bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of ATP on the Oxidation of Succinate in Rat Brain Mitochondria
Authors: Tuena M. , Gómez‐Puyou A. , Peña A. , Chávez E. , Sandoval F.
Published: March 2005

The effect of ATP on the aerobic oxidation of succinate and on the succinic dehydrogenase activity of isolated rat brain mitochondria has been studied.

The addition of dinitrophenol to brain mitochondria with succinate as substrate produced a small activating effect of oxygen uptake which was followed by inhibition of respiration; the further addition of ATP produced a high rate of respiration.

This effect of ATP was enhanced by added Mg++ and oligomycin and was diminished, but not abolished, by inorganic phosphate.

Experiments were conducted which indirectly indicated that oxaloacetate may be involved in the inhibition of the aerobic oxidation of succinate in fresh brain mitochondria incubated with dinitrophenol; however, attempts to find out if ATP removed oxaloacetate through a particular ATP requiring metabolic pathway were unsuccessful.

In aged, sonicated, and frozen and thawed brain mitochondria incubated with dinitrophenol, ATP still activated the aerobic oxidation of succinate.

The assay of succinic dehydrogenase (EC 1.3.99.1) in aged brain mitochondria revealed that ATP diminished the Km for succinate and increased the Vmax of succinic dehydrogenase. ATP also produced a shift in the pH optimum of succinic dehydrogenase and a partial protection of the enzyme against heat denaturation.
Isolation of an Ionic Strength Dependent Cell Agglutinin from Bovine Seminal Plasma
Authors: Forrester J. A. , Arnold R. , Ruhenstroth‐Bauer G.
Published: March 2005

A basic protein of bull seminal plasma is described. The method of preparation involves precipitation at high concentrations of ammonium sulphate and subsequent cation exchange chromatography. The protein has cell agglutinating properties and these are described in relation to erythrocytes and some other cell types from several mammalian species. Amino acid analyses reveal a high proportion of basic residues and also of sulphur containing amino acids. The molecular weight is about 48000 and end group determinations suggest that it is composed of two peptide chains. From measurement of the optical rotatory dispersion of the material it appears that an unusually small proportion of the protein exists in the α‐helical conformation.
Interaction of Cysteamine Oxygenase with o‐Phenanthroline
Authors: Cavallini D. , Cannella C. , Barboni E. , Fiori A. , Marcucci M.
Published: March 2005

The addition of o‐phenanthroline to a solution of cysteamine oxygenase produces only a slight increase of the absorbancy at 510 nm. The absorbancy is much higher when o‐phenanthroline is added in the presence of urea or in the presence of sodium dodecyl sulfate. The further addition of dithiothreitol exhibits a cooperative effect with the denaturant which has been explained with the cleavage of enzymic disulfide bonds. Absorbancies at 327 and 342 nm are also seen when o‐phenanthroline is added in the presence of sodium dodecyl sulfate. These are typical of the complex of o‐phenanthroline with Zn, in agreement with the analytical data indicating the occurrence of Zn in the enzyme. The need of denaturants for the reaction of the enzymic metals suggests that these metals are buried into the protein and should be regarded as an integral part of the enzyme structure.
Separation of Two Types of Light Polypeptide Chains from Rabbit γ‐Globulins
Authors: De Vries G. M. , Lanckman M. , Hamers R.
Published: March 2005

The L chains from rabbit γ‐globulins can be separated by gel filtration into two populations (L1 and L2) differing markedly in their structural and antigenic proporties. The L1 and L2 chains from pooled IgG have different amino‐acid composition and half‐cystine content. In addition, each population can be further resolved in two subfractions called L1a and L1b, L2a and L2b, respectively. The b allotypic markers are located exclusively in L2 chains.

These results indicate that the L chains from rabbit IgG are constituted by two populations of different genetic origin.
Biochemistry of the K Antigens of Escherichia coli
Authors: Olson Anita C. , Schmidt G. , Jann K.
Published: March 2005

Initial work on the determination of the mechanisms of the biosynthesis of the K antigens of E. coli is described. Mutants defective in the biosynthesis of the K‐polysaccharide were used for this study. Acapsular K– mutants were isolated from E. coli 08:K27(A):H– and characterized serologically and through their sensitivity towards specific phages.

Previous studies have shown the K27 antigen to be an acidic polysaccharide consisting of glucuronic acid, glucose, galactose and l‐fucose. The 08‐antigen contains mannose as the main constituent of its specific polysaccharide moiety. The influence of mutation on the biosynthetic pathways leading to the K27 polysaccharide was investigated. For this purpose wild strain and various K– mutants were analyzed in this study for the presence of enzymes catalyzing the synthesis of the nucleotide sugar precursors: UDP‐glucose, UDP‐galactose, UDP‐glucuronic acid, GDP‐mannose and GDP‐l‐fucose. The following bacterial enzyme activities were tested in cell free system: hexokinase, phosphoglucoisomerase, phosphoglucomutase, UDP‐glucose pyrophosphorylase, UDP‐galactose‐4‐epimerase, UDP‐glucose dehydrogenase, GDP‐mannose pyrophosphorylase, GDP‐mannose hydrolyase.

