bluuebunny/crossref_metadata_2025 huggingface dataset:
Terminal Nucleotide Sequences of 17‐S Ribosomal RNA and Its Immediate Precursor 18‐S RNA in Yeast
Authors: DE JONGE Peter , KLOOTWIJK Jacobus , PLANTA Rudi J.
Published: March 2005

The 5′ and 3′‐terminal nucleotide sequences of 17‐S rRNA and its immediate precursor 18‐S RNA from the yeast Saccharomyces carlsbergensis have been analysed. Identification of the terminal oligonucleotides, as present in Ti ribonuclease digests, was performed by diagonal procedures.

The major (molar yield 0.9) 5′‐terminal oligonucleotide of mature 17‐S yeast rRNA proved to be pU‐A‐U‐C‐U‐G. In addition a minor 5′‐terminal oligonucleotide (molar yield 0.15) with the overall composition pU(U2,C2)G was observed. 18‐S precursor RNA was found to contain the same 5′‐terminal sequences as 17‐S rRNA. However, the 3′‐terminal sequences of the two types of RNA appeared to be different. The 17‐S rRNA yields the oligonucleotide A‐U‐C‐A‐U‐U‐AOH while at least half of the 18‐S RNA molecules contain the sequence U‐U‐U‐C‐A‐A‐U‐AOH. In addition 18‐S RNA yields several minor 3′‐terminal oligonucleotides which appear to be structurally related to the major 3′‐terminal sequence. These results demonstrate that the extra nucleotides in 18‐S RNA relative to 17‐S RNA are located exclusively at the 3′‐terminus of the 18‐S RNA molecule.

The possibility that the 3′‐terminal nucleotide sequence of 18‐S RNA plays a role in the maturation process is discussed.
2′‐Deoxy‐2′‐azidocytidine, a New Inhibitor of DNA Replication in Mammalian Cells
Authors: SKOOG Lambert , BJURSELL Gunnar , THELANDER Lars , HÄGERSTRÖM Torsten , HOBBS John , ECKSTEIN Fritz
Published: March 2005

Cell growth is reversibly inhibited by the nucleoside analogue, 2′‐deoxy‐2′‐azidocytidine and the inhibition is a result of interference with DNA replication. The 5′‐diphosphate of the analogue was earlier shown to specifically inactivate the enzyme ribonucleotide reductase in vitro. However, measurements of the pools of deoxyribonucleoside triphosphates in cells incubated in azidocytidine showed only minor changes which appeared to result from and not to be the cause of the inhibition of DNA replication. The DNA synthesized in polyoma‐infected cells after incubation in azidocytidine showed a sedimentation pattern quite different from that seen after inhibition of DNA synthesis with arabinosyl cytosine or hydroxyurea. Experiments with nuclei isolated from azidocytidine‐inhibited, polyoma‐infected cells indicated (a) that the number of replicating molecules is decreased during the inhibition and (b) that upon incubation of the nuclei there is a rapid synthesis of DNA occurring in a new class of DNA molecules which are at a very early state of replication.

Neither the 5′‐triphosphate of azidocytidine nor the nucleoside itself inhibit DNA synthesis in vitro in isolated nuclei from polyoma‐infected cells and at present the nature of the DNA‐synthesis‐inhibiting compound acting in the cells after addition of azidocytidine is unknown. Taken together the results suggest that azidocytidine inhibits DNA synthesis at an early stage, possible by blocking the initiation of DNA synthesis at the origin or by interfering with the elongation of newly initiated DNA molecules.
Fate of Histone Messenger RNA in Synchronized HeLa Cells in the Absence of Initiation of Protein Synthesis
Authors: STAHL Hans , GALLWITZ Dieter
Published: March 2005

The fate of cytoplasmic histone mRNA was studied under conditions in which initiation of protein synthesis in synchronized HeLa cells in S phase was blocked by increasing the osmolarity of the growth medium with NaCl. In contrast to the interruption of DNA replication with hydroxy‐urea, which results in an exponential degradation of translatable histone mRNA with a half‐life of about 10‐13 min, blocking the initiation of protein synthesis leads to only a marginal loss of biologically active histone mRNA in the cytoplasm. When the initiation of protein synthesis was interrupted by treating cells with 150 mM NaCl, 40‐50% of the total cytoplasmic histone mRNA previously translated in polyribosomes appears in the cytoplasm integrated into mRNA. protein particle(s) sedimenting between 15 S and 30 S. On the other hand, in untreated S‐phase cells or in cells blocked with hydroxyurea only 3‐6% of the total translatable histone mRNA is found in the cytoplasm not bound to ribosomes or their subunits. In addition, the degradation of histone mRNA in hydroxyurea‐blocked S‐phase cells is prevented when the initiation of protein synthesis is inhibited with NaCl.

These studies clearly indicate that the inhibition of initiation of protein synthesis per se is not the cause for the rapid degradation of cytoplasmic histone mRNA observed when DNA replication is turned off and that the inactivation of these mRNAs is a process dependent on continuous protein synthesis.
Purification of Bovine Adrenal‐Cortex Plasma‐Membrane Vesicles Containing a Highly Corticotropin‐Sensitive Adenylate‐Cyclase System and Angiotensin‐II‐Binding Sites
Authors: SCHLEGEL Werner , SCHWYZER Robert
Published: March 2005

The purpose of this experimental investigation was to provide a purified plasma membrane fraction containing a highly hormone‐responsive adenylate cyclase system. Bovine adrenal cortex was homogenised and a washed pellet (450000 × g · min) was fractionated by zonal centrifugation in a sucrose and dextran gradient.

