bluuebunny/crossref_metadata_2025 huggingface dataset:
Spectrum and Iron Content of Protein B2 from Ribonucleoside Diphosphate Reductase
Authors: Brown N. C. , Eliasson R. , Reichard P. , Thelander L.
Published: March 2005

Protein B2, one of the two non‐identical subunits of ribonucleoside diphosphate reductase from Escherichia coli B, contains two atoms of inorganic iron per molecule of protein. The metal could be removed from the protein either by prolonged dialysis against 8‐hydroxyquinoline or by precipitation with acid ammonium sulphate. This resulted in loss of enzyme activity which was recovered after treatment of the apoprotein with Fe2+ or Fe3+ but not with any other metals tested. Removal of iron did not change the behaviour of the apoprotein on sucrose gradient centrifugation or on polyacrylamide gel electrophoresis as compared to native protein B2.

No “labile sulphide” could be demonstrated in protein B2.

Protein B2 had a characteristic spectrum with a very sharp peak at 410 nm, a broader one around 360 nm and a steep shoulder at approximately 325 nm. The two peaks disappeared on removal of iron and reappeared on reactivation of the enzyme with the metal.

When protein B2 was treated with hydroxyurea or hydroxylamine the peak at 410 nm (but not that at 360 nm) disappeared in parallel with enzyme activity. Such treatment did not result in the removal of iron.

It is suggested that the very sharp peak at 410 nm originates from an unidentified component of protein B2. Furthermore, this component, together with iron, may be the functional equivalent in the E. coli system of cobamide coenzyme in ribonucleoside triphosphate reductase of Lacto‐bacillus leichmannii.
On the Synthesis of RNA in Lymphocytes Stimulated by Phytohemagglutinin
Authors: Hausen P. , Stein H. , Peters H.
Published: March 2005

The experimental conditions to study lymphocyte transformation in cultures derived from bovine lymph nodes are described. Both deoxyribonucleoprotein‐bound and soluble RNA polymerase can conveniently be tested in cell lysates. During the course of transformation the activities of the two enzymes begin to rise at the same time when the cells begin to accumulate stable RNA. At 30 h after addition of phytohemagglutinin both enzyme activities are enhanced by a factor of two over the controls.
The Effect of Diethylether upon the Function of the Vesicles of Sarcoplasmic Reticulum
Authors: Fiehn W. , Hasselbach W.
Published: March 2005

The calcium permeability of the sarcoplasmic vesicles is greatly increased when vesicular suspensions are extracted with ether.

A 7–8% v/v aqueous ether solution abolishes the calcium concentrating ability of the vesicles completely. The calcium remaining in the solution permanently activates the calcium magnesium dependent ATPase.

In parallel to the change of the permeability of the vesicular membranes membranal lipids are removed by the ether treatment. Concentrations of ether which produce a maximal permeability increase only extract cholesterol esters. Higher concentrations of ether which inhibit the transport ATPase remove neutral lipids and phospholipids. The basic ATPase is not affected.
Urate Oxidase in Primate Phylogenesis
Authors: Christen Philipp , Peacock Wendell C. , Christen Anita E. , Wacker Warren E. C.
Published: March 2005

Several genera of New World monkeys have high uric acid concentrations in serum and urine comparable to man and the higher apes. Like man these animals lack urate oxidase activity in liver tissue. The purine metabolism of these non‐human primates appears to be similar to that of man as exemplified by fasting‐induced hyperuricemia. These South American monkeys should provide an easily accessible experimental system for the study of purine metabolism in a species closely related to man.

Old World monkeys and prosimians have low uric acid concentrations in serum and urine. The urate oxidase activity of prosimian liver is remarkably stable whereas that of Old World monkeys was found to be quite unstable, a probable reflection of the gradual degeneration of this enzyme in primate phylogenesis.
The Properties of Mitochondria Enriched in vitro with Cholesterol
Authors: Graham J. M. , Green C.
Published: March 2005

The unesterified cholesterol content of rat liver mitochondria can be raised from 0.04—0.06 to 0.1—0.13 μg per μg phospholipid by incubation for 16 h at 25° with plasma lipoproteins.

The extra cholesterol taken up by the mitochondria is roughly equally divided between outer and inner membrane fractions.

The NADH oxidase, rotenone‐insensitive, NADH‐cytochrome c reductase, fatty acid oxidase and monoamine oxidase activities of cholesterol‐enriched mitochondria are similar to those of control mitchondria stored at 4° for 16 h.

Dinitrophenol‐stimulated, Mg2+‐dependent ATPase and respiratory control measurements indicated that the functional integrity of the cholesterol‐enriched mitochondria is as good as that of the control mitochondria although in both respiratory control is less marked than in freshly‐isolated mitochondria.

Succinate‐cytochrome c reductase activity is much greater in cholesterol‐enriched than in control mitochondria. When the mitochondrial cholesterol level is alternately raised and lowered, the succinate‐cytochrome c reductase activity increases and decreases with it.

Cholesterol‐enriched mitochondria are much more resistant to large amplitude swelling than control mitochondria, apparently because of the increased strength of the outer membrane.