When two K– mutants were crossed and selections were made for his+, a small but significant number of K+ strains appeared among the recombinants. Thus, it was concluded that the parent K– strains had different blocks in the synthesis of the K27 antigen which mapped at different sites on the chromosome. Repair occurred due to crossing over within a small (his‐linked) region of the chromosome. Biochemically, the repair of the K– character by genetic crossing of the two K– mutants was reflected in the capability for GDP‐l‐fucose synthesis which was deficient in the donor strain as well as in the larger portion of the K– recombinants. In two of the K– mutants all enzymes tested were found to be operative. Since synthesis of the nucleotide precursors was possible, these mutants probably have a defect in the K27 specific transferase system. One K– mutant, which serves as the recipient in the genetic crossing, showed some serological R character. This mutant possessed a very low GDP‐mannose pyrophosphorylase activity; however, some O specific (mannose containing) polysaccharide was formed. Since all other synthetases are active in this mutant it is assumed that also in this case the K27 transferase system may be defective.
15‐Hydroxy‐Prostanoate Dehydrogenase. Prostaglandins as Substrates and Inhibitors
Authors: Nakano J. , ÄNggard E. , Samuelsson B.
Published: March 2005

The Michaelis constants and maximum reaction rates were determined for a series of substrates for 15‐hydroxyprostanoate dehydrogenase (prostaglandin dehydrogenase). The results show that the enzyme is stereospecific with regard to the configuration at C‐15. The nature of the substituents of the cyclopentane ring did not markedly affect the properties as a substrate, whereas the nature of the carboxyl side chain was important. Prostaglandin B compounds and a synthetic epimer of prostaglandin E1, 15‐R‐prostaglandin E1, produced a non‐competitive inhibition.
Dietary and Hormonal Control of Enzymes of Amino Acid Catabolism in Liver
Authors: Pestaña A.
Published: March 2005

A high glucose diet fed ad libitum decreased the activities in rat liver of histidase, serine dehydratase, homoserine dehydratase, ornithine transaminase, mitochondrial aspartate transaminase and cytoplasmic alanine transaminase, as compared to normal fed animals.

Casein hydrolysate or tryptophan alone given to protein deplted rats increased the hepatic levels of serine dehydratase, alanine transaminase and tyrosine transaminase. Histidase and homoserine dehydratase were increased by casein hydrolysate but not by tryptophan. Aspartate transaminase was increased by tryptophan, but not by casein hydrolysate.

Glucose administered intragastrically partly or totally counteracted the induction of serine dehydratase, tyrosine transaminase, cytoplasmic alanine transaminase by cortisol of glucagon administration to protein depleted rats. The similar induction of ornithine transaminase and cytoplasmic aspartate transaminase by cortisol was also counteracted by glucose.
The Subunit Structure of α‐Crystallin
Authors: Schoenmakers J. G. G. , Gerding J. J. T. , Bloemendal H.
Published: March 2005

The present results, together with physicochemical evidence presented elsewhere, indicate that α‐crystallin from calf lens is composed of two acidic and two basic polypeptide chains. The acidic polypeptides could be isolated by chromatography on SE‐Sephadex at pH 5.5 in the presence of 7 M urea. It could be demonstrated that both S‐carboxymethylated acidic polypeptides are of equal molecular weight and amino acid composition. In both chains N‐acetylmethionyl‐aspartate was found as the N‐terminal and serine as the C‐terminal residue. Peptide mapping reveals one unique peptide region for each polypeptide.

In embryonic α‐crystallin only one acidic polypeptide can be detected. From comparative amino acid analysis, peptide maps as well as physicochemical behaviour it may be deduced that the polypeptide present in the early embryonic stages is identical with the adult acidic polypeptide A‐2.
Comparative Structural Studies of the Active Site of ATP‐Guanidine Phosphotransferases
Authors: der Terrossian E. , Pradel L. A. , Kassab R. , Thoai N. V.
Published: March 2005

The purification and the amino acid sequence of the peptide containing the essential sulphydryl group of arginine kinase from Homarus vulgaris muscle is described.

The fractionation of the tryptic digest of arginine kinase labelled with N‐[1‐14C]ethylamaleimide has been carried out using gel filtration, ion‐exchange chromatography and paper chromatography. The composition of the radioactive peptide, isolated from the digest, is reported together with its amino acid sequence. A structural comparison is made between the peptide of arginine kinase and the corresponding active‐cysteine containing peptide isolated from rabbit muscle creatine kinase.
Interconversion in vitro of Two Forms of Liver Phosphorylase Phosphatase
Authors: Merlevede W. , Goris J. , Brandt C. De
Published: March 2005

When a partially purified dog liver phosphorylase phosphatase preparation was incubated with ATP and Mg ions, its activity increased several fold. A slower activation was observed in the presence of Mg ions only. When the same preparation was preincubated with ATP alone, complete inactivation was obtained. The ATP pretreated enzyme could then be reactivated by Mg++, as well as by ATP and Mg++. The activation and the inactivation of the phosphorylase phosphatase, were shown to be dependent on time, temperature and the concentration of ATP and Mg ions. Neither on the activation nor on the inactivation of the phosphorylase phosphatase could and effect of cyclic 3′ :5′ ‐AMP be observed.
The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K 12
Authors: Janin Joël , Iwatsubo M.
Published: March 2005

The stopped‐flow and temperature‐jump techniques are applied to the study of the relaxations of an allosteric protein, the aspartokinase I‐homoserine dehydrogenase I of E. coli K 12. A concerted conformation change is known to occur in this protein under the influence of aspartate, of the K+ ions and of threonine, all of which are effectors of the catalytic activities. The kinetics of the conformation change can be followed by making use of effects on the ultraviolet absorption and on the fluorescence of the protein itself. Stopped‐flow experiments demonstrate that the relaxation observed after addition of any of the three effectors described is an exponential process, which appears to correspond to the slower of two temperature relaxations. In the absence of added ligands, the rates of these relaxations are approximately 1000 sec–1 and 14 sec–1 at 28°. The effects of the ligands on the processes observed in the stopped‐flow and in the temperature‐jump experiments are interpreted and the mechanism of the conformation change is shown to include at least two isomerization steps.
The Activation of Phosphorylase in Neonatal Rat Liver
Authors: Cake M. H. , Oliver I. T.
Published: March 2005

