bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hydrolyse trypsique de dérivés d'amino‐acides non substitués en α
Authors: Seydoux François 
Published: March 2005

The tryptic hydrolysis of l‐lysine methyl ester, l‐lysine paranitroanilide and l‐tyrosine methyl ester has been studied as a function of pH.In addition, these reactions have been studied in the presence of methanol as a nucleophilic reagent wich competes with water in the deacylation step.With lysine methyl ester, a bell‐shaped profile for Kcat vs pH has been observed. The optimum pH for this substrate occurs at pH 5.8. Km decreases 200‐fold when the pH is increased from pH 5 to 8.5. From the nucleophilic competition data, it can be deduced that the k2/k′3 ratio increases between pH 4.8 and 5.8 with deacylation as rate‐determining step.With tyrosine methyl ester, the profiles of both kcat and Km vs pH are bell‐shaped, but the pH optimum in this case occurs at a pH higher than with lysine methyl ester (pH 7.5). The nucleophilic competition data indicate that the acylation step is rate‐limiting for this substrate.With lysine paranitroanilide, kcat increases when the pH is increased from 5 to 6.5, but at higher pH, the kinetic behavior of this substrate is complicated by a non‐Michaelian saturation phenomenon, probably related to transpeptidation.From the quantitative interpretation of our experimental data, the following conclusions could be drawn:

The conjugate acids are more reactive than their corresponding α‐amino acid derivatives.
In the acylenzyme formed with bicationic conjugate acid of lysine methyl ester, the active imidazole pK is strongly displaced from its normal value to the acidic region. (This pK is lowered from 6.90 in the free enzyme to 4.80 in this acylenzyme.)These two first points explain the anomalous value of the pH optimum for this substrate.
The pK shift is less pronounced in the Michaelis complex formed with the conjugate acid of tyrosine methyl ester, lysine methyl ester and lysine paranitroanilide. Since this pK shift can be interpreted as resulting from a repulsive electrostatic interaction between the imidazolium group of enzyme and the α‐ammonium group of the substrate, our data suggest a change of the active site geometry (including substrate) after the acylation step.

The comparison of the kinetic parameters obtained in this work with those of corresponding α‐N‐acyl derivatives suggests that the α‐peptidic linkage is an important structural factor for the acylation step but not for the deacylation.Finally, the effect of possible transpeptidation reaction on the kinetic data is discussed.
The Cu2+‐Alkaline Phosphatase of Escherichia coli
Authors: Lazdunski Claude , Chappelet Danielle , Petitclerc Claude , Leterrier François , Douzou Pierre , Lazdunski Michel 
Published: March 2005

The Cu2+‐alkaline phosphatase is considerably less active than the Zn2+‐ and Co2+‐enzymes. It also differs from the native Zn2+‐alkaline phosphatase by its sensitivity to nucleophiles and ionic strength. However, in spite of these differences, there remains an important common property, the considerable analogy between the kinetics parameters (Km and Vmax) for substrates with very different chemical structures.The Cu2+‐enzyme, like the Zn2+‐enzyme, is phosphorylated on both sites at acidic pH with the substrate AMP or the product inorganic phosphate. No phosphorylation occurs at alkaline pH where phosphorylation of the active centers is the rate‐limiting step. Only non‐covalent complexes containing one mole of inorganic phosphate per mole of enzyme could be isolated. The binding of another phosphate on the second site appears to be very difficult and to occur only at high concentrations of inorganic phosphate. Anticooperativity, an important property of the Zn2+‐phosphatase is preserved in the Cu2+‐phosphatase.Electron paramagnetic resonance indicates that the 4 coppers of the Cu2+‐alkaline phosphatase belong to 2 different classes. There are 2 tight metal sites of type I and 2 loose metal sites of type II. Orthophosphate binding to the Cu2+‐phosphatase alters both the visible and the electron paramagnetic resonance spectra. Electron paramagnetic resonance provides direct evidence for the participation of the coppers of type I in substrate binding. This is further evidence that each active site in the metallo‐phosphatases contains 2 metal atoms, one of type I and one of type II.
Ein Beitrag zur Isolierung und Charakterisierung des C‐Inaktivators aus Humanplasma
Authors: Haupt Heinz , Heimburger Norbert , Kranz Theodor , Schwick H. Gerhard 
Published: March 2005