Adenylate cyclase activity was purified up to 60‐fold to a specific activity of 55, 340 and 210 pmol of adenosine 3′: 5′‐monophosphate (cyclic AMP) produced/minute per mg of protein at 380C for the basal, adrenocorticotrophin and fluoride‐activated states, respectively. The time course of the adenylate cyclase activity is linear. The concentration necessary for half‐maximal stimulation by adrenocorticotrophin‐(1‐24)‐tetracosipeptide is 0.5 μM. The high hormone‐responsiveness of the membrane preparation allows one to demonstrate activation of adenylate cyclase by very weakly agonistic adrenocorticotrophin fragments. The F−‐activated state can be detergent‐dispersed by Lubrol and shows a Km (ATP) different from that of either the basal or adrenocorticotrophin‐stimulated state.

Other marker enzymes such as 5′‐nucleotidase, glucose‐6‐phosphatase and cytochrome oxidase were followed during purification. The plasma membrane fraction shows rather homogeneous, relatively large vesicles (mean diameter 0.5 μm). It contains high‐affinity binding sites for angiotensin II (about 2 pmol per mg protein) with an apparent association constant of 2 × 107 (1/mol) at 120C. The yield, 20 mg of membrane protein per preparation, may make it a tool in either affinity‐labelling studies with the peptide hormones mentioned or the starting point for solubilisation and purification of adenylate cyclase.
Subcellular Distribution of Aminoacyl‐tRNA Synthetases in Various Eukaryotic Cells
Authors: USSERY Michael A. , TANAKA Wesley K. , HARDESTY Boyd
Published: March 2005

The total amount, size distribution and binding of aminoacyl‐tRNA synthetases to ribosomes in a variety of mammalian and avian cells was studied under standard conditions of sample preparation and assay. Aminoacyl‐tRNA synthetases appear to exist in three general forms; ‘free’ enzyme of about 4–9 S, one or more ‘enzyme complexes’ of about 18–25 S, and in association with ribosomes. The aminoacyl‐tRNA synthetase activity for many individual amino acids was surprisingly similar in cell types chosen to be diverse with respect to differentiation state, transformation, and growth rate. Total activity for all amino acids varied about 4‐fold, based on a constant volume of cells. Embryonic tissues had a comparatively high proportion of total synthetase activity associated with ribosomes, whereas this value was relatively low for mouse liver. Distinctive distribution patterns with common and variable features were observed for individual enzymes. The only aminoacyl‐tRNA synthetases found not to be associated in significant amounts with either 18–25‐S enzyme complexes or ribosomes in any of the cell types examined were the enzymes for alanine, histidine, and serine. All cell types evidenced 18–25‐S synthetase activity for arginine, aspartic acid, glutamine, glutamic acid, isoleucine, leucine, lysine, methionine, proline, and valine, although in quite variable proportions of the total activity observed for these amino acids. For example, of the valyl‐tRNA synthetase activity not associated with ribosomes, 35% and 100% were found to sediment at 18–25 S in Friend leukemia cells and mouse liver respectively. All cells had two easily distinguishable peaks of arginyl‐tRNA synthetase activity at 4–9 S and 18–25 S respectively; however, the relative proportion of enzyme activity in the peaks differed between cell types. Phenylalanyl‐tRNA synthetase was not observed to occur in an 18–25‐S complex in any of the cell types examined but was bound to ribosomes in variable but generally relatively high proportions. Numerous other specific differences are described.

No underlying physiological or biochemical principle has been recognized to account for the specific distribution patterns observed. However, they may reflect variations in cellular architecture that may be related to regulation of protein synthesis.
Characterization and Partial Purification of an Inducible Protein Related to Hexose Proton Cotransport of Chlorella vulgaris
Authors: FENZL Friedrich , DECKER Michael , HAASS Dieter , TANNER Widmar
Published: March 2005

Cells of Chlorella vulgaris induced for hexose transport contain a membrane‐bound protein component that is missing in non‐induced cells. This was shown by double labelling experiments with [14C]phenylalanine and [3H]phenylalanine applying the method of Kolber and Stein [Nature (Lond.) 209 (1966) 691]. The specific protein is completely absent from soluble fractions.

An enrichment of the double‐labelled peak was observed in membrane fractions enriched in plasmalemma. The best purification was obtained when cell walls were purified; the residual membranes attached to the walls contained the transport protein with a 12‐times‐higher specific activity than the crude extract.

The transport protein had the characteristics of an intrinsic membrane protein. Its molecular weight was found to be 30000 on sodium dodecylsulphate gels. The protein did not show sugar binding activity, however.

Induced cells lose their state of induction with a half life of about 4 h; 7 h after induction the double‐labelled transport protein is no longer detectable.
Purification and Properties of a Periplasmic Protein
Authors: BOOS Winfried , HARTIG‐BEECKEN Imme , ALTENDORF Karlheinz
Published: March 2005

Protein GLPT, a periplasmic protein previously recognized as closely related to the active transport of sn‐glycerol‐3‐phosphate in Escherichia coli was isolated by the cold osmotic shock procedure. It was purified by Sephadex chromatography and isoelectric focussing. The purified protein does not exhibit any detectable binding activity toward sn‐glycerol‐3‐phosphate. It has no activity as a glycerol phosphatase nor as a glycerol kinase. Polyacrylamide gel electrophoresis in the presence of dodecylsulfate of the protein subsequent to treatment in urea, boiling in dodecyl‐sulfate and crosslinking indicates that it occurs as an oligomeric protein composed of four identical subunits of 40 000 molecular weight.

Membrane vesicles of wild‐type strains that contain protein GLPT in whole cells loose it during vesicle preparation. However, they still exhibit high transport activity toward sn‐glycerol‐3‐phosphate. Membrane vesicles prepared from glp T mutants that may or may not contain protein GLPT do not transport sn‐glycerol‐3‐phosphate.