The percentage of α‐glucan phosphorylase in the active form was measured in the liver of premature rats following surgical delivery. The enzyme is 30% active in the late foetal liver and reaches 45% activation four hours after birth. Thereafter there is a slow decline to the new‐born level. Puromycin and actinomycin D injected at delivery are without effect on the enzyme activity and activation of preformed inactive enzyme therefore occurs. These results are interpreted to mean that there is a transitory increase in the concentration of 3′ :5′‐cyclic AMP in postnatal rat liver. Glucose and ergotamine tartrate partially inhibit phosphorylase activation when injected at delivery and their effects are additive, implicating hypoglycaemia and hormonal mechanisms in the postnatal activation. Fructose and galactose also inhibit phosphorylase activation while mannose is without effect. Foetal liver phosphorylase is activated in utero by injection of 3′ :5′‐cyclic AMP and in postnal rats a transitory increase in phosphorylase activity after injection of the cyclic nucleotide indicates a life time of the injected compound of less than 1 h. These results are discussed in relation to the induction of enzymes in postnatal rat liver by 3′ :5′‐cyclic AMP.
Regulatory Properties of a Plant NAD: Isocitrate Dehydrogenase
Authors: Coultate T. P. , Dennis D. T.
Published: March 2005

Some properties of NAD: isocitrate dehydrogenase from swede (Brassica napus L.) are described. This enzyme shows simpler regulatory properties than that from animals or fungi.

Isocitrate, citrate and tricarballylate are positive effectors but neither ADP nor AMP have any effect. Pyruvate, acetate, α‐ketoglutarate, succinate and dl‐malate do not stimulate or inhibit at 2 mM.

ATP and other nucleoside triphosphates inhibit this enzyme but the inhibition appears to be due to the formation of a complex with the activating cation.

The principal control of the enzyme is a potent inhibition by NADH competitive with NAD. Elevation of either isocitrate or activating cation concentration lowers the Km for NAD and in this respect MnSO4 is more effective than MgSO4. In contrast, the Ki for NADH is 0.05 mM with 1 mM MgSO4 and 0.16 mM with 1 mM MnSO4.

The enzyme is inhibited by high concentrations (> 20 mM) of inorganic anions. This inhibition is apparently competitive with isocitrate but anions most likely cause a conformational change.

The interactions between isocitrate and activating cation have been examined. Elevation of the concentration of either isocitrate or cation raises the affinity of the enzyme for the other to a limiting value. The nature of the cation has only a slight effect on the minimum Km for isocitrate (0.35 mM with MgSO4, 0.25 mM with MnSO4) but the minimum value of the Km for MgSO4 is 100 μM compared with 7 μM for MnSO4.

It is concluded that the enzyme binds one molecule each of isocitrate, activating cation and NAD or NADH at the catalytic site and one molecule of citrate or isocitrate at an effector site.
Sur la biosynthèse de la chaîne latérale éthyle des stérols du Myxomycète Dictyostelium discoideum
Authors: Lenfant M. , Ellouz R. , Das B. C. , Zissmann E. , Lederer E.
Published: March 2005

Previous work of several laboratories has shown that the two carbon atoms C‐28 and C‐29 of the ethyl or ethylidene side chains of phytosterols are introduced on a C‐24 unsaturated side chain by two successive C‐methylation steps. A scheme, based on previous work of Nes et al. and Goodwin et al., gives the various intermediates which can be formed by stabilisation of the intermediate carbonium ion (II). It was usually thought that ethylidene compounds such as (III) were the precursors of ethyl compounds such as (VIII) and this has been proved recently in the case of poriferasterol (IX) which contained four deuterium atoms when Ochromonas malhamensis was grown in presence of [Me‐2H3] methionine [8].

In the present paper it is reported that the sterol of the slime mold Dictyostelium discoideum [Δ 22‐stigmasten‐3‐β‐ol (Xa)] contains five deuterium atoms, when extracted from D. discoideum fed on cells of a methionine‐less strain of E. coli grown in presence of [Me‐2H3] methionine. The unsaturated sterol (Xa) is accompanied by the saturated sterol (XIa) which was not previously found in this organism.

Mass spectrometry of the pentadeuterated sterols (Xa) and (XIa) seemed to show conclusively that all five deuterium atoms are located in the ethyl side chain [11], but it was thought necessary to confirm this conclusion by a detailed analysis of the side chain; thus, the sterol (Xa) was ozonised and part of the side chain isolated as a crystalline p‐phenylphenacyl ester (XIIIa). Mass spectrometry of this compound and of a synthetic non‐labelled (XIIIb) and the corresponding monodeuterated derivative (XIIIc) confirms that all five deuterium atoms of the Dictyostelium sterol (Xa) are in the ethyl side chain.

These results exclude an ethylidene compound (such as III) as a precursor of the sterol (Xa); they do not allow us to choose between other possible routes illustrated in the scheme. Our experiments also show that there is no hydrogen shift during the second methylation step.
Isolation and Partial Characterization of a Carboxypeptidase from Barley
Authors: Visuri K. , Mikola J. , Enari T.‐M.
Published: March 2005

A carboxypeptidase has been purified from germinated barley. The preparations are homogeneous in ultracentrifugal analysis. In disc electrophoresis traces of impurities can be detected at high concentration. The purified enzyme liberates carboxyl‐terminal amino acid residues from a wide range of carbobenzoxy (Z)‐dipeptides. Peptides containing proline are not attacked. It does not possess any endopeptidase, aminopeptidase, or dipeptidase activity. The enzyme has a pH optimum at pH 5.2, is inhibited by di‐isopropylphosphofluoridate and p‐chloromercuribenzoate, and has a molecular weight of about 90,000.
The Purification and Properties of Cytidine Aminohydrolase from Sheep Liver
Authors: Wisdom G. B. , Orsi B. A.
Published: March 2005

Cytidine aminohydrolase was purified about 280‐fold with an overall 8% yield from the cytoplasmic fraction of sheep liver cells by the use of protamine sulphate precipitation, salt fractionation using trisodium citrate, acidification and chromatography on DEAE‐and CM‐celluloses.

