bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Nitrogen Fixation by Fe‐S Protein II in Azotobacter vinelandii
Authors: SCHERINGS Gerard , HAAKER Huub , VEEGER Cees
Published: March 2005

1. Several low‐potential electron carriers from different sources can be photoreduced by the system 3,10‐dimethyl‐5‐deazaisoalloxazine/N‐tris(hydroxymethyl)methylglycine. The carriers studied were flavodoxin, ferredoxin I and iron‐sulfur protein II from Azotobacter vinelandii and the flavodoxins from Desulfovibrio vulgaris and Peptostreptococcus elsdenii.

2. Electron transport to A. vinelandii nitrogenase was studied, employing different preparations of the enzyme: a crude complex; a complex reconstituted from the 0.27 M and 0.38 M NaCl fractions after DEAE‐cellulose chromatography; a complex reconstituted from the 0.27 M and 0.38 M NaCl fraction plus iron‐sulfur protein II purified from the 0.15 M NaCl fraction. Of all photoreduced carriers tested, only flavodoxin hydroquinone from A. vinelandii catalyzes significant electron transport to these complexes.

3. The time course of oxidation of substrate‐amounts of A. vinelandii flavodoxin hydroquinone by catalytic amounts of crude nitrogenase complex shows three characteristic phases: an initial lag phase (1), a phase with constant rate over a range of redox potentials (2) and a final phase with rapidly declining rate (3). It was shown that the Fe‐S protein II is responsible for the lag phase; the potential where phase 2 changes into phase 3 is at a higher value in the presence of Fe‐S protein II. Pre‐reduction of the enzyme photochemically abolishes phase 1 and causes phase 2 to proceed at a higher rate. The rate in phase 2 can be enhanced also by lowering the ‘starting potential’ of the flavodoxin hydroquinone/semiquinone couple.

4. A. vinelandii flavodoxin shuttles between its hydroquinone and semiquinone forms during steady‐state electron transfer. Over 90% of the reducing equivalents is recovered in ethylene formed from acetylene. The concentrations of flavodoxin hydroquinone to give half‐maximum rate in the acetylene reduction assay is 3–6 μM. A scheme is proposed for the regulation of electron donation to nitrogenase.
Biochemical and Immunological Studies on Larval Serum Protein 1, the Major Haemolymph Protein of Drosophila melanogaster Third‐Instar Larvae
Authors: WOLFE Jonathan , AKAM Michael E. , ROBERTS David B.
Published: March 2005

Larval serum protein 1 is the major haemolymph protein just before puparium formation in Drosophila melanogaster. It has been purified and characterised as a family of hexamers (molecular weight 450000–480000) of three immunologically related polypeptides (molecular weight 75000–81000). Homologous proteins, present in other dipterans, are similar in structure, amino acid composition and developmental profile.
Purification and Properties of the NAD+: Protein ADP‐ribosyltransferase Responsible for the T4‐Phage‐Induced Modification of the œ Subunit of DNA‐Dependent RNA Polymerase of Escherichia coli
Authors: SKÓRKO Romuald , ZILLIG Wolfram , ROHRER Hermann , FUJIKI Hirota , MAlLHAMMER Reinhard
Published: March 2005

The NAD+: protein ADP‐ribosyltransferase responsible for the bacteriophage‐T4‐induced modification of RNA polymerase has been purified 100–200‐fold from Escherichia coli B/r infected with the phage T4.

The enzyme has a molecular weight around 26000. It does not require magnesium ions and is protected by sulfhydryl reagents. The temperature optimum is around 20°C. The reaction is inhibited at increased ionic strength and by the reaction products nicotinamide and T4‐modified RNA polymerase. The reaction is irreversible. The only reaction products formed to a significant extent in a crude extract are T4‐modified a subunit and one smaller ADP‐ribosylated peptide. As in vivo, the reaction is complete. Both œ subunits in the RNA polymerase monomer are ADP‐ribosylated in one and the same position.

The product of modification in vitro appears to be identical with that of modification in vivo. ADP‐ribose has been obtained by hydrolysis both of T4‐modified œ subunit and of the reaction product of egg‐white lysozyme, NAD+ and the transferase responsible for bacteriophage‐T4‐induced alteration of host proteins [Rohrer, H., Zillig, W., and Mailhammer, R. (1975) Eur. J. Biochem. 60, 227–238]. The amino acid carrying the ADP‐ribosyl residue appears to be arginine. T4‐modified œ subunit and the major species of T4‐altered œ subunit are identical.

An NAD+: protein ADP‐ribosyltransferase using E. coli proteins and egg‐white lysozyme but no RNA polymerase subunit as acceptor(s) has been purified from uninfected E. coli cells.
Substrate Specificity via Ternary Complex Formation with Glutamate Dehydrogenase
Authors: KOEKOEK Henk , ROBILLARD George T.
Published: March 2005

Very little discrimination is observed in the binary binding of dicarboxylic acid substrate analogues to glutamate dehydrogenase as monitored by proton nuclear magnetic resonance. Variation in length, charge, bulkiness and conformational rigidity resulted in only a factor of five variation in KD and apparent relaxation time, T2.

Upon titration of the binary enzyme‐ligand complex with coenzyme to form the ternary enzyme‐ligand‐coenzyme complex strong discrimination is observed. Coenzyme binds tightly only when the correct substrate is present, otherwise it binds 10 to 150 times more weakly.
The Effects of Histidine Starvation on the Methylation of Ribosomal RNA
Authors: GRUMMT Ingrid
Published: March 2005

