bluuebunny/crossref_metadata_2025 huggingface dataset:
On the Mechanism of Na+‐ and K+‐Stimulated Hydrolysis of Adenosine Triphosphate
Authors: Schoner W. , Beusch R. , Kremer R.
Published: March 2005

The nucleotide specificity of membrane‐bound Na+‐ and K+‐activated ATPase from ox brain was compared with that of the Na+‐dependent phosphorylation reaction of ATPase protein. [32P]Phosphate is incorporated into enzyme protein from terminally‐labelled [32P]nucleotide triphosphates (ATP, ITP, GTP), a process dependent on the presence of Na+. Subsequent addition of K+ ions to the test system is followed by a rapid decrease in incorporated radioactivity. Enzyme‐bound phosphate exchanges its label rapidly with adenine nucleotides and slowly with inosine and guanosine nucleotides.

ATP, ITP, and GTP are substrates of Na+‐ and K+‐activated ATPase. The ratio of the reaction rates is: ATP: ITP: GTP = 27: 2:1. An almost identical activation pattern of nucleotide triphosphate hydrolysis by Na+ ions in the presence of saturating concentrations of K+ is found with all substrates (Km= 10 mM NaCl). On the other hand, the optimal stimulation by K+ at saturating concentrations of Na+ is variable: hydrolysis of ATP is increased 10 times, that of ITP 4 times and that of GTP 1.2 times.

ADP inhibits both ATP and ITP hydrolysis. Only high concentrations of IDP (50 mM) inhibit ATP hydrolysis, whereas low concentrations of IDP inhibit ITP hydrolysis.

Identical nucleotide triphosphate specificities were found for Na+‐ and K+‐activated ATPase from ox brain and from human red blood cells.

It is concluded that all nucleotide triphosphates investigated are hydrolyzed via the same acceptor group. The Na+‐dependent phosphorylation reaction of ATPase protein is considered as part of the overall reaction of membrane‐bound Na+‐ and K+‐activated ATPase.
Structure and Function of Carbamoylphosphate Synthase
Authors: Guthöhrlein G. , Knappe J.
Published: March 2005

Carbamoylphosphate synthase of rat liver has been purified to a homogeneous state. Its molecular weight is about 250,000.

When the catalytic activity is measured at 10°, using an optical method, an activation phase is detectable, which could be assigned to the N‐acetyl‐l‐glutamate induced transition of the enzyme from an inactive conformation to the active conformation. The half‐time of the process is about 1 min at 10 mM acetyl‐glutamate. The specific effect of the cofactor was found to be the labilization of the inactive conformation (as opposed to a stabilization of the active conformation).

At low temperatures the enzyme dissociates reversibly into probably two identical subunits. The dissociation seems to occur at the stage of the active conformation. ATP was found to hold back the dissociation by specifically stabilizing the active conformation.
Purification and some Properties of Leucyl‐tRNA Synthetase from Bacillus stearothermophilus
Authors: Vanhumbeeck J. , Lurquin P.
Published: March 2005

The leucyl‐tRNA synthetase from Bacillus stearothermophilus has been purified 350‐fold. The enzyme is stable when stored at 4° in the presence of sulfhydryl protecting compounds.

The Km values for leucine and ATP are respectively 240 and 0.84 μM. The ATP‐PPi exchange and the aminoacylation reaction satisfy the Arrhenius law up to 45°.

After thermal denaturation the enzyme has been partially renatured.
Studies on the Mechanism of Galactosamine Hepatitis: Accumulation of Galactosamine‐1‐Phosphate and its Inhibition of UDP‐Glucose Pyrophosphorylase
Authors: Keppler D. , Decker K.
Published: March 2005

d‐Galactosamine has been shown to produce a liver damage closely related to human viral hepatitis. d‐Galactosamine‐1‐phosphate and UDP‐galactosamine were identified as the predominant early metabolites of galactosamine in rat liver. The conversion of galactosamine‐1‐phosphate to UDP‐galactosamine is shown to be catalyzed by UDP‐glucose: α‐d‐galactose‐1‐phosphate uridylyltransferase. The low affinity of this enzyme for galactosamine‐1‐phosphate explains in part the high levels of this compound found in galactosamine treated livers. Under these conditions galactosamine‐1‐phosphate accumulation is enhanced by the strongly reduced levels of UDPG. Galactosamine‐1‐phosphate inhibits the UDPG‐pyrophosphorylase reaction, the type of inhibition being mainly competitive with glucose‐1‐phosphate. In the presence of the concentrations of galactosamine‐1‐phosphate and glucose‐1‐phosphate found in vivo after galactosamine treatment, UDPG‐pyrophosphorylases from rat and calf liver are strongly inhibited in vitro. By these mechanisms galactosamine‐1‐phosphate counteracts its own conversion to UDP‐galactosamine. The influence of the strongly diminished UDPG levels on the UDPG‐linked syntheses of glycogen, heteropolysaccharides and glucuronides as well as the trapping of uridine phosphates by formation of UDP‐hexosamines may play an important role in the induction of galactosamine hepatitis.
The pH 6 Acetolactate‐Forming Enzyme from Aerobacter aerogenes
Authors: Tveit B. , Störmer F. C.
Published: March 2005

Crystalline pH 6 acetolactate‐forming enzyme from Aerobacter aerogenes contains 4 thiol groups per enzyme molecule, determined with 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) in 50 mM sodium dodecyl sulfate, or acetate.

In the presence of 50 mM pyruvate, propionate, as well as acetamide, 3 thiol groups are accessible for titration with DTNB. In the presence of phenylpyruvate, 2.3 thiol groups reacted.