The changes in light absorbance associated with small‐amplitude swelling and shrinking of cholesterol‐enriched mitochondria are much smaller than those of control mitochondria.
Complementation in vitro of Phenylalanyl‐tRNA Synthetases of Escherichia coli
Authors: Kosakowski M. H. J. E. , Neidhardt F. C. , Böck A.
Published: March 2005

Complementation in vitro has been studied between the phenylalanyl‐tRNA synthetase subunits present in cell‐free extracts of temperature‐sensitive mutants of Escherichia coli. These mutant subunits, which judged from their size are dimers, form a hybrid enzyme with the dissociation products present in urea treated wild type enzyme preparations. The enzymatically active complementation product possesses the same size as the native enzyme but differs from it in its higher heat sensitivity. Complementation between the phenylalanyl‐tRNA synthetase cross‐reacting material of the different mutants alone could not be demonstrated under the conditions tested.
Flavin Radical Chelates as Studied by Electron Spin Resonance and Isotopic Substitution
Authors: Müller F. , Eriksson L. E. G. , Ehrenberg A.
Published: March 2005

The complexes formed between Zn2+ or Cd2+ and half‐reduced flavins (isoalloxazines) in neutral aqueous as well as non‐aqueous media were studied by electron spin resonance. Formation of 1:1 radical chelates between anionic radicals and Zn2+ as well as Cd2+ were found to occur. The stability constants at 20° for the chelates were estimated. A number of flavin derivatives were investigated. The spin density distribution was mapped by means of isotopic substitutions (2H, 15N, 64Zn, 67Zn, 111Cd, 113Cd, and 114Cd). The spin distribution within the ligand is similar to that found earlier for the anionic flavin radical. Spin delocalization to the metals was observed and hyperfine coupling constants for magnetic isotopes determined by direct measurements and spectra simulations. The difference in spin delocalization between Zn and Cd is discussed.
Thyroid Cytoplasmic Ribonucleoprotein Particles of Very High Size Containing Messenger‐Like and Ribosomal Ribonucleic Acids
Authors: Lissitzky S. , Poirée J.‐C. , Cartouzou G. , Grégoire J.
Published: March 2005

Cytoplasmic giant‐size ribonucleoprotein particles has been purified from the deoxycholate treated 15000 ×g supernatant of sheep thyroid slices incubated in the presence of [3H]uridine for 0.5 to 8 hours. After treatment of unclarified polyribosomes with 10 mM EDTA, 400 to 1000 S ribonucleoprotein particles have been isolated free of ribosomal subparticles by differential sedimentation. They are heterogeneous in size and buoyant density in CaCl gradients (from 1.38 to 1.52 g/ml, with maxima at 1.52, 1.46, and 1.42).

Polyribosome‐associated 400 to 1000 S ribonucleoprotein particles deprived of free ribosomal subparticles contain messenger‐like and ribosomal RNA as shown by the rate of labelling and sucrose gradient centrifugation, polyacrylamide gel electrophoresis and base composition.

Particle proteins obtained by 3 M LiCl‐6 M urea treatment separate in polyacrylamide gel electrophoresis into 3 to 4 basic components which disclose the same electrophoretic mobility as some ribosomal proteins. These 3 to 4 protein bands are distinct from total thyroid soluble proteins and from histones. Similar protein species are present in ribonucleoprotein particles diffusing spontaneously from thyroid nuclei in vitro.

The artifactual formation of these structures during EDTA exposure or slice incubation has been rendered unlikely by appropriate controls. Although tentative, the possible physiological significance of these particles is briefly discussed.
The Effect of Uncouplers of Oxidative Phosphorylation on Oxygen Uptake, Ubiquinone Redox Status and Energy‐Rich Phosphate Levels of Isolated Atria*
Authors: Lechner V. , Siess M. , Hoffmann P. C.
Published: March 2005

The effects of 2,4‐dinitrophenol and carbonyl‐cyanide‐m‐chlorophenylhydrazone on contractile amplitude and frequency, oxygen consumption, and ubiquinone redox status have been examined in spontaneously‐beating isolated guinea pig atria incubated with glucose. Dinitrophenol (2 to 100 μM) and carbonylcyanide‐chlorophenylhydrazone (0.02–0.4μM) lead to depression of contractile amplitude to 10% of control values accompanied by decreases in oxygen consumption of up to 20%. The ubiquinone redox status was shifted maximally from 32–35% oxidized ubiquinone to 66% by dinitrophenol and to 57% by carbonylcyanide‐chlorophenylhydrazone.

The effect of dinitrophenol on oxygen consumption and ubiquinone redox status was further examined in non‐beating atria (low Ca++) incubated with glucose, malate or pyruvate. With glucose dinitrophenol led to a depression of oxygen consumption, while with malate or pyruvate a stimulation to 219% and 235% of the predrug level, respectively, was observed. At 100 μM dinitrophenol, ubiquinone redox status was shifted to the oxidized side and correlated with the rate of respiration with all three substrates. At lower concentrations of dinitrophenol, ubiquinone was oxidized to a lesser extent with malate while no significant shift was observed with pyruvate or glucose.

Levels of creatine phosphate, ATP and pyruvate were decreased with increasing concentrations of dinitrophenol in non‐beating atria incubated with glucose and malate. No significant difference in these effects was detected with regard to substrate, although the trend indicated that the levels of these three compounds were more sensitive in atria incubated with glucose. Lactate levels were unaffected with low concentrations of dinitrophenol and elevated at 100 μM regardless of substrate.

It was concluded that the effect of uncouplers of oxidative phosphorylation on ubiquinone redox status and oxygen consumption in intact atria is dependent upon contractile activity and exogenous substrate. However, the responses expected from studies with isolated mitochondria, namely, stimulation of oxygen uptake and ubiquinone oxidation over a range of dinitrophenol concentrations, can be obtained in non‐contractile atria incubated with malate.
The Quantitative Measurement of DNA Hybridization from Renaturation Rates
Authors: Ley J. De , Cattoir H. , Reynaerts A.
Published: March 2005

A new method is proposed to measure relatedness amongst bacteria, based on renaturation rate determinations of DNA types and their mixture. The equations, relating the degree of binding with renaturation rates, are calculated for several cases. The optimal conditions for measurements are given. The method was checked in several ways. The advantages are summarized.
Molecular Weight and Quaternary Structure of Yeast l‐Lactate Dehydrogenase (Cytochrome b2)
Authors: Pajot P. , Groudinsky O.
Published: March 2005

A new determination of the extinction coefficient of the Soret band of reduced cytochrome b2 provides a value of 183 mM−1cm−1 instead of the generally accepted figure of 232. Four methods were used: pyridine hemochromogen, dicyanide complex, spectrum in formic acid and iron titration.

