously, and consists of about 450 nucleotides near the 3′ end of the 16‐S RNA, but lacking the 3′‐terminal 150 nucleotides. The two small free RNA fragments (above) partly account for these missing 150 nucleotides; both fragments arose from section A of the 16‐S RNA, but section J (the 3′‐terminal 50 nucleotides) was not found. This result suggests that the 3′ region of 16‐S RNA is not involved in stable interactions with protein.
Puromycin and Some of Its Analogues: Conformational Properties in Solution
Authors: DE LEEUW Harry P. M. , DE JAGER Jenny R. , KOENERS Herman J. , VAN BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

The intramolecular conformation of puromycin in 2H2O solution at 25 °C and 65 °C was studied using vicinal coupling constants and chemical shifts obtained at 360 MHz. From vicinal coupling constants a 73% preference for the N‐type (C‐3′‐endo) ribose conformer is deduced. The reference compound, 6‐N‐dimethyl‐3′‐deoxy‐3′‐(acetamido)adenosine, displays even greater preference (80% N). Around the C‐4′– C‐5′ bond of the sugar residue and the C‐α–C‐β bond of the amino acid moiety the gauche+ rotamer (backbone notation) is preferred by 78% and 63% at 25 °C and 73% and 60% at 65 °C, respectively.

From the proton magnetic resonance parameters of the equilibrating species 6‐N‐dimethyl‐3′‐(2′)‐O‐(methoxyacetyl)adenosine it is deduced that the gauche+ orientation of the exocyclic 5′‐CH2OH group is again preferred, but the nature and the position of the substituent exert an influence on the ribose conformational equilibrium. The change from 3′‐amide to 3′‐ester decreases the percentage of N‐type ribose to 34% in the latter whereas the 2′‐ester shows 55% preference for type N.

Eight puromycin conformers are found to occur in significant amounts in solution, the most abundant conformer (36% at 25 °C and 31% at 65 °C) is the one identical with the solid‐state conformation of puromycin dihydrochloride. Calculated time‐averaged shielding values due to the phenyl ring of the amino acid moiety are in reasonable agreement with experiment and are dominated by the preferred conformer which is characterized by the proximity of the phenyl ring to the H‐4′, H‐5′ and H‐5” protons of the ribose moiety. The results substantiate the Remin and Shugar assignment of the C‐5′ methylene hydrogens in purine nucleosides. The possible biological significance of these findings is discussed.
On the Stereochemistry of Activation of Phenylalanine by Phenylalanyl‐tRNA Synthetase from Baker's Yeast
Authors: VON DER HAAR Friedrich , CRAMER Friedrich , ECKSTEIN Fritz , STAHL Kurt‐Wilhelm
Published: March 2005

Because of its chiralic α‐phosphorus atom adenosine 5′‐O‐(1‐thiotriphosphate) (ATPαS) exists in two diastereomeric forms, arbitrarily named (A) and (B).

For phenylalanyl‐tRNA synthetase ATPαS (A) is a substrate whereas ATPαS (B) is neither a substrate nor an inhibitor. During the ATPαS (A)/PPi exchange reaction with phenylalanyl‐tRNA synthetase the configuration at the α‐phosphorus atom is retained. The mechanistic implications of these findings are discussed.

Preliminary investigations with several other aminoacyl‐tRNA synthetases show that the stereochemical requirement with respect to the α‐phosphorus of ATP is not identical for all aminoacyl‐tRNA synthetases.
Protein‐Bound Adenosine 3′:5′‐Monophosphate in Liver of Glucagon‐Treated Rats
Authors: SCHWOCH Gerhild , HILZ Helmuth
Published: March 2005

The adenosine 3′:5′‐monophosphate (cyclic AMP) fraction bound to high‐affinity proteins of rat liver cytosol was determined by homogenization of the frozen tissue with a charcoal suspension followed by centrifugation. Protein‐bound cyclic AMP in the supernatant appears to represent mainly or exclusively cyclic AMP bound to the regulatory subunit of protein kinases as indicated by its identity with the phosphodiesterase‐resistant cyclic AMP fraction in homogenates and its close correlation with protein kinase activation in vivo.

