bluuebunny/crossref_metadata_2025 huggingface dataset:
An Improved Purification of Cell‐Bound Pullulanase from Aerobacter aerogenes
Authors: Mercier Christiane , Frantz B. May , Whelan William J.
Published: March 2005

This communication describes a method of preparing crystalline Aerobacter aerogenes pullulanase free from α‐amylase. The preparation is suitable for structural studies of starch and glycogen. Two forms of pullulanase have been observed, differing in molecular weight the proportions varying according to the conditions of growth of the organism. The crystalline enzyme is that of lower molecular weight, concentration‐dependent values of 108000 to 145000 being found.

The action of the enzyme on polysaccharide substrates has been compared. The rate of action on branched substrates is most rapid when the outer chains have been shortened by α‐ or β‐amylase. Under comparable conditions, action on rabbit liver glycogen is almost negligible.
The Primary Structure of a Monoclonal Human λ‐Type Immunoglobulin L‐Chain of Subgroup II (Bence‐Jones Protein NEI)
Authors: Garver Fred A. , Hilschmann Norbert
Published: March 2005

Protein NEI, which has a molecular weight of 23 500 and contains 216 amino acids, was isolated from the urine of a patient with multiple myeloma by salt‐precipitation and purified by ion‐exchange chromatography, block electrophoresis and gel filtration. 23 tryptic peptides have been isolated from the aminoethylated protein and 13 chymotryptic peptides from the carboxymethylated protein by ion‐exchange chromatography. Sequence studies have mainly been carried out with the tryptic peptides; the chymotryptic peptides have been used for overlaps in the variable region, since the alignment of the tryptic peptides by homology was not unambiguous.

Protein NEI belongs to subgroup II of the λ‐chains. It has a series of subgroup specific exchanges and a subgroup specific deletion at position 96. Protein NEI differs from all other λ‐chains of subgroup II by possessing a sixth cysteine residue. This additional cysteine residue at position 88 is so far unique, as it is adjacent to an invariant cysteine at position 87. Protein NEI also contains a carbohydrate moiety attached to an aspartic acid or asparagine (Asx) residue at position 93, one of the hypermutable areas of the variable part.

Furthermore we conclude by comparison with other λ‐chains that all completely elucidated λ‐chains can be ordered into four subgroups. The subgroups and linkage groups within subgroups are in complete agreement with an evolutionary origin of antibody variability and are incompatible with somatic hypermutation models.
C‐Terminal‐Sequence Determination by Carboxypeptidase C from Orange Leaves
Authors: Tschesche Harald , Kupfer Sigrid
Published: March 2005

Carboxypeptidase C isolated from orange leaves has been investigated for its use in C‐terminal sequence determinations. The sequence of the first six C‐terminal residues of the denatured protein ribonuclease could be deduced unequivocally from a time‐release plot of an incubation mixture containing 1 mole % of the enzyme. No release of free amino acids by autodigestion of the enzyme could be detected after prolonged periods of incubation. Three different peptides have been degraded demonstrating sufficient and comparable rates of release of all protein amino acids, including proline. Thus the enzyme reveals a broad specificity for acidic, neutral and basic amino acids and combines the activity and specificity of the pancreatic carboxypeptidases A and B, but in addition releases the imino acid proline.
Interaction of RNA Polymerase from Escherichia coli with DNA Selection of Initiation Sites on T7 DNA
Authors: Dausse Jean‐Pierre , Sentenac André , Fromageot Pierre
Published: March 2005

Rifampicin‐resistant complexes were formed on T7 DNA at various molar ratios of RNA polymerase to DNA using the asymmetry of transcription as a criterion of specific initiation. Totally asymmetric transcription was only obtained with less than four RNA polymerases per T7 DNA molecules. Under these conditions incorporation of [γ‐32P]ATP or [γ‐32P]GTP indicated that there is a strong promoter site initiating with ATP on the H strand of the T7 genome. At higher enzyme : DNA ratios (≥4) rifampicin‐resistant RNA synthesis increases with the preincubation temperature of the binary enzyme · DNA complex, more chains are initiated, both by ATP and GTP, and transcription also occurs on the L strand of the DNA.
Molecular Characterization of Pullulanase from Aerobacter aerogenes
Authors: Eisele Bernhard , Rasched Ihab R. , Wallenfels Kurt
Published: March 2005

By means of ultracentrifugal studies and acrylamide gel electrophoresis in the presence of sodium dodecylsulfate, pullulanase of Aerobacter aerogenes was shown to be a single polypeptide chain molecule with a molecular weight of 143000. Cyanogen bromide cleavage yields 18 to 20 individual bands in the electrophoresis. Also the amino acid analysis suggests a chemical unit with a minimum molecular weight of 110000 to 120000.

Previous results, indicating lower molecular weights and a subunit structure of the enzyme are explained by partial proteolytie degradation of the enzyme. This is shown by dodecyl sulfate electrophoresis under different conditions.

