bluuebunny/crossref_metadata_2025 huggingface dataset:
Physico‐Chemical Properties of Mitochondrial Ribosomal RNA from Fungi
Authors: Edelman Marvin , Verma Inder M. , Herzog Rita , Galun Esra , Littauer Uriel Z.
Published: March 2005

Several physico‐chemical properties of mitochondrial and cytoplasmic ribosomal RNA from the filamentous fungi, Trichoderma viride and Neurospora crassa were determined and compared with those for Aspergillus nidulans.

The thermal denaturation patterns of the mitochondrial rRNAs differed markedly from those of their homologous cytoplasmic rRNAs. The two rRNAs, therefore, possess distinct conformations in solution.

The guanine + cytosine content of Trichoderma mitochondrial rRNA was shown to be very low (31.5 and 35.5% for the heavy and light components) and to differ considerably from that of its cytoplasmic rRNA (50 and 49%). Such nucleotide compositions are characteristic of fungal mitochondrial rRNA and account, at least in part, for the unusual physical characteristics which these polynucleotides exhibit.

Polyacrylamide gel electrophoresis of mitochondrial and cytoplasmic rRNA was performed in buffers of different ionic strenghts. In low‐salt buffer, the relative mobilities of the mitochondrial rRNAs were severely retarded, causing mitochondrial rRNA to migrate more slowly than cytoplasmic rRNA. In Mg buffer, mitochondrial and cytoplasmic rRNA co‐electrophoresed and were easily distinguished from Escherichia coli rRNA. Thus, under certain conditions, the conformational peculiarities of fungal mitochondrial rRNAs are expressed with this technique as well. It is concluded that accurate extrapolations from electrophoretic mobilities to molecular weight values cannot be made for these rRNA samples.

Mitochondrial rRNAs sediment more slowly than their corresponding cytoplasmic rRNAs. However, under the same experimental conditions the sedimentation velocity patterns for the various mitochondrial rRNAs are not identical.

While the above measurements clearly distinguish mitochondrial rRNAs, as a group, from cytoplasmic and bacterial rRNAs, they also point out a considerable physical heterogeneity among the fungal mitochondrial rRNAs.
Excretion of Endogenous Steroids and Metabolites of [4‐14C]Pregnenolone in Bile of Female Rats
Authors: Cronholm Tomas , Eriksson Håkan , Gustafsson Jan‐Åke
Published: March 2005

Gas‐liquid chromatography, gas chromatography‐mass spectrometry and radio‐gas chromatography were used to identify endogenous steroids and metabolites of [4‐14C]pregnenolone in bile from female bile fistula rats.

About 70% of the administered radioactivity was recovered in the bile during the first 24 h, and less than 1% during the following three days. 76% of the biliary radioactivity was extracted and was separated according to conjugation. The free steroid fraction contained 2%, the glucuronide fraction 22%, the monosulphate fraction 45%, and the disulphate fraction 28% of this radioactivity. The following steroids were identified by gas chromatography‐mass spectrometry and were found to be labelled: glucuronide fraction: 3α‐hydroxy‐5α‐androstan‐17‐one, 3α‐hydroxy‐5α,17β‐pregnan‐20‐one and 3α,16α‐dihydroxy‐5α‐pregnan‐20‐one; monosulphate fraction: 3α,7α‐dihydroxy‐5α‐androstan‐17‐one and/or 3α,15α‐dihydroxy‐5α‐androstan‐17‐one, 3α,11β‐dihydroxy‐5α‐androstan‐17‐one and 3α,16α‐dihydroxy‐5α‐pregnan‐20‐one; disulphate fraction: 3α,7α‐dihydroxy‐5α‐androstan‐17‐one, 3α,11β‐dihydroxy‐5α‐androstan‐17‐one, 5α‐pregnane‐3β,20β‐diol and 3α,21‐dihydroxy‐5α‐pregnan‐20‐one. The main part of radioactivity in the steroid monosulphate fraction was associated with pregnenolone that had a very high specific activity. The amount of steroids excreted during the first 24 h after bile duct cannulation was about 700 μg, when no correction for losses was made. The large quantity of steroid metabolites excreted in the bile confirms the importance of this excretory pathway also for endogenous steroids. The quantitatively most important steroids were found to be unlabelled corticosterone metabolites: 3α‐ and 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one and 3α,11β,15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one. Several other unlabelled steroids were identified. The heterogenous distribution of radioactivity among the metabolites is in contrast to the more uniform labelling previously obtained with intact rats. This shows that the metabolism of injected labelled steroids in bile fistula rats is not representative of the conditions in the intact animal, where enterohepatic circulation results in a continuous labelling of precursor steroid(s).
The Peroxidase Activity of Deuterohemin
Authors: Portsmouth David , Beal Elizabeth A.
Published: March 2005

The reaction of hydrogen peroxide with deuterohemin and the reaction of the products with peroxidase substrates in neutral aqueous solution have been investigated. Qualitative observations indicate that, prior to oxidative degradation of the porphyrin ring system, a peroxidatic hemeperoxide complex is formed which can react with hydrogen donors to regenerate free deuterohemin. Quantitative data support this conclusion and show that this complex formation can account for the peroxidase activity of deuterohemin. The kinetics of formation of this complex and its reaction with peroxidase substrates have been studied and indicate that the principal difference between the catalytic mechanisms of deuterohemin and peroxidase is the very much more rapid rate of formation of the primary active complex in peroxidase. The overall rate of conversion of the primary active complex to the free catalyst is approximately the same in both enzyme and model systems, although in the latter case no intermediate analogous to compound II of peroxidase is formed.