Parallel determination of total cyclic AMP, protein‐bound cyclic AMP and protein kinase activation in the livers of rats sacrificed between 2 and 120 min after glucagon injection made it possible to correlate these parameters at different levels of total cyclic AMP in vivo. In non‐stimulated tissue, total cyclic AMP was 0.7 pmol/mg tissue, about 50 % being present in the form of protein‐bound cyclic AMP. At a total cyclic AMP level of 1.1–1.2 pmol/mg, half‐maximal values for bound cyclic AMP as well as half‐maximal activation of protein kinases were found. This shows not only a close correlation between these two parameters but also indicates that rather small changes in total cyclic AMP are required in vivo to effect binding of cyclic AMP to the regulatory subunit, R, with the concomitant activation of protein kinase.

The reliability of protein‐bound cyclic AMP as an indicator of protein kinase activation was further documented in the livers of stressed animals, in which total cyclic AMP was elevated nearly two‐fold while bound cyclic AMP and protein kinase remained at basal values.
Factors Influencing the Apparent Molecular Weight of Collagenase Produced by Human‐Skin Explants
Authors: FIEDLER‐NAGY Christa , COFFEY John W. , SALVADOR Richard A.
Published: March 2005

When the collagenase from the culture medium of human skin explants was concentrated at 4 °C either by (NH4)2SO4 precipitation, ultrafiltration or lyophilization, the enzyme chromatographed on Sephadex G‐150 at 4 °C as a heterogeneous population of molecules. However, when the crude collagenase was preincubated for several hours at 25 °C (pH 7.6) prior to chromatography, the collagenase eluted from a Sephadex G‐150 column as a homogeneous peak corresponding to a molecular weight of approximately 40000. Incubation of the crude collagenase preparation at 25 °C also resulted in a decrease in the apparent molecular weight of the hydroxyproline‐containing peptides, which were contaminants of the crude collagenase preparation and in a decrease in the levorotation (300 nm) of the crude collagenase solution. The shifts in the apparent molecular weights of collagenase and of collagen fragments caused by incubation at 25 °C for several hours were partially reversed when the crude collagenase was reincubated at 4 °C for 24 h. Incubation of the crude collagenase for longer periods (1–7 days) resulted in the gradual proteolytic degradation of the collagen peptides without appreciable alterations in the apparent molecular weight or enzyme activity of collagenase. EDTA inhibited this proteolytic degradation of the collagen fragments. After 1–7 days of incubation at 25 °C the shifts in the molecular weights of collagenase and collagen fragments were no longer reversible at 4 °C. These data suggest that collagenase may be bound at 4 °C to aggregated helical fragments of collagen. The reversible disruption of these helical fragments at 25 °C may account for the observed changes in the apparent molecular weight of collagenase following incubation at 25 °C.
Activity of Protein Kinase Dependent on Adenosine 3′:5′‐Monophosphate and of Its Thermostable Protein Inhibitor in Rat Hepatoma (HTC) Cells
Authors: ROUSSEAU Guy G.
Published: March 2005

Normal expression of a variety of hormonal effects which depend on cyclic AMP (adenosine 3′:5′‐monophosphate) requires the presence of glucocorticoids. Our hypothesis was that glucocorticoids control directly or indirectly the activity of cyclic‐AMP‐dependent protein kinase. This has been investigated in cultured hepatoma (HTC) cells in which N6,O2′‐dibutyryladenosine 3′:5′‐monophosphate increases the activity of tyrosine transaminase only after glucocorticoid treatment.

In these cells, we have determined the concentration and half‐life of protein kinase, the sensitivity of this enzyme in vitro to cyclic AMP and to its thermostable protein inhibitor, the state of dissociation of protein kinase holoenzyme in vivo and its sensitivity, in the intact cell, to dibutyryladenosine 3′:5′‐monophosphate and to the inhibitor diamide, and we have also determined the concentration of endogenous thermostable protein inhibitor of protein kinase.

None of these parameters were influenced by glucocorticoids under conditions where these hormones stimulate the activity of tyrosine transaminase and restore sensitivity to dibutyryladenosine 3′:5′‐monophosphate.

