bluuebunny/crossref_metadata_2025_split huggingface dataset:
Uniformity and Species‐Specific Features of the N‐Terminal Amino‐Acid Sequence of Porcine Immunoglobulin λ‐Chains
Authors: Novotný Jiři , Dolejš Ladislav , Franěk František 
Published: March 2005

Cleavage with cyanogen bromide was employed to investigate the N‐terminal amino acid sequence of the λ‐chains of normal porcine immunoglobulin. The N‐terminal homoserine‐containing nonapeptide was isolated from all fractions of the λ‐chains in a yield of 60% of theory. Its amino acid sequence was determined by mass spectrometry and was found to be uniform. Other homoserine‐containing fragments were obtained from the cyanogen bromide hydrolyzate of the λ‐chains with split disulfide bonds. Partial amino acid sequence of these fragments provided evidence that methionine in position 9 is present in more than 60% of variants and obviously has an invariant character. Variants of the λ‐chains exist (by estimate not more than 15% of the total λ‐chains) which, in addition to the invariant methionine residue in position 9, possess a variable methionine residue in the section between positions 46 and 51.The data obtained indicate that the sequence of 23 amino acid residues from the N‐terminus of porcine λ‐chains is uniform with the exception of a single replacement (in position 13). Moreover, in comparison with the sequence of λ‐chains of man, mouse and birds, the sequence displays several species‐specific replacements and a deletion of threonine which occupies position 5 in the chains of the other species. In the pig, the λ‐chains represent the predominant type of light chains (some 70%). Hence it follows that some 50% of the light chains in this species possess a uniform amino acid sequence in the N‐terminal section. These facts are difficult to reconcile with the germline theories of genetic control so that mechanisms of somatic diversification of immunoglobulins must be envisaged.
N‐Acetylglutamate Synthetase of Pseudomonas aeruginosa
Authors: Haas Dieter , Kurer Verena , Leisinger Thomas 
Published: March 2005

N‐Acetylglutamate synthetase (acetyl‐CoA: l‐glutamate N‐acetyltransferase), the first enzyme of arginine biosynthesis, has been detected in extracts from Pseudomonas aeruginosa by a specific and sensitive assay in vitro.
The enzyme, partially purified by chromatography on hydroxyapatite, was dependent on l‐glutamate and acetyl‐CoA. l‐Aspartate could not replace l‐glutamate as a substrate; acetyl phosphate and N2‐acetyl‐l‐ornithine were not utilized as acetyl donors.
The enzyme was inhibited by l‐arginine (approx. 50% inhibition by 0.1 mM l‐arginine). N2‐Acetyl‐l‐ornithine, l‐ornithine, and l‐citrulline were not effective as inhibitors. Since N‐acetylglutamate 5‐phosphotransferase of P. aeruginosa is known to be inhibited by arginine, the first and the second enzymes of arginine synthesis in this organism are subject to feedback inhibition by the end‐product.
Arginine did not repress the formation of N‐ acetylglutamate synthetase.
Studies on the Metabolism of Steroids in the Foetus
Authors: Lisboa Belisário Penna , Plasse Jean‐Claude 
Published: March 2005

The hydroxylation of androstenedione in the human foetal liver at midgestation was investigated in vitro. After incubations carried out with 105000 ×g microsome‐preparations of hepatic tissue using a medium supplemented with NADPH‐regenerating system the C19O3‐metabolites were isolated by thin‐layer and gas chromatography and identified by mass‐spectrometry.The presence in the liver microsome preparations of 1β‐, 2β‐, 2α‐, 6α‐, 6β‐ and 18‐hydroxylase activities was demonstrated by the isolation and identification for the first time in the human of six α,β‐unsaturated C19O3‐steroids. Two of them, 1β‐hydroxy and 2α‐hydroxy‐androstenedione have not been previously reported in animal tissue. Three C19O2‐steroids (testosterone, 3α‐ and 3β‐hydroxy‐4‐androstene‐17‐one) have been also isolated in this investigation.
The Intracellular Location of Phenol Oxidases, Peroxidase and Phosphatases in the Leaves of Spinach Beet (Beta vulgaris L. Subspecies vulgaris)
Authors: Parish Roger W. 
Published: March 2005