We conclude from these results that protein GLPT does not participate in the energy‐dependent active transport through the cytoplasmic membrane but could be involved in facilitating the diffusion of sn‐glycerol‐3‐phosphate through the outer layers of E. coli.
On the Involvement of Cytochrome P‐450 in the Binding of Ribosomes to a Subfraction of Rat‐Liver Rapidly Sedimenting Endoplasmic Reticulum
Authors: OHLSSON Rolf , JERGIL Bengt
Published: March 2005

Rat liver endoplasmic reticulum has been separated into four ribosome‐containing subfractions, two from rapidly sedimenting endoplasmic reticulum and two from the microsomes, by differential centrifugation and sucrose density centrifugation. Ribosomes from one of the rapidly sedimenting subfractions were extracted by Triton X‐100 as a complex with cytochrome P‐450, optimally at a detergent protein ratio of 2/1 (w/w). Upon extraction approximately 50% of the cytochrome P‐450 in the membrane appeared complex‐bound to ribosomes, and, maximally, 6‐7 subunit molecules of the cytochrome were attached per ribosome. The specific concentration of cytochrome P‐450 on these ribosomes was 2.5‐times higher than in the parent membrane. Cytochrome b5, glucose‐6‐phosphatase, NADPH‐cytochrome c reductase, NADH‐ferricyanide reductase, cytochrome oxidase and phospholipids were present in small or trace amounts on the ribosomes in relation to cytochrome P‐450. Ribosomes extracted from other subfractions contained much less bound cytochrome P‐450. Phenobarbital treatment induced an increase in the cytochrome P‐450 content that was different for the various subfractions. This increase could not be correlated with changes in the amounts of cytochrome‐ribosome complexes released by detergent. We propose that cytochrome P‐450 is part of a specific binding site in the membrane for a fraction of the ribosomes attached to the endoplasmic reticulum. The ribosomes may be anchored to cytochrome P‐450 via nascent chain proteins.
The Amino‐Acid Sequence of the Dihydrofolate Reductase of a Trimethoprim‐Resistant Strain of Escherichia coli
Authors: STONE David , PHILLIPS Anthony W. , BURCHALL James J.
Published: March 2005

The determination of the amino acid sequence of the dihydrofolate reductase from Escherichia coli RT500 is described. The sequence, comprising 159 residues, has been derived from automatic sequencing of the intact protein in conjunction with manual sequencing of lysine‐blocked tryptic peptides, Staphylococcus aureus protease peptides, and α‐lytic protease peptides. Comparison of the sequence with that of the dihydrofolate reductase from a methotrexate‐resistant strain of E. coli (MB1428) shows that 145 of the residues are identical. The distribution of the differences along the length of the molecule is discussed.
Use of Affinity Elution Chromatography for the Microassay of Adenosine 3′: 5′‐Monophosphate
Authors: SINHA Asru K. , COLMAN Robert W.
Published: March 2005

Using the principle of affinity elution chromatography, a microassay has been developed for the determination of picomolar and even smaller quantities of adenosine 3′: 5′‐monophosphate (cyclic AMP) in biological samples. In principle, cyclic AMP along with the other anions is adsorbed onto an anion exchange cellulose and subsequently, the cyclic nucleotide is specifically eluted with cyclic‐AMP‐dependent protein kinase. In the actual assay cyclic AMP is determined by competitive displacement of radiolabelled cyclic AMP by the non‐labelled cyclic nucleotide. The described procedure results in a linear dependence for the displacement of the radioactive nucleotide on cyclic AMP concentration and obviates the usual procedure of sample enrichment by lyophilization. The degree of interference of different nucleotides including cyclic GMP was found to be minimal and in this respect the use of affinity elution chromatography was comparable to radioimmunoassay.
Calf‐Ovary Protein Kinases Dependent on Adenosine 3′: 5′‐Monophosphate
Authors: SALOKANGAS Anneli , TALMADGE Kenneth , BECHTEL Elke , EPPENBERGER Urs , CHRAMBACH Andreas
Published: March 2005

High resolving power and quantitative application of polyacrylamide‐gel electrophoresis at various pore sizes and electrofocusing provide resolution of a calf‐ovarian protein‐kinase system at an increased level of magnification, as well as optimal preparative routes.

Three protein kinases dependent on adenosine 3′:5′‐monophosphate are distinguished by polyacrylamide gel electrophoresis in calf ovarian cytosol. These enzymes which are observed in the pH range 7.5–10.2, appear to be aggregates of a common subunit or monomer. The three kinases are, by the criteria of polyacrylamide gel electrophoresis, distinct from three adenosine‐3′:5′‐monophosphate‐binding proteins found in the calf ovarian system. Analysis by electrofocusing on polyacrylamide gel shows that conventionally purified preparations of the major kinase of cytosol contain an overwhelming majority of contaminant proteins.
A Polyvalent Proteinase Inhibitor from Horse‐Blood‐Leucocyte Cytosol
Authors: DUBIN Adam
Published: March 2005

Cytosol of horse blood polymorphonuclear leucocytes contains an inhibitor active against neutral proteinases from the granules of these cells and against chymotrypsin, elastases I and II from pig pancreas, but not against trypsin. A method has been elaborated to isolate and purify this inhibitor by means of salting out with ammonium sulphate (45–70% saturation), followed by chromatography and rechromatography on a column of DEAE‐Sephadex A‐50. The preparation obtained is homogeneous during polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecylsulphate after reduction with 2‐mercaptoethanol. It is a thermolabile protein soluble at its isoelectric point (pH 5.38) and having a molecular weight of 35200 but aggregating in solutions of low ionic strength and irreversibly precipitating during dialysis at pH 4.0. The results obtained show the similarity of this inhibitor isolated from horse blood leucocyte cytosol to other inhibitors isolated from human or pig blood leucocytes.
Bromopyruvate as an Affinity Label for Baker's Yeast Flavocytochrome b2
Authors: MULET Christine , LEDERER Florence
Published: March 2005

Bromopyruvate was shown to completely inactivate cytochrome b2 in a reaction that obeyed the kinetic criteria required for affinity labels: it inactivated flavocytochrome b2 according to saturation kinetics, and the inactivation reaction was competitively inhibited by the substrate or competitive inhibitors. Inactivation was irreversible. The behaviour of both forms of flavocytochrome b2 (intact and proteolytically cleaved) was examined. It was found that the reduced cleaved enzyme was not inactivated by bromopyruvate; this phenomenon can probably be ascribed to a structural change undergone upon reduction.