It is concluded that the permissive action of glucocorticoids probably results from a control of cyclic‐AMP‐dependent processes exerted at a level beyond the protein kinase system.
Inhibition of Cholesterol Synthesis in vitro and in vivo by ML‐236A and ML‐236B, Competitive Inhibitors of 3‐Hydroxy‐3‐methylglutaryl‐Coenzyme A Reductase
Authors: ENDO Akira , TSUJITA Yoshio , KURODA Masao , TANZAWA Kazuhiko
Published: March 2005

1. ML‐236A and ML‐236B, potent inhibitors of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase, were readily absorbed into the liver, the major site of cholesterogenesis, after oral administration in mice; peak concentration of these inhibitors in the liver, which were attained within 60 min after administration, were 4% of the dose for ML‐236A and 10% for ML‐236B, respectively.

2. ML‐236B reduced sterol synthesis from both [14C]acetate and [14C]octanoate in rat liver slices to 50% of control at a concentration of 0.05 μg/ml (0.12 μM) but not that from [14C]mevalonate at higher concentrations up to 1.25 μg/ml. Inhibition of sterol synthesis was also observed in liver slices obtained from rats which had previously received this compound.

3. ML‐236B was also inhibitory in vivo to cholesterol synthesis in various tissues of rats in which [14C]acetate was intraperitoneally injected to the animals and labeled digitonin‐precipitable sterols synthesized were isolated and determined. Sterol synthesis in the liver was far more strongly inhibited (over 90 % at 4h after oral administration of 50 mg/kg of ML‐236B) than that in other organs tested including kidney, lung, adrenal, spleen, and testis.

4. These findings strongly suggest that hypocholesterolemic activity of ML‐236A and ML‐236B is owing largely to specific inhibition of hepatic cholesterol synthesis.
The Involvement of a Complex between Formylmethionyl‐tRNA and Initiation Factor IF‐2 in Prokaryotic Initiation
Authors: VAN DER HOFSTAD Gerard A. J. M. , FOEKENS John A. , BOSCH Leendert , VOORMA Harry O.
Published: March 2005

A complex between initiation factor IF‐2 and fMet‐tRNA can be formed under ionic conditions, which are optimal for initiation complex formation. The complex can be retained on cellulose nitrate filters after fixing with glutaraldehyde.

The IF‐2 · fMet‐tRNA complex formation is not influenced by GTP and GDP. Other nucleoside di or triphosphates also have no effect. Evidence is presented that this complex acts as an intermediate in polypeptide chain initiation. The IF‐2 · fMet‐tRNA complex formation is not influenced by initiation factors IF‐1 and IF‐3.

The binary complex can be bound to the 30‐S subunit in the absence of GTP, which indicates that there is no concomittant binding of the IF‐2 · fMet‐tRNA complex and the nucleotide moiety to the 30‐S subunit. The binding of the binary complex is stimulated by GTP. The influence of some inhibitors of initiation on the IF‐2 · fMet‐tRNA complex formation has been tested. Aurin tricarboxylic acid appeared to be a strong inhibitor, whereas the sulfhydryl reagents N‐ethylmaleimide and p‐chloromercuribenzoate had no effect.
Biosynthesis of the Blood‐Group‐B‐Specific Trisaccharide in a Rhesus Monkey
Authors: CHESTER M. Alan , HALLGREN Peter , LINDBERG Bo S. , LUNDBLAD Arne
Published: March 2005

A Rhesus monkey, serologically grouped as B, has been shown to excrete low‐molecular‐weight carbohydrate material in urine closely related to that found in human urine. Galactose feeding resulted in the excretion of a trisaccharide which was shown to be identical to the trisaccharide isolated from the urine of group B humans under the same conditions. Experiments in which [14C]galactose was administered both orally and via an intestinal vein demonstrated that the intestine is the site of biosynthesis.
Hydrolysis and Isomerization of trans,trans‐Farnesyl Diphosphate by Andrographis Tissue‐Culture Enzymes
Authors: MACKIE Hugh , OVERTON Karl H.
Published: March 2005

1. Incubation of (3R,5S)‐[5‐3H1]mevalonate + (3RS)‐[2‐14C]mevalonate with Andrographis cell‐free extract leads to trans,trans‐farnesol and cis,trans‐farnesol which both totally retain tritium.