The effect of amino acid starvation on the control of ribosome biosynthesis at the post‐transcriptional level has been studied in Ehrlich ascites cells. A comparison of the turnover rates of ribosomal precursor RNA (pre‐rRNA) and the degree of methylation of ribosomal RNA after histidine deprivation revealed that the slow down of ribosome formation is accompanied by a significant inhibition of rRNA methylation. Analysis of nucleolar and cytoplasmic RNA double‐labelled with L‐[Me−3H]methionine and [14C]uridine, as well as a quantitative determination of alkali‐stable dinucleotides on DEAE‐Sephadex, showed that methylation of rRNA species was inhibited by about 50% under shift‐down conditions. This decrease in RNA methylation does not reflect an inhibition of rRNA methylases caused by amino acid starvation but is rather brought about by a shrinkage in the pool size of S‐adenosylmethionine, the donor of methyl groups. It is suggested that amino acid starvation might exert its blocking effect on proper ribosome maturation by affecting the methylation of 45‐S RNA.
Hydrogen‐Ion Binding by Tobacco‐Mosaic‐Virus Protein Polymers
Authors: DURHAM Anthony C. H. , VOGEL Dieter , DE MARCILLAC Guy D.
Published: March 2005

Hydrogen ion titration curves of tobacco mosaic virus protein have been measured in various conditions of protein concentration, temperature, ionic strength, and rate of pH change. The polymers present at each stage are deduced from turbidity and sedimentation data, plus published information. A simple semi‐quantitative analysis of the curves is given, and the pK values of the two abnormal carboxylates in single helix are estimated as 6.4 and about 7.0. Disks, and some faster‐forming unknown polymers in the same size range, have one abnormal carboxylate with pK 6.9. These results are most easily interpreted in terms of electrostatic interactions between carboxylates, probably at the axial ends of the protein subunits.
Metastable Aggregates in the Polymerisation of Tobacco‐Mosaic‐Virus Protein
Authors: VOGEL Dieter , DURHAM Anthony C. H. , DE MARCILLAC Guy D.
Published: March 2005

Metastable ‘overshoot’ aggregates are formed when tobacco mosaic virus A‐protein (4–8 S) polymerises to disks (20–22 S) and stacks of disks (27–30 S) at pH 6.8–7.6, 20–25 °C. Two cases are discernible. First, at ionic strength 0.1 M, below pH 7.1, a transient, definite 26‐S peak appears: it transforms in a matter of hours into stable 22‐S (disk) aggregates, at a rate that decreases with decreasing pH and increasing temperature; above pH 7.1, there are only 4–8‐S and 22‐S aggregates. Second, after a rapid increase of ionic strength from 0.1 to 0.75 M, below pH 7.6 a broad distribution of very large overshoot aggregates forms (maximum at 60 S, at pH 6.9). With decreasing pH, the maximum size of the overshoot aggregates increases and reequilibration to stacks of two disks (28 S) becomes slower. The 4–8‐S and 26‐S aggregates can both form the very large overshoot aggregates, but disks and stacks of disks alone cannot.

Under these conditions, disks undergo only limited stacking (27–30 S) and a very slow conformational change. A similar conformational change parallels the decomposition of the very large overshoot aggregates. At pH 8, overshoot does not occur, probably because a higher pH inhibits the growth of rods.

Though the structure of the overshoot‐forming aggregates is still unknown, three‐layer aggregates (8 S and 26 S) are one possibility. We present a scheme including all reversible polymerisations so far observed for TMV protein and discuss their implications for the reconstitution of the virus in vitro.
Reaction of Yeast Fatty Acid Synthetase with Iodoacetamide
Authors: KRESZE Georg‐Burkhard , STEBER Liesel , OESTERHELT Dieter , LYNEN Feodor
Published: March 2005

Yeast fatty acid synthetase possesses very low malonyl‐CoA decarboxylase activity. Treatment with iodoacetamide, while abolishing synthetase activity, induces a strong malonyl decarboxylase activity which, in turn, can be inhibited by N‐ethylmaleimide. Kinetic analysis shows that the emergence of the decarboxylase activity is synchronized to the disappearance of the fatty‐acid‐synthesizing activity and thus, is due to carboxamidomethylation of the peripheral SH‐groups of the multienzyme complex. Strong decarboxylase activity was also found after treatment of the synthetase with methylmalonyl‐CoA. A hypothetical scheme is proposed which explains the origination of the decarboxylase activity as a consequence of conformational changes of the condensing enzyme component which happen when the peripheral SH‐group is acylated or alkylated.
Inhibition of Papain by N‐Acyl‐aminoacetaldehydes and N‐Acyl‐aminopropanones
Authors: BENDALL M. Robin , CARTWRIGHT Ian L. , CLARK Peter I. , LOWE Gordon , NURSE David
Published: March 2005

N‐Acyl‐aminoacetaldehydes are potent inhibitors of the proteolytic enzyme, papain. Although they exist predominantly in their hydrated form in aqueous solution only the aldehyde is an effective inhibitor. The binding constants for related amides and methyl ketones confirm that it is principally the lower steric requirement of the aldehyde rather than its increased electrophilicity which is responsible for its powerful inhibitory properties. Using nuclear magnetic resonance spectroscopy, evidence is provided for an N‐acetyl‐aminoacetaldehyde‐papain complex. Using a cross‐saturation technique evidence is also provided for a hemithioacetal, formed from the aldehyde and the active‐site thiol group. Hemithioacetal formation has also been detected between N‐benzoyl‐aminoacetaldehyde and papain. This provides the first direct evidence for a tetrahedral adduct with papain and supports the proposed involvement of such intermediates in papain‐catalysed hydrolyses.
Processive and Synchronous Mechanism of Polynucleotide Phosphorylase Phosphorolysis
Authors: GUISSANI Annie
Published: March 2005

Polynucleotide phosphorylase is'an exonuclease which shows, under appropriate conditions, two extreme mechanisms of action: progressive or synchronous. In progressive phosphorolysis the enzyme leaves the substrate only when the degradation is complete, whereas in the synchronous mechanism there are as many dissociations as phosphorolysable nucleotides. In order to determine the number of detachments of the enzyme from its substrate, when either the enzyme or the substrate vary, we have undertaken a theoretical study of the phosphorolysis mechanism. We have deduced two series of relationships: on the one hand the percentage, P, of phosphorolysis compared to the percentage of the liberation of the 3′‐OH terminal nucleotide, pN1, for different degrees of progressivity; on the other hand, the respective percentages of liberation of the successive nucleotides pN1 and pN2, independently of the degree of progressivity of the enzymic mechanism.