The enzyme catalyses the deamination of several aminopyrimidine nucleosides. Its pH optimum occurred at 5.0 and it was relatively acid and heat stable. The high sensitivity of the enzyme to compounds known to react with sulphydryl groups and the presence of a substrate‐binding group of pKa 9.2 in the enzyme suggests that a sulphydryl group may be important in the catalytic process. dTTP and dUTP were found to inhibit and dCTP to activate the enzyme respectively. The complex type of inhibition produced by dTTP indicates that this aminohydrolase may have a regulatory role similar to that of dCMP aminohydrolase.
Binding of Bromthymol Blue by Mitochondrial Structural Protein
Authors: Saris N.‐E. L. , Sappälä A. J.
Published: March 2005

The binding of bromthymol blue by structural protein from beef heart mitochondria has been studied. In 20 mM Tris‐acetate (pH 7.4, temperature 240) three moles of the indicator were bound per mole structural protein with equal binding energies. The dissociation constant was 3 μM. Binding decreases the millimolar absorbance coefficient at 618 nm by two thirds, to approximately 13 cm−1 mM−1, and shifts the peak towards a longer wavelength. Increase in pH or ionic strength of the medium displaces the equilibrium towards the dissociated free species. Monovalent cations have little effect on this displacement, and divalent cations even less. The effectiveness of anions increases with increasing charge, and, within the halogenides, from fluoride to iodide. Dinitrophenol is very effective. These data indicate that both electrostatic binding and binding through the non‐polar parts of the molecule are involved. In mitochondrial systems, an increase in absorbance at 618 nm may be due to an increase in the ionization of the free and the bound species and a release of the bound species. All three are a function of pH; the release is also a function of the anionic composition of the compartment involved.
Die O2‐Bindungseigenschaften einiger Larvalhamoglobine von Chironomus th. thummi
Authors: Sick H. , Gersonde K.
Published: March 2005

Some chromatographically separated hemoglobins from Chironomus th. thummi were tested for their oxygen binding properties. In all cases the O2‐affinity showed a characteristic pH‐dependency which can be described as a “simple” Bohr effect. The amplitude of the Bohr effect curves differs from hemoglobin to hemoglobin. The degree of polymerization of these hemoglobins is known from experiments of the Braunitzer group and it becomes evident that this Bohr effect is found both in monomer and in dimer hemoglobins. From the correspondence of the mathematical model with the experiment it can be shown that the Bohr effect is caused by proton dissociation of only one group, having negative interaction to O2‐binding. The possibility is discussed that this dissociable group is localized far from the O2‐binding site and influences the affinity constant through a change of conformation. The half‐value pressure determined experimentally in relation to the pH is therefore the half‐value pressure of the mixture of two “conformation isomers”, each of which is dependent on the pH. The mathematical examination of the model shows the disproportion between the steepness and the amplitude of the Bohr effect curves to be the consequence of a Hill coefficient deviating from n= 1. This coefficient proved to be independent from the pH and shows no relation to the degree of polymerization. The equation describing the Bohr effect curves can be applied also for other hemoglobins of the same type (Gyclostomata) as is shown by the lampetra hemoglobin.
Nature of RNA Synthesized at Low and High Ionic Strength by DNA‐Dependent RNA‐Polymerase from Escherichia coli
Authors: Qasba P. K. , Zillig W.
Published: March 2005

By competition hybridization experiments it is shown, that RNA synthesized in vitro by RNA polymerase on T4‐DNA at high ionic strength, like that synthesized at low ionic strength, contains only early RNA species. However, the product obtained at high ionic strength contains sequences not synthesized at low ionic strength. This is attributed to the early arrest of synthesis at low ionic strength.
Determination of the N‐Terminal Amino Acids in Proteins Using Fluoro‐Nitropyridines
Authors: Signor A. , Biondi L. , Tamburro A. M. , Bordignon E.
Published: March 2005

A new procedure for the determination of the N‐terminal groups in peptides and proteins is described. The free amino‐groups are allowed to react with 2‐fluoro‐3‐nitropyridine or 2‐fluoro‐5‐nitropyridine in slightly alkaline solution; the resulting derivatives are easily hydrolyzed in dilute hydrochloric acid in sealed tubes to give the corresponding nitropyridyl amino acids. Most of these compounds are quantitatively recovered under these conditions: in particular it is possible to estimate proline and glycine as N‐terminal residues.

After isolation the nitropyridyl amino acids can be estimated spectrophotometrically; in some cases it is possible to regenerate the parent amino acid by treating the derivatives with strongly alkaline solutions. The adequacy of the present techniques in relation to those of other workers was tested on insulin, lysozyme and aldolase; the advantages and peculiarities in comparison with these procedures are discussed.
Studies on Renin with Synthetic Substrates
Authors: Reinharz A. , Roth M.
Published: March 2005

Renin has been shown to catalyze the hydrolysis of the protected octapeptide, Z‐Pro‐Phe‐His‐Leu‐Leu‐Val‐Tyr‐Ser‐β‐naphthylamide, at the leucyl‐leucyl bond. When the octapeptide substrate is incubated with renin and an excess of the auxiliary enzyme, aminopeptidase M, the fluorescent β‐naphthylamine is liberated at a rate related to renin concentration. A chemical assay of renin based on these facts is described in detail. When assayed with this method, kidney renin from man and pig shows pH optima of 5.4 and 5.7 respectively. Renin from submaxillary glands of mice, which also attacks the substrate, is optimally active at pH 5.4.