2. This conflicts with our previous results which predict one third tritium loss in the cis,trans‐farnesol. Inversion at C‐1 during hydrolysis of trans,trans‐farnesyl diphosphate to trans,trans‐farnesol could explain this anomaly.

3. (1S)‐trans,trans‐[1‐3H1]Farnesyl diphosphate and phosphate and (1R)‐trans,trans‐[1‐3H1]‐farnesyl diphosphate and phosphate, all prepared chemically, were hydrolysed with Andrographis phosphatase, and alkaline phosphatase and hydrogenolysed with lithium aluminium hydride and the product alcohols exchanged with liver alcohol hydrogenase.

4. Both Andrographis phosphatase and alkaline phosphatase hydrolyse trans,trans‐farnesyl diphosphate and trans,trans‐farnesyl phosphate with retention.

5. Hydrolysis of trans,trans‐[1‐18O]farnesyl diphosphate in H218O with both phosphatases supports P‐O fission.

6. The C‐1 configuration in (1S)‐trans,trans‐[1‐3H1]farnesyl diphosphate and phosphate and (1R)‐trans,trans‐[1‐3H1]farnesyl diphosphate and phosphate is progressively racemised in 0.01 M NH4OH/MeOH (1/9) at –20°C.
Reduction in β‐Adrenergic Response of Cultured Glioma Cells Following Depletion of Intracellular GTP
Authors: FRANKLIN Trevor J. , TWOSE Patricia A.
Published: March 2005

1. When C6 glioma cells were incubated with mycophenolic acid, a potent and specific inhibitor of IMP:NAD oxidoreductase (EC 1.2.1.14) there was a marked depletion of the cellular content of GTP. The viability of the cells was unaffected.

2. The adenosine 3′:5′‐monophosphate (cyclic AMP) response of C6 glioma cells to the β‐adrenergic stimulant, (±)isoprenaline, was considerably reduced after treatment with mycophenolic acid. The diminished response to (±)isoprenaline was prevented by the inclusion of guanine in the culture medium along with mycophenolic acid.

3. The adenylate cyclase response to (±)isoprenaline of whole homogenates from C6 cells treated with mycophenolic acid was also depressed; the response was restored to normal by the addition of GTP.

4. The adenylate cyclase response to (±)isoprenaline of a membrane fraction prepared from homogenates of C6 cells was almost totally dependent on the presence of added GTP. Membrane fractions from control and mycophenolic‐acid‐treated C6 cells gave similar adenylate cyclase responses to (±)isoprenaline in the presence of GTP.

5. It is concluded that mycophenolic acid may depress the β‐adrenergic sensitivity of C6 cells by depleting the cellular content of GTP.
Degradation of Nucleic Acids in Cell Lysates by S1 Nuclease in the Presence of 9 M Urea and Sodium Dodecylsulfate
Authors: ZECHEL Kaspar , WEBER Klaus
Published: March 2005

Single‐strand‐specific nuclease S1 from Aspergillus oryzae is shown to degrade DNA and RNA in lysates of HeLa cells in the presence of 9 M urea and sodium dodecylsulfate. Free dodecylsulfate inhibits S1 nuclease. However, if the detergent is complexed with proteins prior to the addition of the enzyme, S1 nuclease can degrade nucleic acids at dodecylsulfate concentrations which would inhibit the enzyme completely if no other proteins were present. In lysates prepared from HeLa cells by treatment with dodecylsulfate and urea, the detergent is complexed by cellular proteins and therefore S1 nuclease can be used to digest DNA and RNA. DNA can be completely degraded but, even after heat‐denaturation, only 60% of the cellular RNA is converted into acid‐soluble material. Analysis of the acid‐insoluble RNA fragments by gel filtration reveals that the majority of the degradation products is approximately of tRNA size.
Purification and Characterization of Vicilin Peptidohydrolase, the Major Endopeptidase in the Cotyledons of Mung‐Bean Seedlings
Authors: BAUMGARTNER Bruno , CHRISPEELS Maarten J.
Published: March 2005

1. Vicilin peptidohydrolase, the major endopeptidase present in the cotyledons of bean seedlings, has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis and isoelectrofocusing, from the cotyledons of mung bean [Vigna radiata (L.) Wilczek] seedlings.