The shape factors are calculated from the hydrodynamic data and indicate an oblate rotational ellipsoid.
Biological Function of the Ammonia‐Induced Inactivation of Glutamine Synthetase in Escherichia coli
Authors: Schutt Hermann , Holzer Helmut
Published: March 2005

In the first 15–30 see after addition of 10 mM NH4+ to Escherichia coli cells, one observes a 20‐fold increase in the concentration of glutamine, an approximate one‐third reduction in the stationary concentration of glutamate, an approximate 90% decrease in the concentration of ATP and a decrease in the activity of glutamine synthetase to a few percent of the initial value. In the subsequent 30 see, glutamine synthetase remains inactive, glutamine concentration decreases, glutamate concentration increases 2 to 3‐fold, and the ATP concentration rises slowly. The above‐mentioned changes after addition of NH4+, together with previous observations on properties of purified glutamine synthetase and adenylyl transferase, make the following sequence of events probably: NH4+ enters the cells and is quickly incorporated into glutamate yielding glutamine, with a concomitant utilization of ATP. The accumulated glutamine stimulates the adenylyl transferase resulting in an inactivation of glutamine synthetase. Thus, 15 sec after addition of NH4+, further synthesis of glutamine is prevented. Consequently, glutamate concentration increases, glutamine concentration decreases, and ATP concentration increases. On the basis of these findings and conclusions, the following biological functions of NH4+ inactivation of glutamine synthetase are discussed: (a) prevention of a too high level of glutamine and (b) prevention of a sustained decrease in the ATP concentration which would be harmful to the cell.
Étude des interactions entre l'acide désoxyribonucléique et le polypeptide alterné (l‐lysyl‐l‐alanyle)n
Authors: Privat Jean‐Paul , Spach Gérard , Leng Marc
Published: March 2005

The interactions between DNA and sequential polypeptide poly(L‐lysyl‐L‐alanyl) have been studied as a function of salt concentration. Most of the work was performed with a polypeptide of molecular weight 8000. The reaction is observed to be cooperative when NaCl concentration is above 0.1 M and almost completely selective for AT‐rich fractions of heterogeneous samples. This selectivity is reversed in the presence of tetramethylammonium ions. When NaCl concentration is below 0.1 M, no preferential interaction is observed. The main result is that this polypeptide shows the same preferential interaction with DNA as polylysine does. The presence of adjacent lysyl residues is not therefore required to induce the selectivity relative to the DNA‐base composition.
Kinetic Study of the Activation Process of β‐Galactosidase from Escherichia coli by Mg2+
Authors: Tenu Jean‐Pierre , Viratelle Odile M. , Yon Jeannine
Published: March 2005

The action of Mg2+ ions on β‐galactosidase activity has been studied under strictly controlled conditions with different substrates. The activation effect does not depend on the substrate.

In the absence of Mg2+ ions, a residual activity has been found and the catalytic properties of Mg2+‐free enzyme have been determined.

The activation process induced by Mg2+ is a slow process, the rate of which depends on the Mg2+ concentration. The deactivation process obtained by adding EDTA is also a slow process. Activation by Mg2+ is not a cooperative process. The apparent dissociation constant of the Mg2+ enzyme complexe is rather small, 0.65 ± 0.05 μM.

The kinetics of activation and deactivation have been studied with phenyl galactoside as substrate.

The results indicate that activation by Mg2+ operates through a binding with free enzyme. This binding involves at least two steps: the first and more rapid one is a single metal‐protein binding, the following slow step probably involves a conformational change of the enzyme induced by the metal binding.
Purification and Properties of Rat‐Liver Thioredoxin
Authors: Larson Göran , Larsson Agne
Published: March 2005

Rat liver thioredoxin, which could be reduced nonenzymatically by dithiothreitol, was identified by its ability to function as hydrogen donor in the conversion of ribonucleotides to deoxyribonucleotides catalyzed by ribonucleotide reductase both from the same tissue and from Escherichia coli. The latter enzyme was used to detect the reduced form of the thioredoxin during its purification which involved treatment at pH 5, chromatography on Sephadex G‐50, heat treatment, and DEAE‐cellulose chromatography. The isolated material was approximately 50% pure as determined by polyacrylamide electrophoresis. Liver thioredoxin is a heat‐stable protein of low molecular weight.
Preparation and Characterization of Fragments from Yeast tRNAphe
Authors: Harbers Klaus , Thiebe Rainer , Zachau Hans G.
Published: March 2005

The preparation of 13 fragments from yeast tRNAPhe is described. The fragments were prepared by partial digestion with T1‐, T2‐, N1‐, or pancreatic RNAase, by endonuclease and subsequent exonuclease digestion and by a combination of a chemical chain scission with endoor exonuclease degradation. In order to obtain high yields of fragments, in each case several conditions of digestion were investigated by analytical disc electrophoresis. The fragments were purified by column chromatography and preparative gel electrophoresis. They were characterized by their mobility in disc electrophoresis and by analysis of the mono‐ and oligonucleotides after complete digestion with T1‐RNAase.

In the presence of MgCl2 three regions of tRNAPhe seem to be particularly exposed to an attack by endonucleases. These regions are the anticodon loop, the dihydrouridine loop and the nucleotides at the 3′‐end of the molecule. The T‐Ψ‐C loop of tRNAPhe seems to be less exposed. Fragments resulting from a cleavage in this region could be obtained in relatively good yield by digestion with N1‐RNAase or T1‐RNAase, the latter one in the presence of ethidium bromide instead of MgCl2.

The specificity of the various nucleases towards yeast tRNAPhe is briefly discussed.
Stereochemical Course of Decarboxylation of S‐Malate on Malic Enzyme
Authors: Clifford Kenneth H. , Cornforth John W. , Donninger Cyril , Mallaby Richard
Published: March 2005

A preparation of malic enzyme was made from chicken liver.

The enzyme was incubated, in tritiated buffer containing NADP, with 2S, 3S‐[2,3‐2H2]malate. A third malate, 2S, 3R‐[3‐3H1]malate, was incubated with the enzyme, NADP, and unlabelled buffer. Conditions were chosen so that the initially formed pyruvate was converted into lactate, which was isolated.

The lactate from each of the three incubations was oxidized chemically to acetate, which was purified.

Each specimen of acetate was assayed for chirality by the previously established method of conversion into S‐malate by condensation with glyoxylate on malate synthase, and measuring the fractional loss of tritium on incubation of the S‐malate with fumarate hydratase.