The limit of sensitivity of the assay is 0.001 Goldblatt units for human renin and 0.01 Goldblatt units for pig renin. Trials with other possible synthetic substrates showed that Z‐Pro‐Phe‐His‐Leu‐Leu‐β‐naphthylamide is poorly hydrolyzed by renin, and Z‐Leu‐Leu‐β‐naphthylamide not at all.
On the Fatty Acids of Brucella abortus and Brucella melitensis
Authors: Thiele O. W. , Lacave C. , Asselineau J.
Published: March 2005

The main fatty acids of the lipids of Brucella abortus (strain Scherle II, or 1119) and of Brucella melitensis (strain M‐15) are palmitic acid, a C18 monoenoic acid an a C19 cyclopropane fatty acid. By chemical degradation, it is shown that the unsaturated C18 acid is cis‐vaccenic acid. The C19 cyclopropane acid has been identified with lactobacillic acid, either by permanganate oxidation according to Murray, or by mass spectrometry of the keto‐esters obtained by chromic acid oxidation.
Changes in the Short Wavelength Absorption Spectrum of Tyrosine as a Result of its Participation in Peptide, or Similar Bonds
Authors: Delius A. E. , Franze de Fernández M. T. , Paladini A. C.
Published: March 2005

The perturbation of the spectrum of free tyrosine that occurs when this amino acid is combined to other amino acids or in the form of acetyl derivative, acetyl derivative amide or acetyl derivative ethyl ester was studied.

The difference spectrum of each compound was obtained by measuring the absorbance of its solution against that of a mixture of its component free amino acids.

A red‐shift of the 2,400 Å absorption band, in 0.1 M NaOH, or 2,230 Å, at pH 7, was clearly detected.

Maximum perturbation is produced when tyrosine has both its amino and carboxyl groups involved in peptide bonds.
Extraction and Purification of DNA‐Dependent RNA Polymerase from Rat Liver Nuclei
Authors: Seifart K. H. , Sekeris C. E.
Published: March 2005

The enzyme RNA polymerase is difficult to extract from most mammalian cells due to its firm association with chromatin. By a combination of intensive ultrasonic treatment and extraction of the nuclei with 0.75 M NaCl at 0° and pH 8.4 for 1.5 hours approximately 80% of the enzyme can be extracted in a soluble, DNA‐dependent form. The extracted enzyme has been purified by DEAE‐cellulose chromatography and centrifugation on sucrose gradients (5–20%, w/v) containing 10% glycerol. A purification of approximately 250‐fold has been achieved in the peak fraction of the gradient. The enzyme requires all four nucleoside triphosphates, mercaptoethanol, a DNA template and either Mg++ or Mn++, the latter being the preferred ion. Enzyme activity can be stimulated by (NH4)2SO4. No direct stimulation of enzyme activity by cortisol phosphate could be observed.
Partial Purification and Properties of an Induced β‐d‐Xylosidase of Bacillus pumilus 12
Authors: Kersters‐Hilderson H. , Loontiens F. G. , Claeyssens M. , de Bruyne C. K.
Published: March 2005

A highly specific β‐d‐xylosidase with absolute glycon‐substrate specificity was induced in Bacillus pumilus 12 by xylose. It was purified 34‐fold by a procedure involving lysis of the cells with lysozyme, ammonium sulfate fractionation, G‐75 Sephadex gel filtration and hydroxylapatite chromatography. The optimal activity was in the pH region 7.0–7.3. The isoelectric point was found to be 4.4. In contrast to its broad complexing affinity, the hydrolytic activity was restricted to aryl β‐d‐xylopyranosides and lower xylo‐oligosaccharide derivatives. From the dependence of Km and Vmax on pH, it could be deduced that two dissociable groups were necessary for catalytic activity. The enzyme was competitively inhibited by Tris at pH 7.2 and proved sensitive to ionic environment. p‐Chloromercuribenzoate and other SH‐reagents were inhibitory but substrate analogs and competitive inhibitors protected the enzyme against inactivation by p‐chloromercuribenzoate. The results indicate that a histidine residue or imidazolium group and a sulf‐hydryl group might participate in the enzymatic hydrolysis of p‐nitrophenyl β‐d‐xylopyranoside.
Relationship between Sites 1, 2 and Acceptor, Donor Sites for the Binding of Aminoacyl tRNA to Ribosomes
Authors: Igarashi Kazuei , Kaji Akira
Published: March 2005

There appear to be at least two sites on the ribosome for the specific binding of Phe‐tRNA. We have designated these as site 1 and site 2. From the complex of [14C]Phe‐tRNA, poly U, and ribosomes polyphenylalanine was made with [12C]Phe‐tRNA. We have defined that those [14C]phenylalanine which are bound to site 2 will end up in the NH2‐terminal and those that are bound to site 1 will end up in the position next to the NH2‐terminal position of polyphenylalanine. These two sites have often been called the donor site and the acceptor site. When aminoacyl tRNA or peptidyl tRNA is bound to the donor site it can donate its amino acid or peptide to puromycin forming aminoacyl puromycin, or peptidyl puromycin. The acceptor site is the site where ribosome accepts aminoacyl tRNA during polypeptide synthesis. In an attempt to correlate our sites 1 and 2 with acceptor and donor sites, the effects of magnesium ions and tetracycline on these sites were studied. In low Mg++, the donor site, as well as site 2 were occupied. Tetracycline (0.5 mM) preferentially inhibited the binding of phenylalanyl tRNA to site 1 as well as to the acceptor site. From these studies we have concluded that site 1 is the acceptor site and site 2 is the donor site.
Coenzyme Binding in Yeast Pyruvate Decarboxylase
Authors: Wittorf John H. , Gubler Clark J.
Published: March 2005

The formation of a fluorescent complex between apopyruvate decarboxylase of Saccharomyces carlsbergensis and thiochrome pyrophosphate, a competitive inhibitor with thiamine pyrophosphate, and Mg(II) ions is reported. A similar complex between thiochrome and the apoenzyme could not be detected, demonstrating the importance of the pyrophosphate group of the coenzyme in binding to the protein. Complex formation between the apoenzyme and thiochrome pyrophosphate in the absence of Mg(II) could also not be demonstrated. The role of magnesium in coenzyme binding is discussed, especially in reference to interaction with the pyrophosphate moiety. The apparent Km for Mg(II) ions, after correction for residual apoenzyme activity, is reported to be 87 μM.