The apparent Michaelis kinetics, obtained in the presence of acetate, is not altered after 1–3 thiol groups are allowed to react with DTNB.
The pH 6 Acetolactate‐Forming Enzyme from Aerobacter aerogenes
Authors: Störmer F. C. , Solberg Y. , Hovig T.
Published: March 2005

The diffusion and sedimentation constants of the pH 6 acetolactate‐forming enzyme, determined by analytical ultracentrifugation, were used to calculate a molecular weight of 200 000 for the enzyme.

The amino acid composition of the enzyme has been worked out, and 3 molecules of cocarboxylase, and 6 atoms of phosphorus were found to be tightly bound to one molecule of the enzyme. In addition, the effect of divalent cations was studied, results indicated that the enzyme has a requirement for manganese. An absolute requirement for cocarboxylase and manganese was not established.

Two crystalline forms of the enzyme were studied by electron microscopy using negative staining technique. The plate‐shaped crystals were composed of rows of particles measuring approximately 100 × 60 Å. These particles consisted of sub‐units, and in some areas a helical organization of the crystal could be seen. The molecular organization was different in the two different crystalline forms of the enzyme.
On the Biosynthesis of Cyclopenin and Cyclopenol, Benzodiazepine Alkaloids from Penicillium cyclopium Westling
Authors: Nover L. , Luckner M.
Published: March 2005

By feeding of isotope labelled compounds to cultures of Penicillium cyclopium Westling and degradation of the cyclopenin (3‐benzyl‐3,10‐epoxy‐4‐methyl‐2,3,4,5‐tetrahydro‐1 H‐1,4‐benzodiazepine‐2,5‐dione) and cyclopenol [3‐(m‐hydroxy‐benzyl)‐3,10‐epoxy‐4‐methyl‐2,3,4,5‐tetrahydro‐1 H‐1,4‐benzodiazepine‐2,5‐dione] formed the biosynthesis of these alkaloids was investigated:

The carbon skeletons of both alkaloids originate from all the C‐atoms of anthranilic acid and l‐phenylalanine and from the methyl group of methionine.

N(1) and N(4) of the diazepine ring derive from all the nitrogen atoms of anthranilic acid and l‐phenylalanine respectively.

For cyclopenol, of several phenylpropane compounds tested, only l‐phenylalanine is a direct precursor. Thus, the introduction of the m‐hydroxy group by a mixed functional oxygenase as one of the later steps of its biosynthesis appears very likely.
A Quantitative Study of the Biosynthesis of Cytochrome c
Authors: Kadenbach B.
Published: March 2005

Sucrose‐density gradient experiments have shown it impossible to isolate microsomes from rat liver homogenate free from contaminated mitochondrial cytochrome c.

The incorporation of l‐[14C]lysine into cytochrome c from rat liver mitochondria and microsomes in vivo has been studied. The kinetics of the specific radioactivities of free l‐lysine, that of microsomal cytochrome c and that of mitochondrial cytochrome c show precursor‐product relationships.

From the kinetics of radioactivity of free l‐lysine and microsomal cytochrome c, the true pool‐size of the latter was calculated to be 1.02 nmoles/g microsomal protein, that is about 1/270 of total rat liver cytochrome c.

The turnover‐time of cytochrome c in the endoplasmic reticulum was found to be 102 min. From this data a turnover‐time of total rat liver cytochrome c of 19 days and a half‐life of 13.2 days was calculated.
Comportement du N‐acétylphénylalanyl‐tRNA dans un système acellulaire de Mammifère permettant la synthèse de polyphénylalanine
Authors: Reboud J. P.
Published: March 2005

The behavior of N‐acetylphenylalanyl‐tRNA as an initiator of phenylalanine polymerisation in an Escherichia coli cell‐free system shows some analogy with the behavior of formylmethionyl‐tRNA during the synthesis of natural proteins. At low Mg++ concentrations, polyphenylalanine chain initiation depends on N‐acetylphenylalanyl‐tRNA and the same initiation factors which are required for protein chain initiation with formylmethionyl‐tRNA.

However in a rabbit reticulocyte cell‐free system, N‐acetylphenylalanine is not incorporated into polyphénylalanine at any Mg++ concentration. In this paper, we showed that:

a) N‐acetylphenylalanyl‐tRNA did bind to reticulocyte ribosomes in the presence of poly U. This binding was GTP dependent and needed a factor which was extracted by washing ribosomes with salt (NH4Cl 1 M);

b) the bound N‐acetylphenylalanyl‐tRNA was partially released by puromycin or by addition of phenylalanyl‐tRNA. In this last case, incorporation of N‐acetylphenylalanine into polyphenyl‐alanine was noticed (ratio N‐acetylphenylalanine/phenylalanine = 1/20);

c) the ribosomal wash fluid also greatly enhanced phenylalanyl‐tRNA binding to salttreated ribosomes, in the presence of poly U and GTP. The maximum binding was obtained with the same concentrations of Mg++ and K+ as for N‐acetylphenylalanyl‐tRNA binding. It was not possible to separate on a DEAE‐cellulose column the factors that were required for phenylalanyl‐tRNA and acetylphenylalanyl‐tRNA binding. On the other hand, a preparation of the binding enzyme from reticulcyte supernatant enhanced both phenylalanyl‐tRNA and acetyl‐phenylalanyl‐tRNA binding to ribosomes;

d) the optimum Mg++ concentration for phenylalanine polymerisation was higher with salt‐washed ribosomes than with sucrose‐washed ribosomes. Adding the wash fluid shifted the Mg++ optimum back to the lower value. N‐acetylphenylalanyl‐tRNA was not required for this shift as noticed in Escherichia coli.