A simple procedure for the isolation of C‐Inactivator from fresh human ACD plasma is described consisting of the following fractionation steps:DEAE chromatography, ammonium sulfate precipitation, Sephadex G‐150 gel filtration and zone electrophoresis. A 250‐fold concentration was achieved in good agreement with an immunologically determined C‐Inactivator concentration of 25 mg/100 ml serum.C‐Inactivator is a glycoprotein containing 35% carbohydrates; it migrates with the α2‐globulins at pH 8.6 and in the PS‐1 zone at pH 4.4. The isoelectric point is between 2.7–2.8. It had s20,w of 3.67 and a molecular weight of 104000 according sedimentation equilibrium patterns. C‐Inactivator could not be dissoziated in subunits even not by reduction with 2‐mercaptoethanol and subsequent alkylation with iodoacetamide. 1 mg C‐Inactivator neutralizes 17 C‐Inactivator units respectively 5 Remmert and Cohen plasmin units. Only weak affinities for trypsin and chymotrypsin were found.
Characterization of a 72 S Mitochondrial Ribosome from Saccharomyces cerevisiae
Authors: Schmitt Henri 
Published: March 2005

A 72 S ribosome can be isolated from EDTA‐washed mitochondria of Saccharomyces cerevisiae.It easily dissociates into 38 S and 50 S subunits. Incorporation of amino acids into the protein of mitochondria purified in this way is shown to be inhibited by chloramphenicol, while cycloheximide has no effect.When EDTA treatment of the mitochondria is omitted, the mitochondrial fraction contains in addition a special class of 80 S ribosomes, presumably membrane‐bound, which dissociate less easily than the “free” cytoplasmic ribosomes.Incorporation of amino acids into protein of these mitochondria is only partially inhibited by either chloramphenicol or cycloheximide and is stimulated by the addition of a cytoplasmic supernatant.
Effect of Cholestyramine and Diet on Hydroxylations in the Biosynthesis and Metabolism of Bile Acids
Authors: Johansson Gunnar 
Published: March 2005

The effects of biliary drainage, cholestyramine treatment, starvation and a semisynthetic diet on nine different hydroxylations in the biosynthesis and metabolism of bile acids were studied. Biliary drainage and feeding a diet containing 3% cholestyramine resulted in a five‐ to seven‐fold stimulation of the 7α‐hydroxylation of cholesterol. Biliary drainage resulted in a five‐fold stimulation of the 12α‐hydroxylation of 7α‐hydroxy‐4‐cholesten‐3‐one. The other hydroxylations as well as the formation of 7α‐hydroxy‐4‐cholesten‐3‐one from 5‐cholestene‐3β,7α‐diol were unaffected by biliary drainage and cholestyramine treatment. Starvation stimulated the 12α‐hydroxylation of 7α‐hydroxy‐4‐cholesten‐3‐one about four‐fold. Feeding a fat‐free, semisynthetic diet resulted in a decrease in the rate of all reactions.The results support the concept that the 7α‐hydroxylation of cholesterol is a major rate‐limiting reaction in the biosynthesis of bile acids and indicate that also the 12α‐hydroxylation of 7α‐hydroxy‐4‐cholesten‐3‐one may be rate limiting to some extent.
Messenger RNA in HeLa Cells:
Authors: Spohr Georges , Granboulan Nicole , Morel Carlos , Scherrer Klaus 
Published: March 2005