The two series of curves thus obtained relating P to pN1 released and pNi to pNi‐1 released are valid, independent of the length of the substrate considered: comparison of the experimental and theoretical curves relating P to pN1 released enables the determination of the number of detachments of the enzyme under our experimental conditions; comparison of the curves relating pNi to pNi‐1 released permits the location, in a given sequence, of the nucleotides for which the mechanism of phosphorolysis is synchronous or not.

The experimental study applied to a synthetic polymer (A)n‐1‐[14C]U shows that the mechanism becomes less progressive when the enzyme is no longer intact.

The interpretation of the phosphorolysis of oligonucleotides within the framework of this theo‐retical study leads to the conclusion that in fact the mechanism is simple only in case of homo‐oligonucleotides; with hetero‐oligonucleotides the length, the sequence, the structure and the possible imprint of the original substrate have to be taken into account. We find that, to a first approximation, a synchronous mechanism is obtained with oligonucleotides containing no more than twelve mononucleotides.

This study can obviously be generalized to the attack (or the synthesis) of other substrates by exonucleases that react according to either mechanism.
Ionization Behaviour of Native Apolipoproteins and of Their Complexes with Lecithin
Authors: SOETEWEY Frederik , LIEVENS Marie‐José , VERCAEMST Raphaël , ROSSENEU Maryvonne , PEETERS Hubert , BROWN Virgil
Published: March 2005

A comparison of the ionization behaviour of the human apoA‐II, apoC‐I, apoC‐III proteins and of their complexes with dimyristoyl lecithin is based on potentiometric titration of the basic and acidic residues and spectrophotometric titration of the phenolic groups. Experimental data suggest that a number of lysine, arginine, aspartic acid and glutamic acid residues are masked in the complexes. For each of these amino acids and in all three proteins the number of masked residues is consistent with the content of those regions predicted to be involved in lipid binding by the model of Segrest et al. [FEBS Lett. 38, 247–253 (1974)]. These data taken together with the results of calo‐rimetric and titration experiments with the apoA‐I protein reported in the accompanying article [Rosseneu et al. (1977) Eur. J. Biochem. 79, 251–257] strongly support the general nature of the proposed model and further suggest that ionic interactions have some role in the formation of the dimyristoyl lecithin/apolipoprotein complexes.
Quantitative Determination of Glycosphingolipids Illustrated by Using Erythrocyte Membranes of Various Mammalian Species
Authors: OULEVEY Jacques , BODDEN Edna , THIELE Otto Wolfgang
Published: March 2005

A procedure for the quantitative determination of glycosphingolipids is described, involving extraction of total lipids, fractionation on a column, acid cleavage and photometry of a complex formed between sphingosine and methyl orange. The level of glycosphingolipid sphigosine of erythrocyte membranes of a variety of mammals ranges from 59.1 (rat) to 410.1 (pig) nmol/ml packed cells.
Dependence of tRNA Structure in Solution upon Ionic Condition of the Solvent
Authors: LABUDA Damian , HAERTLÉ Tomasz , AUGUSTYNIAK Jacek
Published: March 2005

Barley phenylalanine tRNA (tRNAphe) exhibits a 4.7‐fold enhancement of fluorescence intensity in response to the addition of MgCl2 in 0.01 M Tris‐HCl, pH 7.5, 0.001 M Na2 EDTA. In the case of 2.0 M NaCl about 60% of this effect was observed. The change of fluorescence intensity was used as a measure of bound ligand concentration. From fluorescence Scatchard plots a different character of Mg2+ binding to tRNA was revealed, depending upon the initial conformation of tRNA in solution submitted to titration. In NaCl‐free tRNAphe solution strong interacting (Ks= 1.5 × 105 M−1n= 2.4) and weak independent (Kw= 9 x 103 M−1) Mg2+ binding were observed. In solution containing 0.1 M NaCl both the strong and the weak Mg2+ binding demonstrated independent behaviour. Ks and Kw, in the presence of NaCl, had lower values than in NaCl‐free solution, since Na+ was found to be a competitive inhibitor of Mg2+ binding. Fluorescence titrations of tRNAphe solutions containing Mg2+ evidenced 2 Na+ binding sites/Mg2+ binding site (at least in the region of weak sites of Mg2+) characterized by inhibitory binding constants K̄i1 Na= 45 M−1 and K̄i 1 Na= 8.5 M−1 (mean values) for first and second Na+ bindings respectively. Na+ exerts a destabilizing effect on tRNA conformation induced by Mg2+. A model of Mg2+ binding and concomitant conformational changes of the tRNA molecule is discussed: one class of Mg2+ binding sites in unfolded tRNA and two classes of sites, strong and weak, in folded conformation are postulated.
Affinity Labelling of Alcohol Dehydrogenases
Authors: DAHL Knut H. , McKINLEY‐McKEE John S.
Published: March 2005

dl‐α‐Bromo‐β(5‐imidazolyl)‐propionic acid is a potential affinity labelling reagent for metallo enzymes. It has been used with the alcohol dehydrogenases from liver and yeast. The liver enzyme is chemically modified and inactivated in a Michaelis‐Menten‐type reaction, where one molecule of the reagent is bound per subunit. The enzyme is protected from the inhibitor in a competitive manner by imidazole, 2,2′‐dipyridyl, 1,10‐phenanthroline and cyclohexanone, which all combine with the active‐site zinc. The protection by chloride, acetate and NADH, which are considered to bind at the general anion binding site, is not strictly competitive. Inactivation has an optimum at pH 8.5. For the liver enzyme, the reagent was found to decrease the initial rate of ethanol oxidation. Prior to the irreversible alkylation of Cys‐46, reversible binding is shown to occur at the active‐site zinc atom. The yeast enzyme was extremely resistant to the reagent and no specific modification was found.