No significant differences are observed in the heme spectral properties of cytochrome b2 and its low molecular weight derivative “cytochrome b2 core”.

Dry weight associated with iron and heme content determinations lead to a minimal molecular weight per heme of 58600. This result combined with hydrodynamic and crystallographic studies by other authors allows one to propose a tetrameric structure for cytochrome b2.
Isolation, Purification and Physico‐Chemical Characteristic of Shigella sonnei Phase I Free Endotoxin
Authors: Romanowska E. , Pelczarska A. , Wnuk W. , Mulczyk M. , Godzińska H. , ŠLopek S.
Published: March 2005

The specific extracellular material with endotoxic properties (free endotoxin) was isolated from liquid culture of Shigella sonnei phase I.

Bio‐Gel P‐300 filtration and subsequent ultracentrifugation at 10500μg was used to purify and separate free endotoxin into two fractions: the sediment and the supernatant.

The sediment fraction was identified as lipopolysaccharide‐protein complex of the molecular weight of several millions, containing the same constituents as cell lipopolysaccharide of Shigella sonnei phase I, prepared by phenol–water extraction. This substance was a better endotoxin than cell lipopolysaccharide; it showed strong toxicity (L.D.50= 15 μg), pyrogenicity and elicited local and general Shwartzman reaction.

The supernatant fraction represents phase I specific substance practically free of heptose, 2‐keto‐3‐deoxyoctonic acid and phosphorus. It contained glucose, galactose and glucosamine as does phase I lipopolysaccharide, and also galactosamine and small amounts of mannose. Phase I specific substance was very active serologically and revealed very weak endotoxic properties (L.D.50= 1800 μg). The homogeneity of the supernatant fraction was proved in sedimentation experiments, free electrophoresis and rechromatography on Bio‐Gel P‐300 and Sephadex G‐200 columns. The examination of hydrodynamic properties (sedimentation coefficient, limiting viscosity number, diffusion constant and partial specific volume) of the supernatant fraction showed that this substance has a linear structure with molecular weight 90800, and diameter and length of the molecule of about 15 Å and 800 Å, respectively.
Cooperative Binding to Linear Biopolymers
Authors: Schwarz Gerhard , Klose Sigmar , Balthasar Wolff
Published: March 2005

The binding of proflavine to poly(α‐l‐glutamic acid) at pH 7.5 has been investigated by means of absorbance measurements at 444 nm. Equilibrium properties were registered as a function of the total dye concentration and the polymer to dye ratio. The results agreed very well with the predictions of a basic model theory of binding to a linear lattice where cooperative interaction is restricted to nearest neighbor binding sites. The parameters g (number of binding sites per amino acid residue), K (cooperative binding constant), and q (factor measuring the strength of cooperativity) could be determined for various concentrations of added KCl electrolyte. While g of approx. 1.1 and q of approx. 200 were found to be little affected by increasing the ionic strength, a considerable decrease of K was observed. The latter is interpreted as the effect of competitive inhibition due to non‐cooperative binding of potassium ions (Ki= 160 M–1). Extrapolation to zero electrolyte concentration yields K→Ko= 8 × 104 M–1. It is concluded that the binding between the ligands and the polymer is of essentially electrostatic nature. Cooperative interaction, on the other hand, must be attributed to the stacking tendency of neighboring bound dye molecules.
Choline Esters, their Precursors and Metabolites in the Hypobranchial Gland of Prosobranchiate Molluscs, Concholepas concholepas and Thais chocolata
Authors: Roseghini Marisa , Erspamer Vittorio , Luis Ramorino M. , Gutierrez J. E.
Published: March 2005

Extracts of the hypobranchial body of two prosobranchiate molluscs of the Muricidae family were examined for their content of choline esters, as well as of precursors and metabolites of these esters.

The hypobranchial gland of Concholepas concholepas contained, in addition to large amounts of urocanylcholine (murexine) and senecioylcholine, appreciable amounts of free choline, free urocanic acid and ethyl urocanate. All these compounds, and especially the choline esters, were predominantly localized in the dye‐secreting area of the hypobranchial gland. As much as 3% of the wet weight of the purple area was accounted for by murexine and seneciocylcholine.

The only choline ester found in the hypobranchial body of Thais chocolata was senecioylcholine; derivatives of urocanic acid were lacking.

The occurrence of ethyl urocanate in the hypobranchial gland of Concholepas concholepas demonstrates that not only choline but also other alcohols may be esterified by urocanic acid.
Purification and Studies of Catechol‐O‐Methyltransferase of Rat Liver
Authors: Assicot Marcel , Bohuon Claude
Published: March 2005

Rat liver catechol‐O‐methyltransferase was purified approximately 450‐fold by salt fractionation, get filtration, hydroxyapatite treatment, and ion‐exchange chromatography. The final enzyme preparation is homogeneous when subjected to disc electrophoresis and sedimentation analysis in the ultracentrifuge. The purified enzyme is very labile; reversible inactivation occurs upon storage. Reactivation is obtained by incubation with dithiothreitol. The molecular weight, determined by gel filtration, is 24000.
Malate Dehydrogenase of Ox Kidney. Comparison of the Reactivity of Sulfhydryl Groups and their Relationship to Activities of the Mitochondrial and Cytoplasmic Enzymes
Authors: Skilleter David N. , Lee Nancy M. , Kun Ernest
Published: March 2005