The mechanism of action of peroxidases and the nature of their active peroxide complexes is discussed in the light of these findings.
An Unstable Factor from Rabbit Reticulocytes with Activity in Cell‐Free Protein Synthesis
Authors: Gee‐Clough Judith P. , Arnstein Henry R. V.
Published: March 2005

Fresh enzyme preparations obtained from the cytoplasm of rabbit reticulocytes contain an unstable factor which stimulates cell‐free protein synthesis. The active material can also be obtained by extraction of unwashed reticulocyte ribosomes with 0.5 M KCl, but it is different from the previously described transfer factors TF‐I and TF‐II. It has been partially purified by precipitation at 60% ammonium sulphate saturation followed by chromatography with Sephadex G‐200. The properties of this factor are compared with those of others known to participate in mammalian protein biosynthesis.
Intra‐ and Extramitochondrial Isozymes of (NADP) Malate Dehydrogenase
Authors: Brdiczka Dieter , Pette Dirk
Published: March 2005

The intracellular distribution of (NADP) malate dehydrogenase (decarboxylating) was studied by means of fractional extraction of extra‐ and intramitochondrial enzymes in various tissues of mouse, rat, rabbit, guinea pig, pig, beef, sea gull and pigeon. Absolute activity levels of (NADP) malate dehydrogenase in the same tissues of different species vary greatly. In white adipose tissue and adrenal medulla, (NADP) malate dehydrogenase is located exclusively within the extramitochondrial compartment. In liver, 95% of (NADP) malate dehydrogenase activity is extramitochondrial and only 5% intramitochondrial. Mitochondrial subfractionation reveals a location of this enzyme within the inner mitochondrial compartment. Brain, heart, adrenal cortex and kidney also contain extra‐ and intramitochondrial (NADP) malate dehydrogenase. In kidney and adrenal cortex, the ratio of extra‐ and intramitochondrial isozymes varies between different species. In heart, a relatively constant distribution is found, i. e. 30% of the enzyme activity is extramitochondrial and 70% is intramitochondrial. Extra‐ and intramitochondrial (NADP) malate dehydrogenases show different electrophoretic mobility in all tissues examined and can be characterized as two distinct isoenzymes. Extra‐ and intramitochondrial isozymes of (NADP) malate dehydrogenase are probably related to different metabolic functions in various tissues, e. g. extra‐intramitochondrial hydrogen transfer (malate shuttle) or transhydrogenation reactions.
Massenspektrometrische Identifizierung substituierter Phenylthiohydantoine aus dem Edman‐Abbau von Petiden
Authors: Weygand Friedrich , Obermeier Rainer
Published: March 2005

The mass spectroscopic identification of substituted phenylthiohydantoins from the Edman degradation of peptides is described. For better recognition of the PTH peaks within the spectrum, p‐bromophenylisothiocyanate was used predominantly. The mass numbers, characteristic for each of the common amino acid PTH derivatives, are shown. The whole procedure, named MS‐Edman degradation was tested by degradation of the octapeptide Pro‐Phe‐Phe‐Leu‐Ile‐Ile‐Leu‐Val and the N‐terminal sequence of glucagon (16 cycles). This procedure has the following advantages:

a) Direct identification of phenylthiohydantoins by mass numbers instead of indirect, e.g. chromatographic comparison.

b) Unequivocal assignment of the amide groups by differences between the phenylthiohydantoins of Asn and Asp, or Gln and Glu and easy differentiation of Leu and Ile.

c) High sensitivity.

d) Simultaneous observation of side reactions and peptide fragmentation during the Edman procedure.
Solid‐Phase Edman Degradation
Authors: Laursen Richard A.
Published: March 2005

A modification of the Edman degradation is described whereby peptides are degraded sequentially on a solid support by a machine, the solid‐phase peptide sequencer. Reagents are pumped through a column containing the resin‐bound peptide, and the amino acid thiazolinones released after each degradative cycle are collected on a fraction collector for subsequent analysis. A complete cycle requires 2 h, and all operations are controlled automatically by a programmer. An improved peptide resin is described, to which peptides can be attached in yields ranging from about 70% for the 30 amino acid insulin B‐chain to nearly 100% for smaller peptides. Of the 18 amino acids examined so far, glycine, alanine, phenylalanine, tyrosine, valine, leucine, isoleucine, proline, methionine, asparagine, glutamine, histidine, lysine, threonine, serine and cysteic acid are degraded normally; the phenylthiohydantion of threonine is partially dehydrated and that of serine is almost completely destroyed by trifluoroacetic acid, however. The phenylthiohydantion of glutamic acid is not detected, because it remains bound by its γ‐carboxyl group to the resin. When aspartic acid is encountered in peptides, further degradation is completely blocked, presumably as a result of the β‐carboxyl group having formed a cyclic imide. Using the sequencer and unpurified reagents, it was possible to degrade entirely a 0.24 μmole sample of the 21 amino acid insulin A‐chain (oxidized) and to analyze the products within a 3‐day period. Degradative yields averaged 94% per cycle. Similarly, 0.1 μmole of the insulin B‐chain was degraded through 18 cycles before identification of the phenylthiohydantoins became ambiguous. The sequencer is simple in design and operation, and is inexpensive to construct. The scope and limitations of the method are discussed.
Optical Rotatory Dispersion and Circular Dichroism of Cytochrome Oxidase
Authors: King Tsoo E. , Bayley Peter M. , Yong Fook Choy
Published: March 2005

Optical rotatory dispersion of the oxidized and the reduced cytochrome oxidase has been measured from 196 to 550 nm. The experimental data have been analyzed in order to separate the extrinsic cotton effects from the rotations of the protein backbone. From the resolution, two cotton effects of similar sign are obtained in the Soret region. For the reduced enzyme, the cotton effect centered at 446.5 nm shows a molar amplitude of 192 k‐degrees with a band width of 5.3 nm and rotational strength of 0.25 Debye magneton; the second cotton effect centered at 444 nm shows a molar amplitude of 45 k‐degrees with a band width of 12.9 nm and rotational strength of 0.14 Debye magneton. Upon oxidation, the centers of these cotton effects shift to 432 and 423.7 nm. The amplitudes become 114 and 122 k‐degrees with band widths of 6.1 and 11.2 nm and rotational strengths of 0.18 and 0.35 Debye magneton, respectively. In addition, a less defined third cotton effect centered at 376 nm appears with an amplitude of 28 k‐degrees, band width of 18 nm, and rotational strength of – 0.15 Debye magneton. The circular dichroic curves constructed from the optical rotatory dispersion data by the Kronig‐Kramers transform are in general agreement with the experimentally determined spectra.