The value of the lactate dissociation constant of intact cytochrome b2 was determined in competition experiments with bromopyruvate. By comparison with the divergent published values for the Ks of the cleaved form, it appears that only those that differ from the Km by a factor of two or three are reasonable.

This study opens the way for the identification of an active site residue and its localization in the peptide chain of the bifunctional enzyme.
DNA Polymerases from Intracisternal A‐Type Particles of the Mouse Plasmacytoma MPC 11
Authors: SCHMIDT Jörg , PRAGNELL Ian B. , WEIMANN Bernd J.
Published: March 2005

Intracisternal A particles from mouse plasma cell tumours were isolated from microsomal vesicles by detergent treatment and separated in linear sucrose gradients. Four peaks of DNA polymerase activities banding at densities of 1.30, 1.24, 1.20–1.22 and 1.13 g/cm3 were observed assaying on poly(rA) · oligo(dT). Solubolized DNA polymerases of the 1.30 g/cm3 fraction were eluted from phosphocellulose at salt concentrations around 0.30 M, 0.5 M and 0.65 M KCl. They could be distinguished by their different responses towards poly(rA) · oligo(dT), poly(rC) · oligo(dG) and poly(dA‐dT).

Serological experiments on the three DNA polymerases with antisera directed against Rauscher leukaemia virus reverse transcriptase showed that only enzymes responding to poly(rA) · oligo(dT) and poly(rC) · oligo(dG) could be inhibited.
Membrane‐Dependent Cleavage of the Human Placental Lactogen Precursor to Its Native Form in Ascites Cell‐Free Extracts
Authors: BOIME Irving , SZCZESNA Elzbieta , SMITH Donna
Published: March 2005

Messenger RNA derived from term placenta directs the synthesis of a precursor to human placental lactogen (prelactogen, molecular weight 25000) in an ascites cell‐free system containing ribosome‐free supernatant and preincubated purified ribosomes. The processing of prelactogen to native lactogen (molecular weight 22200) was only observed when a microsomal membrane preparation was added prior to the synthesis of complete protein, i.e. before release.

Placental mRNA directed the synthesis of prelactogen in a system containing free polysomes, whereas in a comparable system containing membrane‐bound polysomes both prelactogen and lactogen were synthesized. The prelactogen synthesized in the latter system could be cleaved by the addition of membranes at the start of incubation.

Preprotein cleavage activity was inhibited 100% by 0.04% Triton X‐100, while protein synthesis was inhibited only about 30%. Using Triton to block cleavage specifically at intervals after mRNA and membrane additions, it was determined that the overall cleavage reaction required about 15 min.

When the ascites system was incubated with charged initiator [35S]methionyl‐tRNAfMet prelactogen was formed. The labeled prelactogen was processed when membranes were added during the first few minutes of incubation, but no processing occurred when membranes were added after 60 min of incubation.

The results indicate that prelactogen is the primary gene product, and cleavage activity is apparently associated only with the membrane‐bound ribosomal fraction of the cell.
Secondary and Conformational Specificities of Trypsin and Chymotrypsin
Authors: WRIGHT H. Tonie
Published: March 2005

Specific and non‐specific trypsin substrates of known structure have been examined for common features. This analysis suggests that trypsin has a specificity for a particular conformation near the scissile bond which I denote as a conformational specificity. This conformation is a bent left‐handed helix at the third and fourth (P3 and P4) amino acid positions toward the amino terminus from the scissile bond which I denote as a conformational specificity. This conformation is a bent left‐handed show a high frequency of proline and glycine at these positions consistent with the left‐handed helical conformation. This apparent secondary specificity for a particular substrate residue other than that at the primary position is not related to the nature of the residues at the third and fourth positions. Rather, these residues determine the bend of left‐handed helix which has the effect of exposing main chain hydrogen‐bonding groups of the substrate peptide chain to hydrogen‐bonding groups on the enzyme. Thus, the secondary specificity of trypsin is not sequence‐specific, but is for peptide main chain in the third and fourth positions and is determined by the tertiary structure of the substrate. This hypothesis for conformational and secondary specificity in trypsin can be extended to chymotrypsin. It also provides a means for the regulation of certain processes in vivo catalyzed by other proteases.
Prostaglandin E2 Production in 3T3 Cells Transformed by Polyoma Virus Raises the Intracellular Adenosine 3′: 5′‐Monophosphate Levels
Authors: CLAESSON Hans‐Erik , LINDGREN Jan Åke , HAMMARSTRÖM Sven
Published: March 2005

High cellular adenosine 3′: 5′‐monophosphate (cyclic AMP) levels were found in a polyoma‐virus‐transformed 3T3 fibroblast line, which produces comparatively high prostaglandin E2 concentrations. Prostaglandin E2 and cyclic AMP increased with time during 6 days of growth. Both effects were prevented by three different cyclo‐oxygenase inhibitors. Addition of prostaglandin E2, at a concentration which would have been synthesized in the absence of inhibitor, reversed the effect of indomethacin, one of the cyclo‐oxygenase inhibitors, on cyclic AMP. It is concluded that endogenous prostaglandin E2 production in these transformed cells influences their cellular cyclic AMP levels.
Chemical and Pharmacological Characterization of Toxic Polypeptides from the Venom of Bungarus caeruleus
Authors: BON Cassian , CHANGEUX Jean‐Pierre
Published: March 2005