The potential affinity labelling and crosslinking reagent, symmetrical 1,3‐dibromoacetone although unstable, has also been used for chemical modification. With the liver enzyme, concentrations below 5 mM gave a reaction of the Michaelis‐Menten‐type at pH 7.0. Several ligands known to complex with the active‐site region protect the enzyme against the reagent. Dibromoacetone gave rapid inactivation of the yeast enzyme. Despite the fact that a pseudo‐first‐order reaction was observed with respect to enzyme as well as inhibitor, no saturating effect was found. In this work, dibromoacetone reacted like a monofunctional reagent.
Chick‐Embryo DNA Polymerase γ
Authors: BERTAZZONI Umberto , SCOVASSI Anna I. , BRUN Gilbert M.
Published: March 2005

The level of DNA polymerase γ as compared to DNA polymerases α and β has been determined in chick embryo by means of specific tests: the amount of γ‐polymerase in the 12‐day‐old chick embryo reaches about 15% of the total polymerase activity. This enzyme is mainly localized in nuclei and mitochondria, where it represents the prevailing if not the unique DNA polymerase activity. The mitochondrial DNA polymerase γ is likely to be associated with the internal membrane or the matrix of this organelle since it is not removed by digitonin treatment.

The γ‐polymerases have been purified from chick embryo nuclei and mitochondria 500–700 times by means of DEAE‐cellulose, phosphocellulose and hydroxyapatite chromatographies. The purified mitochondrial DNA polymerase γ is closely related to the homologous enzyme purified from the nuclei of the same cells. So far, they cannot be distinguished on the basis of their sedimentation, catalytical properties and response to inhibitors or denaturating agents. The purified γ enzymes are distinct from the chick embryo DNA polymerases α and β and are not inhibited by antibodies prepared against the latter enzymes. The nuclear and mitochondrial γ‐polymerases do not respond to the oncogenic RNA virus DNA polymerase assay with natural mRNAs.
Physicochemical Parameters and Subunit Composition of Yeast Phosphofructokinase
Authors: KOPPERSCHLÄGER Gerhard , BÄR Jörg , NISSLER Karl , HOFMANN Eberhard
Published: March 2005

The properties of proteolytically non‐modified phosphofructokinase from baker's yeast were investigated by means of hydrodynamic, electrophoretic, and chemical methods. The sedimentation coefficient shows a linear dependence on the protein concentration down to 0.01 mg/ml. The sedimentation coefficient has been determined to be S020,w= 20.81 S. At concentrations of the enzyme at less than 0.01 mg/ml a significant decrease of this value was found.

The partial specific volume of the enzyme as determined by three different methods at 20°C is v2= 0.742 ml/g.

From equilibrium sedimentation a molecular weight of 835000 ± 32000 was calculated for the native enzyme.

Amino acid analysis of the enzyme was performed and the number of half‐cystine residues determined as 11/100000 g is in good agreement with the number of titratable –SH groups after denaturation of the enzyme.

The subunit molecular weight was determined by applying equilibrium sedimentation in the presence of sodium dodecylsulphate. Taking into account that 0.37 g of dodecylsulphate is bound per g of the enzyme protein as found by equilibrium dialysis, a subunit molecular weight of 104000 could be calculated. This value is about 15% smaller than that obtained by gel electrophoresis in the presence of sodium dodecylsulphate.

Evidently, yeast phosphofructokinase seems to be octameric. Limited proteolysis in the presence of proteinase B from yeast was investigated by following the alterations of the sedimentation coefficient, the molecular weight, and the subunit pattern of the enzyme. During partial proteolytic degradation the molecular weight of the native enzyme decreases by about 230000. This diminution is in accordance with the difference in the molecular weight per subunit if an octameric composition is considered.
Metal‐Free and Metal‐Substituted Cytochromes c. Use in Characterization of the Cytochrome c Binding Site
Authors: VANDERKOOI Jane M. , LANDESBERG Regina , HAYDEN Geoffrey W. , OWEN Charles S.
Published: March 2005

The luminescent properties of metal‐free, tin(IV) and zinc(II) cytochromes c have been used to characterize the interaction of cytochrome c with mitochondria and cytochrome oxidase. Diminution in the fluorescence yields of tin and zinc cytochrome c occur when these derivatives bind to cytochrome oxidase or mitochondria. Based upon spectral overlap and quantum yield, the distance between the porphyrin rings of cytochrome a and cytochrome c is estimated according to Forster theory to be in the neighborhood of 3.5 nm. Measurements of the polarized emission of metal‐free ‘porphyrin’ cytochrome c when bound to oriented layers of cytochrome c oxidase indicate that the porphyrin is bound obliquely to the plane of the oxidase layers with an angle of about 70°C from heme plane to membrane plane. It is proposed that these data have significance for elucidation of electron transfer mechanisms.
Inhibition of Chloramphenicol Binding to Escherichia coli 70‐S Ribosomes by 2′(3′)‐O‐Aminoacyl‐dinucleoside Phosphates Derived from the Aminoacyl‐tRNA Acceptor Terminus
Authors: GOLDBERG Ronald , RINGER David , CHLÁDEK Stanislav
Published: March 2005

The effect of 2′ and 3′‐O‐aminoacyl‐dinucleoside phosphates cytidylyl(3′‐5′)‐2′(3′)‐O‐l‐phenylalanyladenosine (I), cytidylyl(3′‐5′)‐3′‐deoxy‐2′‐O‐l‐phenylalanyladenosine (IIa), cytidylyl(3′‐5′)‐2′‐deoxy‐3′‐O‐l‐phenylalanyladenosine (IIIa), cytidylyl(3′‐5′)‐3′‐deoxy‐2′‐O‐glycyladenosine (IIb), cytidylyl(3′‐5′)‐2′‐deoxy‐3′‐O‐glycyladenosine (IIIb), cytidylyl(3′‐5′)‐3′‐deoxy‐2′‐O‐l‐leucyladenosine (IIc), cytidylyl(3′‐5′)‐2′‐deoxy‐3′‐O‐l‐leucyladenosine (IIIc), cytidylyl(3′‐5′)‐3′‐O‐methyl‐2′‐O‐l‐phenylalanyladenosine (IId) and cytidylyl(3′‐5′)‐2′‐O‐methyl‐3′‐O‐l‐phenylalanyladenosine (IIId) as analogs of the 2′(3′)‐aminoacyl‐tRNA termini, on chloramphenicol binding to 70‐S Escherichia coli ribosomes was investigated. The association constants (Kb) of the investigated compounds were determined by the equilibrium dialysis method. Based on the constancy of Kb over the range of inhibitor concentration, it was determined that the binding site of the 2′ isomers IIa–IIc overlaps with the chloramphenicol site, whereas the variability of Kb for the 3′ isomers IIIb, IIIc and especially IIIa seems to indicate that they do not achieve a complete fit. The consistently higher values of the Kb values for the 3′ isomers IIIa–IIIc relative to that of the 2′ isomers IIa–IIc also indicate a stabilization of the binding of the former due to a specific interaction between its amino acid portion and a ribosomal site.
Fate of Histone Messenger RNA in Mengovirus‐Infected Ehrlich Ascites Tumor Cells
Authors: GALLWITZ Dieter , TRAUB Ulrike , TRAUB Peter
Published: March 2005

