bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stereospecificity of the Dihydroorotate‐Dehydrogenase Reaction
Authors: Blattmann Paul , Rétey János 
Published: March 2005

Dihydroorotate dehydrogenase from Zymobacterium oroticum is shown to catalyse the anti‐elimination of hydrogen from the substrate.
In the presence of a catalytic amount of NAD+ the enzyme catalyses the exchange between the abstractable hydrogen atoms of dihydroorotate and solvent protons, the exchange of (5S)‐H atom being twice as fast as that of the 4‐H atom.
Sodium‐ethoxide‐catalysed exchange affects both diastereotopic protons in the methylene group of dihydroorotate, the (5S)‐H atom being exchanged somewhat faster than the (5R)‐H atom.
These findings are discussed in terms of stereospecificity and mechanism of flavin dependent dehydrogenase reactions.
The Mechanism of the Inhibition of Gramicidin‐S Synthesis by d‐Leucine
Authors: Saxholm Harald , Zimmer Trine‐Lise , Laland Søen G. 
Published: March 2005

The results show that enzyme I of gramicidin S synthetase activates d‐leucine in a manner analogous to that of l‐leucine (ATP +d‐leucine ⇌d‐leucyl‐adenylate + PP1) and binds two moles of d‐leucine in thioester linkage. It has therefore two thiol sites for d‐leucine, compared to one for the l‐isomer. One of the sites seems to be the thiol site for L‐leucine which is therefore stereo‐unspecific. The results also indicate that the site for the formation of the aminoacyl adenylates is the same for the two isomers. In contrast to thioester‐bound l‐leucine, enzyme‐bound D‐leucine does not catalyze the ATP‐[14C]AMP exchange reaction.At a molar ratio of l‐leucine: D‐leucine equal to 4, gramicidin S synthesis is almost completely inhibited and the growth of the peptide chain stops at the tetrapeptide stage (D‐Phe‐l‐Pro‐l‐Val‐L‐Orn). Enzyme‐bound D‐leucine is thus not able to replace L‐leucine in the formation of the pentapeptide.
Mouse‐Myeloma RNA Polymerase B Template Specificities and the Role of a Transcription‐Stimulating Factor
Authors: Lentfer Dierck , Lezius Axel G. 
Published: March 2005

A DNA‐dependent transcriptase has been purified from mouse myeloma tissue. The system consists of an RNA polymerase of type B and a basic, heat‐labile protein of Mr 20000–30000 (stimulating factor). The RNA polymerase was characterized by its requirement for Mn2+ and medium ionic strength as well as by its susceptibility to inhibition by α‐amanitin and phosphate ions.The template specificities were studied using native and heat‐denatured DNA as well as synthetic double and single‐stranded polydeoxynucleotides. Alternating polydeoxynucleotides like poly[d(A‐T) · d(A‐T)] and poly[d(I‐C) · d(I‐C)] are excellent templates, the latter exceeding native DNA by two orders of magnitude. With these templates, factor stimulation is less expressed and at most two‐fold. With DNA‐like templates, such as poly[d(A‐C) · d(T‐G)] and poly[d(A‐G) · d(T‐C)], transcription proceeds at a lower rate (though still exceeding native DNA), but factor can stimulate up to 5‐fold. In contrast, the homoduplexes poly(dA) · poly(dT), poly(dG) · poly(dC), and poly(dI) · poly(dC) are transcribed poorly, if at all, irrespective of the presence or absence of stimulating factor. Among the single‐stranded polydeoxynucleotides tested poly[d(A‐C)], poly(dA), poly(dI), and poly(dC) are moderately well transcribed, whereas poly[d(T‐G)] and poly(dI) are completely ineffective.Initiation of transcription was studied using [γ‐32P]ATP. From the results it is concluded that the stimulating factor is involved in the progress of transcription rather than in initiation.
Energy‐Dependent Changes in Membranes of Rhodospirillum rubrum Chromatophores as Measured by 8‐Anilino‐naphthalene‐1‐sulfonic Acid
Authors: Vainio Harri , Baltscheffsky Margareta , Baltscheffsky Herrick , Azzi Angelo 
Published: March 2005