Messenger RNA (mRNA) and messenger like RNA (mlRNA) were investigated in the cytoplasm of HeLa cells while ribosomal RNA synthesis was arrested. Under these conditions, functional mRNA associated in polyribosomes and cytoplasmic free mlRNA are formed and can be labelled selectively to steady state.All cytoplasmic non‐ribosomal RNA sedimenting at more than 6–7 S exists in the form of ribonucleoprotein complexes which pre‐exist in the cell, and are stable upon cell lysis, sedimentation and (after fixation) CsCl density gradient analysis. The functional, true mRNA is contained in a complex of mRNA and protein which bands in association with ribosomes (ϱ= 1.52 to 1.60 g/cm3) in CsCl density gradients or, released by EDTA, at its own intrinsic density of 1.40–1.48 g/cm3. The cytoplasmic free mlRNA bands as a particle of mlRNA‐containing ribonucleoprotein at an identical low density. The molecular weight spectrum of mRNA is identical to that of mlRNA and the sedimentation pattern of the mRNA · protein complex released from polyribosomes is similar to that of the free mlRNA · protein complex.The physico‐chemical separation of mRNA · and mlRNA · protein complexes allowed us to follow their relative kinetics of synthesis and decay. Each type of ribonucleoprotein obeys a different, strictly time‐dependent pattern. Label enters the pool of free mlRNA · protein complexes first and may, in a pulse‐chase experiment, be partially chased into polyribosomes. At steady state (6 h) 40–60% of the labelled RNA remains in the form of free mlRNA · protein particles. These cannot be chased into polyribosomes, the kinetics of mRNA · and mlRNA · protein complexes decay following identical patterns.These findings are in agreement with a model according to which mlRNA from the nucleus first joins the pool of free ribonucleoprotein. Then, the activated mRNA · protein complexes attach to ribosomal subunits and form polyribosomes whereas inactivated mlRNA · protein complexes remain free in the cytoplasm.In order to further strengthen the evidence in favour of the real existence of mRNA · and mlRNA · protein complexes in the cells, the corresponding fractions from sedimentation or CsCl density gradients were observed in the electron microscope. By this method it was possible to see small cytoplasmic particles which have not before been identified. The rounded structures, with diameters ranging from 100 Å to 200 Å, seem to consist of the coiling of a 35 Å wide pearllike chain which may also be identified in polyribosomes. The frequency of these particles is highest in the mlRNA · protein band (ϱ= 1.40–1.48 g/cm3). Thus they may correspond to the mRNA · protein complex. However since they share some morphological features with other known biological structures the evidence is not conclusive.
Mammalian Cell‐Free Protein Synthesis Directed by Viral Ribonucleic Acid
Authors: Mathews Michael B. , Korner Asher 
Published: March 2005

A cell‐free system from mouse Krebs II ascites cells is described which responds by increased amino acid incorporation into protein on addition of encephalomyocarditis virus (EMC‐RNA). RNA from other sources does not produce this response.
The stimulation by EMC‐RNA occurs over a narrow range of Mg2+ concentrations and is maximal at 5 mM, which is optimal for the endogenous incorporation and lower than that required in the presence of poly(U).
The EMC‐RNA‐directed product is distinguished from the endogenous products by its size and composition.
After an initial lag of 4 min, during which the nascent chains are too short to be acid‐insoluble, EMC‐RNA‐directed protein synthesis is active for more than 1 h.
EMC‐RNA‐directed synthesis is particularly sensitive to the inhibitors cycloheximide and dextran sulphate. Using the latter, it has been shown that the initiation of protein synthesis is completed during the first 15 min of incubation.
Only 20–30% of the product of the cell‐free system, whether endogenous or EMC‐RNA‐directed, is released from the ribosomes. Despite the size of the viral RNA, it did not appear to promote the formation of exceptionally large polysomes.
It is concluded that the EMC‐RNA is acting as a message for viral proteins in the ascites cell‐free system.
Sterol Biosynthesis in Euglena gracilis Z
Authors: Brandt Roger D. , Pryce Robert J. , Anding Claude , Ourisson Guy 
Published: March 2005