Phenol oxidase activity was estimated with three substrates: l‐3‐(3,4‐dihydroxyphenyl)ala‐nine, 4‐methylcatechol and p‐coumaric acid (hydroxylase activity). Between 70–85% phenol oxidase activity and 10–20% peroxidase activity was sedimentable in leaf extracts from Beta vulgaris. Some 20–50% of the total phenol oxidase activity in a 6000×g (20 min) fraction could be released by washing with distilled water, incubating at 37°C or freezing and thawing. Acid phosphatase and catalase activities were much more readily released (90%) into solution by these treatments. Disruption of membranes with Triton X‐100 or sonication released the majority of phenol oxidase activity and this paralleled the release of chlorophyll and protein from membranes. Peroxidase was also strongly bound to the membranes.The intracellular location of enzymes was studied exhaustively using a variety of cell fractions and density gradients.Phenol oxidase activity towards all substrates was present in the chlorophyll‐rich chloroplast membranes. Some dihydroxyphenylalanine oxidase and 4‐methylcatechol oxidase was associated with mitochondria. p‐Coumaric acid hydroxylase was restricted to the chloroplasts and supernatant. No phenol oxidase activity sedimented with the peroxisomes.A third membrane fraction containing dihydroxyphenylalanine oxidase (and acid phosphatase) was isolated from a 5 to 20000×g (20 min) fraction, but was apparently not microsomal or mitochondrial in origin. A similar fraction could be isolated when chloroplasts were mechanically disrupted, and it is tentatively suggested this fraction represents the chloroplast envelope.Enzyme distribution among gradient fractions indicated that specific enzyme proteins exist for dihydroxyphenylalanine and catechol/p‐coumaric acid. This does not exclude the existence of isozymes active toward all three substrates.Sedimentable peroxidase was associated with mitochondria and a light membrane fraction. Mixing experiments showed this was not an antifact of homogenization. Chloroplasts were devoid of peroxidase.Different intracellular and intrachloroplastal locations were found for a number of phosphatases.
On the Peptidoglycan of the Cell Walls of Pseudomonas aeruginosa
Authors: Heilmann Hans‐Dietrich 
Published: March 2005

The peptidoglycan from cell walls of Pseudomonas aeruginosa consists of a macromolecular bag‐shaped network of covalently linked repeating units. Metal ions obviously do not participate in conserving its structure, nor could any other component besides glutamic acid, 2,5‐diaminopimelic acid, alanine, glucosamine and muramic acid (the latter two possibly being acetylated) be shown to be a constituent of the peptidoglycan sacculus.Approximately one out of four diaminopimelate residues of the repeating units of the peptidoglycan is cross‐linked to another unit.The protein covalently bound to the peptidoglycan can be removed from the latter by proteolytic digestion. Lysis experiments proved that Tris buffer and EDTA attack the outer layers of the cell wall which protect the peptidoglycan against being hydrolyzed by lysozyme.
Mammalian 3‐Oxosteroid Δ5—Δ4‐Isomerase
Authors: Geynet Philippe , Gallay Jacques , Alfsen Annette 
Published: March 2005

“Solubilization” of a beef adrenal microsomal enzyme: 3‐oxosteroid Δ5‐Δ4‐isomerase has been obtained by high concentrations of divalent cation (0.5–2 M CaCl2). A study of the Solubilization yield as a function of ion concentration and pH has shown that optimum yield is obtained with 1 M CaCl2 near pH 7.After elimination of calcium from the “soluble” enzyme, precipitation occurs and enzymatic activity is irreversibly lost in both precipitate and supernatant.The values of the kinetic parameters determined at pH 8.5 and 35°C are: V= 410 and 920 nmol · min−1· mg−1 and Km= 36 and 11 μM for 5‐androstene‐3,17‐dione and 5‐pregnene‐3,20‐dione respectively. Our results suggest the existence of only one C19 and C21 3‐oxosteroid isomerase in the beef adrenal glands.
The Biosynthesis of Blood‐Group‐B Character on Human O‐Erythrocytes by a Soluble α‐Galactosyltransferase from Milk
Authors: Pacuszka Tadeusz , Kościelak Jerzy 
Published: March 2005