Rhodospirillum rubrum chromatophores enhance the fluorescence of 8‐anilino‐naphthalene‐1‐sulfonic acid (ANS). Energization of the chromatophore membrane by ATP, PPi, or succinate produces a fluorescence increase in ANS; PPi is the most potent in this respect.
It was possible to inhibit the fluorescence increase due to ATP, PPi, and succinate by uncouplers; namely, gramicidin, valinomycin, and tetraphenylboron. The uncouplers did not, however, affect the non‐energy‐dependent ANS fluorescence.
It was possible to inhibit the increase in ANS fluorescence due to ATP by oligomycin, whereas the PPi‐induced ANS fluorescence increase was even slightly stimulated in the presence of oligomycin.
The antibiotic dio‐9 was able to inhibit the fluorescence increase due to both ATP and PPi, without, however, affecting the non‐energy‐dependent ANS fluorescence.
Chaotropic agents such as KSCN and urea, and the detergent deoxycholate all inhibited the PPi‐induced membrane energization, as detected by ANS fluorescence.
Interaction between Pyridoxamine 5′‐Phosphate and Apo‐Aspartate Aminotransferase from Pig Heart
Authors: Arrio‐Dupont Martine 
Published: March 2005

Apoenzyme of aspartate aminotransferase prepared according to various techniques can bind the pyridoxamine‐P coenzyme and recover its full activity. An anticooperativity is observed between the two sites of the dimer; the intrinsic dissociation constants are 20 nM and 200 nM for apoenzymes prepared in the presence of phosphate and less than 1.8 and 6 nM for an apoenzyme prepared without phosphate. The phosphate ions can bind to two sites of the apoenzyme with an intrinsic constant of 1.6 μM; a competition is observed between pyridoxamine‐P and phosphate but it is not sufficient to explain the observed differences between the various apoenzymes. At pH 8.3, pyridoxamine‐P can dissociate from the holoenzyme: the dissociation rate is very slow so that it does not occur in enzymatic transamination.In the presence of succinate, the two sites become equivalent and the affinity for pyridoxamine‐P is smaller, meaning the occurence of an interaction between the substrate‐binding site and the coenzyme‐binding site.
The Subunit Structure of Lactate Dehydrogenase from Streptococcus cremoris US3
Authors: Dynon Maria K. , Jago G. Richard , Davidson Barrie E. , Gething Mary Jane H. , Davidson Barrie E. 
Published: March 2005

Lactate dehydrogenase from Streptococcus cremoris US3, which has a molecular weight in solution of 140000, was found by dodecylsulphate‐gel electrophoresis to have a minimum molecular weight of 37000. It is, therefore, a tetramer.
Approximately 40 tryptic peptides were obtained by fingerprinting and in conjunction with the amino acid composition this indicates that the subunits are similar and probably identical.
There is one thiol group per subunit.
p‐Chloromercuribenzoate inhibits the enzyme.
The Purification and Properties of an NADPH‐Linked Aldehyde Reductase from Pig Brain
Authors: Turner Anthony J. , Tipton Keith F. 
Published: March 2005

An NADPH‐linked aldehyde reductase has been purified some 600‐fold from the supernatant fraction of a pig brain homogenate.
Subcellular distribution studies show that the enzyme is almost exclusively located in the cytosol.
The enzyme has a molecular weight of 29000 and an isoelectric point of 5.8.
The enzyme reduces a wide range of aliphatic and aromatic aldehydes, including some biogenic aldehydes. Ketones are not reduced.
Specificity studies carried out on the enzyme suggest that substituted phenylglycolaldehydes are reduced far more readily than the corresponding substituted phenylacetaldehydes.
Ethanol, reserpine and several aldehyde dehydrogenase and liver alcohol dehydrogenase inhibitors do not affect the activity of brain NADPH‐linked aldehyde reductase. 0.1 mM sodium barbitone markedly inhibits the enzyme.
Differences between the ATP/ADP Ratios in the Mitochondrial Matrix and in the Extramitochondrial Space
Authors: Heldt Hans W. , Klingenberg Martin , Milovancev Mirjam 
Published: March 2005

The relationship between the ATP/ADP ratios inside and outside the mitochondria was studied using silicon‐layer centrifugation. As predicted from the properties of the adenine nucleotide translocation, the ATP/ADP ratio in the extramitochondrial space was found to be higher than in the mitochondria when in the state of respiratory control. Taking into account the content of inorganic phosphate, the phosphorylation potential in the mitochondria is calculated to be 2.3 kcal lower than in the medium.This difference between the internal and external ATP/ADP ratios is abolished if uncoupler is added, When there is also no Mg2+ present in the medium, the internal ATP/ADP ratio may be even higher than the external ratio, provided that there was oligomycin added in order to inhibit ATP hydrolysis.It is shown that the ADP is transported against a gradient from the extramitochondrial space into the mitochondria. The discussion of a model suggests that the physiological role of the energy‐requiring transport of ADP into the mitochondria is to maintain a constant level of the ATP in the cytosol.
Reaction Mechanism of l‐Glutamate Dehydrogenase
Authors: di Franco Ann , Iwatsubo Motohiro 
Published: March 2005