In this way, the acetate derived from 2S, 3R‐[3‐2H1]malate was shown to have S chirality and the acetate derived from 2S, 3S‐[2,3‐2H2]malate was shown to have R chirality. As expected, the acetate from 2S, 3R‐[3‐3H1]malate was found non‐chiral.

It is deduced that when S malate is decarboxylated on malic enzyme, carboxyl is replaced by hydrogen without net change of configuration. This confirms a result obtained by Rose in 1970 by different methods.
Multiple Phases in the Reduction of Xanthine Oxidase by Substrates
Authors: Swann John C. , Beay Robert C.
Published: March 2005

A sytematic study has been made using electron paramagnetic resonance spectroscopy of the effects of enzyme concentration and relative proportions of active and inactivated enzyme on the anaerobic reduction of xanthine oxidase at pH 8.2 by the substrates xanthine, purine and NADH. Changes in intensity of two molybdenum (V) signals (earlier designated Rapid and Slow) and iron‐sulphur and flavin radical signals were monitored over the time‐range 0.1 sec to 2 days. A few measurements with the inhibitory substrate allopurinol were also made.

Reduction can occur in as many as four phases, all of which are clearly seen with xanthine as substrate. The Rapid type of signals from active enzyme first appear in a time comparable to the turnover time (phase I), then decrease in intensity over a somewhat longer time (phase II). This is followed by a further increase in intensity over periods of up to a few hours (phase III), to levels higher than those attained in phase I. Finally with a half‐life of several hours (phase IV) the Slow signal, which is known to be derived from inactivated xanthine oxidase, appears also. Fewer phases can be clearly distinguished with the other substrates.

In accordance with other work, results are interpreted in terms of redox equilibria among different enzyme species with molybdenum signal intensity controlled by interconversions between the paramagnetic Mo(V) and the diamagnetic Mo(IV) and Mo(VI) oxidation states. The observed kinetics indicate that Mo(V) produced in the catalytic phase I is “over reduced” in phase II to Mo(IV) in a bimolecular reaction in which inactivated enzyme is not involved and for which the reducing agent may possibly be a substrate radical. This Mo(IV) of active xanthine oxidase is then slowly reoxidised to Mo(V) by inactivated enzyme, which remains inert in the catalytic phase but undergoes reduction in the slower phases. The reducing equivalents appear in the inactivated form, first in the flavin and iron‐sulphur and then in the molybdenum. However, with NADH, where catalytic reduction is known to occur via flavin instead of via molybdenum, both active and inactivated enzymes are reduced simultaneously, thus confirming that the difference between these enzyme forms is minimal.
Function of Met‐tRNAMetf and Met‐tRNAMet in Peptide‐Chain Elongation in Cell‐Free Systems from Mouse‐Liver and Ascites‐Tumor Cells
Authors: Drews Jürgen , Grasmuk Hans , Weil Rudolf
Published: March 2005

The two methionine‐accepting tRNA species from mouse ascites tumor cells were investigated with respect to their function in peptide chain elongation, using a mouse liver polysome system and a T‐factor dependent ribosomal‐binding assay. In support of results published previously it was found that in a non‐initiating cell‐free system Met‐tRNAMetf inserts methionine into internal positions of nascent peptide chains, even in the presence of Met‐tRNAMet.

Correspondingly, T factor from ascites tumor cells stimulated the binding of both methionine‐accepting tRNAs to washed ribosomes. When Met‐tRNAMet and Met‐tRNAMetf were introduced together into the ribosomal binding assay, a strong preference for the T‐factor‐induced attachment of Met‐tRNAMet became visible. Formylated initiator tRNA was not recognized by T factor Whereas the ribosomal binding of formylated or unformylated initiator tRNA promoted by initiation factors functioned equally well with poly(A,U,G) and the triplet ApUpG, the T‐factor‐induced attachment of both Met‐tRNAMet species required the presence of the polynucleotide.

Competition experiments with high concentration of Val‐tRNAVal and Met‐tRNAMetf revealed the occurrence of a partial mutual interference of these two tRNA species during chain elongation.

The magnitude of this effect, however, was not sufficient to attribute the function of Met‐tRNAMetf in chain elongation to miscoding. No competition was observed between Val‐tRNAVal and Met‐tRNAMet.
Active and Inactive Forms of Pyruvate Dehydrogenase in Rat Liver
Authors: Wieland Otto H. , Patzelt Christoph , Löffler Georg
Published: March 2005

Active form and total activity of pyruvate dehydrogenase were measured in rat liver homogenates. The activity obtained immediately after homogenization was considered to represent the active form, i.e. dephospho pyruvate dehydrogenase, originally present in the liver. Enzyme activity found after incubation of the homogenate with Mg2+ and partially purified pyruvate dehydrogenase phosphatase from pig heart, whereby inactive dehydrogenase is dephosphorylated to give the active form, was regarded as total pyruvate dehydrogenase activity. In the livers from normal fed rats, the active form accounts for only one sixth of total pyruvate dehydrogenase activity. This differs from other tissues such as heart muscle, kidney, brain and adipose tissue where about two thirds of total pyruvate dehydrogenase are present as the active form (Wieland et al., 1971; Siess et al., 1971).