The backbone rotations are analyzed and expressed in the terms of Shechter‐Blout's parameters. The effective helicity thus computed is 50% for the reduced and 44.5% for the oxidized form.

The sources of the Soret cotton effects are discussed along with the possibilities of the components present (cytochromes a and a3 and copper), the intrinsic property of the Soret transition, possible splitting of the Soret band, interactions with the protein helix, and heme‐heme and heme‐copper couplings. The fact that the optical rotatory dispersion and circular dichroism behavior is dependent upon the oxidation state of cytochrome oxidase could arise either from an altered conformation of the protein backbone and/or from an altered interaction with the heme group. Regardless of the possibilities, the geometrical conformation of cytochrome oxidase and the electronic relation between the prosthetic group and the apoenzyme is dependent upon the oxidation state of the enzyme.
Flip‐Flop Mechanisms in Enzymology
Authors: Lazdunski Michel , Petitclerc Claude , Chappelet Danielle , Lazdunski Claude
Published: March 2005

A new mechanism is proposed which permits a clear‐cut interpretation of the properties and features characteristic of the alkaline phosphatase of Escherichia coli. It is called the Flip‐Flop mechanism. The Flip‐Flop mechanism implies alternating functions for each of the two active sites of the phosphatase. The phosphorylation of one site is concurrent with the dephosphorylation of the other site. The alkaline phosphatase is considered to be a model. The functional and evolutive advantages of the Flip‐Flop mechanism suggest that it might be fairly common among polymeric enzymes with Michaelian kinetics. Data already reported in the literature for other enzymes are discussed in light of the Flip‐Flop mechanism.
Bovine Proinsulin: Amino Acid Sequence of the C‐Peptide Isolated from Pancreas
Authors: Salokangas Anneli , Smyth Derek G. , Markussen Jan , Sundby Finn
Published: March 2005

The amino acid sequence of the proinsulin C‐peptide isolated from bovine pancreas has been determined and the experimental data is presented. Digestion of C‐peptide with chymotrypsin provided three fragments, the compositions of which accounted for the whole structure. Digestion of the three peptides with papain gave rise to a variety of small peptides, which were isolated. The sequences of the chymotrypsin and papain peptides allowed the primary structure of bovine C‐peptide to be assigned. The similarity of the sequence obtained for C‐peptide isolated from bovine pancreas with that written for C‐peptide from bovine proinsulin provides strong evidence for their identity. The sequence presented contains two differences from that cited previously.
The Amino Acid Sequence of the C‐Peptide of Human Proinsulin
Authors: Ko Arthur S. C. , Smyth Derek G. , Markussen Jan , Sundby Finn
Published: March 2005

The determination of the amino acid sequence of the C‐peptide of human proinsulin is presented. Two constituent fragments of C‐peptide, Chy 1 + 2 and Chy 3 (residues 1–24 and 25–31, respectively) were liberated by mild chymotryptic digestion and isolated by gel filtration and ion exchange chromatography. From Chy 1 + 2 was obtained seven smaller peptides by further digestion with chymotrypsin. The sequence analysis of these fragments allowed an overall structure to be assigned. Papain digestion of the intact C‐peptide provided 12 peptides, the compositions of which provided unequivocal confirmation for the proposed sequence.
The pH6 Acetolactate‐Forming Enzyme from Aerobacter aerogenes
Authors: Huseby Nils‐Erik , Størmer Fredrik C.
Published: March 2005

The pH6 acetolactate‐forming enzyme from Aerobacter aerogenes is competitively inhibited by 2‐oxobutyrate with an inhibitor constant of approximately 5 mM. 2‐Oxobutyrate is able, to a lesser extent, to participate in the reaction with pyruvate, yielding a mixture of acetolactate and acetohydroxy‐butyrate. These products are identified as their respective pteridines.
Light‐Induced Structural Transitions of Chloroplasts Studied by the Spin‐Probe Method
Authors: Tzapin A. I. , Mmolotkovsky Y. G. , Ggoldfield M. G. , Dzjubenko V. S.
Published: March 2005

The light‐induced structural state changes of chloroplast (swelling and shrinkage) are compared with the change of activity (ferrcyanide reduction and photophosphorylation) and the state of hydrophobic membrane parts, characterised by the electron spin resonance spectra of iminoxyl radical‐probe, introduced in the membrane.

Shrinkage of chloroplasts in sodium acetate medium is accompanied by non‐essential changes of activity and conformational state. Moreover light‐induced swelling in sodium chloride medium is accompanied by transition of chloroplasts to the uncoupled state and by change of conformational state.

The regulatory function of conformational transitions in the chloroplasts is discussed and the corresponding scheme of regulation of photophosphorylation is suggested.
Ribosomal Proteins
Authors: Homann Heide E. , Nierhaus Kund H.
Published: March 2005

The 43 S precursor of the 50 S ribosomal subunit shows a protein pattern very similar to that of a “core” particle derived from 50 S subunits on CsCl gradients.