Histone mRNA was isolated from mengovirus‐infected Ehrlich ascites tumor cells at various times postinfection and quantitated in a reticulocyte cell‐free protein‐synthesizing system. The amount of translatable histone mRNA decreases during the first hour postinfection by 30%, rises during the following 1–1.5 h by 10–15%, drops progressively in the further course of infection, and reaches 20% of the control at the end of the infectious cycle (8–9 h postinfection). On the basis of the relative histone mRNA contents, the histone‐synthesizing potentials of mengovirus‐infected Ehrlich ascites tumor cells are substantially higher throughout infection than actually expressed in vivo. This result indicates that the virus‐induced shutoff of histone synthesis is not directly the consequence of inactivation or degradation of histone mRNA.

Most of the histone mRNA recovered from mengovirus‐infected Ehrlich ascites tumor cells is bound to ribosomes.

Late in infection, certain mRNAs are co‐isolated with histone mRNAs, very likely due to loss or shortening of poly(A) occurring after release of the mRNAs from polyribosomes.
Conformational Features of Bradykinin
Authors: LINTNER Karl , FERMANDJIAN Serge , REGOLI Domenico , BARABÉ Jean
Published: March 2005

The circular dichroism (CD) of the peptide hormone bradykinin and its analogues, [Phe(H4)5]‐bradykinin, [Phe(H4)8]bradykinin, [Phe(H4)5,8]bradykinin, [TyrOMe5]bradykinin, [TyrOMe8]bradykinin and [TyrOMe5,8]bradykinin, is described. The comparison of the CD spectra of these analogues with each other, recorded under a variety of conditions (pH, solvent, temperature), allows the monitoring of the behaviour of the aromatic side‐chains (phenylalanine, tyrosine) and an estimation of their respective spectral contributions in both spectral regions (320–250 nm, 250–190 nm) with good precision. Conformational non‐equivalence of the residues Phe‐5 and Phe‐8 together with some overall conformational features of bradykinin are thus established.
Acyl‐Acceptor Specificities of 1‐Acylglycerolphosphate Acyltransferase and 1‐Acylglycerophosphorylcholine Acyltransferase Resolved from Rat Liver Microsomes
Authors: MIKI Yoshinobu , HOSAKA Kohei , YAMASHITA Satoshi , HANDA Hajime , NUMA Shosaku
Published: March 2005

The acyl‐acceptor specificities of the 1‐acylglycerolphosphate acyltransferase and 1‐acyl‐glycerophosphorylcholine acyltransferase resolved from rat liver microsomes have been investigated with various synthetic 1‐acyl‐sn‐glycerol 3‐phosphate and 1‐acyl‐sn‐glycero‐3‐phosphorylcholine derivatives differing in their fatty acid constituent (myristic, palmitic, stearic, oleic, linoleic or arachidonic acid) as well as with various 1‐acyl‐sn‐glycerophospholipid analogues carrying different polar head groups (phosphorylcholine, phosphoryldimethylethanolamine, phosphorylmonomethyl‐ethanolamine, phosphorylethanolamine, phosphorylethanol or phosphate).

The activities of the two acyltransferases are virtually not affected by the fatty acid constituent of the respective acyl acceptors, except that the 1‐stearoyl and 1‐arachidonoyl derivatives are somewhat less effective acyl acceptors for both enzymes.

The 1‐acylglycerophosphorylcholine acyltransferase utilizes several acyl acceptors differing in the polar head group with the following order of effectiveness: 1‐acyl‐sn‐glycero‐3‐phosphoryl‐choline > 1‐acyl‐sn‐glycero‐3‐phosphoryldimethylethanolamine > 1‐acyl‐sn‐glycero‐3‐phosphoryl‐monomethylethanolamine ∼ 1‐ acyl‐sn‐glycero‐3‐phosphorylethanolamine. 1 ‐Acyl‐sn‐glycero‐3‐phosphorylethanol and 1‐acyl‐sn‐glycerol 3‐phosphate are ineffective. In contrast, the 1‐acylglycerolphosphate acyltransferase is highly specific for 1‐acyl‐sn‐glycerol 3‐phosphate.

The fatty acid constituent of the acylacceptors exerts essentially no effect on the acyl‐donor specificity of each enzyme except in the case of 1‐arachidonoyl‐sn‐glycero‐3‐phosphorylcholine. Furthermore, the acyl‐donor specificity of the 1‐acylglycerophosphorylcholine acyltransferase is virtually not affected by the polar head group of the acyl acceptor.