The shift in the fluorescence emission spectrum of thiochrome pyrophosphate of 23–24 nm toward lower wavelengths, upon complex formation, coincided with the behavior of thiochrome in solvents of lower polarity than water. A hydrophobic region at the coenzyme binding site of pyruvate decarboxylase is proposed. Enhancement of thiochrome fluorescence occurred concomitantly with the blue shift in the emission spectrum upon complex formation.

The excitation wavelength of 290 nm employed to demonstrate complex formation suggested energy transfer from the tryptophan to the thiochrome chromophore. Quenching of fluorescence in the 340–350 nm region, however, occurred not only in the protein emission spectrum upon complex formation, but also when free tryptophan and thiochrome interacted with each other. The quenching of the protein fluorescence appears to be independent of hydrophobic interaction at the coenzyme binding site. It is suggested that protein tryptophan residues relatively exposed to solvent interact individually with the thiochrome molecule by an energy transfer mechanism to produce the quenching in pyruvate decarboxylase.
An Investigation of the Conformation of Bovine α‐Lactalbumin by Proton Magnetic Resonance and Optical Spectroscopy
Authors: Cowburn D. A. , Bradbury E. M. , Crane‐Robinson C. , Gratzer W. B.
Published: March 2005

Proton magnetic resonance spectra are reported of α‐lactalbumin at 100 and 220 MHz. The extensive sequence homologies between this protein and lysozyme have led to the postulate of a close conformational similarity. Our results on optical rotatory dispersion, circular dichroism and infrared spectra of the two proteins are consistent with this hypothesis. The proton magnetic resonance spectra of the native proteins show differences in the upfield region of the spectrum associated with aliphatic side chain protons. Calculations of ring‐current shifts induced in these protons, in terms of the atomic coordinates for the putative α‐lactalbumin structure, as fitted by Phillips and co‐workers to the lysozyme model, account semi‐quantitatively for the observed spectrum. The discrepancies between the α‐lactalbumin and lysozyme spectra are, moreover, almost wholly explicable in terms of specific sequence differences between the two proteins.
Synthesis and Charactrisation of a Copolymer Consisting of Alternating Deoxyadenosine‐ and 2‐Thiodeoxythymidine Nucleotides
Authors: Lezius Axel G.
Published: March 2005

Starting from 2‐thiodeoxythymidine, the mono‐ and triphosphate of the nucleoside analog were prepared by enzymatic phosphorylation with thymidine kinase and thymidylate kinase, respectively. 2‐Thiodeoxythymidine triphosphate was also obtained by chemical pyrophosphorylation of the monophosphate. The analog nucleoside‐triphosphate is a substrate for Escherichia coli DNA polymerase. A copolymer consisting of alternating deoxyadenosine‐ and 2‐thiodeoxythymidine nucleotides is synthesized extensively by the enzyme. The polymer was characterized by nearest neighbour analysis, sedimentation and buoyant density in the analytical ultracentrifuge, ultraviolet spectra and stability against thermal and alkaline denaturation.
Specific Dissociation of Ribosomes from Saccharomyces cerevisiae by a Protein Factor
Authors: Pêtre Jean
Published: March 2005

A ribosome fraction enriched in “native” subunits by differential centrifugation yields, upon washing in a buffer containing ammonium chloride and magnesium ions, a protein mixture which can dissociate cytoplasmic ribosomes from Saccharomyces cerevisiae into 40 S and 60 S subunits.

This heat dependent reaction cannot be related to any marked ribonuclease activity in the protein mixture.

Furthermore, the mixture is unable to dissociate Escherichia coli or rabbit reticulocyte ribosomes; this property is unique, since proteolytic enzymes readily dissociate reticulocyte ribosomes as well as yeast ribosomes.

Within 30 min of incubation, the factor‐dependent dissociation reaches a plateau, due either to the stoichiometric mode of action of the factor, or to the inability of ribosomes to support complete dissociation under the ionic conditions of the experiments.

These two points are discussed.
Relations structurales entre les acides mycoliques insaturés et les acides inférieurs insaturés synthétisés par Mycobacterium phlei
Authors: Asselineau Cécile P. , Lacave Charlotte S. , Montrozier Henri L. , Promé et Jean‐Claude
Published: March 2005

Mycobacterium phlei synthesizes a complex mixture of saturated and unsaturated fatty acids, with a normal or a branched chain. We have isolated the unsaturated fatty acids R–CH = CH–(CH2)n–CO2H, with twenty to twenty seven carbon atoms. These acids only make up a very small amount of the total fatty acids. Their structures were established by means of the oxidation method of Lemieux, followed by gas chromatographic analysis of the lower acids thus obtained.

Some of these unsaturated acids (4‐eicosenoic, 6‐docosenoic, 8‐hexacosenoic acids) are likely to be metabolically related, whereas 5‐enoic fatty acids (normal chain 5‐enoic acids with 22, 24 or 26 carbon atoms and branched chain 5‐enoic acids with 25 and 27 carbon atoms) could arise through a common peculiar process.