A comparison of the protein patterns of “core” particles from 50 S subunits after CsCl equilibration runs or incubation with increasing LiCl concentrations over the molarity range 0.4–6 M revealed that the proteins are successively released from the “core” in five distinct groups.

The protein pattern of the 21 S precursor of 30 S ribosomal subunits was compared with that of the reconstituted intermediate particle and LiCl‐treated “core” particles of 30 S subunits. The 21 S is similar to both the reconstituted intermediate and the “core” particles derived by a 1 M LiCl treatment, whereas the reconstituted intermediate and this core particle differ rather more.
Synthetic Antigens with Sequential and Conformation‐Dependent Determinants Containing the Same l‐Tyrosyl‐l‐alanyl‐l‐glutamyl Sequence
Authors: Schechter Bilha , Schechter Israel , Ramachandran Janakiraman , Conway‐jacobs Abigail , Sela Michale , Benjamini Eli , Shimizu Meikyo
Published: March 2005

Two synthetic copolymers have been used in order to investigate the role of conformation in immunogenicity and antigenic specificity. The first one is a high molecular weight ordered copolymer composed of the repeating sequencel‐tyrosyl‐l‐alanyl‐l‐glutamyl. This polymer exists as an α‐helix under physiological conditions. In the second polymer, the tripeptide l‐tyrosyl‐l‐alanyl‐l‐glutamic acid is attached to branched poly‐dl‐alanine. In this branched polymer the tripeptide exists as a random coil. These polymers elicit the formationof antibodies with distinct specificities, since there was no cross‐precipitation reaction between the two systems. Furthermore, antibodies against the branched polymer were efficiently inhibited by the tripeptide l‐tyrosyl‐l‐alanyl‐l‐glutamic acid, whereas antibodies against the helical polymer were inhibited neither by the tripeptide, nor by other small peptides of sequences related to the primary structure of the helical polymer. However, inhibition was obtained with oligopeptides possessing the general formula (Tyr‐Ala‐Glu)n (with n = 3, 4, 7, and 9), the inhibitory capacity increasing with the value of n. Peptides with n values of 7 and 9 were efficient inhibitors, whereas (Tyr‐Ala‐Glu)13 precipitated with the antibodies. The dependence of inhibitory capacity on a suggests that the peptides with n = 2–9 undergo transconformation (from random coil to α‐helix) upon reaction with the antibody combining site.

The immunological studies showed that the antigenic determinats of the α‐helical polymer are conformation‐dependent, whereas the branched polymer contains sequential determinants.
pH Dependence of the Activity of β‐Galactosidase from Escherichia coli
Authors: Tenu Jean‐Pierre , Viratelle Odile M. , Garnier Jean , Yon Jeannine
Published: March 2005

The “specific reactivity” of β‐galactosidase from Escherichia coli for different substrates has been studied at the optimal pH value. The pH dependence of the enzyme activity has been reinvestigated in highly controlled conditions with respect to Mg2+ and Na+ concentrations and ionic strength. The different kinetic parameters kcat, km and the kcat/km ratio have been determined with o‐nitrophenyl‐β‐d‐galactoside and o‐nitrophenyl‐β‐d‐fucoside, at different pH values between 5.16 and 10, for both Mg2+‐enzyme and Mg2+‐free enzyme, since a residual activity has been found in the absence of Mg2+.

The activity of both types of enzyme is controlled by a protonated group which ionizes in the alkaline range and by at least one unprotonated group which ionizes in the acidic range. This latter group has a pK smaller than 6 in both types of enzyme; it is plausible to assume that this group is a carboxylate.

In the alkaline range, the pK of the involved group shifts from about 6.5 in Mg2+‐free enzyme to 8.4 in the Mg2+ enzyme. Either the substrate or the Mg binding induces this shift in the ionization of the group.

The β‐galactosidase catalyzed reactions proceed via two intermediary complexes. For o‐nitrophenyl‐β‐d‐galactoside substrate the limiting process is not the same for the Mg2+ enzyme and for the Mg2+‐free enzyme.
Studies on “Nonspecific” Binding
Authors: Andersson Lars‐Olov , Rehnström Ann , Eaker David L.
Published: March 2005

The binding of flourescein to bovine serum albumin was studied by equilibrium dialysis under various conditions. At pH 8.0 and 5°C there are three binding sites with association constants of 2.8 × 104 M−1. Specific labelling of the binding sites using fluorescein‐isothiocyanate showed that two of the binding sites are immediately adjacent. The labelled peptides were isolated and the sequence around one of the binding regions was determined revealing the presence of three lysines in this eight amino acid sequence. The binding is mainly casused by hydrophobic interactions and to lesser extent by electrostatic forces. The electrostatic contribution is much less than expected compared with other substances which are bound to bovine serum albumin. This is probably related to the fact that fluorescein is a nonphysiological compound and is “nonspecifically” bound to bovine serum albumin.

Studies on binding properties of various peptic fragments of bovine serum albumin were performed. They showed that even slight fragmentation caused a drastic decrease in the ability to bind fluorescein.
Coordination de la transcription des RNA 5 S et 23 S et coordination de la maturation du RNA 5 S et de la sous‐unité reibosomique 50 S chez Escherichia coli
Authors: Galibert Francis , Tiollais Pierre , Sanfourche Françoise , Boiron Michel
Published: March 2005