One likely explanation of our results with reticulocytes is the possibility that the “binding enzyme” of Schweet et al., which was present in our ribosome wash fluid, could work with both N‐acetyl as well as with phenylalanyl‐tRNA. Thus, the inability to incorporate N‐acetylphenyl‐alanine might be due to a competition between these two compounds for the same enzyme and the same ribosomal site. If this is indeed the case, it is in marked contrast to results obtained with Escherichia coli and could reflect a basic difference in the mechanism of protein chain initiation.
Enzymes Involved in Fructose Metabolism in Liver and the Glyceraldehyde Metabolic Crossroads
Authors: Sillero M. A. G. , Sillero A. , Sols A.
Published: March 2005

Fructokinase, the B isoenzyme of fructosediphosphate aldolase and triokinase, the three enzymes of the Hers' pathway for fructose metabolism, are constitutive in adult rats.

A quantitative model of the metabolic crossroads of d‐glyceraldehyde has been developed. This model permits a prediction of the relative contributions of the phosphorylative, oxidative and reductive pathways in different conditions. The main pathway for d‐glyceraldehyde metabolism in liver is phosphorylation by triokinase.
An Optical Rotatory Dispersion Investigation of Fungal Laccase
Authors: Bossa F. , Rotilio G. , Fasella P. , Malmström B. G.
Published: March 2005

The optical rotatory dispersion curves for fungal laccase A and B have been determined in the wavelength region 200–750 nm.

Both proteins show pronounced Cotton effects in the visible region. These are discussed in terms of possible chromophores (the so‐called Type 1 and Type 2 Cu2+ as well as the two diamagnetic copper ions). Various chemical treatments indicate that the anomalous dispersion is mainly associated with the blue, Type 1 Cu2+, which is thus coordinated in an asymmetric manner, but a small contribution from the Type 2 Cu2+ cannot be excluded.

There are several Cotton effects in the ultraviolet region, probably associated with aromatic amino acids and peptide bonds in α‐helical configuration, but possible contributions from carbohydrates and disulfide bridges are also considered. The curves in the ultraviolet region do not change on complete reduction of the enzyme, indicating that there are no gross conformational changes on reduction and that the groups giving rise to the anomalous dispersion in this region are not associated with the electron‐accepting sites.
Suche nach einem Metaboliten bei Vergiftung mit Desmethylphalloin (DMP)
Authors: Puchinger H. , Wieland Th.
Published: March 2005

Various observations cited at the beginning of this paper and in the Discussion suggested that phalloidine, a poisonous constituent of the green mushroom Amanita phalloides, would not be toxic by itself, but would be rendered toxic previously in the liver. Using a tritiated toxic derivative of phalloidine ([3H]desmethylphalloin) we found that:

(a) in rats, 60% of the administered substance are excreted unchanged in the urine within the first day. A second radioactive substance of low concentration, moving more slowly in paper chromatography, turned out as a product of the self‐degradation of [3H]desmethylphalloin;

(b) respectively 98% and 99,9% of the radioactivity were extracted with methanol from homogenates of livers of rats or mice which had received [3H]desmethylphalloin 1 to 2 hours before. Aqueous homogenates of liver of poisened rats also contained radioactivity firmly bound to a high molecular weight substance, as shown by gel chromatography. The radioactive substance was totally removed from the carrier, presumably ribosomal material, by adding methanol, and was identified as [3H]desmethylphalloin;

(c) [3H]desmethylphalloin was not metabolized on incubation with oxygen and rat liver ribosomes in a NADPH‐regenerating system. A radioactive substance extracted from liver microsomes of a poisoned rat also proved to be original toxin.

As a result of these experiments it can be concluded that [3H]desmethylphalloin and most probably also phalloidine are not metabolized in the livers of rats and mice.
Glutamic‐Aspartic Transaminase — Antitransaminase Interaction:
Authors: Patramani I. , Katsiri K. , Pistevou E. , Kalogerakos T. , Pavlatos M. , Evangelopoulos A. E.
Published: March 2005

Transaminase was cross‐linked with various proteins after the addition of glutaraldehyde or ethyl chloroformate. The water insoluble conjugates were used for the purification of antitransaminase and antiapotransaminase. A method is described.

Pig heart transaminase and ox heart transaminase react differently with anti‐pig‐heart transaminase and apotransaminase. Slight differences were also observed in the reactions between holoenzyme and apoenzyme with their homologous and heterologous antibodies.

l‐Aspartate and α‐ketoglutarate protect transaminase from the inhibitory action of antitransaminase. Pyridoxal‐5′‐phosphate has no effect on the enzyme‐antienzyme binding when added in the system in stoichiometric amounts. Higher concentrations of the coenzyme, however, activate the complex antigen‐antibody by facilitating its solubilization.

The intact conformation of transaminase is not essential for enzyme‐antienzyme‐binding. Peptides in the dialysate of a tryptic or a chymotryptic hydrolysate of transaminase were able to react with the antibody and form a precipitate.
The Oxidation and Reduction of Glyoxylate by Lactic Dehydrogenase
Authors: Duncan R. J. S. , Tipton K. F.
Published: March 2005

Crystalline rabbit muscle lactate dehydrogenase has been shown to catalyze the oxidation of glyoxylate to oxalate and its reduction to glycollate. Both these reactions have been shown to be catalyzed by all the isoenzymes present. Kinetic constants for both these reactions have been calculated. Oxalate inhibition patterns suggest that glyoxylate may be acting in its hydrated form as a substrate for oxidation and in its non‐hydrated form as a substrate for reduction.
Determination of Elastolytic Activity with 125I and 131I Labelled Elastin
Authors: Robert B. , Robert L.
Published: March 2005

A method is described for the preparation of iodine labelled elastin of high specific activity using 131I or 125I. The labelled elastin is used as substrate for the study of the kinetics of elastolysis with crystalline elastase. Its high sensitivity renders the method suitable for the detection and determination of low levels of elastolytic activity. This method is compared to the one using azo‐dyed elastin (“azoelastin”). Similar kinetics, with a lag period are obtained with both methods. The enzyme concentration v. s. activity curve is a straight line with both methods up to a ratio of enzyme to substrate of 1:50 at least. The advantages of this method over the others proposed is its greater sensitivity and its safe use in colored and turbid media.