Simultaneously, by use‐of the same oxidation method, we have identified the radical R in the mycolic acids R–CH = CH–X–CH(OH)–CH(R')–CO2H. These radicals R are identical to the radicals R of the five different 5‐enoïc fatty acids. This observation suggests the hypothesis that these unsaturated acids play a role in the synthesis of the mycolic acids in Mycobacterium phlei. By a critical analysis of previous work in this field, we show that this hypothesis is consistent with all the results found in the literature.
Specific Alkylation of the Sarcoplasmic Reticulum ATPase by N‐Ethyl‐[1‐14C]maleimide and Identification of the Labeled Protein in Acrylamide Gel‐Electrophoresis
Authors: Panet Rivka , Selinger Zvi
Published: March 2005

The total sulfhydryl content of sarcoplasmic reticulum determined in the presence of sodium dodecylsulfate is 14 sulfhydryl equivalents per 105 g of membrane protein, but in the absence of sodium dodecylsulfate only 10 sulfhydryl groups are titrateable with 5,5′‐dithiobis (2‐nitrobenzoic acid). The rate of ATPase inactivation by N‐ethylmaleimide in the presence of ATP yields an ATP‐ATPase dissociation constant of 45μM. When the sulfhydryl group essential for ATPase activity was reacted with N‐ethyl‐[1‐14C]maleimide after all the sulfhydryl groups unrelated to ATPase activity had been reacted previously with unlabeled reagent, only one protein band in electrophorograms was strongly labeled. The presence of ATP during trypsin proteolysis preserves ATPase activity and the band that can be specifically labeled with N‐ethylmaleimide is demonstrated in gel electrophorograms. When trypsin proteolysis was carried out in the absence of ATP this band can no longer be seen. The N‐ethyl‐[1‐14C]maleimide labeledband is tentatively identified with the ATPase.
An Investigation of the Conformational Changes of Histones F1 and F2a1 by Proton Magnetic Resonance Spectroscopy
Authors: Boublík Miloslav , Bradbury E. Morton , Crane‐Robinson Colyn
Published: March 2005

High resolution nuclear magnetic resonance spectroscopy is used to study conformational changes in histone fractions F2a1 and F1 when the ionic strength of aqueous solutions is raised. Increasing line widths of certain resonance peaks, in particular those of apolar and aromatic amino acids, together with sequence data lead to the conclusion that the C‐terminal half of F2a1 and a central portion of F1 are involved in the conformational changes. The proportion of amino acid residues incorporated into secondary structure (as indicated by optical rotatory dispersion) is less than that involved in the conformational changes indicated by the nuclear magnetic resonance results. Intermolecular interactions are therefore postulated to explain this difference and these are specific in as much as they involve only a part of the histone molecule and include the regions of the chain having high potential for secondary structure formation.
On the Active Sites of Proteases
Authors: Schechter Israel
Published: March 2005

The degree of Stereospecificity exhibited by carboxypeptidase A is dependent on the structural features of the substrate employed. Carboxypeptidase A can effectively split the C‐terminal d‐alanine residue from peptides that are strongly bound to the enzyme through interactions distant from the catalytic center (in subsites S2 and S3). From studies using strongly interacting substrates it may be concluded that carboxypeptidase A shows a higher degree of stereospecificity toward the penultimate amino acid residue than toward the C‐terminal residue.
Horse Liver Alcohol Dehydrogenase
Authors: Jörnvall Hans
Published: March 2005

The ethanol‐active isoenzyme of horse liver alcohol dehydrogenase has been carboxymethylated with iodo[14C]acetate and different samples of the labelled protein treated with trypsin (both with and without previous maleylation of the substrate), chymotrypsin, pepsin or cyanogen bromide. In each case most of the resultant peptides have been purified and characterized.

Data are given for those peptides originating from an N‐terminal region comprising about 40% of the protein chain and the amino acid sequence of the first 140 residues is deduced.

It is established that over this region the two chains of the enzyme have an identical sequence. The N‐terminus is an acetylated serine residue and the “active site” cysteine residue occupies position no. 46. One of the two tryptophans of the chain, but none of the four tyrosines, is in the sequence given. The region around position 100 is rich in cysteine residues. Some serine residues were found to have reacted during maleylation of the protein.

The amino acid sequence is compared to the previously known sequences of glyceraldehyde 3‐phosphate dehydrogenase from two species. Similarities strongly suggest a true homology, at least of an N‐terminal region, and support theories of a common ancestral origin for these enzymes. The reactive cysteine residues are, however, at widely different positions in the primary structures.
The Identification of a Reactive Lysine Residue in Lobster Glyceraldehyde‐3‐phosphate Dehydrogenase
Authors: Davidson Barrie E.
Published: March 2005

Lobster glyceraldehyde‐3‐phosphate dehydrogenase was found to contain a reactive lysine residue that is specifically acetylated when the NAD‐deficient form of the enzyme is incubated with [1‐14C]acetyl phosphate at pH 8.5. The residue was identified by sequence analysis as number 182 in the polypeptide chain of the enzyme.