The stepwise biosynthesis of Escherichia coli ribosomes can be characterized by a number of precursor ribonucleoprotein particles. It has been shown that 5 S RNA exists as a precursor, pre‐RNA 5 S, prior to its integration into the 40 S relax particle. The present study is concerned with the regulation of 5 S RNA biosynthesis. We report here that:

a) the pre‐5‐S‐RNA fraction containing the precursor of 5 S RNA is heterogeneous and consists of a mixture of three (or possibly four) RNA's each precursor to 5 S RNA and each corresponding to a different step in the pathway of 5 S RNA biosynthesis;

b) amino acid starvation of an auxotrophic strain of Escherichia coli arrests the maturation of ribonucleoprotein and of 5 S RNA. 23 S and 16 S ribosomal RNA appear in 25 S and 18 S particles and in 38 S and 21 S particles, depending on whether RCrelaxed or RCstringent strains were used. 5 S RNA accumulates as 5 S relax RNA or 5 S stringent RNA. The electrophoretic, chromatographic and denaturation analysis of these accumulated RNA's reveal that 5 S relax RNA is less mature than 5 S stringent RNA. The results demonstrate that the maturation of ribosomal particles and 5 S RNA are coordinated together, as the arrest of ribosomal particle maturation is accompained by the arrest of 5 S RNA maturation at a comparable stage;

c) by utilizing different methods of cell lysis one can demonstrate that the precursor of 5 S RNA and the immature ribonucleoprotein particles are not found free in the cell but are attached to cellular structures which may be involved in the coordination of the maturation of 5 S RNA and ribosomes;

d) kinetic studies of uridine incorporation into RNA during amino acid starvation of an Escherichia coli RCstringent auxotrophic strian reveals that an equimolar amount of 23 S RNA and 5 S RNA are synthesized and one may conclude that these syntheses are coordinated.
The meta Cleavage of Catechol by Azotobacter Species
Authors: Sala‐trepat Jose M. , Evans W. Charles
Published: March 2005

Catechol was metabolized through 2‐hydroxymuconic semialdehyde by cell‐free extracts of benzoate‐grown Azotobacter Strains. Some properties of catechol 2,3 oxygenase preparations from Azotobacter vinelandii 206 are described.

Two different enzymatic activities able to attack 2‐hydroxymuconic semialdehyde have been found in crude extracts from benzoate‐grown cells; one catalyses a hydrolytic release of formate from the semialdehyde and the other a dehydrogenation of this compound to 4‐oxalocrotonate. However, the low, non‐inducible levels of 2‐hydroxymuconic semialdehyde hydrolase activity appear negligible for metabolic purposes and the semialdehyde seems to be dissimilated almost exclusively via 4‐oxalocrotonate, by the action of a NAD+‐dependent dehydrogenase, in Azotobacter strains.

A tautomerase activity responsible for the interconversion of the enol and keto forms of 4‐oxalocrotonic acid was found in extracts from benzoate‐grown cells.

4‐Oxalocrotonate was stoicheiometrically converted to CO2 and 4‐hydroxy‐2‐oxovalerate by a partially purified extract, with the transient formation of a compound that appears to be 2‐oxopent‐4‐enoic acid. The 4‐oxalocrotonate decarboxylase activity was stimulated by Mg2+ or Mn2+ ions and was inhibited by EDTA.

Cell‐free extracts from Azotobacter strains converted synthetic 4‐hydroxy‐2‐oxovalerate to acetaldehyde and pyruvate.

A reaction sequence, termed the 4‐oxalocrotonate pathway, for the dissimiation of catechol to acetaldehyde and pyruvate by Azotobacter species is presented. All the enzymes operative in this pathway were inducible, except the 4‐hydroxy‐2‐oxovalerate aldolase.

The findings described here are discussed in connection with the two previously reported meta cleavage pathways for the oxidation of catechol in Pseudomonas strains.
A Reinvestigation of the Anomeric Configuration of Mannose in the Antigens of Salmonella Groups B, D and E
Authors: Fukuda Minoru , Egami Fujio , Hämmerling Günter , Lüderitz Otto , Bagdian Geneviève , Staub Anne‐Marie
Published: March 2005

The anomeric configuration of mannose is α in the O antigens of Salmonella groups B and D, and β in group E, i.e. directly opposite to what was anticipated earlier from data obtained with mannosidase from rat epididymis. The present results were obtained with the aid of α‐mannosidase from jack bean meal and of purified α‐ and β‐mannosidase from a marine gastropod, which were allowed to act upon the disaccharides mannosyl‐rhamnose isolated from partial hydrolysates of the respective antigens. The new findings, however, do not alter the validity of previous conclusions.
Anaerobic Oxidation‐Reduction Titrations of Human Ceruloplasmin
Authors: Carrico Robert J. , Malmström Bo G. , Vänngård Tore
Published: March 2005

Anaerobic oxidation‐reduction titrations of human ceruloplasmin were performed with ascorbate and NADH as reductants. Under most conditions examined one electron per copper ion was required to diminish completely the electron paramagnetic resonance (EPR) signals and the 330 and 610 nm optical absorption bands. In sodium phosphate buffer at pH 7.4 the same changes were attained by addition of only 0.5 electron per copper ion. However, one equivalent NADH was oxidized per g‐atom of copper indicating that the electron accepting capacity of the protein was unchanged. Three kinds of electron acceptors were apparent from titration curves based on optical absorption and EPR parameters. One acceptor is the “blue” copper which gives the Type 1 EPR signal. The second is the remaining paramagnetic copper, Type 2, and this does not have detectable absorption bands in the visible spectrum. The third is a chromophore with an optical absorption maximum near 330 nm and is probably associated with the diamagnetic copper.