To elucidate the possible role of pyruvate dehydrogenase interconversion in the regulation of pyruvate metabolism in liver, the activity of active and total pyruvate dehydrogenase were determined in livers of rats subjected to various metabolic conditions. After fasting and refeeding with glucose as well as after treatment with nicotinic acid or insulin there were significant changes of active dehydrogenase activity without gross alterations of total activity. In general, metabolic states associated with decreased plasma fatty acids resulted in an increase of active pyruvate dehydrogenase whereas a rise in plasma fatty acids led to a lowering. It is concluded that in liver, similar to heart muscle and kidney (Wieland et al., 1971), fatty acids play an important role in the control of pyruvate dehydrogenase interconversion. The significance of this mechanism for the regulation of pyruvate metabolism in liver relative to the feedback control of pyruvate dehydrogenase by acetyl‐CoA is discussed.
Study of the Thermal‐Denaturation Mechanism of Hen Egg‐White Lysozyme through Proteolytic Degradation
Authors: Matthyssens Gaston E. , Simons Gaston , Kanarek Louis
Published: March 2005

The all‐or‐none character for denaturation of gloublar proteins was studied, in the case of hen egg‐white lysozyme, through pepsin digestion of the enzyme at pH 2.0. Finding complete agreement of the thermodynamic values obtained through proteolysis and through direct analysis of the thermal transition curve, we conclude that, in the case of lysozyme, the thermal native ⇄ denatured transition is of the all‐or‐none type. In addition it was possible to obtain accurate ΔH° and percentage denaturation values in the low temperature range. Studies on the enzymatic degradation of lysozyme by pepsin, suggested that at different stages of the digestion, there is no indication of formation of any stable high‐molecular weight intermediates possessing enzymatic activity. It is therefore concluded that the breakdown of lysozyme proceeds according to a typical one‐by‐one mechanism.
Studies on there‐Citrate Synthase Reaction
Authors: Gottschalk Gerhard , Dittbrenner Susanne , Lenz Helmut , Eggerer Hermann
Published: March 2005

re‐Citrate synthase catalyzes the hydrolysis ofR‐citryl‐CoA andR‐malyl‐CoA. TheSdiastereomers are not attacked. The stereospecificity of the hydrolase activity is an agreement with the stereochemical course of citrate synthesis as catalyzed by this enzyme. The cooperative action of 2‐oxoglutarate and metal ions (Co2+) induces maximum acetyl‐CoA enolase activity ofre‐citrate synthase.R‐Malate is not effective in this reaction. SinceS‐malate is the best inducer of the enolase activity ofsi‐citrate synthase this suggests differences in the architecture of the active sites of these enzymes.

Whenre‐citrate synthase is incubated with citrate and coenzyme A in tritiated water, tritium is incorporated into citrate. This indicates that there‐citrate synthase reaction is reversible.
Occurrence and Particle Character of Aminoacyl‐tRNA Synthetases in the Post‐Microsomal Fraction from Rat Liver
Authors: Vennegoor Claudia , Bloemendal Hans
Published: March 2005

A post‐microsomal fraction (Fraction X) was isolated from cell sap from rat liver by centri‐fugation at 105000 ×g for 15 h. The activity of a number of aminoacyl‐tRNA synthetases was considerably higher in the resulting pellet than in the supernatant. These enzymes (glutaminyl‐, isoleucyl‐, leucyl‐, lysyl‐, and methionyl‐tRNA synthetase) were purified by gel filtration on Sephadex G‐200, chromatography on DEAE‐Sephadex A‐50 and on hydroxyapatite. As compared with crude cell sap a 120‐ to 170‐fold purification was achieved.

In all purification steps, including additional centrifugation on sucrose gradients or isoelectric focusing, the major peak of activity of the five enzymes coincided.

In the electron microscope circular and rectangular particles, 11–14 nm in size, could be visualized. From these findings we suggest that in rat liver cells, if not in all animal cells, part of the aminoacyl‐tRNA synthetases occur in a particulate state.
Nuclear‐Magnetic Resonance and Optical‐Spectroscopic Studies of Conformation and Interactions in the Cleaved Halves of Histone F2B
Authors: Bradbury E. Morton , Cary Peter D. , Crane‐Robinson Colyn , Riches Paul L. , Johns Ernest W.
Published: March 2005

In a previous study it was proposed that salt‐induced intrachain and interchain interactions in histone F2B were located largely in the carboxyl half of the molecule. To confirm this proposal F2B was cleaved at the methionines 59 and 62 to give the two halves of the molecule. Optical rotatory dispersion studies show that the induced secondary conformation is located entirely in the carboxyl half of the molecule and that the amino half of the molecule remains in the random coil from at all ionic strengths. Nuclear magnetic resonance studies show that the residues whose mobilities are restricted by the secondary conformation and interchain interactions lie between 66 and 102 in the carboxyl half. Although the amino half of the molecule remains in the random coil conformation throughout the range of ionic strengths 0 to 2.5 M NaCl, between 1.5 and 2.5 M NaCl nuclear magnetic resonance studies show that interchain interactions take place in the segment 30 to 50. The behaviour of the whole molecule is the average of the behaviours of its constituent halves showing that the different sections of the molecule are probably functionally independent.
Thermodynamics and Kinetics of Base‐Stacking Interactions
Authors: Pörschke Dietmar , Eggers Frieder
Published: March 2005

The thermodynamics and kinetics of the self‐association of N6, N9‐dimethyladenine has been studied as a model system for base stacking. Molal osmotic coefficients have been determined by vapor pressure osmometry in aqueous solutions at four different temperatures. These data, indicating a very high degree of association much beyond the dimer stage, are consistent with a series of consecutive equivalent association processes. The analysis is performed with an isodesmic model, which allows for cooperativity of the first binding step. Cooperativity can almost be neglected in the present case; the thermodynamic parameters for the formation of stacks are ΔH°=– 8.7 (° 1.5) kcal/mol and ΔS°=– 21.6 cal/K resulting in a stability constant K= 60 at 20°C.