This method was used for the detection of elastolytic activity in a number of animal, plant and fungal enzyme preparations. High activity was associated with pepsin, papaïn and pronase. Low, but distinct activity, was present in α‐chymotrypsin and ficin preparations. A purified bacterial collagenase had an intermediary activity. Crystalline trypsin had a very low, hardly detectable activity and plasmin had no activity.
Étude directe, par dialyse sur Sephadex G‐25, de la formation d'un complexe entre l'hexokinase de levure et ses substrats
Authors: Noat G. , Ricard J. , Borel M. , Got C.
Published: March 2005

A hexokinase has been isolated from Baker's Yeast, purified and obtained in crystalline form. Its specific activity is between 600 and 700 units/mg. This enzyme is homogeneous on DEAE‐cellulose chromatography. The molecular weight, estimated by molecular sieving on Sephadex G‐100, is around 49000.

The authors of the present article have been able to show, by equilibrium dialysis on Sephadex G‐25, that one molecule of the enzyme (MW 49000) binds one molecule of [14C]glucose in the absence of ATP. The value of the affinity constant for [14C]glucose, under the experimental conditions used and in the absence of ATP, is 10000 M–1. This value is nearly identical, within experimental errors, to 1/Km obtained from the measurement of hexokinase activity.

The binding of the Mg[14C]ATP2– chelate on the enzyme cannot be detected in the absence of glucose and under the experimental conditions used. This result implies that the affinity constant of the hexokinase for MgATP2– is lower than 800 M–1. The value of 1/Km for the ATPase reaction, determined under the same experimental conditions, is around 200 M–1.

These results are compatible with an ordered mechanism of hexokinase catalysis, in which the enzyme binds glucose first. They seem difficult to reconcile with a rapid equilibrium random mechanism.
Evidence for Two Molecular Forms of Streptococcal Erythrogenic Toxin
Authors: Nauciel C. , Blass J. , Mangalo R. , Raynaud M.
Published: March 2005

Two molecular forms of streptococcal erythrogenic toxin have been isolated by DEAE‐cellulose chromatography.

These two forms exhibit the same biological and immunological properties. They have the same sedimentation coefficient s20= 2.52. Molecular weight is about 30 000. The protein is made of a single polypeptide chain with glutamic acid as N‐terminal amino acid.

Exposure to 2‐mercaptoethanol induces conversion to a single form.

The possibility that the different molecular forms arise from the binding of small molecules of different charge through a sulfhydryl group of the protein is discussed.
Amino Acid Sequence of a 22‐Residue Section from the Variable Part of Pig Immunoglobulin λ‐Chains
Authors: Franěk F. , Keil B. , ŠOrm F.
Published: March 2005

The section which represents a part of a larger fragment of the variable part of pig immunoglobulin λ‐chains is constituted by two peptides (18 residues and 4 residues) of variable character. These peptides were fractionated and their amino acid sequence determined. At certain positions two to four different amino acids were found. There is a close relation between the found amino acid sequence

Ala‐Thr‐Leu‐Thr‐Ile‐Thr‐Gly‐Ala‐Gln‐Ala‐Glu‐Asp‐Glu‐Ala‐Asp‐Tyr‐Phe‐Cys‐Ala‐Leu‐Tyr‐Lys

Ala Leu Ala Asn Ser Gly Arg Gly Gly Thr Gly and the amino acid sequence of the corresponding region of human type L Bence‐Jones proteins.
Effects of Trypsin on the Electron Transport Systems of Liver Microsomes
Authors: Orrenius S. , Berg A. , Ernster L.
Published: March 2005

Treatment of rat liver microsomes with low amounts of trypsin resulted in an inactivation of NADPH‐cytochrome P‐450 reductase parallel to the release of NADPH‐cytochrome c reductase. Associated herewith there was an inhibition of the NADPH‐linked drug hydroxylation and lipid peroxidation activities, as well as an inactivation of the reduction of cytochrome b5 by NADPH. The NADH‐cytochrome c reductase system, on the other hand, was considerably less sensitive to trypsin treatment of the microsomes. The inhibition of the NADH‐cytochrome c reductase activity at higher concentrations of trypsin was, however, likewise correlated with a solubilization of cytochrome b5. High concentrations of trypsin also caused a conversion of cytochrome P‐450 into cytochrome P‐420 and a disappearance of the spectral shift characteristic of the interaction of cytochrome P‐450 with substrates of the hydroxylating system.
Über die Substrat‐ und Hormoninduktion der Tryptophan‐Oxygenase in der isoliert perfundierten Rattenleber
Authors: Staib R. , Thienhaus R. , Ammedick U. , Staib W.
Published: March 2005

This paper deals with investigations in isolated perfused rat livers on tryptophan‐oxygenase activity under various experimental conditions. Enzyme‐activity showed a linear rise with rising amounts of tryptophan (0; 125 and 250 mg of tryptophan/kg) in the perfusate. Adrenalectomized and sham‐operated animals have been compared and activity in the adrenalectomized rats were significantly lower in all cases.

A significant decrease in the substrate induced increase of tryptophan‐oxygenase‐activity occured 12 h after adrenalectomy. Substrate‐concentration was 250 mg/kg in these experiments.

The influence of substrate and cortisol on tryptophan‐oxygenase‐activity has been investigated 7 days after operation. Combined application of both substrate and steroid resulted in no difference to sham‐operated animals when compared to substrate only in the same concentration. On the other hand, in livers from adrenalectomized rats values were significantly higher with combined application than in livers of adrenalectomized animals which received substrate only.