Five toxic fractions have been purified to homogeneity from the venom of Bungarus ceruleus by chromatography on CM‐Sephadex, QAE‐Sephadex and Bio‐Rex 70 followed by filtration on Sephadex G‐50 and Biogel P‐30. One of them blocks synaptic transmission at the pre‐synaptic level and is identified as a β‐bungarotoxin similar to that described by Lee from the venom of B. multi‐cinctus. Three other ones are short‐chain (type I) α‐toxins which block the response of the isolated electroplaque from Electrophorus electricus to bath‐applied carbamylcholine. These basic poly‐peptides of 61–62 amino acids contain four disulfide bridges. A postsynaptically acting toxin, different from the α‐toxins, has also been purified from the same venom. This ceruleotoxin is acidic and made up of several subunits.
Inhibition by CO of Hepatic Benzo[a]pyrene Hydroxylation and Its Reversal by Monochromatic Light
Authors: COOPER David Y. , SCHLEYER Heinz , ROSENTHAL Otto , LEVIN Wayne , LU Anthony Y. H. , KUNTZMAN Ronald , CONNEY Allan H.
Published: March 2005

Inhibition by CO of benzo[a]pyrene hydroxylation was studied in hepatic microsomes from rats pretreated with phenobarbital, 3‐methylcholanthrene or 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin, from animals treated with vehicle (saline or corn oil, respectively), and in a reconstituted microsomal cytochrome P‐448 system prepared from rats treated with 3‐methylcholanthrene.

In all preparations the hydroxylation was inhibited by CO, and this inhibition was most effectively reversed by irradiation with monochromatic light of 450 nm wavelength. These observations provide direct evidence that the oxygen‐activating component of all the examined benzo[a]pyrene hydroxylase systems is a P‐450‐type heme protein.

The only striking difference observed in these systems was the low CO sensitivity of the benzo‐[a]pyrene hydroxylase reaction in microsomes from animals treated with 3‐methylcholanthrene or 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin. Half‐maximal inhibition occurred at CO/O2 ratios of 9–12, rather than at 1–2, which is the usual range for P‐450‐linked mixed‐function oxidase reactions.

In contrast, the reconstituted benzo[a]pyrene hydroxylase system, with purified cytochrome P‐448 from 3‐methylcholanthrene‐induced rats, exhibited a considerably higher sensitivity towards CO (CO/O2 ratio ∼ 1), well within the range for mixed‐function oxidase reactions.

It is concluded that the observed diminished CO sensitivity of microsomal benzo[a]pyrene hydroxylase in 3‐methylcholanthrene‐ or 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐treated rats results from alterations in the composition and/or structural organization of the microenvironment of cytochrome P‐448 in the endoplasmic reticulum in response to the inducing action of polycyclic aromatic hydrocarbons and related agents, and is not related to changes in the heme protein P‐448 per se. The detailed nature of these changes is the subject of ongoing studies.
A Correlation between Glutathione Levels and Cellular Damage in Isolated Hepatocytes
Authors: HÖGBERG Johan , KRISTOFERSON Annika
Published: March 2005

To obtain high levels of glutathione in isolated hepatocytes an isolation procedure shorter than 16 min was used. This procedure gave a moderately high yield of viable cells (200–300 × 106 cells/ 10 g liver) with 44 ± 3 nmol of glutathione/106 cells. Incubation in Krebs‐Henseleit solution containing 2% albumin resulted in a continuous loss of reduced glutathione from the cells, while incubation in a medium containing amino acids and horse serum resulted in increased levels, suggesting active synthesis for 5 h. A short and apparently harmless depletion of reduced glutathione was induced by diethylmaleate or cumene hydroperoxide. A depletion of reduced glutathione lasting more than 1 h was accompanied by an increased cellular leakage. The depletion was induced by either diethylmaleate plus paracetamol or diethylmaleate alone in higher concentrations. A common mechanism for these toxic responses is suggested.
Interaction of RNA Polymerase with Promoters from Bacteriophage fd
Authors: SEEBURG Peter H. , NÜSSLEIN Christiane , SCHALLER Heinz
Published: March 2005

Replicative form DNA of bacteriophage fd, which had been fragmented with the restriction endonuclease II from Hemophilus parainfluenzae (endo R ·HpaII), was reacted with Escherichia coli RNA polymerase; the resulting stable preinitiation complexes were analysed using the filter binding assay followed by gel electrophoresis. At 120 mM KCl the first‐order rate constants for complex decay were determined to be 10−2–10−6s−1. The second‐order rate constants for complex formation were found to be about 106–107 M−1 s−1. From these values association constants for the individual promoters were calculated to be 2 × 10−8–2 × 10−11 M−1.

The rate of formation and the stability of promoter complexes was enhanced in superhelical DNA. No evidence was found for stable promoter‐specific closed complexes consisting of enzyme and helical DNA. This and the kinetic data suggest that the unwinding of base pairs is already important early in promoter selection, and not only for the formation of the final open complex.

The initiation of RNA synthesis from the preinitiation complex was faster than complex dissociation and complex formation for all promoters. Consequently, the initiation efficiency of a promoter is determined by the rate of complex formation, and not by its ‘affinity’ for the enzyme.

No correlation was found between the relative order of the fd promoters for the binding and the dissociation reaction. This is explained by different structural determinants, for the two reactions, which are located in different parts of the promoter DNA.
The Metabolism of trans‐Cyclohexan‐1,2‐diol by an Acinetobacter Species
Authors: DAVEY John F. , TRUDGILL Peter W.
Published: March 2005

Acinetobacter TD63 was one of some thirty organisms isolated by elective culture with trans‐cyclohexan‐1,2‐diol as sole source of carbon. The great majority of these isolates displayed the same growth spectrum as Nocardia globerula CL1 and Acinetobacter NCIB 9871 being capable of utilizing trans‐cyclohexan‐1,2‐diol, 2‐hydroxycyclohexan‐1‐one, cyclohexanol, cyclohexanone, 1‐oxa‐2‐oxocycloheptane and adipate and were assumed to use well described metabolic pathways.