The kinetics of base stacking has been investigated by means of sound absorption techniques in the 10–100 MHz frequency range. The excess absorption profiles are only slightly broader than theoretically expected for a single relaxation process. The data can be explained by an isodesmic kinetic model. Formation of stacks proceeds with a rate constant of about 109 M−1 sec−1 and an activation enthalpy of + 6 kcal/mol. Evaluation of the amplitudes leads to a volume change of 6.8 ml (at 25°C) per mol of stacking sites. The volume change decreases with increasing temperature. Optical measurements in a pressure cell show that the volume decreases with increasing number of stacks.

These results are discussed with respect to current theories of base stacking. Both ΔH and ΔV found for the stacking equilibrium are in contrast to the thermodynamic parameters of hydro‐phobic interactions according to the standard view. The present data demonstrate a characteristic difference of hydrophobic interactions between unpolar, unpolarisable particles like simple hydrocarbons and between compounds like nucleic acid bases with high polarisability and a dipole structure.
Studies on the Active Site of IRC‐50 Arvin, the Purified Coagulant Enzyme from Agkistrodon rhodostoma Venom
Authors: Collins J. Peter , Jones J. Gareth
Published: March 2005

A number of the properties of the active site of IRC‐50 arvin, a coagulant enzyme from Agkistrodon rhodostoma, have been studied. The results indicate that the enzyme is in many ways similar to the mammalian serine proteinases.

IRC‐50 arvin will hydrolyse α‐N‐benzoyl‐L‐arginine esters and a study of the rates of hydrolysis of a number of these esters indicates that the hydolysis may proceed via a common acylenzyme intermediate.

IRC‐50 arvin is inactivated by di‐isopropylfluorophosphate and phenylmethanesulphonyl fluoride but at much lower rates than the mammalian serine proteinases.

Both the catalytic activity and rate of inactivation of the enzyme by di‐isopropylfluorophosphate and phenylmethanesulphonyl fluoride are dependent on the ionisation of a group with pK(app)∼ 7.0; this group is probably a histidine residue.

Evidence is presented which suggests that the esterase and coagulant activity of the enzyme are associated with the same active site.

Titration of the active site of the enzyme with p′‐nitrophenol p′‐guanidinobenzoate demonstrates that IRC‐50 arvin has one active site per mole, based on a molecular weight of 55000.
Enzyme Activities Related to Fatty‐Acid Synthesis in Liver and Adipose Tissue of Rats Treated with Triiodothyronine
Authors: Diamant Sophia , Gorin Erela , Shafrir Eleazar
Published: March 2005

The effect of triiodothyronine treatment on fatty acid synthesis was investigated in liver and adipose tissue of fed and 48‐h fasted rats. In the liver, the thyrotoxic state was accompanied by an increased incorporation of [1‐14C]acetyl‐CoA to fatty acids and a rise in the activity of acetyl‐CoA carboxylase and fatty acid synthetase, both in the fed and fasted rats. The activity of glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase, NADP‐malate dehydro‐genase and ATP citrate lyase was also enhanced by triiodothyronine treatment. Liver malate and citrate content was not changed in fed thyrotoxic rats, but was increased in fasted triiodo‐thyronine‐treated animals. Palmitate oxidation was increased in the liver of the same animals, either fed or fasted.

The rise in acetyl‐CoA carboxylase activity due to triiodothyronine administration was apparent when assayed both before and after citrate activation in vitro. Maximal activity of this enzyme in the presence of citrate was reached earlier in the liver of triiodothyronine‐treated rats.

In adipose tissue, triiodothyronine treatment induced an increase in the activity of acetyl‐CoA carboxylase, fatty acid synthetase and in glucose‐6‐phosphate dehydrogenase and 6‐phosphogluconate dehydrogenase. The activity of NADP‐malate dehydrogenase and ATP citrate lyase was slightly but insignificantly increased. Malate and citrate levels in adipose tissue were unchanged by triiodothyronine treatment.

It is suggested that the triiodothyronine‐induced simultaneous stimulation of opposing pathways (the synthesis of fatty acids and their oxidation) creates a futile metabolic cycle which contributes to the general energy waste and thermogenesis characteristic of thyrotoxicosis.
The DNA‐RNA Hybridisation of [3H]Methylated Mouse‐Globin mRNA in Conditions of RNA Excess
Authors: Morrison Marcelle R. , Paul John , Williamson Robert
Published: March 2005

Milligram quantities of mouse reticulocyte 9‐S RNA, which has been demonstrated to contain the mRNA for globin, have been isolated using the zonal ultracentrifuge. In vitro methylation of the 9‐S RNA with dimethylsulphate has allowed investigation of its hybridisation to mouse DNA. The RNA saturates between 0.084% and 0.15% of the DNA.

The hybridisation is rapid, being complete in under 3 min. Repeated hybridisations of the RNA eliminate the possibility of the high saturation levels being due to the presence of contaminating traces of other RNA species. Similar saturation levels are found for mouse sperm DNA and total mouse embryo DNA. Slightly higher values are found for 14‐day mouse embryo liver DNA. There was no hybridisation with Escherichia coli DNA.

The 9‐S RNA formed hybrids which had a Tm 28 °C lower than that predicted from the 50.7% (G + C) content of the RNA. The size of the hybridised RNA was small after elution from RNAase treated filters, the largest fragments being approximately 60–120 nucleotides in length.