The substrate‐dependent rise in tryptophan‐oxygenase‐activity could not be influenced by actinomycin D in contrast to the cortisol effect which was significantly inhibited under these conditions. Both “inducers” were inhibited by cycloheximid.

These inhibition‐experiments suggest and confirm two different mechanisms of the substrate‐activation and steroid‐induction of tryptophan‐oxygenase.
Activité de treize enzymes du métabolisme intermédiaire, dans le foie du jeune rat qui récupère d'une malnutrition protidique
Authors: Camus J. , Vandermeers‐Piret M.‐C. , Wodon C. , Christophe J.
Published: March 2005

100 g rats, submitted for 3 weeks to protein malnutrition (mostly lysine and threonine deficiency) with a diet offered ad libitum but containing 7% gluten as the sole protein, were starved for 24 h and allowed to recover for 3 days on a diet containing 18% casein and 0.3%dl‐methionine. These animals were compared to control rats refed on the gluten diet.

The food intake as well as the level of immunoreactive plasma insulin increased immedieately. Among the free amino acids of the plasma, lysine, threonine and tyrosine rose rapidly (4‐ to 6‐fold), others were also significantly elevated while serine, glycine and histidine decreased.

In the liver, the concentration of taurine was multiplied 7‐fold, that of threonine tripled, those of lysine, leucine, isoleucine, valine, tyrosine and phenylalanine doubled approximately, while the level of serine was markedly decreased. After 3 days, the total protein and DNA contents had increased by 70% and 2% respectively. Protein concentration on a wet weight basis was slightly higher than that observed in rats maintained on gluten whereas glycogen concentration levelled at a significantly lower (–43%) value. Of the 13 enzymatic specific activities investigated for their contribution to the intermediary metabolism, some did not vary significantly (α‐amylase bound to glycogen, hexokinases 1, 2 and 3, NADP:malate dehydrogenase, ATP:citrate lyase, arginase). Five activities increased (glucokinase 4‐fold, glucose‐6‐phosphate dehydrogenase 6‐fold, 6‐phosphogluconate dehydrogenase 2‐fold, serine dehydrogenase 2‐fold and ornithine aminotransferase 8‐fold) and three activities decreased somewhat (pyruvate kinases 0.60‐fold, glucose‐6‐phosphatase 0.65‐fold and fructose‐1,6‐diphosphatase 0.70‐fold).

These rapid modifications might favour in the liver (a), a faster utilization of glucose in the two main glycolytic pathways; (b), a decrease in the hepatic glucose output and (c), the removal of the amino group of amino acids supplied in excess of the needs for protein storage.

From these results and previously published experiments it thus appears that within the 3 days following a period of amino acid imbalance, insulin hypersecretion could not only have general effects on protein synthesis and turnover and on mitotic activity but might also be specifically involved in the induction of glucokinase and glucose‐6‐phosphate dehydrogenase and in the repression of glucose‐6‐phosphatase and fructose‐1,6‐diphosphatase. The influx of free amino acids plays the main role in the induction of serine dehydratase and ornithine aminotransferase and could share the responsability with insulin in the induction of glucokinase and glucose‐6‐phosphate dehydrogenase.
Localization and Function of External and Internal Carnitine Acetyltransferases in Mitochondria of Rat Liver and Pig Kidney
Authors: Brdiczka D. , Gerbitz K. , Pette D.
Published: March 2005

Subfractionation of rat liver mitochondria by the “digitonin method” revealed that carnitine acetyltransferase is present in two different compartments. The major part of the mitochondrial activity of this enzyme is found to be located in the inner mitochondrial compartment which contains also the soluble enzymes of the citric acid cycle, fatty acid oxidation and amino metabolism. At least 25% of the total mitochondrial activity of carnitine acetyltransferase can be assigned to the outer mitochondrial compartment. The localization of this “external” carnitine acetyltransferase corresponds thus to that of mitochondrial adenylate kinase. Carnitine dependent oxidation of acetyl‐CoA was studied in pig kidney mitochondria after treatment with low digitonin concentrations. It was found that oxidation of acetyl‐CoA in the presence of carnitine depends on the amount of carnitine acetyltransferase present in the outer mitochondrial compartment. Extraction of external carnitine acetyltransferase decreases the oxidation rate of acetyl‐CoA + carnitine. This may be partly restored by re‐addition of extracted external carnitine acetyltransferase. Relatively high digitonin concentrations (0.75 mg/10 mg mitochondrial protein) render inner mitochondrial membrane permeable to acetyl‐CoA. It is concluded that the inner membrane is a barrier to acetyl‐CoA and that transport of acetyl‐CoA across this membrane is mediated by the function of external and internal carnitine acetyltransferase.
The Utilization of Exogenous Pyrimidines and the Recycling of Uridine‐5′‐Phosphate Derivatives in Saccharomyces cerevisiae, as Studied by Means of Mutants Affected in Pyrimidine Uptake and Metabolism
Authors: Grenson Marcelle
Published: March 2005

Mutants impaired in cytosine uptake (cyt‐p) or in cytosine deamination (cyt‐da) are unable to use cytosine as a source of pyrimidines or as a source of nitrogen. The cyt‐p mutation makes it possible to define a specific cytosine permease.

A mutation (ura‐p) affecting exclusively uracil uptake delineates a specific uracil permease.

The study of mutants lacking uridine‐kinase activity (urid‐k) has shown that, whereas uridine can no longer be used as a source of pyrimidines in the absence of this enzymic activity, uridine kinase is not an obligatory step in the formation of UMP from uracil.

A mutation (urid‐p) affecting specifically uridine uptake delineates a uridine permease.

The behaviour of mutants (ups) blocked in UMP formation from uracil indicates that they have lost an enzyme catalyzing this reaction in one step.