Free and bound (esterified and water solubilised) sterols of light grown (green) and dark grown (white) Euglena gracilis Z have been characterised by thin layer chromatography, gas chromatography and mass spectrometry. Differences in sterol metabolism by these two types of E. gracilis are reflected in profound quantitative and qualitative differences in their sterol compositions. Free sterols predominate over bound sterols in green E. gracilis whereas in white E. gracilis the reverse is true. The free sterols of green E. gracilis consist almost exclusively (98%) of Δ7‐sterols while in white E. gracilis they are a mixture containing Δ5‐ (31%) and Δ7‐sterols (47%). However, in both types of E. gracilis only Δ5‐sterols are bound in esterified and water soluble forms.The following sterols were found in green and white E. gracilis: Δ7‐cholestenol, Δ5,7‐cholestadienol, cholesterol, episterol, chalinasterol, Δ7‐ergostenol, 5‐dihydroergosterol, Δ7‐chondrillasterol, Δ5,7‐chondrillasterol, Δ22‐dihydrobrassicasterol, clionasterol and poriferasterol. Chondrillasterol, the major sterol in green E. gracilis, was not found in white E. gracilis. Cholesterol, a minor constituent of free sterols of green (1%) and white (12%) E. gracilis, is the major constituent of their bound, water soluble sterols.
Hefe‐Phosphofruktokinase
Authors: Freyer Renate , Kubel Martin , Hofmann Eberhard 
Published: March 2005

The kinetic behaviour of different preparations of yeast phosphofructokinase is described, predominantly regarding the interactions between substrate‐ and effector‐binding sites.The substrate‐binding site for ATP reacts with ITP and UTP too, whereas the ATP‐inhibitor site possesses higher specifity: other nucleoside triphosphates do not compete with ATP for this site.Binding of protons to the enzyme produces a decrease in cooperativity towards fructose‐6‐phosphate with ATP as well as with ITP and UTP as phosphate donors, accompanied by a lowering of the fructose‐6‐phosphate concentration necessary for half‐maximal activity. At the same time the inhibitory action of ATP is descreased.AMP is bound to an effector site evidently different from the ATP‐inhibitor site.With ITP as phosphate donor, AMP shows no activating effect. AMP decreases the apparent Km value for fructose‐6‐phosphate too without affecting cooperative interactions. Magnesium and ammonium ions are necessary for full enzymatic activity. The latter increases maximal activity and decreases the half‐maximal concentration of fructose‐6‐phosphate.Free ATP seems to be a stronger inhibitor for yeast phosphofructokinase than the magnesium‐ATP‐chelate.
On the Biosynthesis of Laminitol (d‐4‐C‐Methyl‐myo‐inositol) in Porphyridium sp.
Authors: Wöber G. , Hoffmann‐Ostenhof Otto 
Published: March 2005

Of all tested presumable precursors of laminitol (d‐4‐C‐methyl‐myo‐inositol) which were applied in labelled form to the alga Porphyridium sp., l‐gluco‐heptulose showed by far the highest rate of incorporation. It seems probable that in this organism l‐gluco‐heptulose is derived from l‐arabinose by C2 addition in a way similar to which it is formed in some higher plants.The occurrence of stationary concentrations of both l‐gluco‐heptulose and 1‐deoxy‐l‐gluco‐heptulose in the algae was established.On the basis of these results a biosynthetic pathway for laminitol which comprises as key reaction an intramolecular condensation between the carbon atoms 2 and 7 of 1‐deoxy‐l‐gluco‐heptulose or of a close derivative of this deoxysugar is suggested.
ω‐ and (ω‐1)‐Oxidation of Fatty Acids by Rat Liver Microsomes
Authors: Björkhem Ingemar , Danielsson Henry 
Published: March 2005