Acinetobacter TD63 was distinctive in being incapable of growth with cyclohexanol, cyclohexanone or 1‐oxa‐2‐oxocycloheptane and because of this it was hoped that it would display an alternative pathway for the oxidation of trans‐cyclohexan‐1,2‐diol.

Studies with cell extracts have shown the presence of inducible dehydrogenase for the conversion of trans‐cyclohexan‐1,2‐diol to 2‐hydroxycyclohexan‐1‐one and cyclohexan‐1,2‐dione and of 6‐oxohexanoate to adipate. These enzymes are linked into a metabolic sequence by the action of a monooxygenase of broad specificity but efficiently capable of converting 2‐hydroxycyclohexan‐1‐one into the lactone 1‐oxa‐2‐oxo‐7‐hydroxycycloheptane that spontaneously rearranges to yield 6‐oxohexanoate.

An enzyme capable of attacking cyclohexan‐1,2‐dione (mono‐enol) in the absence of an electron donor or oxygen has also been detected. Evidence has been presented indicating that this enzyme catalyses a keto‐enol tautomerization between cyclohexan‐1,2‐dione (mono‐enol) and cyclo‐hexan‐1,2‐dione (mono‐hydrate) and is not involved in the pathway of ring cleavage.

The failure of Acinetobacter TD63 to grow with cyclohexanol, cyclohexanone or 1‐oxa‐2‐oxocycloheptane is due not to this organism possessing a distinctive metabolic sequence but to a narrow inducer specificity coupled with an inability to form a lactone hydrolase enabling it to cleave the stable 1‐oxa‐2‐oxocycloheptane which is an intermediate in the established pathway of cyclohexanol and cyclohexanone oxidation.
RNA Sequences in Ribonucleoprotein Fragments of the Complex Formed from Ribosomal 23‐S RNA and Ribosomal Protein L24 of Escherichia coli
Authors: BRANLANT Christiane , SRI WIDADA Johanes , KROL Alain , EBEL Jean‐Pierre
Published: March 2005

Upon digestion of the complex formed from the 23‐S ribosomal RNA and the 50‐S ribosomal protein L24 of Escherichia coli, two fragments resistant to ribonuclease were recovered; these fragments contained RNA sections belonging to the 480 nucleotides at the 5′ end of 23‐S RNA. By determining the sequence of 70% of this latter region we were able to localise the sections which, in the presence of the protein, are resistant to ribonuclease. Our results suggest that the region encompassing the 480 nucleotides starting at the 9th nucleotide from the 5′ end of 23‐S RNA has a compact tertiary structure, which is stabilised by protein L24.
The Ribitol‐Phosphate‐Containing Lipopolysaccharide from Proteus mirabilis, Strain D52
Authors: GMEINER Jobst
Published: March 2005

A soluble hydrophilic lipopolysaccharide, termed lipopolysaccharide II, isolated from Proteus mirabilis, strain D52 contained N‐acetylglucosamine, glucose, galactose, ribitol phosphate and ethanolamine phosphate as constituents of the O‐specific polysaccharide.

Periodate oxidation studies were carried out on the polymer before and after dephosphorylation with hydrofluoric acid and on oligosaccharides derived from the polymer by partial acid hydrolysis. The results obtained indicate that the polysaccharide chain consists of the chemical repeating unit Gal‐1,3(4)‐GlcNAc‐1,3‐Glc‐1,3‐GlcNAc‐, where GlcNAc stands for N‐acetylglucosamine. Whereas the galactose residue is substituted at C‐3 by ribitol phosphate, the glucose is substituted by ethanolamine phosphate at C‐6.
Studies of the Interaction between Aminoacyl‐tRNA Synthetase and Transfer Ribonucleic Acid by Equilibrium Partition
Authors: HUSTEDT Helmut , KULA Maria‐Regina
Published: March 2005

The partition behavior of isoleucyl‐tRNA synthetase, leucyl‐tRNA synthetase and tRNA in aqueous two‐phase systems composed of the polymers poly(ethyleneglycol) and dextran was investigated. From the results of this investigation a two‐phase system could be derived which can be employed for the study of the interactions between synthetases and their cognate tRNAs by equilibrium partition. These measurements show that in each case one molecule of cognate tRNA is bound per molecule of enzyme. The binding constants were in the range 1–5 μM1. It could be demonstrated that equilibrium partition is a useful method for the study of interactions between macromolecules.
Interacting Binding Sites of Isoleucyl‐tRNA Synthetase from Escherichia coli Studied by Equilibrium Partition
Authors: HUSTEDT Helmut , FLOSSDORF Josef , KULA Maria‐Regina
Published: March 2005

The binding of tRNAIle to isoleucyl‐tRNA synthetase in the presence of isoleucine or ATP was investigated using the equilibrium partition method. Isoleucine decreased the affinity of tRNAIle for the enzyme by a factor of about 5. For the free standard energy of interaction a value of about 1 kcal/mol (4.2 kJ/mol) was calculated. ATP exhibits qualitatively the same effect as isoleucine.

A binding of two molecules isoleucine per molecule of enzyme could not be demonstrated even in the presence of ATP and pyrophosphatase.
Studies on the Accessability of Ribosomes to Inactivation by the Toxic Lectins Abrin and Ricin
Authors: FODSTAD Øystein , OLSNES Sjur
Published: March 2005

The rate of protein synthesis in HeLa cells was measured at various periods of time after addition of abrin and ricin to the medium and compared with the concurrent ability of the isolated ribosomes to support poly(U)‐stimulated synthesis of polyphenylalanine in a cell‐free system. Similarly, the endogenous synthesis in unfractionated cell‐free systems from HeLa cells and rabbit reticulocytes was compared with the ability of the isolated ribosomes to support poly(U)‐stimulated polymerization of phenylalanine. In the intact cells and the unfractionated cell‐free systems protein synthesis decreased progressively with the time after addition of toxins or toxin A chains. In contrast, the ability of the isolated ribosomes to support polyphenylalanine synthesis was only moderately reduced initially and then remained constant or even increased.