2. The protease has an apparent molecular weight of 23 000 as estimated by gel filtration and polyacrylamide gel electrophoresis, an isoelectric point of 3.75 and belongs to the class of the sulfhydryl proteases as judged by the effect of various protease inhibitors on the activity of the enzyme.

3. The purified enzyme readily hydrolyzes synthetic esters between amino acids and p‐nitrophenol coupled to N‐carbobenzoxy and N‐tert‐butoxycarbonyl residues. Esters with asparagine and glutamine residues are hydrolyzed most readily.

4. The purified enzyme readily degrades vicilin, the principal reserve protein present in mung bean seeds. Degradation is optimal at pH 5.1. The various polypeptides of vicilin are gradually digested to smaller polypeptides and oligopeptides. Vicilin peptidohydrolase and carboxypeptidase cooperate in the complete digestion of vicilin with the resulting release of amino acids.
The Binding Site of Ribosomal Protein S8 on the 16‐S Ribosomal RNA from Bacillus stearothermophilus
Authors: STANLEY John , EBEL Jean‐Pierre
Published: March 2005

The region of Bacillus stearothermophilus strain NCA 1503 16‐S ribosomal RNA protected from ribonuclease digestion by the 30‐S ribosomal subunit protein S8 from Bacillus stearothermophilus has been established. The nucleotide sequence is compared to the analogous region in Escherichia coli and a possible base‐pairing scheme proposed that is consistent with the data of ribonuclease susceptibility.
Initiation of RNA Synthesis in Isolated Nuclei
Authors: GILBOA Eli , SOREQ Hermona , AVIV Haim
Published: March 2005

Ribonucleotide triphosphates, labeled at the β position, were synthesized and used directly to quantify RNA chain initiation in nuclei isolated from Friend cells grown in tissue culture. At the optimal salt concentration, low‐molecular‐weight RNAs (4–5S) synthesized by RNA polymerase III were the predominant species initiated. Less than 5% of the molecules were initiated by polymerase II. We calculate that 50–80% of the small RNA molecules synthesized in vitro were also initiated in vitro. Assuming that a substantial fraction of the nuclei were active in vitro, the number of 4–5S RNA molecules initiated per nucleus was about 100 molecules/min.
Cellulases from Chaetomium thermophile var. dissitum
Authors: ERIKSEN Jonny , GOKSØYR Jostein
Published: March 2005

When cultivated in a liquid medium with cellulose, Chaetomium thermophile var. dissitum produced extracellular cellulolytic enzymes. By concentration of the culture filtrate, followed by ion‐exchange chromatography on DEAE‐Sephadex A‐50 and gel filtration on Biogel P‐100, three electrophoretically pure components were obtained. Of these, one was a typical Cx enzyme (endoglucanase) causing rapid decrease of the viscosity of carboxymethylcellulose solutions, while showing low effect on native cellulose.

The other was active towards native cellulose, but had little effect on the viscosity of carboxymethylcellulose. It is concluded that this enzyme is an exoglucanase (C1 enzyme), possibly a cellobiohydrolase.

The third component showed only cellobiase (β‐glucosidase) activity, and had no effect on cotton or carboxymethylcellulose. The three components, when mixed, showed synergistic effects on highly ordered cellulose. The endo‐ and exoglucanases were characterized with regard to molecular size and iso‐electric point (pI). Both cellulases had pI near 4.55, but their molecular weights were different: 67 000 (exoglucanase) and 41 000 (endoglucanase). The effect of temperature on the activity of the cellulases was examined with both cotton and carboxymethylcellulose as substrate. Arrhenius activation energies, Q10, and temperature optima for the different reactions were determined.
Preparation of N6‐[N‐(6‐Aminohexyl)carbamoyl]‐Adenine Nucleotides and Their Application to Coenzymically Active Immobilized ADP and ATP, and Affinity Adsorbents
Authors: YAMAZAKI Yoshimitsu , MAEDA Hidekatsu , SUZUKI Hideo
Published: March 2005

Reaction of ADP with hexamethylene diisocyanate in hexamethylphosphoramide followed by treatment in an acidic medium afforded three new adenine nucleotide analogues, N6‐[N‐(6‐aminohexyl)carbamoyl]‐ADP, N6‐[N‐(6‐aminohexyl)carbamoyl]‐ATP, and N6‐[N‐(6‐aminohexyl)carbamoyl]‐AMP in yields of 13%, 12% and 17%, respectively. The occurrence of the ATP analogue may be interpreted in terms of the equilibrium, 2ADP = ATP + AMP.