Rat liver microsomes fortified with NADPH catalyzed the conversion of laurate, palmitate and stearate into the corresponding ω‐ and (ω‐1)‐hydroxy derivatives. In addition, small amounts of the dicarboxylic acids were formed from palmitate and stearate. The ratio between ω‐ and (ω‐1)‐oxidized products was about 1 in incubations with laurate, about 2 in those with palmitate and about 3 in those with stearate. Addition of NAD to incubations with palmitate and stearate increased the relative amount of dicarboxylic acids and also the total yield of ω‐oxidized products showing the presence in microsomes of ω‐hydroxy‐fatty acid dehydrogenases. This conclusion was confirmed by the finding that 16‐[1‐14C]hydroxypalmitic acid and 18‐[9,10‐3H2]hydroxystearic acid were converted efficiently into the corresponding dicarboxylic acids by the microsomal fraction fortified with NAD. The same reactions occurred also in the presence of the 100000 ×g supernatant fluid. The microsomal fraction fortified with NAD as well as the 100000 x g supernatant fluid were found to catalyze the oxidation of 17‐hydroxystearic acid into 17‐oxostearic acid.Administration of phenobarbital had no significant effect on microsomal ω‐hydroxylation of laurate, whereas ω‐hydroxylation of palmitate and stearate was inhibited. (ω‐1)‐Hydroxylation of laurate, palmitate and stearate was stimulated about two to three‐fold. Carbon monoxide inhibited ω‐ as well as (ω‐l)‐hydroxylation of laurate, palmitate and stearate.
The Structure of Legumin, a Storage Protein of Broad Bean (Vicia faba) Seed
Authors: Bailey Christopher J. , Boulter Donald 
Published: March 2005

Legumin from broad bean seed has been carboxymethylated and analysed. The protein contained three N‐terminal residues, leucine, glycine and threonine. Three polypeptide chains were found by polyacrylamide gel electrophoresis of the dissociated protein; their molecular weights were 56000, 42000 and 23000. 140 peptides were found on fingerprints of tryptic digests of S‐carboxymethyl‐legumin. Of these 14 were radioactive in digests of [14C]carboxymethylated legumin, and 16 were radioactive in digests of legumin prepared from material labelled in vivo with [35S]sulphate. Three new end groups were found after cyanogen bromide cleavage corresponding to Met‐Val, Met‐Phe and Met‐Pro sequences. Taken in conjunction with the amino acid analyses these peptide mapping experiments suggest that the protein contains a total sequence weight of 115000, which is in agreement with the finding of three polypeptide chains whose weights total 121000.On the basis of dye binding studies and of the incorporation in vivo of uniformly 14C‐labelled amino acid mixture, it is suggested that the polypeptide chains of molecular weights 56000, 42000 and 23000 are present in molar ratios of 1:4:6 or 1:3:6, respectively. From consideration of experimental accuracy the 1:3:6 structure is preferred. The proposed 10 chains model for legumin has a molecular weight of 320000.
The Essential Thiol Group of Arginine Kinase from Homarus vulgaris Muscle Studied by Difference Spectrophotometry and N‐Ethyl‐[l‐14C]Maleimide Labelling
Authors: Roustan Claude , der Terrossian Elisabeth , Pradel Louise‐Anne 
Published: March 2005

Interaction of N‐ethylmaleimide with the essential cysteinyl residue of ATP:l‐arginine phosphotransferase from Homarus vulgaris muscle has been studied by means of difference spectrophotometry and labelling with radioactive N‐ethyl‐[1‐14C]maleimide.Cysteine and glutathione were used as model compounds reacting with the inhibitor. The difference spectrum at 238 nm observed with the l‐arginine‐arginine kinase complex might be ascribed to the modification of ionized – SH groups. The absorption change observed near 287 nm. produced by tyrosyl perturbation, is the same when the enzyme reacts with the inhibitor or with l‐arginine.Labelling data give evidence of a significant protection of the essential thiol group by l‐arginine against N‐ethyl‐[1‐14C]maleimide. This cysteinyl residue is included in the tryptic peptide T1A the amino acid sequence of which, recently determined, is very similar to that to the homologous peptides of muscle and brain rabbit creatine kinases and of earth‐worm muscle lombricine kinase.
Purification of Animal Neurotoxins
Authors: Miranda François , Kupeyan Charles , Rochat Hervé , Rochat Catherine , Lissitzky Serge 
Published: March 2005