The activity of isolated monosomes decreased progressively with time after addition of toxin A chain, whereas polysomes were only partly inactivated and the extent of inactivation varied from one experiment to another.

The results indicate that the inactivation of one or a few ribosomes per polysome stops the translation of mRNA. It is suggested that the intact ribosomes thus trapped are inaccessible to the toxins and that the isolation of polysomes results in release of functionally intact ribosomes capable of supporting poly(U)‐directed polymerization of phenylalanine.
Lipopeptidophosphoglycan from Trypanosoma cruzi
Authors: DE LEDERKREMER Rosa M. , TANAKA Cecilia T. , ALVES Maria Jûia M. , COLLI Walter
Published: March 2005

Lipopeptidophosphoglycan, extracted from whole cells of epimastigote forms of Trypanosoma cruzi, has now been shown to contain 12.6% of fatty acids in addition to the previously identified content of neutral sugars (60%), glucosamine (0.8%), peptide (9.5%) and acid‐hydrolyzable phosphate (2%). The main fatty acids are palmitic (6.9%) and lignoceric (4.6%) acids. Stearic (0.55%), oleic (0.15%) and myristic (0.18%) acids were also found. One third of the fatty acids are bound in the lipopeptidophosphoglycan as esters (14 mmol%) and two thirds as amides (28 mmol%). Lignoceric acid was found to be bound only as amide. Two ninhydrin‐positive compounds, obtained by chloroform extraction of a total acid hydrolysate of the lipopeptidophosphoglycan, were tentatively identified as sphingosine bases.
Thymidine Uptake in Bacteria: The Effect of Purine Nucleosides
Authors: VYACHESLAVOV Lev G. , MOSEVITSKY Mark I.
Published: March 2005

The kinetics of thymidine uptake by Escherichia coli and Bacillus subtilis cells in the presence of adenine and guanine nucleosides was investigated. The initial concentration of thymidine in the growth medium was 0.35 ug/ml while the initial concentration of purine nucleosides ranged from 25 to 250 ug/ml. Adenine nucleosides when present at a concentration more than 50 ug/ml. strongly inhibit thymidine uptake by the bacteria. The duration of the inhibition depends on the initial concentration of adenine nucleoside in the growth medium. At an initial con‐centration of deoxyadenosine (or adenosine) of 250 ug/ml the time of inhibition of thymidine uptake was about 60 min. During this period thymidine is almost completely preserved from the action of bacterial thymidine phosphorylase.

Guanine nucleosides (guanosine or deoxyguanosine) do not markedly inhibit thymidine uptake by bacteria even at a concentration of 250 ug/ml. It is shown that they do protect thymidine from the phosphorolytic action of the thymidine phosphorylase although much less effectively than adenine nucleosides.

It is suggested that some areas in the bacterial membrane where thymidine phosphorylase is located are not available to guanine nucleosides.
Analysis of the α‐Satellite DNA from African Green Monkey Cells by Restriction Nucleases
Authors: FITTLER Friedrich
Published: March 2005

By the use of restriction endonucleases the organization of the α‐satellite DNA from African green monkey cells (Cercopithecus aethiops) has been analyzed. With endo R ·HindIII, endo R ·AluI and with endo R ·EcoRI at conditions of low salt and high pH (endo R ·EcoRI*) all of the satellite was digested while only a part of the satellite was cleaved with endo R ·Bsu and endo R ·EcoRI under standard conditions. With each of the four nucleases a series of fragments was formed which were multiplies in size of a basic repeat unit linked in tandem arrays in the intact satellite. The quantitative evaluation of the digestion with each nuclease as well as with combinations of two nucleases yielded information about the distribution of the cleavage sites. While the arrangement of the endo R ·HindIII cleavage sites conforms to a random distribution across the entire satellite, the results from the endo R ·Bsu and endo R ·EcoRI cleavage patterns are consistent with a picture where the cleavage sites are clustered in fractions of the satellite.

Since endo R ·AluI recognizes the central four nucleotide pairs of the endo R ·HindIII cleavage site, the redigestion of the endo R ·HindIII dimer with endo R ·AluI gave information about the distribution of mutations in the satellite. The results of these experiments together with the comparison of the sequence divergence determined from digestion with endo R ·HindIII and endo R ·EcoRI* lend support to the hypothesis that mutations have affected all bases in the satellite evenly.

The γ‐satellite, another fraction of the African green monkey DNA, could be separated by Ag+/ CsSO4 density gradient centrifugation into two components. With the three restriction nucleases used both components gave a background of fragments of heterogenous length on gel electrophoresis with some faint bands of no apparent regularity in one case.
Studies of Glutamate Dehydrogenase
Authors: RASCHED Ihab R. , BOHN Anneliese , SUND Horst
Published: March 2005

Cross‐linking of the unimer of glutamate dehydrogenase from beef liver (consisting of six polypeptide chains each having a molecular weight of 56000) with dimethyladipimidate and subsequent analysis by sodium dodecylsulfate electrophoresis shows predominantely the trimeric species (molecular weight 168000). Treatment with dimethylimidates of other chain length yields significantly less trimeric species indicating that the amino groups being cross‐linked are within a distance of about 0.85 nm. Comparison of the molar amount of incorporated [14C]dimethyladipimidate with the number of modified amino groups (determined with trinitrobenzenesulfonic acid) shows that although 8–9 of the 34 amino groups have reacted, only 2–3 of them are involved in cross‐links.