Out of 10 to 11 cysteine residues per mole of kidney mitochondrial malate dehydrogenase, 3 to 4 exhibit time‐dependent reactivity with 5,5′‐dithio‐bis‐(2‐nitrobenzoic acid) (DTNB) at pH 9.5 to 9.8, when incubated over a period of 3 hours. Under the same conditions, 1.5 to 2.0 out of a total of 6 cysteine residues of the cytoplasmic malate dehydrogenase became DTNB‐reactive. The process of unmasking of unreactive thiols is initiated by a relatively rapid (10 min) reaction on one thiol with DTNB. Beyond this initial reaction, DTNB is not required and unmasking of thiol groups proceeds spontaneously in an alkaline medium. Enzymatic activity is unaffected by the reaction of thiol groups with DTNB. Both the rate and the extent of the thiol‐DTNB reaction is greatly increased in the presence of 2 M urea, which by itself has no effect on the availability of thiol groups. Thiol unmasking in the mitochondrial enzyme is completely inhibited by 1 mM NADH (or to a lesser extent by ADP), whereas the cytoplasmic enzyme does not respond to these agents. Enzymatic activity of the cytoplasmic malate dehydrogenase is more resistant than the mitochondrial enzyme to 4 M urea. ADP or NADH tend to stabilize enzymatic activities of both isoenzymes. There is no simple relationship between enzymatic activity and reactive cysteine thiols. The specific effect of NADH (or ADP) on the mitochondrial malate dehydrogenase by preventing thiol liberation, is interpreted as an expression of a specific nucleotide binding site which controls small scale unfolding of the enzyme protein.
Inhibition of Brain Glutamate Decarboxylase by Adenosine Triphosphate
Authors: Turský Timotej
Published: March 2005

ATP acts as an inhibitor of brain glutamate decarboxylase in a water brain tissue extract with low ATPase activity. This inhibition is not caused by competition of ATP with glutamate or pyridoxal‐5‐phosphate.

The concentration of ATP, which causes a 50% inhibition of glutamate decarboxylase, [I]50%, depends on the pyridoxal‐5‐phosphate concentration. At 15 μM pyridoxal‐5‐phosphate the [I]50% is 0.3 mM ATP. At 75 μM pyridoxal‐5‐phosphate the [I]50% is 1.3 mM ATP. AT 375 μM pyridoxal‐5‐phosphate glutamate decarboxylase is not inhibited to 50% even with 10 mM ATP.

ADP, GTP and UTP are strong inhibitors of glutamate decarboxylase at high concentration. At lower concentrations ATP is the most potent inhibitor. AMP and adenosine do not effect glutamate decarboxylase, nor do they affect the inhibitory action of ATP.

Inorganic phosphate in concentrations of 5—40 mM does not influence the activity of glutamate decarboxylase. At the same concentrations Pi diminishes the inhibitory effect of ATP. The [I]50% increases from 0.3 mM ATP (without Pi) to 1.7 mM ATP in the presence of 20 mM Pi.
Partial Purification and Properties of Acyl‐CoA Synthetase of Escherichia coli
Authors: Samuel D. , Estroumz J. , Ailhaud G.
Published: March 2005

A purification of acyl‐CoA synthetase of Escherichia coli is described. The enzyme behaves as an acid: CoA ligase (AMP) (EC 6.2.1.3), being active on C4–C18 fatty acids. A multienzyme complex (120000 molecular weight) seems to be involved; although the ratios of activities on different fatty acids remain constant during the purification procedure, heat treatment, kinetic experiments and column chromatography on hydroxyapatite indicate the presence of two enzymes, one acting on short and medium chain fatty acids, and the other on long chain fatty acids.
Biosynthese von 6‐Methylsalicylsäure
Authors: Dimroth Peter , Walter Hilde , Lynen Feodor
Published: March 2005

An extract was prepared from Penicillium patulum grown in submerged culture, which converted acetyl‐CoA and malonyl‐CoA into 6‐methylsalicylic acid in the presence of NADPH. The enzyme was very labile in the crude extract, but appeared stable after acetone precipitation. A 100‐fold purification was achieved by ammonium sulfate fractionation, sedimentation in the ultracentrifuge, and sucrose‐density gradient centrifugation. The enzyme was a single particle with a molecular weight of 1.1–1.5 × 106 as determined by gradient centrifugation with catalase and fatty acid synthetase as standards. In the first steps of purification, fatty acid synthetase from Penicillium patulum migrated with the methylsalicylic acid synthetase complex. A separation of the two enzyme systems was accomplished by sucrose‐density gradient centrifugation. The purified enzyme represents a new multienzyme complex which shares several common properties with fatty acid synthetase. Methylsalicylic acid synthetase is inhibited by iodoacetamide and N‐ethylmaleinimide. The incubation of enzyme with acetyl‐CoA and malonyl‐CoA in the absence of NADPH led to triacetic acid lacton. This indicates that in 6‐methylsalicylic acid synthesis reduction occurs at the triacetic acid level before the final condensation with malonyl‐CoA. Triacetic acid lacton was produced by the enzyme at 10% of the rate of 6‐methylsalicylic acid synthesis in the complete system. This rate of lacton synthesis, however, is an order of magnitude greater than that catalysed by fatty acid synthetase. A possible explanation for this difference in behavior is given. Covalent linkage of substrates and intermediates to SH‐groups of the protein during the synthesis of 6‐methylsalicyslic acid is suggested. A reaction scheme leading to 6‐methylsalicylic acid is proposed and the partial reactions of this synthesis are discussed with respect to similar reactions of fatty acid biosynthesis.
Interference of Ethanol and Sorbitol with Hepatic Ketone Body Metabolism in Normal, Hyper‐ and Hypothyroid Rats
Authors: Lindros Kai O.
Published: March 2005

Rats pretreated with triiodothyronine or propylthiouracil were compared with normal rats with respect to the changes induced by ethanol or sorbitol in the levels of ketone bodies and acetyl coenzyme A and in the mitochondrial redox state of the NAD couple in the intact liver after overnight fasting.