Using the same method of purification of small molecular weight proteins worked out to obtain pure scorpion neurotoxins, four neurotoxins have been isolated from the venom of the cobra Naja haje. N. haje venom from the Miami Serpentarium contains two neurotoxins (I and II) whereas the venom of the same species obtained from the Institut Pasteur contains three neurotoxins (I, II′ and III). Toxins I, II and II′ consist of 61 amino acid residues and toxin III of 71. All are basic proteins containing four or five disulphide bridges. It is likely that the difference observed in neurotoxin composition of N. haje venoms from two different sources has a taxonomical counterpart, each venom originating from a different sub‐species.
Chromatographic Separation of Membrane Proteins on Lipophilic Ion Exchange Resins
Authors: Weiss Hanns , Bücher Theodor 
Published: March 2005

Aliphatic amines of increasing chainlength (butyl, capryl, lauryl, and palmityl) and increasing degree of unsaturation (stearyl, oleyl, and linoleyl) were bound by amide bonds to polyacrylic acid resin. Mitochondrial membrane proteins solubilized with cholate and deoxycholate were chromatographically separated on such resins.The separation efficiency as measured by the number of protein fractions and their specific cytochrome content was highest with capryl or linoleyl resins while the free polyacrylic acid resin or the stearyl resin proved to be the most ineffective ones.These effects are discussed in terms of free thermal mobility of the aliphatic chains necessary for hydrophobic interaction with the membrane proteins.
Influence of Bile Acids on the Activity of Rat Liver 3‐Hydroxy‐3‐methylglutaryl Coenzyme A Reductase
Authors: Hamprecht Bernd , Nüssler Christa , Waltinger Gudrun , Lynen Feodor 
Published: March 2005

The rate limiting enzyme of cholesterol biosynthesis, 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, is found in the microsomal fraction of a liver homogenate. It is markedly inhibited in vitro by the cholesterol metabolites cholate, deoxycholate, chenodeoxycholate and their taurine conjugates only at unphysiologically high concentrations.
The inhibition by taurochenodoxycholate is non‐competitive and only partially reversible. Therefore, the effect of bile salts in vitro is interpreted as a non‐specific detergent effect.
Feeding of cholic acid in the diet prevents the diurnal rhythm of the activity of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase. The activity decreases with increasing time of feeding of cholic acid.
The specific activity of the enzyme decreases with increasing age of the animal.
It is concluded that the effect of bile salts in vitro on this enzyme cannot account for their effect in vivo. Rather, it is suggested that in vivo bile salts interfere with the de novo‐synthesis and/or the degradation of the enzyme.
Influence of Bile Acids on the Activity of Rat Liver 3‐Hydroxy‐3‐methylglutaryl Coenzyme A Reductase
Authors: Hamprecht Bernd , Roscher Rudolph , Waltinger Gudrun , Nüssler Christa 
Published: March 2005

Cholic acid feeding strongly depresses the activity of the rate limiting enzyme of cholesterol synthesis in liver 3‐hydroxy‐3‐methylglutaryl CoA reductase. Experiments were undertaken to decide between (a) a direct influence of the bile acid itself in liver or (b) an indirect effect of the bile acid by improving the intestinal resorption of cholesterol, which itself then would depress the activity of hepatic 3‐hydroxy‐3‐methylglutaryl CoA reductase.
For this purpose lymph fistula rats were fed with cholic acid. Compared with the control animals, which did not obtain cholic acid, the experimental groups showed strong depression of 3‐hydroxy‐3‐methylglutaryl CoA reductase activity. This was only observed with animals killed during the dark period, i.e. when the rhythmically changing activity of the enzyme was high.
A partial depression of the enzyme by fasting can be excluded, because animals fed with or without cholic acid, respectively, ingest the same amount of food.