The results obtained in this work and in our previous investigations support the view that the substrate specificities of glycerolphosphate acyltransferase, 1‐acylglycerolphosphate acyltransferase and 1‐acylglycerophosphorylcholine acyltransferase make an essential contribution to the nonrandom fatty acid distribution in naturally occurring glycerolipids.
Hydrodynamic Properties of 2‐Mercaptoethanol‐Modified Glycogen
Authors: GEDDES Robert , HARVEY John D. , WILLS Peter R.
Published: March 2005

Treatment of glycogen with 2‐mercaptoethanol and iodoacetamide gives rise to a modified glycogen which resembles the original glycogen in its hydrodynamic behaviour but has a pronounced tendency to aggregate. The modified glycogen can be distinguished easily, by its diffusion coefficient, from glycogen degraded by more traditional methods of extraction. The ‘fundamental’ glycogen particle appears to be composed of two or three glycogen β‐particles linked by a single protein chain.
Detection of Guanosine‐Nucleotide · Elongation‐Factor‐G Complexes Produced during the Decay of Guanosine‐Nucleotide · Elongation‐Factor‐G · Ribosome Complexes
Authors: GIRBES Tomás , VÁZQUEZ David , MODOLELL Juan
Published: March 2005

The mechanism of formation and decay of complexes containing guanosine nucleotide, elongation factor G (EF‐G) and the ribosome (with or without fusidic acid) has been examined in a purified Escherichia coli system. With a [14C]GDP · [3H]EF‐G · ribosome · fusidic‐acid complex in the presence of either unlabelled GDP plus EF‐G or the antibiotic thiostrepton, it has been found that [14C]GDP and [3H]EF‐G are simultaneously released, which suggests that they dissociate from the ribosome as a binary complex. In fact, guanosine‐nucleotide · EF‐G complexes, which are too unstable to be isolated, seem to be present in mixtures containing either the [3H]GPD · EF‐G · ribosome · fusidic‐acid complex, the [3H]guanyl‐5′‐yl‐methylene‐diphosphonate · EF‐G · ribosome complex, or the [3H]guanyl‐5′‐yl‐imidodiphosphate · EF‐G · ribosome complex, since ribosomes added to these mixtures, together with a 100‐fold excess of the corresponding unlabelled guanosine nucleotide, specifically bind substantial amounts of radioactive guanosine nucleotide.

The binary complexes in these mixtures seem to proceed exclusively from the decay of ternary or quaternary complexes, since the same detection procedure fails to reveal binary complexes in mixtures containing only EF‐G and either [14C]GTP, the labelled GTP analogs or [3H]GDP. Moreover, only very small amounts of [3H]GDP · EF‐G complexes are detected in mixtures where either the EF‐G plus ribosome‐dependent GTP hydrolysis or the interaction of [3H]GDP with EF‐G and ribosomes in the absence of fusidic acid is taking place. The results suggest that EF‐G, after binding to the ribosome in the presence of either GTP or its nonhydrolyzable analogs (but not GDP), undergoes a conformational change and retains guanosine nucleotide more strongly. With GTP, the hydrolysis of this nucleotide would reverse this conformational change, facilitating both the release of GDP · EF‐G from the ribosome and the dissociation of this binary complex. Fusidic acid would interfere with the conformational transitions by preserving the prehydrolytic conformation after GTP hydrolysis and/or by inducing it after formation of the GDP · EF‐G · ribosome complex.
Effects of Cations, Antibiotics and Other Agents on the Turnover of Guanosine‐Nucleotide · Elongation‐Factor‐G · Ribosome Complexes
Authors: GIRBES Tomás , CAMPUZANO Sonsoles , VÁZQUEZ David , MODOLELL Juan
Published: March 2005

The turnover of complexes containing elongation factor (EF) G, the ribosome and either GDP plus fusidic acid or guanyl‐5′‐yl methylene diphosphonate or guanyl‐5′‐yl imidodiphosphate has been measured by chasing the complexed, radioactive forms of these guanosine nucleotides with either an excess of unlabelled guanosine nucleotide or the antibiotic thiostrepton. With the three different complexes, the decay promoted by thiostrepton was substantially faster than that induced by the guanosine nucleotide and, under both decay conditions, no simple first‐order kinetics were observed. Assuming that the dissociation of the complexes proceeds via a guanosine‐nucleotide · EF‐G binary complex and a free ribosome, this result suggests that dissociation was reversible and that after it the inactivation of free ribosomes by thiostrepton was faster than the decay of the newly formed binary complex. Increasing the NH4+ concentration from 10 to 180 mM or decreasing the Mg2+ concentration from 30 to 10 mM during measurement of turnover accelerated the chase of [3H]GDP from a [3H]GDP · EF‐G · ribosome · fusidic‐acid complex. Since these variations did not affect the amount of complex present in solution, they evidently accelerated the reactions leading to formation of the complex. In contrast, increasing the NH4+ concentration of the wash buffer used to isolate the complexes on nitrocellulose membranes sharply decreased their recovery.

The antibiotics tetracycline, viomycin and several aminoglycosides that induce misreading (streptomycin, neomycin, kanamycin, tobramycin, gentamicin and paromomycin) inhibited the decay of the GDP · EF‐G · ribosome · fusidic acid complex, but other aminoglycosides like spectinomycin and kasugamycin had no effect. Several peptidyl transferase inhibitors (chloramphenicol, lincomycin, viridogrisein, griseoviridin, spiramycin, gougerotin and althiomycin), the EF‐Tu‐inactivating kirromycin and other antibiotics of less well defined mode of action (erythromycin, bottromycin and micrococcin) were also ineffective. Streptomycin and neomycin were effective at concentrations lower than 10 μM thus indicating that they were probably acting specifically and not as a result of a Mg2+ ‐like action caused by their polycationic nature. The antibiotics did not completely block turnover and their effect could be explained by an increase in the affinity of the ribosome for the binary GDP · EF‐G complex. Since all the effective antibiotics bind to the 30‐S subunit and affect functions related to the ribosomal A‐site, the results emphasize the important role of this subunit for EF‐G interaction and suggest that EF‐G binds to the ribosome in a region near the 30‐S portion of the A‐site.

A ternary complex containing Phe‐tRNA, EF‐Tu and GTP did not modify the chase rate of [14C]GDP from a [14C]GDP · EF‐G · ribosome · poly(uridylic acid) · fusidic‐acid complex.
Kinetic Studies on the Transport of Cytoplasmically Synthesized Proteins into the Mitochondria in Intact Cells of Neurospora crassa
Authors: HALLERMAYER Gerhard , ZIMMERMANN Richard , NEUPERT Walter
Published: March 2005

The transport of cytoplasmically synthesized mitochondrial proteins was investigated in whole cells of Neurospora crassa, using dual labelling and immunological techniques.