Both the acetylation behaviour and the position of the lysine residue are identical with that found for mammalian glyceraldehyde‐3‐phosphate dehydrogenases. In addition the amino acid sequence around lysine 182 was found to be identical, except for a few conservative changes, with the corresponding sequence in pig glyceraldehyde‐3‐phosphate dehydrogenase.
Steroids in Germfree and Conventional Rats
Authors: Gustafsson Jan‐Åke
Published: March 2005

Gas chromatography‐mass spectrometry was used to identify steroids in the monosulphate fraction of urine from germfree and conventional, male and female rats. No steroid monosulphates were found in urine from male rats. Germfree rats had a total daily excretion of about 110 μg including the following steroids: 5α‐androstane‐3α,17β‐diol, 3α,11β‐dihydroxy‐5α‐androstan‐17‐one, 3α,7α‐dihydroxy‐5α‐androstan‐17‐one, 5α‐androstane‐3α,7α,17β‐triol, 3α,16α‐dihydroxy‐5α‐pregnan‐20‐one, 3α,15α‐dihydroxy‐5α‐pregnan‐20‐one, 3α‐ and 3β,17α‐dihydroxy‐5α‐pregnan‐20‐one, 5α‐pregnane‐3α,16α,20α‐triol, 11β,21‐dihydroxy‐5α‐pregnane‐3,20‐dione, 3α,11β,21‐tetrahydroxy‐5α‐pregnan‐20‐one, 3α,15α,21‐trihydroxy‐5α‐pregnan‐11,20‐dione and 3α, 11β,15α,21‐tetrahydroxy‐5α‐pregnan‐20‐one. Conventional female rats excreted about 110 μg of monosulphurylated steroids in 24 h. This steroid fraction in conventional rats contained the same steriods as urine from germfree rats and in addition the following steroids were found: 3α,19‐dihydroxy‐5α‐androstan‐17‐one, 3α‐hydroxy‐5α,17α‐pregnan‐20‐one, 3α‐hydroxy‐5α‐pregnan‐20‐one, 3α‐hydroxy‐5α‐pregnan‐20‐one, 3α‐hydroxy‐5α‐pregnan‐20‐one, 3α‐hydroxy‐5α‐pregn‐16‐en‐20‐one, 3α,19‐dihydroxy‐5α,17α‐pregnan‐20‐one and 3β,15α‐dihydroxy‐5α‐pregnan‐20‐one.
The Necessity of an Electric Potential Difference and its Use for Photophosphorylation in Short Flash Groups
Authors: Junge Wolfgang , Rumberg Bernd , Schröder Hartmut
Published: March 2005

The ATP‐synthesis and the electric phenomena on the thylakoid membrane have been studied under excitation of photosynthesis with short flash groups. The light induced electric potential difference has been measured by means of field indicating absorption changes around 523 nm.

It is shown that upon excitation with short flash groups a certain electric potential difference is required before ATP can be synthesized. Moreover it is confirmed that the ATP‐synthesis is accompanied by an additional flux of protons down their electrochemical potential gradient. Correlation between the number of protons which have flowed across the ATPase coupled pathway, and the number of ATP‐molecules which have been formed, yields a ratio H+/ATP = 3.

Since this ratio includes only those protons, which have interacted with the ATPase complex, it represents the lower limit for H+/ATP‐ratios determined by any other method.

The necessity of a certain electric potential difference and the fact that 3 H+ have to be translocated down their electrochemical potential difference before one molecule of ATP can be synthesized is consistent with the chemiosmotic postulate, that the ADP ∼ P bond energy is derived from the electrochemical potential difference of the proton across the coupling membrane.

With respect to the alternative hypothesis for phosphorylation, the results impose grave restriction on a phosphorylation driven only by the free energy of a chemical intermediate, if this does exist at all.
The Critical Electric Potential Difference for Photophosphorylation
Authors: Junge Wolfgang
Published: March 2005

The physical meaning of the observed critical electric potential difference necessary for photophosphorylation is discussed. An interpretation of this phenomenon on the basis of the equilibrium thermodynamic formulation of the chemiosmotic hypothesis requires a reversible ATPase system. Contrary to this, ATP hydrolysis experiments seem to confirm that the Mg++‐dependent ATPase system of class II‐chloroplasts is irreversible in the absence of SH‐compounds. This sheds doubt on the equilibrium thermodynamic interpretation of the critical electric potential difference for phosphorylation. However it can be shown, that the seeming irreversibility may be due to some trigger requirements of an intrinsically reversible ATPase system.

This leads to an identification of the critical electric potential difference with the triggering level of the enzyme system.

Two operational models for an ATPase system, whose activity is modulated by the electric potential difference, are derived. These account quantitatively for a set of experiments on the critical electric potential difference.
Reaction of Puromycin with Chemically Prepared Peptidyl Transfer RNA
Authors: DE Groot Nathan , Panet Amos , Lapidot Yehuda
Published: March 2005

Chemically synthesized peptidyl‐tRNAphe reacts with puromycin when bound non‐enzymatically to Escherichia coli ribosomes in the presence of poly U. Guanosine triphosphate and G factor have no influence upon the puromycin reaction, when the magnesium ion concentration is 0.01 M or lower, but stimulate the puromycin reaction when the binding of peptidyl‐tRNA is carried out at higher magnesium ion concentration. In this stimulation β,γ‐methylene‐guanosine triphosphate cannot replace GTP, and fusidic acid inhibits the stimulation. The percentage of bound peptidyl‐tRNA which reacts with puromycin in the absence of G factor and GTP decreases upon increasing the magnesium ion concentration. From all these observations one can conclude that at magnesium ion concentrations below 0.01 M, peptidyl‐tRNA (such as Gly2‐Phe‐tRNA) binds preferentially to the donor site, but at higher magnesium ion concentrations (such as 0.025 M) the ribosomal aceptor site is preferred.

Peptidyl‐tRNAPhe can react with puromycin in the absence of the proper mRNA at rather highmagnesium ion concentration (0.02 M and higher) but this interaction in the absence of mRNA may be not a common feature of other peptidyl‐tRNAs, except tRNAMetf.

Phe‐tRNA interacts with puromycin but the rate of this interaction is much slower than that of peptidyl‐tRNAPhe such as Gly2‐Phe‐tRNA.