Control experiments with [3H]methylated 32P‐labelled 5‐S ribosomal RNA showed that, although the saturation values obtained for hybridisation of methylated and non‐methylated RNA are identical, the methylated RNA‐DNA hybrid melts at a lower temperature. This difference in itself cannot, however, explain the low Tm of the mRNA‐DNA hybrids.
Steroid Metabolism in Rats Given [1‐2H2]Ethanol
Authors: Cronholm Tomas
Published: March 2005

The rate of excretion of different corticosterone metabolites in rat bile after administration of [1‐2H2]ethanol, and the transfer of deuterium to these metabolites have been studied by gas chromatography‐mass spectrometry. The excretion of corticosterone metabolites increased after injection of ethanol, but the relative amounts of different steroids remained essentially constant. The deuterium excess in 3α,11β, 15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one and 3β(and 3α),11β,21‐trihydroxy‐5α‐pregnan‐20‐one in female rats and of 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one, 5α‐pregnane‐3β‐(and 3α),11β,20β,21‐tetrol and 5β‐pregnane‐3α,11β,20β,21‐tetrol in male rats varied between 7 and 20 atoms per cent. Only monodeuterated molecules were found. All the deuterium in 3β,11β,21‐trihydroxy‐5α‐pregnan‐20‐one was present in the 3α‐position, and little or no deuterium was found in the 20α‐position of the corresponding 20β‐hydroxysteroid.

When 4‐androstene‐3,17‐dione was given to male bile‐fistula rats, 3α‐hydroxy‐5α‐androstan‐17‐one, 3β‐hydroxy‐5α‐androstan‐17‐one, and 3α‐hydroxy‐5β‐androstan‐17‐one, and various hydroxylated metabolites were excreted in bile. Of the former compounds, the 3α,5β‐epimer was predominant in the glucuronide fraction, whereas the 3β,5α‐epimer was the major epimer in the sulphate fraction. During metabolism of [1‐2H2]ethanol the 3β‐ and 3α‐positions of these compounds contained 5–20 atoms per cent of deuterium. No deuterium was found in the 5α‐ or 5β‐positions.

The results indicate that the coenzyme pool(s) used in different reductions at C‐3 is metabolically related to NADH formed in the alcohol dehydrogenase reaction. Since little or no deuterium was found at C‐5 and C‐20 other coenzyme pools are probably used for the reductions of these positions.
The Oxidation of Fatty Acids by Rabbit Reticulocytes and Their Isolated Mitochondria
Authors: Schultze Maria , Rost Johann , Augustin Wolfgang , Gellerich Frank , Rapoport Samuel
Published: March 2005

The oxidation of fatty acids by rabbit reticulocytes and their isolated mitochondria has been studied with [1‐14C]octanoate, [U‐14C]palmitate and [U‐14C]oleate.

The fatty acids, particularly octanoate, represent good respiratory substrates.

They are activated both by carnitine‐dependent and carnitine‐independent systems, the former predominating for palmitate, the latter for octanoate.

The oxidation of palmitate presents a nearly hyperbolic substrate‐saturation curve. The limitation lies presumably in the capacity of the activating enzymes for the long‐chain fatty acids on in their translocation through the mitochondrial membrane. With octanoate no indication of saturation was observed. It may contribute as much as 65% of the respiratory substrate.

Oxaloacetate exerts a double effect on the oxidation of fatty acids. In low concentrations it activates their oxidation as an acceptor for acetyl‐CoA; in high concentrations it serves itself as a source of acetyl‐CoA and produces a competitive inhibition.

It is estimated that fatty acids furnish about 10% of the respiratory substrate of reticulocytes under physiological conditions.
The Interaction of the Bowman‐Birk Inhibitor with Trypsin and Chymotrypsin
Authors: Steiner Robert F.
Published: March 2005

The combination of trypsin and chymotrypsin with the Bowman‐Birk inhibitor results in characteristic changes in absorption and fluorescence emission spectra, indicating an alteration of the microenvironments of the enzyme chromophores as a consequence of the interaction. One of the two tyrosine groups of this inhibitor is accessible to nitration with tetranitromethane. The pK of the resultant nitrotyrosine group is shifted to lower values upon combination of the inhibitor with trypsin or chymotrypsin, suggesting a proximity to the zone of contact.

The reaction of trypsin with Bowman‐Birk inhibitor is biphasic, an initial second‐order combination being followed by a slower first‐order change. The rate constant for dissociation of the complex increases at acid pH. The dissociation has a high activation energy. Bowman‐Birk inhibitor which has been converted to the modified form by prolonged incubation with catalytic amounts of trypsin forms a complex with trypsin which has the same rate constant and activation energy for dissociation as the complex formed by the native inhibitor.
The Dual Genetic Control of Ornithine Carbamoyltransferase in Escherichia coli
Authors: Legrain Christiane , Halleux Paulette , Stalon Victor , Glansdorff Nicolas
Published: March 2005

In Escherichia coli K 12 either of the two genes argF and argI is able to produce a functional ornithine carbamoyltransferase. When both are active in the same cell the two gene products interact to form a family of four hybrid trimeric isoenzymes of undistinguishable molecular weights, a situation presently unique in bacteria where very little genetic redundancy seems to occur. In E. coli B and W only the I gene product is found. Evolutionary implications of the structure and the genetic control of ornithine and aspartate carbamoyltransferase are discussed.
Phospholipid Metabolism of Stimulated Lymphocytes
Authors: Resch Klaus , Ferber Ernst
Published: March 2005