In pulse and pulse‐chase labelling experiments the mitochondrial proteins accumulate label. The appearance of label in mitochondrial protein shows a lag relative to total cellular protein, ribosomal, microsomal and cytosolic proteins.

The delayed appearance of label was also found in immunoprecipitated mitochondrial matrix proteins, mitochondrial ribosomal proteins, mitochondrial carboxyatractyloside‐binding protein and cytochrome c. Individual mitochondrial proteins exhibit different labelling kinetics.

Cycloheximide inhibition of translation does not prevent import of proteins into the mitochondria.

Mitochondrial matrix proteins labelled in pulse and pulse‐chase experiments can first be detected in the cytosol fraction and subsequently in the mitochondria. The cytosol matrix proteins and those in the mitochondria show a precursor‐product type relationship.

The results suggest that newly synthesized mitochondrial proteins exist in an extra‐mitochondrial pool from which they are imported into the mitochondria.
Ornithine Decarboxylase Antizyme Is a Normal Component of Uninduced H‐35 Cells and Rat Liver
Authors: HELLER John S. , KYRIAKIDIS Dimitri , FONG Wang F. , CANELLAKIS E. S.
Published: March 2005

Ornithine decarboxylase antizyme (a non‐competitive protein inhibitor of ornithine decarboxylase induced by the addition of putrescine, spermidine, spermine to a variety of cell lines and rat liver) has been detected in unstimulated H‐35 cells and in normal rat liver. The ornithine decarboxylase antizyme is bound to subcellular components in a nonreactive form from which it can be released as the free antizyme by treatment with high concentrations of salts or low concentrations of diamines or polyamines. Of the latter, putrescine is the most effective; 1 μM putrescine will release free ornithine decarboxylase antizyme from subcellular particles. The molecular weights and general physical properties of the bound ornithine decarboxylase antizyme are the same as those of the induced ornithine decarboxylase antizyme.

In H‐35 cells, 80% of the bound form of the ornithine decarboxylase antizyme is localized in the nucleus whereas only 35% of the bound form of the ornithine decarboxylase antizyme is found in the rat liver nucleus. The remaining activity is not associated with ribosomes or polysomes but is localized in a particulate fraction that sediments through a 5–40% sucrose gradient. This bound form of ornithine decarboxylase antizyme also does not react with ornithine decarboxylase unless it is first liberated by polyamines.

The presence of ornithine decarboxylase antizyme bound to particles in unstimulated cells and its liberation from the particles by low levels of polyamines as well as its induction in the intact cell by polyamines suggests that it may play a role in modulating ornithine decarboxylase activity in vivo.
Quantitative Determination of Myoinositol, Inositol 1‐Phosphate, Inositol Cyclic 1 : 2‐Phosphate and Glycerylphosphoinositol in Normal and Rous‐Sarcoma‐Virus‐Transformed Quail Fibroblasts under Different Growth Conditions
Authors: DIRINGER Heino , KOCH‐KALLNBACH Marianne E. , FRIIS Robert R.
Published: March 2005

Myoinositol and its phosphorylated derivatives have been quantitatively determined in normal and Rous‐sarcoma‐virus‐transformed quail cells under various growth conditions using [2‐3H]‐myoinositol at isotope equilibrium conditions. The following amounts were determined (nmol/μmol phospholipid, as a unit of cell mass): exponentially growing normal and tumor cells contained 25–40 nmol free inositol, 0.40–0.45 nmol myoinositol 1‐phosphate, 0.30–0.50 nmol glyceryl‐phosphoinositol, and 0.03–0.04 nmol myoinositol cyclic 1 : 2‐phosphate. At high cell populations in the absence of serum, conditions which result in cessation of growth by normal but not by tumor cells, changed levels were found for glycerylphosphoinositol and free inositol. In tumor cells the levels of these two compounds increased to 0.64 nmol and 64 nmol, respectively. In normal cells glycerylphosphoinositol increased to 0.95 nmol and free inositol showed highly elevated levels of 144 nmol. At short pulses the specific activities of inositol 1‐phosphate and inositol cyclic 1 : 2‐phosphate were found to be higher than that of phosphatidylinositol. This was not the case for glycerylphosphoinositol.
Identification of the Mammalian DNA‐Binding Protein P8 as Glyceraldehyde‐3‐Phosphate Dehydrogenase
Authors: PERUCHO Manuel , SALAS José , SALAS Maria L.
Published: March 2005

The DNA‐binding protein P8 from transformed hamster fibroblasts (line NIL‐1‐hamster sarcoma virus) has been purified to homogeneity by DNA‐cellulose and phosphocellulose chromatography. The molecular weight of dissociated P8 is 36000, the same as that reported for the subunits of glyceraldehyde‐3‐phosphate dehydrogenase, and the mobility of these proteins in polyacrylamide gels is identical. The amino acid composition of P8 is very similar to that of glyceraldehyde‐3‐phosphate dehydrogenase. When assayed for glyceraldehyde‐3‐phosphate dehydrogenase activity the P8 preparation had a specific activity of 54.6 units/mg, a value comparable to that of the crystalline enzyme from several sources. Furthermore, serum prepared against P8 crossreacts with glyceral‐dehyde‐3‐phosphate dehydrogenase from hamster muscle. These results show that P8 is glyceraldehyde‐3‐phosphate dehydrogenase. The interaction of P8 from transformed fibroblasts and glyceral‐dehyde‐3‐phosphate dehydrogenase from hamster and rabbit muscle with DNA has been studied using a Millipore filtration technique. These proteins have affinity for single‐stranded DNA but not for double‐stranded DNA.
Mechanism of Reactivation and Refolding of Glyceraldehyde‐3‐Phosphate Dehydrogenase from Yeast after Denaturation and Dissociation
Authors: RUDOLPH Rainer , HEIDER Ingrid , JAENICKE Rainer
Published: March 2005

Glyceraldehyde‐3‐phosphate dehydrogenase from yeast has been reversibly dissociated utilizing various denaturants. Applying comparable experimental conditions, dissociation, denaturation, and deactivation run parallel. Dissociation and reassociation occur at different limiting concentrations of the denaturant causing a range of metastability where irreversible aggregation competes with the process of reconstitution. In order to realize a high yield in kinetic reactivation experiments, solvent conditions far from the tetramer ⇌ monomer transition were chosen. Maximum recovery of enzymatic activity was obtained after denaturation in strong denaturants (citrate pH 2.3 + 6 M guanidine · HCl), and reactivation in the presence of 10 mM NAD++ 0.1–10 mM dithiothreitol at pH 7.6, c < 0.2 μM.