Reaction with dimethylimidates inactivates the enzyme. The loss of the activity is partly concomitant to cross‐linking to the trimeric species and not simply due to the modification of essential lysine residues. This is supported by the fact that, although more lysine residues react with monofunctional methylimidates, the loss of activity is reduced.

Purified chymotryptic and tryptic peptides of the radioactive‐labeled trimeric species were subjected to sequence analysis. Six peptides containing 75% of the total label were identified: one involves the amino‐terminal residue alanine‐1 and the others involve lysine‐105, lysine‐154, lysine‐269, lysine‐358 and lysine‐399.

Quantitative analysis of the specific radioactivity of each peptide/mol lysine leads to the conclusion that only lysine‐105, lysine‐154, lysine‐269 and lysine‐358 participate in cross‐links, lysine‐269 and lysine‐358, respectively, being at isologous and lysine‐105 cross‐linked with lysine‐154 at heterologous contact domains of the enzyme.

A model for the planar arrangement of the trimeric species in the quaternary structure of glutamate dehydrogenase is discussed. It includes both isologous and heterologous contact areas between the polypeptide chains.
Poly(A)‐Containing RNA in Neuroblastoma: Immature and Differentiated Cells in Culture
Authors: CROIZAT Bernard , BERTHELOT Francis , FELSANI Armando , GROS François
Published: March 2005

We have analysed the poly(A)‐containing RNA from neuroblastoma cells at two different developmental states: either as round, immature neuroblasts grown in suspension, or as differentiated cells exhibiting the morphological properties of mature neurons, when attached to a culture dish.

Suspension‐grown and monolayer cells were pulse‐labelled with tritiated uridine. The profile of cytoplasmic poly(A)‐containing RNA from suspension cells is highly heterogeneous with peaks ranging from 16–30 S. The profile obtained from differentiated cells appears somewhat distinct from the previous one. This is evidenced by a relative decrease in the 26‐S peak and a virtual disappearance of the 16‐S component.

In order to compare the ‘steady‐state’ patterns of poly(A)‐containing RNA in these two developmental stages, polysomal RNA was prepared from unlabelled cells. Following sucrose gradient sedimentation, each fraction was hybridized to [3H]poly(U). Examination of the two RNA hybridization profiles reveals striking similarities suggesting that ‘steady‐state’ messenger populations include, on the average, the same subspecies. The 16‐S fraction, which was not observed after the pulse‐labelling of the monolayer culture, is detected here by hybridization to [3H]poly(U) when using polysomal poly(A)‐containing RNA from monolayer cells as substrate.

These results suggest that terminal differentiation of neuroblastoma cells is not accompanied by major alterations of the transcription program and is paralleled by a marked stabilization of the 16‐S species.
On the Nature of Chromophore in Pig Kidney Diamine Oxidase
Authors: FINAZZI AGRÒ Alessandro , GUERRIERI Pietro , COSTA Maria Teresa , MONDOVI Bruno
Published: March 2005

The nature of the 500‐nm chromophore in pig kidney diamine oxidase was investigated by absorption spectroscopy and fluorescence in the presence of various chelating or carbonyl‐specific reagents. From the spectroscopic measurements the following conclusions can be drawn. First, the 500‐nm absorption band is not due to copper, the reduction of which is not related to the disappearance of this band. Second, phenylhydrazine and cycloserine give rise, upon reaction with the enzyme, to absorptions very similar to those of a pyridoxal enzyme, aspartate aminotransferase. Third, these enzyme derivatives are unexpectedly non‐fluorescent. Copper removal, obtained after prolonged incubation of cycloserine‐treated enzyme in the presence of reducing and chelating agents, leads to a fluorescence similar to that of cycloserine‐aspartate transaminase. It is proposed that copper is coordinated to the postulated pyridoxal phosphate of diamine oxidase through the pyridine nitrogen.
Methionyl‐tRNA Synthetase from Escherichia coli: Substituting Magnesium by Manganese in the L‐Methionine Activating Reaction
Authors: HYAFIL François , BLANQUET Sylvain
Published: March 2005

While Mg2+ can be efficiently replaced by Ni2+, Co2+ and Mn2+ in the ATP‐PPi isotopic exchange reaction catalysed by methionyl‐tRNA synthetase from Escherichia coli, the latter ion was selected for a detailed analysis of the L‐methionine activation reaction. In order to avoid artefactual results due to the slow aggregation of Mn2+ with pyrophosphate, this process was investigated by electron paramagnetic resonance and conditions were determined where it does not interfere with enzymic experiments. The thermodynamic parameters derived from steady‐state (ATP‐PPi isotopic exchange, fluorescence at equilibrium) or prestationary (fluorescence stopped‐flow) experiments are compared to those obtained in the presence of Mg2+ [Hyafil et al. (1976) Biochemistry, 15, 3678–3685].

While the standard ΔG for the reaction (E · Met · ATP‐Me2+⇄ E · Met ∼ AMP · PPi−Me2+) is close to zero in the case of Mg2+, Mn2+ slows down the rate of adenylate reversion and thus shifts the reaction towards the latter species. The ΔG for the formation of the E · Met ∼ AMP complex does not depend on the metal used, suggesting that the divalent ion does not participate in the structuration of this complex. Substituting Mn2+ for Mg2+ decreases notably the dissociation constant of PPi‐Me2+ from the E · Met ∼ AMP · PPi‐Me2+ species and from its abortive analog E · Met · Ado · PPi‐Me2+. Similarly the dissociation constant of ATP‐Me2+ from another dead‐end analog E · methioninol · ATP‐Me2+ is decreased by Mn2+. Involvement of the purine N7 atom in the binding of the metal ion to the active site of methionyl‐tRNA synthetase is ruled out by the use of 7‐deazaadenosine.

The role of the metal in the catalytic process of methionine activation and its relevance to the specificity of the reaction is then discussed in the light of the results obtained without metal and with Mg2+ and Mn2+.