A study of the mutants ups and urid‐k has shown that, when the utilization of uracil or uridine is decreased as a result of a mutation, the rate of uptake of uracil or uridine, respectively, is diminished by feedback inhibition of internal pyrimidines on the corresponding permeases, and, furthermore, that the enzymes coded or controlled by the ups and the urid‐k genes are not constituents of the transport systems.

The excretion of uracil by several mutants under different conditions has suggested that endogenous UMP liberates uracil in two steps catalyzed by a UMP phosphatase and a uridine‐ribohydrolase respectively. A mutant lacking the uridine‐ribohydrolase activity (urid‐rh) has been isolated. The observations reported suggest that uracil produced endogenously from UMP is continuously recycled.

The results of a chromatographic study of the fate of labelled pyrimidines added to the medium, and accumulated in the soluble pool, are in agreement with the interpretation of the various mutations.
The Antigenic Independence of the Three Aspartokinases of Escherichia coli K 12
Authors: Kaminski M. , Falcoz‐Kelly F. , Truffa‐Bachi P. , Patte J.‐C. , Cohen G. N.
Published: March 2005

The immunological properties of three pure enzyme proteins carrying respectively the two threonine‐sensitive aspartokinase I and homoserine dehydrogenase I activities (Aspartokinase I—homoserine dehydrogenase I), the two methionine‐repressible aspartokinase II and homoserine dehydrogenase II activities (aspartokinase II—homoserine dehydrogenase II), and the lysine‐sensitive aspartokinase III (aspartokinase III), have been investigated by means of double diffusion precipitation in agar gel.

The antigenic structures of these three proteins appeared different: no cross‐reactions were detected.
Metabolic Regulation of Polygalacturonic Acid trans‐Eliminase in Erwinia
Authors: Moran F. , Starr M. P.
Published: March 2005

Strains of Erwinia carotovora and Erwinia aroideae, formerly thought only to be inducible, are constitutive for polygalacturonic acid trans‐eliminase. Maximum enzyme formation, however, occurs in the presence of polygalacturonic acid.

A release from strong catabolic repression enables glucose‐grown cultures to initiate their attack on polygalacturonic acid and, thereby, eliminates the necessity for the entry of the polymer into the cell.

A low constant proportion (less than 25%) of the total polygalacturonic acid trans‐eliminase is present in the culture fluid during the exponential growth‐phase of Erwinia carotovora. The enzyme may be regarded as an exoenzyme. The exact location of polygalacturonic acid trans‐eliminase in the cells was not established; it could be truly intracellular or reside between the cell‐wall and membrane.
The Cytoplasmic DNAs of Cultured Human Cells
Authors: Koch J.
Published: March 2005

In cultured human cells (Chang, liver) labeled thymidine is incorporated during short labeling periods into a cytoplasmic DNA, presumably located in the mitochondria, with a buoyant density of 1.688 g/ml (nuclear DNA main band 1.700 g/ml). This DNA is apparently metabolically labile and becomes completely delabeled after a chasing period of approximately three cell generations. During such a chase period an increasing amount of radioactive label appears in closed circular duplex DNA (buoyant density 1.706 g/ml). Pulse ([Me‐3H]thymidine)–chase–pulse ([2‐14C]‐thymidine) experiments suggest that the “light cytoplasmic” DNA is a precursor of the closed circular duplex DNA.
Replication of Poliovirus RNA Studied by Gel Filtration and Electrophoresis
Authors: ÖBerg B. , Philipson L.
Published: March 2005

The kinetics of synthesis of poliovirus RNA has been studied in suspension cultures of HeLa cells.

Single stranded poliovirus RNA was separated from double stranded structures (replicative form and replicative intermediate) by gel filtration on sphere‐condensed agarose. Electrophoresis on polyacrylamide‐agarose gels was used for analytical separation of the three classes of poliovirus RNA in infected cells i. e. single stranded RNA, replicative form and replicative intermediate.

Continuous labeling experiments confirmed that single stranded RNA is synthesized at a higher rate than the double stranded structures. Net synthesis of the replicative form levels off after 4 h of infection but the replicative form and the single stranded RNA are synthesized at high rates at later times. The replicative form probably precedes the single stranded RNA in the replication cycle.

No free single strands complementary to viral RNA could be detected from 1 to 5 h after infection.

Pulse labeling experiments reveal that the replicative form, the replicative intermediate and the single stranded RNA are synthesized at the highest rates 3–3.5 h post infection. The synthesis of the replicative intermediate and the single stranded RNA decreases at later times, while the replicative form synthesis continues unabated. At 3.5 h post infection a short (1 min) pulse introduces more label into the replicative intermediate than the single stranded RNA and the replicative form. As the length of the pulse increases the relative amount of label in the single stranded RNA increases, the replicative form remains constant and the replicative intermediate decreases. These results indicate a precursor‐product relationship between the replicative intermediate and the single stranded RNA, at 3.5 h post infection. At late times in the replication cycle most of the newly synthesized replicative form is not formed as an end product from the replicative intermediate.
On the Role of Quinones in Bacterial Electron Transport
Authors: Kröger A. , Dadák V.
Published: March 2005

The menaquinone of Bacillus megaterium (14581 ATCC) is localized exclusively in the cytoplasmic membrane as are the cytochromes. Menaquinone is contained in a 5 to 10‐fold molar excess over cytochrome b1.

The constituent menaquinone is almost completely reduced to menaquinole in the anaerobic state with the substrates respired by the membrane fraction. On the addition of oxygen the menaquinole is reoxidized to menaquinone. Menaquinone and menaquinole are the only forms of MK detected in the steady states of the electron transport.

The redox reactions of menaquinone indicate its participation in the electron transport from the substrates α‐glycerophosphate, malate and NADH, to the acceptors oxygen and fumarate. Menaquinone is in a steady state in all the flow systems established with each substrate and both acceptors, and exhibits the steady state response expected for a respiratory component.