Removal of the denaturant by dilution, and separation of high aggregates, e.g. by gelchromatography, lead to 100% regain of specific enzymatic activity and complete recovery of the hydrodynamic and spectral properties characterizing the native tetrameric enzyme.

The analysis of the time course of reactivation and renaturation suggests that transconformation of inactive monomers and their subsequent association are responsible for the sigmoidal kinetic traces. A sequential uni‐bimolecular mechanism with first and second order rate constants k1= (11 ± 2) × 10−3 s−1 and k2= 45 ± 10 mM−1· s−1 (15°C) makes it possible to fit the reactivation kinetics in a quantitative manner. Within the limits of error k1 and k2 hold for the reactivation in the absence and in the presence of NAD+. NADH leads to complex kinetic profiles which cannot be fitted by a simple mechanism due to instability of the enzyme in the presence of the reduced coenzyme.
Regulation in vitro and in vivo of Adenosine 3′:5′‐monophosphate‐Dependent Inactivation of Rat‐Liver Pyruvate Kinase Type L
Authors: FELÌU Juan E. , HUE Louis , HERS Henri‐Géry
Published: March 2005

The cyclic‐AMP‐dependent inactivation of pyruvate kinase L has been studied in a crude Sephadex filtrate of isolated hepatocytes. This inactivation requires the presence of Mg‐ATP (apparent Km= 0.1 mM) and a half‐maximal rate of inactivation was obtained in the presence of 0.15 μM cyclic AMP. It was inhibited by physiological concentrations of phosphoenolpyruvate and by micromolar concentrations of fructose bisphosphate and these inhibitory effects were counteracted by Mg‐ATP and by several l‐form amino acids such as cysteine, alanine, serine, phenylalanine, which are also ligands of pyruvate kinase. As a rule, it appears that effectors that increase the activity of the enzyme are also those which prevent its cyclic‐AMP‐dependent inactivation and vice‐versa.

Considering the potentially important role of phosphoenolpyruvate concentration in the control of gluconeogenesis, experiments were performed to check if the inhibitory action of this metabolite on the inactivation of pyruvate kinase was of importance in vivo. We found that the concentration of phosphoenolpyruvate was higher than normal in the livers of anesthetized rats in conditions like fasting and diabetes in which the rate of gluconeogenesis is known to be higher and the activity of pyruvate kinase lower than normally. The reverse was true upon sucrose feeding. Inactivation of pyruvate kinase was induced in vivo by the administration of glucagon. This inactivation was less important in the livers with high concentrations of phosphoenolpyruvate and vice‐versa. It is proposed that phosphoenolpyruvate exerts an important buffering effect in the variations of the rate of gluconeogenesis.
Heterogeneity in the Kinetics of Oxygen Binding to Partially Reduced Human Methemoglobin
Authors: RAAP Adriaan , van LEEUWEN Johan W. , van ECK‐SCHOUTEN Tony , ROLLEMA Harry S. , de BRUIN Simon H.
Published: March 2005

The pulse‐radiolysis technique has been introduced because it permits a rapid reduction (in a few microseconds) of one heme group of the methemoglobin tetramer by hydrated electrons. The kinetics of the binding of oxygen to this particular valence intermediate (Hb3+) with one reduced α or β subunit has been studied. It appears that the hydrated electrons preferentially reduce one type of subunit of methemoglobin at acid and neutral pH‐values as is shown by the biphasic behaviour of Hb3+ on oxygenation. The second‐order on‐rate constants measured for the binding of oxygen to Hb3+ are 14 ± 3 mM−1 ms−1 and 56 ± 9 mM−1 ms−1, respectively. The relative contribution of the faster fraction is about 0.63 ± 0.08 of the total oxygenation process.

A comparison of the kinetic absorbance difference spectrum for the reduction of methemoglobin with the static difference spectrum of deoxyhemoglobin and methemoglobin in the Soret‐region revealed a decreased absorbance of the unliganded subunit of Hb3+ at 430 nm. This fact suggests that Hb3+ is in the relaxed quaternary conformation, which is in agreement with the observed on‐rate constants.
The Interrelations between the Transport of Sodium and Calcium in Mitochondria of Various Mammalian Tissues
Authors: CROMPTON Martin , MOSER Rolf , LÜDI Hans , CARAFOLI Ernesto
Published: March 2005

Addition of ruthenium red to mitochondria isolated from brain, adrenal cortex, parotid gland and skeletal muscle inhibits the further uptake of Ca2+ by these mitochondria but induces little or no net Ca2+ efflux; the further addition of Na+, however, induces rapid efflux of Ca2+. The velocity of the Na+‐induced efflux of Ca2+ from these mitochondria exhibits a sigmoidal dependence on the [Na+]. Addition of Na+ to mitochondria exhibiting the most active Na+‐dependent efflux of Ca2+ (brain and adrenal cortex) also releases Ca2+ in the absence of ruthenium red and, under these conditions, the mitochondria become uncoupled. It is concluded that the efflux of Ca2+ from these mitochondria occurs via a Na+‐dependent pathway, possibly a Na+‐Ca2+ antiporter, that is distinct from the ruthenium‐red‐sensitive carrier that catalyses energy‐linked Ca2+ influx. The possible role of the Na+‐dependent efflux process in the distribution of Ca2+ between the mitochondria and the cytosol is discussed. In contrast, mitochondria from liver, kidney, lung, uterus muscle and ileum muscle exhibit no Na+‐dependent efflux of Ca2+.