A single dose of ethanol (2.5 g/kg) increased the ketone body level and the acetyl‐CoA content after 75 min in both normal and hyperthyroid rats but not in hypothyroid rats.

Infusion of sorbitol (0.5 g/kg) resulted in decreases in both ketone formation and levels of acetyl‐CoA after 15 min in all types of rats.

The β‐hydroxybutyrate/acetoacetate ratio, which represents the redox state of the NAD couple in the mitochondria, was increased more than three‐fold by treatment with ethanol in normal rats, but only by 50% in hyperthyroid rats. A ten‐fold increase in the redox state of the mitochoondia was caused by ethanol in hypothyroid rats, as the result of a strongly diminished acetoacetate level.

Sorbitol infusion resulted in slight increase of the β‐hydroxybutyrate/acetoacetate ratio in normal and hypothyroid rats but decreased the ratio in hyperthyroid rats.

The different ketone body levels observed in these situations were not found to be related to changes in the mitochondrial redox state, but a highly significant correlation (P < 0.001) was observed between the changes in the content of acetyl‐CoA and the ketone bodies.
Structure des acides corynomycoliques de Corynebacterium hofmanii et leur implication biogénétique
Authors: Welby‐Gieusse M. , Lanéelle M. A. , Asselineau J.
Published: March 2005

The three main components of the mixture of corynomycolic acids (α‐branched, β‐hydroxy acids of the general formula R1—CHOH—CHR2—COOH) isolated from a strain of Corynebacterium hofmanii are:

C32H64O3 (corynomycolic acid); C34H66O3 (corynomycolenic acids); C36H68O3 (corynomycoldienic acid).

By thin‐layer chromatography on silver nitrate‐impregnated Silicagel G, we were able to separate the hydroxy acids having 0, 1 or 2 double bonds. The pyrolytic chromatography of their methyl esters indicates that the mono‐unsaturated acids form a mixture of C34‐acids containing the double bond either in the main chain or in the side chain; the di‐unsaturated acid fraction is constituted solely by a C36‐acid with a double bond in each fragment arising from pyrolysis.

By oxidative cleavage, we find that the position of the double bond is 9, 10 (as in oleic acid). According to the nature of the main chain (aldehyde fragment of pyrolysis) and of the side chains (ester fragment of pyrolysis), some suggestions are made about the selective use of fatty acids in the biosynthesis of corynomycolic acids.
Fluctuation of Hepatic Enzymes Important in Glucose Metabolism in Relation to Thyroid Function
Authors: Böttger I. , Kriegel H. , Wieland O.
Published: March 2005

Enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, pyruvate kinase and of mitochondrial glycerolphosphate dehydrogenase were studied in the livers of rats under the following states of thyroid function: 131I‐thyroidectomized rats, 131I‐thyroidectomized rats treated with triiodothyronine, normal rats treated with excess of triiodothyronine, and normal untreated rats. In the thyroidectomized group all of the enzyme activities listed were significantly diminished. After treatment with a single dose of triiodothyronine the activities of pyruvate carboxylase and of pyruvate kinase were restored to normal whereas phosphoenolpyruvate carboxykinase and — most markedly — glycerolphosphate dehydrogenase were raised above the normal level. After repeated administration of triiodothyronine to normal rats a further increase in the activity of pyruvate carboxylase, phosphoenolpyruvate carboxykinase and of glycerolphosphate dehydrogenase was observed.

The activities of glycerolphosphate dehydrogenase and of pyruvate carboxylase 70 h following triiodothyronine injection into hypothyroid rats were more than 50% lower after treatment with actinomycin C suggesting that the increase in enzyme activities was due to increased synthesis of enzyme protein. Phosphoenolpyruvate carboxykinase activity was not changed significantly by actinomycin C.

The possible significance of these thyroid hormone dependent enzyme fluctuations in controlling the overall rate of hepatic gluconeogenesis is discussed.
Synthèse de RNA stables dans une souche d'Escherichia coli stringent pendant la carence en acide aminé spécifique
Authors: Galibert F. , Eladari M. E. , Larsen C. J. , Boiron M.
Published: March 2005

By definition amino‐acid starvation of an RCstrEscherichia coli strain causes simultaneous arrest of RNA and protein synthesis. However, it has recently been demonstrated that under such conditions mRNA synthesis does not stop and this result showed that RNA synthesis is not a coordinated process. The present study, which was carried out to determine the effects of amino‐acid starvation on net RNA synthesis in an RCstrE. coli strain, has led to the following results:

a) during amino‐acid starvation net RNA synthesis is reduced to about 5% of the normal level;

b) ribosomal RNA appears in 50 S and 30 S ribosomes and in 38 S and 21 S particles which seem to be precursors of 50 S and 30 S ribosomes, respectively;

c) analysis by chromatography on methylated albumin column and by electrophoresis on polyacrylamide gels shows that the 38 S particle is devoid of 5 S RNA;

d) a 5 S RNA appears in the supernatant fraction of ribosome preparations. This RNA, which is slightly different from the true 5 S RNA, is called 5 S stringent RNA. With respect to its chromatographic behaviour on G‐100 Sephadex and methylated albumin column, its mobility on disc electrophoresis, it is identical to 5 S relax RNA and 5 S chloromycetin RNA which are synthesized when protein synthesis is blocked either by amino‐acid starvation of a RCrelE. coli strain or by addition of chloromycetin;

e) from the relative quantity of the different classes of RNA it is concluded that RNA synthesis is not coordinated and from the appearance of rRNA in ribosomal particles, a mechanism concerning the regulation of RNA synthesis is proposed.
The Covalent Murein‐Lipoprotin Structure of the Escherichia coli Cell Wall
Authors: Braun Volkmar , Sieglin Uta
Published: March 2005

After pronase treatment of the murein‐lipoprotein complex (rigid layer) of the cell wall of E. coli B or E. coli K12 (W 945), lysine and arginine remain as the sole amino acids covalently bound to the murein (peptidoglycan, glycopeptide). These amino acids occur in equimolar amounts, each equal to the amount of lysine remaining with the murein after trypsin digestion of the murein‐lipoprotein complex.