The cytochrome system of this strain of B. megaterium consists of a b‐type cytochrome with a redox potential of + 8 mV and absorption bands close to those of pure cytochrome b1, two cytochrome oxidases, and α‐type cytochrome and cytochrome o and probably a small amount of c‐type cytochrome. Cytochrome b1 is completely reducible by each of the substrates.

2‐n‐Heptyl‐4‐hydroxyquinoline‐N‐oxide inhibits the electron flow at about the concentration of the constituent menaquinone between menaquinone and the cytochromes. The electron flow from each of the substrates to both acceptors depends completely on the reaction sensitive to the inhibitor.

The electron transport from α‐glycerophosphate, malate and NADH to oxygen and fumarate as well as the reduction of cytochrome b1 is dependent on the presence of MK. This is demonstrated by pentane extraction and reincorporation of the MK. The reincorporation leads to the reconstitution of the original pathway, since MK and cytochrome b1 participate in the electron transport and the same sensitivity to 2‐n‐heptyl‐4‐hydroxyquinoline‐N‐oxide is found.

The position of menaquinone in the respiratory system of B. megaterium is between the dehydrogenases for α‐glycerophosphate, malate and NADH and cytochrome b1 to which the fumarate reductase and the cytochrome oxidases are linked.
α‐Chymotrypsin‐Catalyzed Hydrolysis of N‐Furylacryloyl‐Tyrosine Derivatives
Authors: Yu S. H. , Viswanatha T.
Published: March 2005

The hydrolysis of furylacryloyl‐tyrosine derivatives by chymotrypsin has been investigated. The reaction of N‐furylacryloyl‐l‐tyrosine methyl ester with the enzyme can be followed spectrophotometrically at 340 nm. The product of the reaction, N‐furylacryloyl‐l‐tyrosine, does not function as an inhibitor. The d‐isomer of the substrate functions as a competitive inhibitor. The reaction of furylacryloyl‐l‐tyrosine methyl ester with chymotrypsin at low pH is accompanied by absorption changes separable into three distinct stages. These studies offer evidence for the occurrence of the reaction via the acylenzyme route in the case of typical substrates. Analysis of the kinetics of the reaction suggest the formation of an additional intermediate prior to the acylation of the enzyme.
Chemical Synthesis and Quantitative Estimation of 3‐Deoxy d‐manno‐Octulosonic Acid
Authors: Charon D. , Sarfati R. S. , Strobach D. R. , Szabó L.
Published: March 2005

3‐Deoxy d‐manno‐octulosonic acid has been synthesised starting from d‐mannose via 2‐deoxy d‐manno heptose, cyanohydrine addition, and selective oxidation of the epimeric hydroxyl group of the resulting 3‐deoxy octonic acids. When treated with periodate in 0.1 N sulphuric acid solution at 0°, the deoxy octulosonic acid and other 3‐deoxy aldulosonic acids all yield one molar equivalent of β‐formyl pyruvate. Conditions are described in which β‐formyl pyruvate can be estimated by the thiobarbituric acid method; hence, when treated successively with periodate and thiobarbiturate, all 3‐deoxy aldulosonic acids have the same molar extinction coefficient [(92 ± 5)103 in dilute acid; 133 × 103 in cyclohexanone] and can be estimated by this method without the use of standards.
On Carbanion Formation in the Process of Enzymatic Transamination
Authors: Shlyapnikov S. V. , Karpeisky M. Y.
Published: March 2005

The rate of decomposition of tetranitromethane in the presence of enzyme‐substrate complexes of l‐aspartate: 2‐oxoglutarate aminotransferase has been studied; the intermediate Shiff base of aspartate aminotransferase and erythro‐β‐hydroxy‐l‐aspartate was shown to undergo nitration. The catalytic course of nitration of the complex is indicative of predominant modification of the substrate part of the carbanion. The presence of carbanion was demonstrated in the course of both model and enzymatic transamination.
L'acétylcholinestérase des organes électriques de Poissons (torpille et gymnote); complexes membranaires
Authors: Massoulié J. , Rieger F.
Published: March 2005

After extraction of the torpedo electric organ, three main molecular species, A, C, D, carrying the acetylcholinesterase activity have been found. This is observed in fresh‐frozen organ extracts, toluene‐autolysed organ extracts, or autolysis exsudate. A fourth species, B, appears during autolysis in the exsudate, or following treatment of the extracts by proteolytic enzymes, trypsin or α‐chymotrypsin. A series of agents such as pH, p‐hydroxymercuribenzoate, or pharmacological drugs, have been tried in order to modify the distribution of activity among the different species; all have failed, demonstrating the stability of these species.

The two species which migrate faster in a sucrose gradient, C and D, show the phenomenon of aggregation in low salt concentration (0.01 M), whereas and A and B, the slower species, do not.

The buoyant density of the active species in our extract is the same as that of the purified enzyme; a high content of lipids seems therefore excluded.

Species B in electric eel extracts (obtained after trypsic treatment) has been identified with the molecular form of the enzyme as recently purified and cristallized.

This raises the question of the biological significance of this preparation, especially since one of the original species, A, has a smaller mass and is also converted into B during proteolysis.
Glutamate déshydrogénase
Authors: Pantaloni D. , Dessen P.
Published: March 2005

The pig liver glutamic dehydrogenase has been studied by difference supectroscopy and shows two diphosphopyridine nucleotide binding sites per protomer of molecular weight 52000. A first NADH molecule binds on an adenylic site (Ka= 42 mM–1), and a second molecule on an nicotinamide site (Kn= 13 mM–1). NADPH binds only on the nicotinamide site (K= 21 mM–1).