A temperature‐dependent electron shift caused the 610 nm absorbance of partially reduced ceruloplasmin to decrease when the temperature was lowered. Fluoride ion prevented this shift and brough about marked changes in the titration behavior of the electron acceptors.
Studies of Glutamate Dehydrogenase
Authors: Pilz Ingrid , Sund Horst
Published: March 2005

The size and shape of beef liver glutamate dehydrogenase in the presence of the allosteric effectors GTP and NADH were investigated by X‐ray small‐angle measurements. These nucleotides cause a dissociation of the associated particles into oligomers. The radius of gyration (R) of the oligomer was found to be 47 Å and a pronounced subsidiary maximum was obtained as is the case with the associated particles. Comparison with theoretical scattering curves shows that the overall shape of the oligomer corresponds well to a rounded cylinder with a ratio diameter to length of 1:1.5, a length of 12.6 nm and a diameter of 8.4 nm. The relatively high value of the volume (V= 668 nm3) indicates that the oligomer is loosely built and from the height of the subsidiary maxima it must be assumed that the oligomer contains larger voids. It is concluded that after interaction with GTP and NADH the conformation of glutamate dehydrogenase is not changed to a great extent although these nucleotides favor the dissociation into the oligomers.

Based on the comparison of calculated scattering curves the eclipsed model for the oligomer proposed by Eigenberg and Reisler can be excluded. The calculated curve for the more staggered model has some similarities but does not coincide completely with the experimental scattering curve.
The Linkage of Phosphate Groups and of 2‐Keto‐3‐deoxyoctonate to the Lipid A Component in a Salmonella minnesota Lipopolysaccharide
Authors: Gmeiner Jobst , Simon Markus , LÜDERITZ Otto
Published: March 2005

From the lipid A backbone of Salmonella lipopolysaccharide, β‐1,6‐linked glucosamine disaccharides had been isolated previously, which were substituted at the non‐reducing glucosamine unit with phosphate and 2‐keto‐3‐deoxyoctonate residues. The results now obtained by periodate degradation show that the phosphate group is linked to the 4 position and the 2‐keto‐3‐deoxy‐octonate oligosaccharide probably to the 3 position of the non‐reducing glucosamine unit.
Étude du rameau dégradatif commun des hexuronates chez Escherichia coli K 12
Authors: Pouysségur Jacques M. , Stoeber François R.
Published: March 2005

The investigation of hexuronate (d‐glucuronate and d‐galacturonate) metabolism in Escherichia coli K 12 has been completed by studying the biochemical properties of the 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase.

The enzyme was obtained from a culture of E. coli K 12 grown on galacturonate followed by a 175‐fold purification which included these steps: sonic extraction of the enzyme, protamine sulfate precipitation, ammonium sulfate fractionation, heat step and DEAE‐Sephadex chromatography. The richest fraction of the chromatographic peak represented 5% of the initial proteins, and has been purified 600‐fold.

The basic biochemical properties of the enzyme have been established using the DEAE‐Sephadex purified extract. The enzyme is stable in a 0.2 M Tris‐HCl buffer, pH 7.6; its pH optimum in this buffer is about 7.6. The aldolase seems to function in the absence of coenzyme. In the above‐mentioned properties it appears to be related to the enzyme which has been studied in Pseudomonas fluorescens. The Michaelis constant for the substrate 2‐keto‐3‐deoxy‐6‐phospho‐gluconate is about 2 × 10−4 M. Numerous compounds tested have no affinity for this aldolase: only the glyceraldehyde‐3‐phosphate (substrate of the reverse reaction) and the gluconate‐6‐phosphate competitively inhibit the aldolase with Ki constants of respectively 4 × 10−4 M and 8 × 10−4 M. The narrow specificity of this aldolase is demonstrated by comparing the compounds with and without affinity for the enzyme; the simultaneous presence of a carboxyl function in position 1 and of a phosphate group in position 6 is necessary for ligand fixation on the enzyme site in the cleavage reaction. The study of denaturation by heat illustrates the thermostability of this enzyme and suggets strongly that at least 99% of the aldolase activity is carried by a homogeneous protein. Half‐denaturation is obtained in 10 min at 78°.

From the point of view of the physiological regulation, we have shown that the aldolase, which is induced by either of the two hexuronates or by gluconate (the metabolism of which involves the same kind of enzymatic cleavage), is controlled by a single structural gene. Extracts of this aldolase which have been induced separately by each of the three compounds, were purified, mixed and chromatographed on DEAE‐Sephadex: the mixture is not dissociable. The kinetics of thermal denaturation of each of these crude extracts are absolutely identical. Finally, the isolation of mutants deficient in aldolase has contributed genetic evidence. These mutants have always simultaneously lost the faculty of metabolizing the three compounds. Spontaneous reversion of these mutants, allowing growth on any of the three substrates, or transduction of any of the three growth phenotypes always leads to the recovery of growth on either of the other two compounds. These different and independent arguments bear out the uniqueness of the aldolase regardless of the inducer employed.
A Deuterium‐Hydrogen Exchange Study of Inhibitor‐Induced Conformational Changes in Ribonuclease A
Authors: Nonnenmacher Geneviève , Viala Eliane , Thiéry Jean Marie , Calvet Paul
Published: March 2005

Deuterium‐hydrogen exchange has been used to detect possible conformational changes accompanying the interaction of ribonuclease A with two competitive inhibitors: cytidine 3′‐phosphate (Cyd‐3′‐P) and cytidine 2′‐phosphate (Cyd‐2′‐P). An improvement of the cryosublimation technique was employed and combined with mass spectrometry measurements of sample deuterium content. Exchange kinetics were followed from 1 min to 24 h, at pH 6.5, and 38 °C (0.1 M NaCl). Two methods were used to present the data: a graphical exponential‐curve‐fitting and a numerical analysis. They both lead to the same conclusions. Four different classes of hydrogens were found. Among them, the one involving the instantaneously exchanged atoms is increased by about ten hydrogens after the binding of these inhibitors; the number of unexchanged hydrogens (within 24 h) is hardly altered in the presence of Cyd‐3′‐P whereas it is decreased by one third after the fixation of Cyd‐2′‐P. These exchange results suggest that both inhibitors induce in ribonuclease A conformational changes which may be correlated with inhibitor strength.
Biosynthetic l‐Threonine Deaminase as the Origin of l‐Serine Sensitivity of Escherichia coli
Authors: Raskó István , Alföldi Lajos
Published: March 2005