From partial acid hydrolysates of such a murein, prepared by pronase digestion of the mureinlipoprotein complex, the following peptides have been isolated: (1) diaminopimelyl‐lysyl‐arginine; (2) alanyl‐glutamyl‐diaminopimelyl‐lysyl‐arginine; (3) glucosaminyl‐muramyl‐alanyl‐glutamyl‐diaminopimelyl‐lysyl‐arginine. Peptide 1 shows that the lipoprotein is bound by the α‐amino group of the presumbaly N‐terminal lysine to the carboxyl group of diaminopimelic acid. Peptide 2 consists of a peptide side chain of the murein to which the two amino acids of the N‐terminal end of the lipoprotein, lysine and arginine, are attached. Peptide 3 constitutes a repeating unit of the murein to which the peptide lysyl‐arginine of the lipoprotein is bound.

The lipoprotein cleaved from the murein by a short trypsin digestion had an amino acid composition similar to the lipoprotein in the untreated murein‐lipoprotein complex and arginine as N‐terminal amino acid. The following structure is proposed: murein‐lysyl‐arginyl‐lipoprotein.

In the rapid reaction of trypsin with the cell wall the enzyme apparently cleaves at the C‐terminal end of the lysime of the murein‐lipoprotein linkage which results in murein‐lysine and arginyl‐lipoprotein. Some cleavage at the C‐terminal end of arginine gives rise to free arginine (14%).

These results support the supramolecular structure of the murein‐lipoprotein complex previously proposed. On the average one lipoprotein molecule is covalently bound to every tenth repeating unit of the murein from which an average distance of 103 Å between two lipoprotein molecules along the polysaccharide chains of the murein is deduced.
Kinetic Study of Yeast Hexokinase
Authors: Noat Georges , Ricard Jacques , Borel Maguy , Got Claude
Published: March 2005

The ionic species ATP4− and Mg2+, at high concentrations, decrease the rate of glucose phosphorylation catalyzed by yeast hexokinase. A theoretical study of all the possible mechanisms (271 models) of this inhibition has been made. The comparison of these mechanisms with the experimental results shows that only one model can explain the inhibition of the reaction by ATP, and that only one can also explain the effect of the high magnesium concentrations.

It was thus possible to show that the ionic species ATP4− can be bound on hexokinase and on the hexokinase‐glucose complex. Its affinity is much stronger for the second form of the enzyme than for the first. The complexes thus formed are devoid of any activity (“dead‐end” complexes). The inhibition of the hexokinase by the high nucleotide concentrations is therefore due to the formation of these complexes. A ternary chelate of the Mg(ATP)26− type does not exist.

Magnesium possesses no affinity for the enzyme. Its role in the catalysis must therefore by indirect. The results presented agree with the idea that the metal polarizes the phosphoryl bond of the ATP that is split when glucose‐6‐phosphate is formed. Magnesium can form with ATP a ternary chelate of the Mg2ATP type, unable to be bound on the hexokinase. The inhibiting effect of high concentrations of the metal can be explained by the formation of this inactive ternary chelate and by the decrease in concentration of the MgATP2− complex.
Fast Membrane H+ Binding in the Light‐Activated State of Chromatium Chromatophores
Authors: Chance B. , Crofts A. R. , Nishimura M. , Price B.
Published: March 2005

A fast and sensitive single‐beam spectrophotometer and a variety of flash and steady‐state illumination sources permit the resolution of bromcresol purple absorbancy changes from background absorbancy changes of Chromatium chromatophores, and identification of these fast absorbancy changes with rapid H+ binding by the chromatophores. A saturating light flash gives a rate several hundred times faster than that obtained in continuous light, the maximal rate being about 50 H+/chlorophyll μsec, with a magnitude of the H+: chlorophyll ratio of 1:30 and a duration for the H+ binding reaction, measured by absorbancy changes of bromcresol purple, of 2.2 msec (half‐time, 800 μsec for a 200 μsec flash). A single turnover flash (10 μsec) yields an H+: chlorophyll value of 1:100, with a duration of the H+ change of 800 μsec and a half‐time of 400 μsec. A double‐flash technique shows the recovery for the fast H+ reaction to have a half‐time of 60 msec. The effects of pH, temperature, uncouplers, and inhibitors are also reported.

While the hydrogen ion binding would appear to be closely associated with the reduction of an electron acceptor, Y, particularly on the basis of the similar amounts of the two substances (roughly one each per hundred chlorophyll) the reduction time for Y of 27 μsec is over ten‐fold shorter than the H+ binding time in single flash experiments. In addition, the pH sensitivities and activation energies of the two reactions differ.