Rabbit lymphocytes from the mesenteric lymph nodes were stimulated by phytohemagglutinin, concanavalin A and goat‐anti‐rabbit‐immunoglobulin serum or antibody. The synthesis of lecithin de novo was followed by incorporation of [14C]choline. The turnover of the fatty acid moieties of individual lipids was measured by conversion of [14C]oleate as well as [14C]acetate. In resting lymphocytes the turnover of the long chain fatty acids of lecithin exceeded the synthesis de novo by a factor of 100. All three stimulants increased choline and oleate uptake to a similar extent starting very early after stimulation. Enhanced oleate incorporation began within 30 min after addition of a stimulant. Synthesis of long chain fatty acids and neutral lipids from acetate was raised similarly (between two‐ and six‐fold) when the lymphocytes were activated by the three stimulants. In contrast incorporation of acetate into phospholipids was much more increased during activation with phytohemagglutinin or concanavalin A than during activation with antiimmunoglobulin, resulting in stimulation indices of 32 (phytohemagglutinin, concanavalin A) and 9 (anti‐immunoglobulin) for lecithin. The relevance of the observed rapid changes in the membrane phospholipids for the mechanisms of lymphocyte activation is discussed.
Protection of Escherichia coli Ribosomes against Streptomycin by Purified Initiation Factors
Authors: Lelong Jean‐Claude , Cousin Marie‐Anne , Gros François , Miskin Ruth , Vogel Zvi , Groner Yoram , Revel Michel
Published: March 2005

The effect of the purified initiation factors F1, F2 and F3 on the binding of radioactive dihydrostreptomycin to 30‐S subunits and 70‐S sensitive ribosomes of Escherichia coli was studied in the absence of other substrates.

Neither of the three factors alone inhibits dihydrostreptomycin binding on 30‐S subunits; the same applies to 70‐S ribosomes except that F2 alone exerts a partial inhibitory effect when present in large excess.

In contrast, certain associations of factors appear very efficient in preventing dihydrostreptomycin from binding on ribosomes: thus a mixture of F1 with either F2 or F3 strongly inhibits radioactive dihydrostreptomycin binding to 70‐S ribosomes; the same is observable with 30‐S subunits although the F1– F3 association exerts the most potent effect, this effect being even more pronounced by the further addition of F2.

The competing effect exerted by factors toward streptomycin binding does not manifest itself at low temperatures. Elongation factors have no effect.

In line with these results, it was also found that combination of the three initiation factors can protect ribosomes against the streptomycin‐induced release of initiator tRNA which is bound in the presence of natural messenger‐RNA and GTP.

The observed competition between initiation factors and streptomycin for the ribosomes indicates that the factors either bind or induce conformational changes in the P10 region of the ribosome.
Regulation of Pyruvate Metabolism in Rat‐Liver Mitochondria by Adenine Nucleotides and Fatty Acids
Authors: Stucki Jörg W. , Brawand Fritz , Walter Paul
Published: March 2005

Fluxes of pyruvate through the pyruvate dehydrogenase and the pyruvate carboxylase reactions in intact rat liver mitochondria were measured under various experimental conditions. When amounts of pyruvate not limiting for pyruvate dehydrogenase were present in the incubation medium, pyruvate carboxylase was found to be regulated essentially by the ATP:ADP ratio within the mitochondrial matrix. No direct correlation between pyruvate carboxylase activity and the level of intramitochondrial acetyl‐CoA could be observed under these conditions.

When the ATP:ADP ratio within the matrix was decreased by elevation of the ADP concentration in the medium, a strong inhibition of the pyruvate carboxylase was observed. After addition of oleate to the medium this low ATP:ADP ratio within the matrix was increased and the inhibition of pyruvate carboxylase was partially reversed. It is demonstrated that this effect of oleate is probably caused by an inhibition of the adenine nucleotide translocase.

The effects of oleate on pyruvate carboxylase were compared to those of octanoate. In contrast to oleate, octanoate diminished the pool size of the exchangeable adenine nucleotides, ATP and ADP, within the matrix and oxidative phosphorylation was stimulated. The reduced level of intramitochondrial ADP and the increased rate of oxidative phosphorylation elevated the ATP:ADP ratio within the matrix and partially reversed the inhibition of the pyruvate carboxylase by ADP. Thus, both fatty acids reversed the inhibition of pyruvate carboxylase by ADP via changes in the intramitochondrial ATP:ADP ratio but they did this by differing mechanisms.

Octanoate was found to be much more rapidly activated and oxidized within the matrix than oleate. Furthermore, octanoate strongly inhibited pyruvate oxidation whereas with oleate this effect was much smaller.

The physiological implications of these findings are discussed in relation to the stimulation of gluconeogenesis by fatty acids in the liver.
3′:5′‐Cyclic Nucleotide Phosphodiesterases in Rat Tissues
Authors: Campbell Mary T. , Oliver Ivan T.
Published: March 2005

The rates of enzymatic hydrolysis of cyclic AMP, cyclic GMP, cyclic IMP, cyclic UMP, cyclic CMP and cyclic dTMP (all 3′:5′‐phosphates) have been assayed in several rat tissues. Subcellular distribution studies indicated that, in all tissues except brain and heart, most of the enzymatic activity is confined to the soluble cell fraction.

The purine cyclic nucleotides are the preferred substrates for soluble 3′:5′‐cyclic nucleotide phosphodiesterase in all tissues. Cyclic AMP is a slightly better substrate than cyclic GMP and cyclic IMP in most tissues. However, spleen, lung and erythrocytes hydrolyse cyclic AMP 2–3 times faster than the other purine cyclic nucleotides. Hydrolysis of the cyclic pyrimidine nucleotides was very low or not detected in most tissues. The exceptions were cyclic UMP and cyclic dTMP hydrolysis by brain, and cyclic dTMP hydrolysis by liver.

Polyacrylamide gel electrophoresis resolved the soluble cyclic nucleotide phosphodiesterase activity into a number of components. Two components, designated B and C, arc present in all tissues except brain although there are quantitative differences between tisues. These two components appear to be specific for purine cyclic nucleotides. A third form, A, is present in spleen, lung and erythrocytes. It hydrolyses all the cyclic nucleotides except cyclic CMP, although cyclic AMP is the best substrate. However, this component also has 5′‐nucleotidase activity which may indicate that A is not specific for 3′:5′‐cyclic nucleotides. A fourth component D, detected only in liver, hydrolyses cyclic AMP and cyclic dTMP at comparable rates.