Bacteria of some Escherichia coli strains are inhibited temporarily by l‐serine in resuming growth at a minimal to minimal medium transfer when this amino acid is present in high enough concentrations in the new medium. The serine effect can be counteracted by l‐threonine in a competitive manner and by isoleucine and by 2‐oxobutyrate in a noncompetitive manner. It is demonstrated that l‐threonine deamination by the biosynthetic l‐threonine deaminase is inhibited in the presence of l‐serine.
Purification and Properties of DNA‐Dependent RNA Polymerase from Bacillus subtilis Vegetative Cells
Authors: Avila Jesús , Hermoso José M. , Viñuela Eladio , Salas Margarita
Published: March 2005

A purification procedure to prepare highly purified DNA‐dependent RNA polymerase from Bacillus subtilis vegetative cells is described. The enzyme consists of four different subunits, β′, β, σ and α (molecular weights 154000 for β′ and β, 56000 for σ and 43000 for α) in a molar ratio 1:1:1:2. By phosphocellulose chromatography RNA polymerase has been disscociated in σ subunit and core enzyme, containing β′, β and α subunits in a molar ratio 1:1:2; the σ subunit stimulates the activity of core polymerase with ϕ 29 DNA but not with poly[d(A—T)]. The general properties of the enzyme are also described.
On the Bactericidal Action of Levallorphan
Authors: Boquet Paul‐Louis , Devynck Marie‐Aude , Aurelle Hélène , Fromageot Pierre
Published: March 2005

Levallorphan behaves as a bactericidal agent at a concentration which equals or exceeds the minimal value to block Escherichia coli growth; the lethal effect of levallorphan results from its action on the plasmic membrane as indicated by permeability changes and lysis of spheroplasts. On whole cells, Mg2+ and Ca2+ loosely bound to the cell surface prevent the penetration of levallorphan and thus act as protecting agents.
Preparation and Properties of Ribosomal Subunits from Mouse Plasmocytoma Tumors
Authors: Mechler Bernard , Mach Bernard
Published: March 2005

Mouse myeloma ribosomes were prepared without exposure to detergent and their behavior with either decreasing concentrations of Mg2+ or increasing concentrations of KCl examined. The minimal conditions for dissociation into subunits were determined and the properties of the subunits obtained under different conditions compared. Ribosomes which are first freed of nascent chains dissociate into 40 S and 60 S subunits at physiological Mg2+ concentrations (5 mM) and at only 0.3 M KCl (K3 subunits). Although they lack one distinct protein present in low Mg2+ subunits, these K3 subunits reassociate spontaneously to form 80 S particles at lower ionic strength (0.05 M KCl) and are highly active in poly(U)‐directed polyphenylalanine synthesis. This activity is maintained after storage at—70 °C for several months. Subunits obtained at a higher concentration of KCl (0.88 M) are less active and less stable, and they lack at least one protein which is present in K3 subunits. Low Mg2+ subunits are unable to reassociate spontaneously, are inactive in the poly(U) system, have lower sedimentation coefficients, and have lost the 5 S RNA.
Studies on Ferricytochrome c
Authors: Greenwood Colin , Wilson Michael T.
Published: March 2005

Titration of the 695 nm band of ferricytochrome c has been fitted by a model involving four ionic species over the pH range 0–11.

A co‐operative effect between protein denaturants and pH has been observed in the bleaching of the 695 nm band. This effect is discussed in terms of a conformational change and protein titration data suggest this to be of a limited nature.

Circular dichroism and magnetic circular dichroism studies have revealed that the 695 nm absorption band has an associated, well defined dichroism. Magnetic circular dichroic spectra are discussed in terms of haem symmetry.
Wechselwirkungen der Hefe‐Phosphofructokinase mit Dextranblau 2000
Authors: Kopperschläger Gerhard , Diezel Wolfgang , Freyer Renate , Liebe Stefan , Hofmann Eberhard
Published: March 2005

Yeast phosphofructokinase binds to Blue Dextran 2000® with remarkable high affinity. Whereas phosphofructokinase from muscle and erythrocytes also have high affinity to Blue Dextran, aldolase, hexokinase, pyruvate kinase from muscle and glyceraldehyde‐3‐phosphate dehydrogenase show no affinity to this chromophore.

The chromophoric component of Blue Dextran seems to be responsible for these interactions, because chromophore‐free dextran 2000 does not show any affinity to phosphofructokinases.

Blue Dextran immobilized by fixation in cross‐linked polyacrylamide gel can be used as a valuable tool for affinity chromatography of phosphofructokinase. The conditions of binding, namely pH dependence, effects of ionic strength and of enzyme effectors have been studied using gel‐fixed phosphofructokinase. ATP (2–3 mM) but not ITP (up to 5 mM) specifically splits the enzyme chromophore complex.

With non‐inhibitory concentrations of ATP Blue Dextran inhibits the enzyme very strongly showing competition with respect to ATP. No correlation between inhibition by Blue Dextran and fructose‐6‐phosphate concentration could be detected.

A model is proposed for the interactions of Blue Dextran with phosphofructokinase, postulating binding of the chromophore to the ATP binding sites of the enzyme.
Kinetic Study of the Dicarboxylate Carrier in Rat Liver Mitochondria
Authors: Palmieri Ferdinando , Prezioso Girolamo , Quagliariello Ernesto , Klingenberg Martin
Published: March 2005

The rate of uptake in mitochondria as catalyzed by the dicarboxylate carrier of dicarboxylates and, in the presence of N‐ethylmaleimide, of inorganic phosphate, has been analyzed by using the inhibitor stop method.