The adenylic site does not only strongly bind ADP (K= 2800 mM–1, but also ADP‐ribose, ATP, AMP, NADH, NAD, ApA, GpA, with a decreasing affinity; ApG and GTP does bind on this site which is specific of the adenosine 5′‐phosphate group. The high ionic strength decreases ADP binding.

The linkage between the nicotinamide site and NADH induces a positive Cotton effect in the range of the absorption band of the reduced nicotinamide group; moreover the absorption spectra of bound NADH is shifted about 5 nm to the greater wavelengths with an hypochromism of 6%, with respect to free NADH.

The evidence for two distinct binding sites is given by: (a) the existence of two difference spectra: the first due to the nicotinamide chromophore perturbation and the second due to the adenylic chromophore perturbation. This last is similar to that obtained with ADP, AMP, ATP; etc.; (b) the competition between ADP and NADH only on the adenylic site; (c) the fact that the saturation curves obtained from measurements made in the absorption range of adenosine and reduced nicotinamide chromophores are different and square with the theoretical saturation curves of two distinct sites per protomer.

NAD also binds on the adenylic site in competition with ADP, whereas NADP and NADPH do not induce any perturbation of the adenosine chromophore.

All these results are in good agreement with the interpretations given by Frieden to explain the effects observed in the steady‐state enzymatic kinetics at high concentrations of coenzyme.
The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K 12
Authors: Janin Joël , Cohen Georges N.
Published: March 2005

Aspartokinase I‐homoserine dehydrogeanase I is an allosteric protein with six subunits. Conformation changes upon addition of a substrate, l‐aspartate, and of an activator, K+, or an inhibitor, l‐threonine, are demonstrated by a study of the ultraviolet absorption and of the fluorescence emission of the protein. The effects of the ligands are compatible with a two‐state model and a three‐by‐three concerted transition of the subunits from the inactive, threonine binding conformation, to the active conformation binding aspartate and the potassium ions.
The Immunochemistry of Shigella flexneri O‐Antigens
Authors: Simmons D. A. R.
Published: March 2005

Structural analysis of a representative group of Shigella flexneri lipopolysaccharides has shown that the O‐specific side chains of these polymers consist of a primary unbranched chain of N‐acetylglucosamine and rhamnose substituted with secondary side chains of glucose. Sh. flexneri Y variants which lack the glucose secondary side chains and are thus structurally identical with the primary side chains of the different serotypes, belong to one of two types namely, Y1 which consists of N‐acetylglucosaminyl‐(1→2)‐rhamnosyl‐(1→4)‐rhamnose repeating units bound together by a (1→6)‐linkage or Y2 which consists of N‐acetylglucosaminyl‐(1→3)‐rhamnosyl‐(1→4)‐rhamnose repeating units bound together by a (1→4)‐linkage. The different smooth Sh. flexneri serotypes are synthesised from these variant Y1 or Y2 precursor structures by the action of specific UDP‐glucose transferases which incorporate the glucose secondary side chains in a position that characterises each serotype. The structural sequences that seem to play an immunodominant role in antigen factors I, II, III, IV, V and 7,8 are α‐glucosyl‐(1→4)‐N‐acetyl‐glucosamine, α‐glucosyl‐(1→4)‐rhamnose, O‐acetyl‐α‐glucosyl‐(1→2)‐rhamnose, α‐glucosyl‐(1→6)‐N‐acetylglucosamine, α‐glucosyl‐(1→3)‐rhamnose and α‐glucosyl‐(1→2)‐rhamnose respectively. The group antigen 3,4 in which rhamnose is the immunodominant sugar, seems to be related to the internal rhamnosyl‐(1→4)‐rhamnose sequence. The serotype 6 antigen (VI) used in this study is so different structurally from its analogues that this organism is no longer regarded as a true Sh. flexneri species. From the structural relationship of the different O‐antigens, X and Y variants are the consequence of enzyme defects that interrupt the biosynthesis of the complete smooth lipopolysaccharides. Y1 variants probably result from defects in the specific UDP‐glucose transferases of serotypes 1a and 2a while Y2 variants probably result from defects in the analogous UDP‐glucose transferases of serotypes 4a, 3a, 5 and variant X. Variant X is an intermediate stage in the biosynthesis of serotypes 3a and 5 from the precursor Y2 structure. Further evidence for the role of UDP‐glucose transferases in conferring type specificity on the cryptic Y structures comes from two sources. Firstly, the type specific or T‐loci of Petrovskaya that map near the lac locus are those in which glucose is the immunodominant sugar and loss of this locus by genetic recombination results in a glucose‐deficient Y‐type hybrid. Secondly, the structures proposed show that all phage conversions of Sh. flexneri reported to date, can be explained in terms of single enzyme changes involving specific UDP‐glucose transferases. From the limited number of antigens studied, it can be predicted that the expression of type specific antigens will depend on the presence of group factors, as the former are always end group determinants which cannot be incorporated into the growing molecule before biosynthesis of the more central regions which determine group antigenicity.
Synthesis of Lens Protein in vitro.
Authors: Konings R. N. H. , Bloemendal H.
Published: March 2005

RNA was isolated from calf lens polysomes and crude nuclear preparations. Template activity, base composition, sedimentation behaviour, and electrophoretic properties were compared with corresponding RNA fractions from rat liver. 8–9‐fold stimulation of amino acid incorporation was obtained in the cell‐free test system derived from Escherichia coli. Template activity was widely distributed throughout sucrose density gradients. However, the major activity consistently was found at the bottom of the tubes. All fractions from the gradient revealed a heterogeneous pattern when subjected to polyacrylamide gel electrophoresis. The bottom fraction constistently showed a diffuse pattern instead of discrete bands. Only RNA fractions extracted at 65° and at pH 8.3 showed a G‐C content which was significantly different from ribosomal RNA.