The enzyme α‐galactosyltransferase from human milk catalysed the synthesis of B‐character on blood‐group‐O erythrocytes. Erythrocytes from secretors and nonsecretors participated in this reaction to the same extent. Substances possessing H‐active structure inhibited the acquisition of B‐character by erythrocytes when added to the reaction mixture.The enzyme also incorporated galactose into isolated, water‐soluble substances of erythrocytes with H‐activity from secretors.
Dissoziationsgleichgewichte von Glutathion
Authors: Jung Günther , Breitmaier Eberhard , Voelter Wolfgang 
Published: March 2005

The dependence of the 13C‐NMR resonances on the pH of solutions is determined for reduced and oxidized glutathione and for the model substances glutamine, cysteine and acetylglycine by pulse Fourier transform (PFT) 13C‐NMR spectroscopy.The 1H‐noise decoupled 22.63 MHz PFT 13C‐NMR spectra of 0.1–0.3 M aqueous solutions were measured on a Bruker HFX‐90 multinuclear spectrometer with an 18” magnet and equipped with a Fabritek 1074 and a PDP‐8‐I computer for accumulating the pulse interferograms and for performing their Fourier transformations.The pH induced variations of charge densities on the various carbon atoms of these molecules can be selectively observed. The pK and pI values obtained by these 13C‐NMR investigations often agree very well with the electrometric and photometric determinations. However, large deviations from classical values are found for other dissociation equilibria.For glutamine we obtained the pK values 2.1 (COOH) and 9.1 (NH3+) and an isoelectric point at 5.65. The pH induced shifts are particularly large for the ammonium dissociations. The titration of cysteine from pH 7 to pH 11 gives total shifts on the Cα of 4.1 ppm, on the β of 6.10 ppm and on the carboxylic carbon of 10 ppm with two macroscopic pK‐values at pH 8.25 and 10.25 corresponding to the ammonium and thiol dissociations. The curves show that cysteine exhibits mixed dissociation equilibria as reported previously. However a comparison of the results of the 13C‐NMR measurements on reduced and oxidized glutathione indicates the thiol dissociation in oxidized glutathione to be largely independent of the ammonium dissociation at the glutamic acid residue and a pK‐value of 9.2 for the thiol dissociation is found.Whereas the usual titrimetric methods register the macroscopic behaviour, e.g. an increase of the proton concentration. 13C‐NMR spectroscopy allows investigations at each carbon atom concerning microscopic events resulting from — NH3+, — SH and — COOH dissociations.
The Temperature‐Dependent Enzymatic Breakdown of rRNA of Reticulocytes
Authors: Prehn Siegfried , Rosenthal Sinaida , Rapoport Samuel M. 
Published: March 2005

The influence of temperature on the enzymatic breakdown of rRNA and ribosomal particles from rabbit reticulocytes by ribosomal ribonuclease and phosphatase was studied. Nucleosides were formed after the initial attack of ribonuclease on single‐stranded regions of the RNA.The following conclusions were reached for free RNA: U and C were distributed in a random manner in the single‐stranded loops at 20 °C. Short (A‐U)‐rich sequences were liberated at 37 °C, the relative share of which appeared to be higher in the 28‐S RNA. It contained correspondingly longer G‐C sequences. Clusters of A‐U sequences of greater length than at 37 °C were melted at 50 °C. It was proved that only ‐U‐U‐U‐ sequences were split, by analysis of of the 3′ and 5′ terminal nucleotides of the primary split products of ribonuclease.The behaviour of the particle‐bound RNA was modified considerably by interactions with proteins. Some of the cytosines appear to be protected against enzymatic attack at 20 °C. The 60‐S subunit shows a strongly predominant melting of short U‐A sequences at 37 °C. The reduction of the effective length of the U‐A clusters may be due to interaction of the RNA with proteins. The 40‐S subunit on the other hand would appear to exhibit a reduction of the effective length of G‐C sequences.The interaction of the subunits with each other in an 80‐S ribosome does not modify the behaviour of the subunits.
Oxidative Deamination of Sulfinyl, Sulfonyl and Thiosulfonyl Amino Acids by D‐Aspartate Oxidase
Authors: De Marco Carlo , Rinaldi Augusto 
Published: March 2005