Thus, either an unidentified electron acceptor is involved in the fast H+ changes, or there is no direct relationship between electron transport and H+ binding. If the latter is the case, we propose that electron transport through the photosynthetic center produces in 800 μsec a state of the membrane (a “membrane Bohr effect”) in which hydrogen ion binding is considerably enhanced.
Magnesium, Potassium, and the Adenylate Kinase Equilibrium
Authors: Blair J. McD.
Published: March 2005

The influence of magnesium and potassium on the equilibrium catalysed by adenylate kinase has been studied. An equation has been derived which describes the effect of these ions on the equilibrium; values for the metal‐nucleotide stability constants, and for the “true” equilibrium constant, [AMP2−] · [ATP4−]/([ADP3−])2, have been calculated, which give the best fit of this equation to the experimental data.

Using these constants, the concentrations of all significant species in an adenine nucleotide pool equilibrated by adenylate kinase can be calculated, for any specified total concentrations of magnesium, potassium, and adenine nucleotide pool, at any pH and any pyrophosphate bond content of the adenine nucleotide pool. Magnesium emerges as a significant feedback signal from an adenine nucleotide pool considered as an isolated system.

The possible significance of this data with respect to the control of metabolic flux in general and of phosphofructokinase in particular is discussed.
Amino Acid Sequence of C‐Terminal Fragment of Hog Pepsin
Authors: Kostka V. , Morávek L. , ŠOrm F.
Published: March 2005

The C‐terminal fragment of hog pepsin, which had been obtained in the preceding study on the cyanogen bromide hydrolysate of this protein, was subjected to sequential studies. The 37‐residue peptide was digested in independent experiments by trypsin, chymotrypsin, and pepsin, and the amino acid sequence of the arising peptides was determined. In parallel experiments the sequence of seventeen amino acid residues in the N‐terminal region of the peptide was established by the Edman degradation technique. The obtained data permit the C‐terminal peptide of hog pepsin to be ascribed the amino acid sequence Asp‐Val‐Pro‐Thr‐Ser‐Ser‐Gly‐Glu‐Leu‐Trp‐Ile‐Leu‐Gly‐Asp‐Val‐Phe‐Ile‐Arg‐Gln‐Tyr‐Tyr‐Thr‐Val‐Phe‐Asp‐Arg‐Ala‐Asn‐Asn‐Lys‐Val‐Gly‐Leu‐Ala‐Pro‐Val‐Ala. These results extend and partly correct our previous knowledge of this region of the pepsin molecule as recorded in literature. The problem of the tryptophan content of pepsin is briefly discussed.
The Metal Complexes of Peptides and Related Compounds
Authors: ÖSterberg Ragnar
Published: March 2005

The complex formation between copper(I) ions and glycylglycylglycine (H2A+) has been studied in 3.0 M NaClO4 medium at 25° by constant current coulometry using copper amalgam and glass electrodes. In each series of measurements the copper ions were generated into the ligand solution by electrolysis of two phases copper amalgam. The data, shown in Table 2, were treated by graphical methods and by the least squares computer program, Letagrop.

It is concluded that the results obtained are consistent by assuming the equilibria

Cu++ Cu2++ 2A−⇄ CuICuIIH–3A22−+ 3H+, log β11–32=–3.35 ± 0.04

Cu++ A−⇄ CuIA, log β1001= 6.2 ± 0.1 where β is an equilibrium constant.

It is assumed that the copper(II)‐equilibria, reported in the previous paper of this series, coexist in solution. On the basis of the equilibrium constants it may be deduced that for pH higher than 6.5 the principal copper(I) complex is CuICuIIH–3A22−. The standard oxidation–reduction potential at pH 7 for the formation of this species from its copper(II) analogue, Cu2H–4A22−, was calculated to be +340 mV.
Tryptophanyl‐tRNA synthétase de pancreas de bœuf
Authors: Cittanova Nicole , Alfsen Annette
Published: March 2005

A simple and rapid method of obtaining, from beef pancreas, a homogeneous tryptophanyl‐tRNA synthetase without tryptophan, is described. Spectrophotometric and fluorimetric methods have provided new insights into the conformation of the enzyme in absence of substrate.

By spectrophotometry, 26 tyrosine groups per mole of enzyme are titrated in the native protein. These chromophores present an abnormally high pK of 11.3 and also have a low accessibility to ethylene glycol and glycerol. In 8 M urea 29 tyrosines are titrated, compared to 36 residues found by amino acid analysis.

By spectrofluorimetry, using a fluorescent probe of conformation, 1‐anilino‐8‐naphtalen sulfonate (ANS), we have demonstrated that there is one binding site for ANS per mole of enzyme. The association constant KA= 0.8 × 105 M–1 has been calculated for the complex ANS‐enzyme. The emission wavelength of the complex at 465 nm (instead of 515 nm for ANS in water) suggests a very hydrophobic environment for the bound ANS. The existence of energy transfer between intrinsic chromophores of the protein and ANS is demonstrated. The presence of tryptamine, a competitive inhibitor of the enzymatic reaction, quenches the fluorescence emission of the bound ANS.

Although the emission fluorescence of the bound dye is affected by the presence of tryptamine, interpretation of the data demonstrates that the binding of ANS does not occur at the same site as tryptamine. This seems to imply that the catalytic site is not directly involved in the binding of ANS to tryptophanyl‐tRNA synthetase.
The Regulation of Synthesis of Krebs Cycle Enzymes in Neurospora by Catabolite and End Product Repression
Authors: Flavell Richard B. , Woodward Dow O.
Published: March 2005

Krebs cycle enzymes were repressed in Neurospora during growth on sucrose as carbon source compared with growth on acetate. In mitochondrial, cytochrome‐deficient strains grown on sucrose, this glucose or catabolite repression was reduced. In mutant strains in which protein synthesis and mitochondrial respiration were inhibited, catabolite repression was enhanced. These data support the hypothesis that ATP or the cellular energy level is involved in catabolite repression. The interrelationships between energy metabolism, the energy distribution in the cell and catabolite repression are discussed.