Presented here are the optical characteristics (absorbance, fluorescence, circular dichroism) of several enzyme · coenzyme complexes of l‐glutamate dehydrogenase, with and without the different substrates (or products). Three of the four complexes studied can be distinguished unambiguously by the following characteristics: the E · NADPH and E · NADPH ·l‐glutamate complexes have similar absorption and fluorescence properties, but markedly differ in their opposite ellipticities; the E · NADPH · 2‐oxoglutarate possesses characteristic absorption and elliptical properties, but its highly quenched fluorescence is its most distinctive feature.A detailed study of the elementary kinetic parameters, k+ and k‐, was carried out with respect to each ligand. A comparison is made of the Kd values calculated from the k−/k+ ratio and from direct binding studies. As far as NADPH is concerned it is clear that its association rate constant is not affected, while its dissociation rate constant is very sensitive to the presence of the substrates. Thus, the stability of each complex is mainly governed by the dissociation rate. These dissociation rate constants are not very different from those obtained for the different substrates.
Fast‐Kinetic Studies of the Oxidative Deamination of Glutamate Catalyzed by Glutamate Dehydrogenase
Authors: D'albis Anne , Pantaloni Dominique 
Published: March 2005

The release of protons, one of the products of the reaction, in the oxidative deamination of glutamate by glutamate dehydrogenase, has been followed in a stopped‐flow spectrophotometer by measuring the absorbance change of an indicator dye. The results obtained at pH 7.8 and 15 °C, in 0.05 M Na2SO4, with NADP+ as coenzyme, indicate that the liberation of the protons occurs after the electron transfer but before the release of NADPH, i.e. before the steady state; the results obtained in the same conditions, but in presence of the inhibitor GTP, indicate moreover that it occurs also before the liberation of 2‐oxoglutarate.The oxidative deamination of glutamate has in addition been studied in the presence of equi‐molar amounts of the reduced coenzyme NADPH. The elimination of the presteady‐state step when all the enzyme is in the form of the enzyme · NADPH · glutamate complex supports the hypothesis that the rate‐limiting step in the overall mechanism is the dissociation of NADPH and the regeneration of the enzyme from this ternary complex.
Interaction of Concanavalin A with Rat Lymphocytes
Authors: Betel Ido , van den BERG Kor J. 
Published: March 2005

Concanavalin A covalently bound to acrylic polymer particles (enzacryl AA), which cannot enter the cells, stimulated rat lymphocytes into DNA synthesis.
Evidence was obtained that concanavalin A binds to the lymphocyte membrane specifically through its sugar‐binding sites. No nonspecific adsorption was found.
The equilibrium constant found for the binding of concanavalin A to the cell was 12 μ−1 at 37°C and 3°−l at 0°C. No heterogeneity in the affinity of the binding sites was detected. The average maximum number of concanavalin A molecules (Mol. wt 108000) that could be bound was about 5 ×106 per cell.
Optimum stimulation of DNA synthesis was obtained when about 5%, of the available sites on the cells were occupied by concanavalin A.
The Theory of Alternative Substrates in Enzyme Kinetics and Its Application to Yeast Hexokinase
Authors: Ricard Jacques , Noat Georges , Got Claude , Borel Maguy 
Published: March 2005

The theory of alternative substrates has been thoroughly investigated. The reciprocal rate equations, for ordered and random kinetic mechanisms, in the presence of an alternative substrate, have been developed. The conditions in which those equations give rise to either hyperbolic or linear reciprocal plots have been established. Degeneracy and constraint conditions of the various kinetic models have also been derived.Definite conclusions concerning the relevant mechanism can be obtained only if the Dalziel coefficients pertaining to the substrate are different from those corresponding to the alternative substrate. It follows from this, that the method termed “isotope competition”, cannot be used to make a choice among various two‐substrate mechanisms, and appears to be unfounded.The theory of alternative substrates has been applied to the study of yeast hexokinase. The results obtained are at variance with a random mechanism. They do agree, on the other hand, with a compulsory order of substrate binding on the enzyme, glucose being bound first.
Metabolism of Isolated Kidney Tubules
Authors: Guder Walter G. , Wieland Otto H. , Stukowski Barbara 
Published: March 2005