C3 and C4α‐amino acids bearing an —SO2H, —SO2SH or —SO3H ω‐group are oxidatively deaminated by partially purified D‐aspartate oxidase preparations obtained from beef, pig or rabbit kidney.From the C3 sulfur‐containing amino acids the partially oxidized sulfur radicals (—SO2H, —SO2SH) are split off during the reaction, originating pyruvate. When a fully oxidized sulfur radical (—SO3H) is present, sulfopyruvic acid is obtained.The C4 sulfur‐containing amino acids all give rise to the corresponding sulfur‐containing ketoacids.The Km and V values observed with the three different enzyme preparations for all the compounds under study are reported.The present results and some observations already known suggest that the substitution of a partially or fully oxidized sulfur radical for the ω‐carboxyl group does not greatly affect the specificity of enzymes acting on dicarboxylic amino acids.
Steps in the Biosynthesis of Ribosomal RNA in Cultured Aedes aegypti Cells
Authors: Dalgarno Lynn , Hosking Dianne M. , Shen Cynthia H. 
Published: March 2005

The continuous Aedes aegypti cell line developed by Grace in 1966 grows in a medium which is free of the non‐defined constituents commonly used in the continuous culture of insect cells. In the work presented, advantage has been taken of this fact to carry out labelling experiments which demonstrate the sequence of steps in the biosynthesis of rRNA in this continuous insect cell line under specified growth conditions.Preliminary experiments show that the large and small rRNAs of A. aegypti cells have molecular weights of 1.5 × 106 and 0.7 × 106, respectively, as determined by polyacrylamide gel electrophoresis; the corresponding sedimentation values are 26 S and 18 S. On incubating cells for 20 min at 30 °C with [3H]uridine four major RNA species are labelled. These have molecular weights of approximately 3.8 × 106 (41 S), 2.1 × 106 (30 S) 1.3 × 106 (25 S) and 0.7 × 106 (18 S). When prelabelled cells are incubated with actinomycin, 41‐S RNA is rapidly converted into the 30‐S and the 25‐S species. The 30‐S RNA thus formed is slowly degraded, probably by exonucleolytic breakdown, to give 26‐S RNA; the large rRNA is first labelled after 45‐min incubation with [3H]uridine. 25‐S RNA is the probable precursor of 18‐S RNA; the small rRNA is first labelled after 20 min incubation with [3H]uridine.The results obtained are discussed in relation to previous studies on the molecular weights and biosynthesis of rRNA in mammalian, insect, amphibian and plant tissues.
Purification and Properties of NADP‐Dependent l(+)‐3‐Hydroxybutyryl‐CoA Dehydrogenase from Clostridium kluyveri
Authors: Madan Vijay K. , HILLMER Peter , Gottschalk Gerhard 
Published: March 2005

l(+)‐3‐Hydroxybutyryl‐CoA dehydrogenase was purified 18 fold with a 29% yield from extracts of Clostridium kluyveri. The enzyme was found to be homogeneous by gel electrophoresis and ultracentrifugation. The analysis of the sedimentation equilibrium and gel filtration experiments gave a molecular weight of 220000 and 215000, respectively. Gel electrophoresis in the presence of sodium dodecylsulfate revealed the presence of 8 subunits per enzyme molecule. The subunits contain approx. 5 sulfhydryl groups per mole of protein (26000×g). l(+)‐3‐Hydroxybutyryl‐CoA dehydrogenase from C. kluyveri is NADP‐dependent. The enzyme does not oxidize 3‐hydroxyvaleryl‐CoA or 3‐hydroxycaproyl‐CoA. The specific activity of the pure enzyme as measured by acetoacetyl‐CoA reduction at the optimum pH of 6.5 is approx. 450 U/mg protein at 25°C. The oxidation of 3‐hydroxybutyryl‐CoA at pH 9.5 proceeds with 7% of the rate of acetoacetyl‐CoA reduction. N‐Acetyl‐S‐acetoacetyl‐cysteamine is reduced with 15% of the rate of acetoacetyl‐CoA.Measurements of the Michaelis constants for the substrates of l(+)‐3‐hydroxybutyryl‐CoA dehydrogenase gave values of 50 μM for acetoacetyl‐CoA and 70 μM. for NADPH2.
On the Structure of Yeast tRNAPhe
Authors: Pongs Olaf , BALD Rolf , Reinwald Erwin 
Published: March 2005