The electrophoretic patterns obtained for brain phosphodiesterase differ from those of all other tissues investigated and suggest that the enzyme(s) probably has markedly different properties from the cyclic nucleotide phosphodiesterases of other tissues.
Unphysiological Breakdown of Fast‐Labelled RNA by Actinomycin D in Primary Chick Fibroblasts
Authors: Scholtissek Christoph
Published: March 2005

Metabolically unstable RNA can be unravelled by pulse‐chase experiments with [3H]uridine in chick fibroblasts, in which the UTP pool has been lowered drastically by incubation with glucosamine. A newly synthesized RNA fraction, which under normal conditions is relatively stable, is broken down rapidly after addition of actinomycin D.
Étude d'un glycolipide polyinsaturé synthétisé par Mycobacterium phlei
Authors: Asselineau Cécile P. , Montrozier Henri L. , PromÉ Jean‐Claude , Savagnac Arlette M. , Welby Michèle
Published: March 2005

Mycobacterium phlei synthesizes an homologous series of polyunsaturated fatty acids, the main member of which is hexatriaconta‐4,8,12,16,20‐pentaenoic acid. These acids will be refered to as “phleic acids”. Natural derivatives of these acids and their subcellular localization are studied in this paper.

A series of esters of trehalose and phleic acids, called “lipids P”, have been isolated from Mycobacterium phlei. They differ by their number of esterified hydroxyl groups. Because of their low stability, it is difficult to make a distinction between natural product and artefact. The main member of this series, lipid P1, has been isolated in an homogeneous form by chromatographic procedures.

Saponification of lipid P1 gives only phleic acids and trehalose. Quantitative estimation by gas chromatography, as methyl phleate and trehalose trimethylsilyl ether, and examination of infrared and nuclear magnetic resonance spectra of the whole lipid P1 suggest the structure of octaphleate of trehalose for this lipid. Then lipid P1 is a glycolipid having a strong lipidic character, that differs from most other natural lipid compounds by its high molecular weight (4400) and its high unsaturation (40 double bonds).

Lipids P appear to be the main form of phleic acid derivatives. They are located in the outer part of the cell, and can be extracted by a simple washing of the bacteria with hexane; bacterial cells are still living after this treatment.

Phleic acids are not obligatory components of mycobacteria, as they have been found in two saprophytic strains (Mycobacterium phlei and Mycobacterium smegmatis), but not in two strains of Mycobacterium tuberculosis and one strain of Mycobacterium bovis (BOG). On the basis of chemical structure, subcellular localization and species distribution, lipids P exhibit analogies with other kinds of esters of trehalose found in some strains of mycobacteria devoid of phleic acids.

Although the content of phleic acids in a culture of Mycobacterium phlei reaches a constant value long before the end of the logarithmic phase of growth, phleic acids do not appear to be simple excretion products. They are not storage compounds: unlike common fatty acids, they are neither used when no carbon source is supplied to the medium, nor accumulated when the main nitrogen source of the medium (asparagin) is omitted. An important decrease of the content of phleic acids is observed when the medium is supplemented with a surfactant, which prevents bacterial aggregation.
Electrofocusing and Gel Electrophoresis of Bovine Neurophysins
Authors: PliŠKA Vladimir , Mckelvy Jeffrey F. , Sachs Howard
Published: March 2005

The isoelectric points of bovine neurophysins I and II were estimated by isoelectric focusing in a sucrose gradient and in polyacrylamide gels. The values found were 4.43 and 4.97 for neurophysins I and II, respectively, and were in agreement with the observed relative electrophoretic mobilities in disc gel electrophoresis. Electrofocusing in gels resolves the components of crude neurophysin better than disc electrophoresis. Neurophysins I and II obtained by electrofocusing in a sucrose gradient were about as pure as preparations obtained by ion‐exchange chromatography.
Membrane‐Bound Cyclic AMP Phosphodiesterase in Chemotactically Responding Cells of Dictyostelium discoideum
Authors: Malchow Dieter , Nägele Brigitte , Schwarz Heinz , Gerisch Günther
Published: March 2005

In cells of the cellular slime mold, Dictyostelium discoideum, that respond chemotactically to cyclic AMP, cell‐bound cyclic AMP phosphodiesterase was studied under two conditions: in suspensions of living cells with external cyclic AMP as a substrate, and in the 27000 ×g sediment of cell homogenates. Under both conditions a drastic increase of cell‐bound phosphodiesterase activity in the developmental phase preceding cell aggregation was found. In non‐aggregating mutants this increase is smaller or absent. The particle‐bound cyclic AMP phosphodiesterase has an optimum at pH 7.0–8.0. In vivo it is resistant to inhibition by external glutathione and to a macro‐molecular phosphodiesterase inhibitor from Dictyostelium discoideum; in particle fractions enzyme activity is partially sensitive to these inhibitors. A possible function of this enzyme in the chemo‐tactic receptor system is discussed.
Angiotensin II Conformations
Authors: Fermandjian Serge , Fromageot Pierre , Tistchenko Anne‐Marie , Leicknam Jean‐Pierre , Lutz Marc
Published: March 2005

Vibrational spectra of angiotensinamide II, some constitutive peptides, and an analog, are presented. Comparison of data for the solid hormone with those for the subunits yielded amide vibrations of the former in the 500–1700 cm−1 range. Angiotensinamide II was shown to adopt a preferential antiparallel β conformation in both the solid state and concentrated aqueous solutions. Conformational information obtained by other methods are discussed together with the present results.