The effects of parathyroid hormone, adenosine 3′:5′‐monophosphate (Ado‐3′:5′‐P) and oleate on renal metabolism have been studied in isolated kidney‐cortex tubules from starved rats. Substrate uptake and glucose formation have been determined with l‐lactate, pyruvate, glutamate, and glycerol as substrates.The results indicate that parathyroid hormone and Ado‐3′:5′‐P increase renal gluconeogenesis by the same mechanism, which however, differs from the effects brough about by free fatty acids:

Parathyroid hormone and Ado‐3′: 5′‐P increased glucose formation from l‐lactate, pyruvate and glutamate. The stimulatory effect of parathyroid hormone was always smaller compared with that of Ado‐3′:5′‐P. Glucose formation from l‐lactate was shown to be stimulated with hormone doses as low as 1–10 nM. Oleate had an increasing effect oil glucose formation from l‐lactate and glycerol, but no effect on glucose formation from 10 mM pyruvate and inhibited gluconeogenesis from glutamate.
The stimulatory action of parathyroid hormone and Ado‐3′:5′‐P was accompanied by an increase in substrate uptake, whereas oleate decreased substrate uptake.
The effects of parathyroid hormone and Ado‐3′:5′‐P on kidney‐tubule metabolism were not additive, when both substances were added together.
All effects of oleate were found to be unchanged in the presence of parathyroid hormone and Ado‐3′:5′‐P or both.

The results suggest two different mechanisms which may regulate renal gluconeogenesis additively.
Thermochemistry of Lysozyme‐Inhibitor Binding
Authors: Bjurulf Carin , Wadsö Ingemar 
Published: March 2005

The binding of saccharide inhibitors to lysozyme has been studied using a microcalorimetric method. Measurements have been performed with N‐acetyl‐d‐glucosamine (pH 5 and 25 and 40°C), tri[N‐acetyl‐d‐glucosamine] (pH 2, 5 and 7.6 at 25°C and pH 5 at 40°C) and with tetra‐[N‐acetyl‐d‐glucosamine] (pH 2, 5 and 7.6 at 25°C). From the results of the calorimetric measurements values for ΔG0, ΔH0, ΔS0, and ΔCp0 were derived for the binding processes.In addition to the saccharide‐binding reactions, some model experiments were performed.
Studies on Rat‐Liver Ribosomes, Ribosomal Subparticles and RNA Fixed with Formaldehyde
Authors: Dessev George N. , Grancharov Konstantin 
Published: March 2005

Rat liver polysomes, single ribosomes, EDTA‐produced subparticles and free ribosomal RNA were treated with formaldehyde and their properties were studied. The following results were obtained.

RNA can be isolated from fixed polysomes or subparticles by exhaustive digestion with pronase. Such RNA shows a normal electrophoretic behaviour in agar gel.
In free RNA treated with formaldehyde the mobility of 18‐S and 28‐S RNA fractions is reduced; latent breaks are not revealed by heating; sensitivity towards ribonuclease is increased. No such effects are observed with RNA treated with formaldehyde while in the particles.
In fixed polysomes or single ribosomes the two subparticles appear to be covalently bound through their proteins. No intermolecular bonding occurs between 18‐S and 28‐S RNA either in fixed polysomes or in free RNA. The fixed ribosomes and subparticles can be fractionated by agar gel electrophoresis and give profiles similar to those of untreated particles. These results may prove useful in combining CsCl density gradient fractionation with gel electrophoretic methods.
Hydrosoluble Complexes of Sterols, Sterol Esters and Their Precursors from Zea mays L.
Authors: Rohmer Michel , Ourisson Guy , Brandt Roger D. 
Published: March 2005

The study undertaken shows the existence of water‐soluble complexes of sterols, sterol esters and their precursors (triterpenes and 4α‐methylsterols) in a higher plant, Zea mays L. The leaves, roots and culture medium were treated separately in order to investigate the role and nature of these complexes. The sterols found in the water‐soluble form represent 0.1–1% by weight of the total sterols in the plant. The composition of the hydrosoluble fraction of the sterols and their precursors is different from that of the lipid‐soluble fraction, cholesterol, sitosterol and isofucosterol being preferentially complexed compared to campesterol and stigmasterol.This complex was found in the microsomal supernatant after homogenisation and also in the culture medium. The sterols are released from the complex by a treatment with pyrogallol under basic conditions or after repeated extraction with petroleum ether. The complex is precipitated with ethanol but not with trichloroacetic acid. The behaviour of several sterols on treatment with a solution of starch was investigated. Starch was able to complex with a large number of phytosterols unspecifically. The implication of this complex for the transport of sterols, for the biosynthesis of steroids and the ecological impact is discussed.
The Effect of Structural Modifications of ATP on the Yeast‐Hexokinase Reaction
Authors: Hohnadel David C. , Cooper Cecil 
Published: March 2005