Coenzymic activities of the ADP analogue against acetate kinase and pyruvate kinase were 82% and 20%, respectively, relative to ADP and those of the ATP analogue against hexokinase and glycerokinase were 63% and 87%, respectively, relative to ATP. These analogues were bound to CNBr‐activated soluble dextran through their terminal amino group to give an immobilized ADP and an immobilized ATP, each of which was recycled in a system comprising acetate kinase and hexokinase, and when placed in a membrane reactor together with the enzymes, functioned as an immobilized coenzyme continuously yielding glucose 6‐phosphate.

A series of chemically defined affinity adsorbents were obtained by coupling these analogues to CNBr‐activated Sepharose, and were used to separate the enzymes in a mixture of hexokinase, pyruvate kinase, phosphoglycerate kinase, lactate dehydrogenase, and alcohol dehydrogenase.
Metabolic Behaviour of Nonhistone Chromosomal Proteins in Proliferating and in Resting Fibroblasts
Authors: DJONDJUROV Lalju Petrov , IVANOVA Emilia Christova , TSANEV Roumen Georgiev
Published: March 2005

The metabolism of nonhistone chromosomal proteins was studied in two lines of cells showing a different degree of contact inhibition: human diploid fibroblasts, which are easily contact‐inhibited, and Chinese hamster fibroblasts, which had been made to stop proliferating by fasting. By following the 3H/14C ratio of [3H]tryptophan‐labelled nonhistone chromosomal proteins and [14C]thymidine‐labelled DNA in chase experiments three main groups of these proteins could be detected with respect to their metabolic behaviour: (a) a metabolically stable group which is acid‐insoluble and represents the bulk of nonhistone chromosomal proteins in proliferating cells; this group is conserved when the cells enter a resting phase; (b) a metabolically labile group which is acid‐insoluble and is observed as a minor fraction in proliferating cells; (c) a metabolically labile group which is acid‐insoluble and accumulates in resting cells; this fraction is much larger in contact‐inhibited cells.

Stimulation of cell proliferation by trypsinization decreases the amount of nonhistone chromosomal proteins in resting cells to the basic level observed in proliferating cells.
Hemoglobin S Travis: a Sickling Hemoglobin with Two Amino Acid Substitutions [β6(A3)Glutamic Acid → Valine and β 142(H20) Alanine → Valine]
Authors: MOO‐PENN Winston F. , SCHMIDT Robert M. , JUE Danny L. , BECHTEL Katherine C. , WRIGHT Jane M. , HORNE McDonald K. , HAYCRAFT Gordon L. , ROTH Eugene F. , NAGEL Ronald L.
Published: March 2005

Hb S Travis is a previously undescribed sickling hemoglobin with two amino acid substitutions in the β chain: β6 Glu → Val and β142 Ala→ Val. The β6 Glu→Val mutation imparts to Hb S Travis the characteristic properties of sickling hemoglobin, namely its association with erythrocyte sickling, the insolubility of the hemoglobin in the reduced form, and a minimum gelling concentration value identical to Hb S. Unlike Hb S, Hb S Travis exhibits an increased oxygen affinity and a decreased affinity for 2,3‐bisphosphoglycerate and inositol hexakisphosphate. In addition, the variant hemoglobin's tendency to autoxidize and its mechanical precipitability suggest that there are conformational differences between Hb S and Hb S Travis.
Thermophilic Polynucleotide Phosphorylase from Thermus thermophilus
Authors: HISHINUMA Fumio , HIRAI Keiko , SAKAGUCHI Kenji
Published: March 2005

A thermophilic polynucleotide phosphorylase lacking polynucleotide phosphorolytic activity was purified from Thermus thermophilus HB‐8 strain.