By comparing the dependence on substrate concentration of the rate of succinate, malonate and malate uptake, it is found that the Km (malate) = 0.23 < Km (malonate) = 0.37 < Km (succinate) = 1.17 mM. The V values, however, are not significantly different for all three dicarboxylates (V∼ 70 μmoles/g protein × min at 9°C).

Succinate, malate and malonate are shown to be competitive with each other in the kinetics of uptake.

The inhibition of the rate of malate uptake by 2–phenylsuccinate is found to be competitive.

Several “permeant” anionic substrates such as acetate, pyruvate, 3‐hydroxybutyrate, glutamate and aspartate have no effect on the rate of dicarboxylate uptake, even when added in large excess.

The rate of malonate uptake is inhibited by 2‐oxoglutarate but unaffected by citrate, while the rates of succinate and malate uptake are decreased by both 2‐oxoglutarate and citrate. Malate is inhibited more than succinate. The inhibition of the rate of malate uptake by citrate and 2‐oxoglutarate is shown to be non‐competitive. This is explained due to a re‐exchange of accumulated malate by citrate or 2‐oxoglutarate.

In the presence of N‐ethylmaleimide, externally added Pi inhibits the rate of dicarboxylate uptake. The Pi inhibition of malate uptake is found to be mixed with a larger share of non‐competitive than competitive inhibition.

The V of the rate of P1 uptake is approximately equal to that of malate uptake in the presence of N‐ethylmaleimide. The K1 is, however, higher for Pi (Ki=Kp= 1.95 mM) than for malate.

It is concluded firstly that the dicarboxylate carrier specifically transports either a dicarboxylate or Pi (but not both together), and secondly that the carrier has two separate binding sites, one specific for Pi and the other specific for the dicarboxylates.
Purification and Properties of a Polynucleotide Ligase from Human Cell Cultures
Authors: Spadari Silvio , Ciarrocchi Giovanni , Falaschi Arturo
Published: March 2005

A polynucleotide ligase has been detected and purified over 300‐fold from human cells cultured in vitro. The enzymic activity is dependent on ATP and Mg2+. The pH curve is bimodal with two optima at pH 7.5 and 8.1. The purified enzyme requires for its activity a heat‐resistant protein factor present in the crude extract.
A Novel Form of Initiation Factors from Escherichia coil which Binds Formylmethionyl tRNA and GTP: “F2 · F3 Complex”
Authors: Groner Yoram , Revel Michel
Published: March 2005

A fraction containing a complex between initiation factors F2 and F3 was isolated from Escherichia coli ribosomes. In contrast to the free factors, this F2 · F3 complex makes a stable ternary association with formylmethionyl‐tRNA and GTP. F2 · F3 complex can be formed on the ribosomes during the process of initiation. It acts preferentially on the 30 S ribosomal subunit to which it confers a much larger affinity for fMet‐tRNA than observed with the free factors. A model for the function of F2 · F3 complex as an intermediate in the initiation cycle is proposed.
Spin Labeling Studies of d‐Glyceraldehyde‐3‐phosphate Dehydrogenase
Authors: Balthasar Wolff
Published: March 2005

d‐Glyceraldehyde‐3‐phosphate dehydrogenase has been reacted with spin labels, which were designed to elucidate the geometry of the active site of the enzyme. Three series of spin labels were used, which represented either substrate and coenzyme analogs or irreversible inhibitors, the separation of the active site directing part from the nitroxide radical being readily adjusted through the length of the intervening methylene chain.

The effect of this variation on the electron paramagnetic resonance spectrum of the labeled protein as well as the effect of coenzyme binding on these spectra have been investigated. All spectra are complex and represent the superposition of at least three different types of spectra. These three types of spectra are correlated to nitroxide radicals in three different types of motion: (a) very fast rotation in a fluid region, (b) medium fast rotation of a hindered radical and (c) slow rotation of an almost immobilized radical. The spin labels differ only in the relative population in the three states related to the three types of motion. Also addition of the coenzyme NAD only alters this relative population. A spin labeled coenzyme analog, which has the nitroxide in the region of the sugar‐nicotinamide bond, is bound to the protein and displays an electron paramagnetic resonance spectrum typical for an immobilized radical. The geometry of the active of d‐glyceraldehyde‐3‐phosphate dehydrogenase is complex and cannot be described as a simple cleft, but instead as a system of narrow and wider spaces, which is changed by the binding of the coenzyme.
γ‐Glutamyl‐glutamic Acid, an Interpeptide Bridge in the Murein of Some Micrococci and Arthrobacter sp.
Authors: Bogdanovsky Daria , Interschick‐Niebler Elfriede , Schleifer Karl‐Heinz , Fiedler Franz , Kandler Otto
Published: March 2005

The mureins of several micrococci and coryneform organisms contain more than one mole of glutamic acid per mole of meso‐diaminopimelic acid (Dpm). The amino acid sequence of these mureins was studied. The peptide subunit consists of Mur‐l‐Ala‐γ‐d‐Glu‐meso‐Dpm‐d‐Ala, where Mur = muramic acid. The γ‐carboxyl group of glutamic acid is substituted in most cases by a glycine amide. The cross‐linkage of the peptide subunits is performed by the dipeptide γ‐d‐glutamyl‐d‐glutamic acid. This peptide is bound by the γ‐carboxyl group of the C‐terminal glutamic acid to the amino group of diaminopimelic acid and to the carboxyl group of d‐alanine of an adjacent peptide subunit by the amino group of the N‐terminal glutamic acid. The mureins of the micrococci studied contain only one mole of amide ammonia per mole of diaminopimelic acid and this is bound to glycine. In the mureins of the coryneform organisms a second mole of amide ammonia was found which is probably bound to diaminopimelic acid. The murein of Staphylococcus lactis CCM 2432 lacks amide ammonia as well as glycine.