The addition of casamino acids to a culture growing on sucrose or acetate, did not cause repression of any of the Krebs cycle enzymes measured, in spite of the fact that many of the constituent amino acids can be considered as biosynthetic products of the Krebs cycle.
The Presence of Both Phage PS8 and Agrobacterium turnefaciens A6 DNA Base Sequences in A6‐Induced Sterile Crown‐Gall Tissue Cultured in vitro
Authors: Schilperoort Robbert A. , van Sittert Nico J. , Schell Jef
Published: March 2005

The presence of both Agrobacterium tumefaciens and phage PS8 DNA base sequences in sterile crown‐gall tissue grown in vitro was demonstrated by hybridization experiments using crown gall DNA and [3H]RNAs complementary to the bacteria and phage DNAs. These experiments were done under stringent conditions minimizing mismatching of the bases. The approximate amount of A. tumefaciens DNA and PS8 DNA base sequences in crown gall DNA was respectively 9 and 900 genome equivalents. Data are presented indicating that the A6 genome equivalents consist of many copies of a small part of the bacterial genome, while the PS8 genomes are present almost completely. The A6 segment must be large enough to code for four proteins specific for A. tumefaciens present in the crown gall cells and also form a fractional part of the PS8 genome. In cells of the A6 strain used in the experiments no prophage is integrated in the genome of the bacteria. It is postulated that PS8–like DNA is present as a plasmid. Further hybridization‐competition experiments using [3H]cRNA from A. radiobacter showed that one‐third of the A. tumefaciens DNA base sequences in crown gall are also present in A. rudiobacter DNA.

No plaque‐forming activity was observed when crown gall tissue homogenates were tested.
Iodoacetic‐Acid‐Resistant Alcohol Dehydrogenase of an Escherichia coli Mutant
Authors: Meyhack Bernd , Pfleiderer Gerhard , Mennigmann H. D.
Published: March 2005

Nitrosoguanidine‐induced mutants of Escherichia coli were tested in vivo (tetrazolium staining assay) for iodoacetic acid resistance of their alcohol dehydrogenase. One mutant was isolated (mutant 2773) which has a fully iodoacetic‐acid‐resistant alcohol dehydrogenase. Mutant 2773 contains two species of alcohol dehydrogenase; one (the enzyme of the mutant strain, wild‐type fraction i.e. wild‐type‐mutant enzyme) has the same characteristics as the enzyme of the parental strain (wild‐type enzyme), including the inhibition by iodoacetic acid; the other species (enzyme of the mutant strain, mutant fraction i.e. the mutant enzyme) however, is iodoacetic acid resistant, has a high molecular weight (> 600000) and is not related to the wild‐type enzyme as detected by immunological methods.

The role of SH‐groups taking part in the reaction at the active center of the alcohol dehydrogenase and the occurrence of several enzyme species are discussed.
Deoxyribonucleotide Pools during Liver Regeneration
Authors: Söderhäll S. Stefan , Larson Agne , Skoog K. Lambert
Published: March 2005

The intracellular pools of deoxyribonucleotides were analyzed during liver regeneration in rats. In normal liver the content of dATP, dCTP, dGTP and dTTP was found to be 0.3, 1.3, 0.3 and 0.4 pmol per μg DNA, respectively. When the liver tissue started to proliferate the pools of the individual deoxyribonucleoside triphosphates increased and reached maxima at 24–27 h after partial hepatectomy. This increase in the levels of deoxyribonucleotides coincided in time with the peak of DNA synthesis as measured by the incorporation of labeled thymidine. A burst of mitotic activity was observed at 32 h. During liver regeneration ribonucleotide reductase is induced; the activity of this enzyme was found to be maximal at 50 h after partial hepatectomy, i.e. when the deoxyribonucleoside triphosphate pools as well as the rate of DNA synthesis had declined, indicating a feed‐back control of the enzyme.
Activity of a Rat‐Liver Glycoprotein: Sialyltransferase Utilizing Desialyzed Prothrombin as an Acceptor in Normal and Hypothrombinemic Animals
Authors: Bernacki Ralph J. , Bosmann H. Bruce
Published: March 2005

A glycoprotein : sialyltransferase which catalyzed the addition of N‐acetyl[14C]neuraminic acid to desialyzed glycoprotein precursors has been partially purified and characterized from rat liver, using desialyzed human prothrombin as an exogenous acceptor. The enzyme was extracted with 0.1% Triton X‐100 and was purified 31‐fold by gel chromatography. The sialyltransferase with desialyzed human prothrombin as acceptor was most active at 37 ° C, pH 7.0, 15 mM Mg2+, and its activity toward other asialoglycoproteins was similar with respect to optimum conditions. The enzyme was inhibited by 10 mM HgCl2, PbCl2 and ZnCl2. Kinetic data were treated according to the model of Cleland for a bisubstrate reaction and the various constants were Kb= 50 μM, Kb= 31 μM, Kia= 10 μM, Kib= 5.8 μM, and V1= 100 nmol × h−1× mg protein−1, where a = CMP‐N‐acetyl[14C]neuraminic acid and b = prothrombin. The activities with various desialyzed acceptors could not be separated by gel chromatography and may be due to a single sialyltransferase. Rats made hypothrombinemic by maintenance on a vitamin‐K‐deficient diet had normal or slightly elevated amounts of sialyltransferase activity using either desialyzed prothrombin or fetuin as glycoprotein acceptors for N‐acetylneuraminic acid. Rat liver obtained from vitamin‐K‐deficient rats also had normal amounts of other glycosyltransferases, including galactosyl, glucosyl and N‐acetylglucosaminyltransferase, as measured with endogenous or exogenous acceptors.