The enzyme is an altered form of the native polynucleotide phosphorylase, probably attacked by the proteinase(s) of this extreme thermophile during the purification process. This modified enzyme lacks phosphorolytic activity to poly(A) while retaining weak activity to phosphorolyse tetranucleotides or hexanucleotides. The purified enzyme was shown to be homogeneous by electrophoretic analysis in polyacrylamide gel. This enzyme had a molecular weight of 190000 as calculated both from electrophoresis on polyacrylamide gel and from the Stokes' radius derived from the gel filtration pattern and the sedimentation coefficient. The enzyme was separated into three polypeptide chains by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate; their molecular weights were calculated to be 92000, 73000 and 35000.

The enzyme was thermophilic and thermotolerant, exhibiting its maximal activity at 70 °C. The four ribonucleoside diphosphates (ADP, GDP, UDP and CDP) were polymerized to the extent of 7‐S size.
Structure and Synthesis of a Lipid‐Containing Bacteriophage
Authors: TSUKAGOSHI Norihiro , SCHÄFER Rolf , FRANKLIN Richard M.
Published: March 2005

Endolysin was induced in Pseudomonas BAL‐31 infected with bacteriophage PM2 and was also associated with the purified virion. This enzyme required divalent cations for its activity, Ca2+ being the most effective cation. Endolysin activity in the virion increased up to three‐fold upon disruption and the activity could be localized in the viral nucleocapsid. Thus the enzyme is localized within the virion. After purification of the structural proteins of bacteriophage PM2, only the nucleocapsid protein (III) had endolysin activity.
Translation of Enzymically Decapped Messenger RNA
Authors: ABRAHAM K. Abraham , PIHL Alexander
Published: March 2005

An enzymic procedure was used to remove the 7‐methylguanosine diphosphate moiety at the 5′ ends of rabbit hemoglobin mRNA and mouse immunoglobulin light‐chain mRNA. Evidence was obtained that the procedure, which involves the use of polynucleotide kinase, does not result in any further degradation of the mRNA.

The enzymically decapped mRNA was as effective as untreated mRNA in supporting protein synthesis in a wheat germ system. This was the case over a wide range of mRNA concentrations and over a considerable period of time. The presence in the incubation mixture of S‐adenosylhomocysteine, an inhibitor of methylation, did not affect the results.

The data indicate that the presence of a 7‐methylguanosine diphosphate residue at the 5′ end of mRNAs is not an obligatory requirement for translation in eucaryotic systems.
Primary Structure of Human Fibrinogen
Authors: GÅRDLUND Bengt , HESSEL Birgit , BLOMBÄCK Birger , MARGUERIE Gerard , MURANO Genesio
Published: March 2005

Five disulfide‐containing fragments obtained from cyanogen‐bromide‐cleaved human fibrinogen (N‐DSK, Hi2‐DSK, Ho1‐DSK, Ho2‐DSK and Ho3‐DSK) have been characterized in this report. The complete primary structure of the ‘N‐terminal disulfide knot’, N‐DSK, has been elucidated earlier. The individual chains of the fragments composed of more than one chain have been purified following reduction and alkylation.

Hi‐2‐DSK, Mr 26600, a one‐chain fragment with one intra‐chain disulfide bond is derived from the Aα chain of fibrinogen. Ho1‐DSK, Mr 43000, consists of five chains linked together by six disulfide bridges. The largest chain of Ho1‐DSK, Mr 21000, is derived from the γ chain of fibrinogen, a smaller chain, Mr 5900, from the Aα chain and the three remaining, Mr 5800, 4300 and 2200, from the Bβ chain. Ho2‐DSK, Mr 5400, is derived from the Bβ chain of fibrinogen and constitutes a single‐chain peptide with one disulfide bond. Ho3‐DSK, Mr 7400, consists of two chains connected with one disulfide bridge. Amino acid analysis of the disulfide knots and their individual chains is presented. Partial amino acid sequence of Hi2‐DSK, the four larger chains of Ho1‐DSK and Ho2‐DSK and the complete sequence of the smallest chain of Ho1‐DSK is shown.

All disulfide knots except N‐DSK were found to be monomeric. One mole of N‐DSK and two moles of each of the other disulfide knots are present per mole of fibrinogen. These five disulfide knots account for all disulfides in human fibrinogen (fraction 1–4). The location of the disulfide knots in the fibrinogen molecule and the arrangement of the disulfide bonds is discussed.