The specificity of yeast hexokinase for ATP was examined with the aid of a number of synthetic and natural analogs. The main features observed are:

The presence of a 2‐amino group either alone or in combination with a 6‐amino or 6‐hydroxyl group inhibits catalysis.
The replacement of one or both hydrogen atoms of the 6‐amino group with methyl groups does not have a large effect on either binding or catalysis.
The replacement of the nitrogen at position 7 with a carbon atom has virtually no effect.
The movement of the glycosidic bond from the N9 to the N3 position has only a moderate effect on both Km and V.
The presence of both of the 2′‐ and 3′‐cis hydroxyls in the ribose is important for catalysis.
The replacement of ribose with arabinose or glucose produces a large decrease in catalysis. Inhibitor experiments were carried out with the ATP analog containing glucose instead of ribose. It was found to be competitive for ATP and uncompetitive for glucose under the conditions used. These results are consistent with an ordered reaction mechanism.
Composition du suc pancréatique humain
Authors: Clemente François , de Caro Alain , Figarella Catherine 
Published: March 2005

The enzyme composition of human pancreatic juice has been studied by immunological techniques, using non‐activated and activated juice.
Antisera were prepared with activated and non‐activated pancreatic juice. Same samples of antisera were adsorbed with human serum. Immunoelectrophoresis was carried out at pH 8.6 and disc immunoelectrophoresis at pH 8.9 and 5.0. Gel‐diffusion media contained benzamidine, to prevent zymogen activation. Precipitation lines were characterized by activating the zymogens on the plates with trypsin, and identifying the enzymes produced using specific synthetic substrates.
Immunoelectrophoresis showed 15–20 antigenic constituents. Although disc immunoelectrophoresis gave slightly different results, those were consistent with the results of immunoelectrophoresis.
The following active enzymes were identified and localized: α‐amylase, lipase and carboxylester hydrolase, which is a lipolytic enzyme structurally and catalytically different from lipase.
Several zymogens were identified: two immunologically distinct trypsinogens, one procarboxypeptidase B, two forms of procarboxypeptidase A, which are immunologically identical, and two proelastases with some common antigenic determinants. After activation, both proelastases showed a weak but different degree of chymotryptic activity. Chymotrypsinogen was localized in two major anionic lines, and one, minor, cationic line. The possibility that one anionic immune precipitate represents a polymer is discussed. No chymotryptic activity was associated with procarboxypeptidase A.
Immunoelectrophoresis of activated pancreatic juice demonstrates electrophoretic and antigenic changes. Activation of one anionic trypsinogen gave rise to cationic trypsin, whereas after activation chymotrypsinogens and procarboxypeptidases A showed no important change.
A 13C Nuclear‐Magnetic‐Resonance Study of the Enzyme Cofactor Flavin‐Adenine Dinucleotide
Authors: Breitmaier Eberhard , Voelter Wolfgang 
Published: March 2005

An assignment of the 13C‐NMR spectrum of the enzyme cofactor flavin‐adenine dinucleotide (FAD) is given by comparing it with the 13C chemical shifts of the constituents of FAD, i.e. adenosine 5′‐diphosphate, riboflavin 5′‐monophosphate, riboflavin, adenosine and adenine. The 13C‐NMR spectrum of FAD shows three groups of signals:a) At highest field the resonances of the methyl groups (‐18 to ‐21 ppm) of the isoalloxazine moiety.b) At lowest field the signals of the carbon atoms of the isoalloxazine ring (‐117 to ‐161 ppm).c) In the range of ‐47 to ‐88 ppm the resonances of the carbon atoms of the ribose and ribitol residue.
Metal‐Combining Properties of Human Lactoferrin
Authors: Teuwissen Bruno , Masson Pierre L. , Osinski Pierre , Heremans Joseph F. 
Published: March 2005

By means of a Spectrophotometric titration method it was found that six of the tyrosyl residues of the metal‐binding proteins lactoferrin and transferrin were titratable by alkali in the metal‐free proteins but not in their iron protein complexes, suggesting the involvement of three tyrosyl groups in each of the two binding sites present in these proteins.Seemingly discordant estimates of the number of binding tyrosyls were obtained using the difference spectra of the lactoferrin · Tb3+vs lactoferrin, lactoferrin · Fe3+vs lactoferrin, transferrin · Tb3+vs transferrin or transferrin · Fe3+vs transferrin. However, such discrepancies as were found could be explained in the light of the ligand‐field theory and need not invalidate the conclusions based on alkali‐titration data.