The availability of (yeast)tRNAPhe for helix formation with complementary oligoribonucleotides has been explored by equilibrium dialysis studies. Oligoribonucleotides complementary to sequences which are double‐stranded, according to the cloverleaf model of tRNAPhe, did not bind. Two regions of tRNAPhe were fully acessible to oligonucleotide binding: (a) the 3′‐terminus; (b) the sequence (3′‐5′)U‐G‐A‐A‐Y (33–37) of the anticodon loop. Two other regions were accessible to oligonucleotide binding: (a) part of the extra arm; (b) the (3′‐5′)T‐Ψ‐C sequence of the ribosylthymine loop. However, the binding constants of trimers and tetramers complementary to these parts of tRNAPhe are very low.The data indicate that the anticodon loop of tRNAPhe has an asymmetric structure, which is not compatible with the Fuller‐Hodgson model. The low binding constants for oligonucleotides complementary to the extra arm or to the TpΨpC sequence are discussed. They seem to be due either to sterical hindrance or to different conformations of tRNA. In the latter case, one conformation would be accessible to complementary oligonucleotides for helix formation, the other would not. Present available oligonucleotide binding data of four tRNAs suggest that each tRNA has an individual pairing scheme, according to which the loops of the cloverleaf contribute to the three‐dimensional structure.
The Oleyl‐Coenzyme‐A Desaturase of Potato Tubers
Authors: Ben abdelkader Ahmed , CHERIF Abdelkader , Demandre Chantal , MAZLIAK Paul 
Published: March 2005

An active oleyl‐coenzyme‐A desaturase was associated with a microsomal fraction from “aged” slices of potato tuber; starting from [1‐14C]oleyl‐coenzyme A as a substrate, in vitro, more than 40% of the final fatty acid radioactivity was recovered in linoleic acid, the most abundant fatty acid of the intracellular membranes of potato tubers.
The presence of serum albumin in the reaction mixture was essential for the desaturation reaction and for preventing the degradation of polyunsaturated fatty acids. This stabilization of the desaturase activity by serum albumin allowed a great resistance upon freezing.
The microsomal oleyl‐coenzyme‐A desaturase showed a pH optimum around 7.0–7.2 and a temperature optimum near 25°C. The apparent Km of the desaturase for oleyl‐coenzyme A was 25 μM.
Oxygen and pyridine nucleotide were required for oleyl‐coenzyme A desaturation (NADH or NADPH were found nearly equally effective in the desaturation). Although NADPH was more effective than NADP, NAD was a effective as NADH for desaturation, suggesting that a NADH‐regenerating system probably exists in the microsomal suspension.
The oleyl‐coenzyme‐A desaturase was specific in vitro for oleyl‐coenzyme A as a substrate; no desaturation was observed with ammonium oleate, which was, however, desaturated in vivo by aged slices of potato tuber.
Oleyl‐coenzyme A (and the newly labelled linoleic acid molecules) were essentially incorporated into phospholipids and particularly phosphatidylcholine.
The oleyl‐coenzyme‐A desaturase was almost entirely associated with the microsomal membranes, as shown by differential centrifugation and sucrose‐gradient separations.
The oleyl‐coenzyme‐A desaturase activity (absent in the freshly cut slices) was induced during the aging of slices; a linear increase with the time of aging was found in many microsomal activities, such as oleyl‐coenzyme A desaturase, NADH‐cytochrome c reductase and protein synthesis. The inhibition of all these events by cycloheximide, or the partial inhibition by actinomycin D indicated that derepression of the genetic activity and synthesis of new proteins were involved in the induction of the oleyl‐coenzyme‐A desaturase.
The Molecular Basis of Mutagenesis by Methyl and Ethyl Methanesulfonates
Authors: Rhaese Hans‐Jürgen , BOETKER Naomi Kay 
Published: March 2005

The molecular basis of the biological effect of alkylation, mutation induction and inactivation, was studied using transforming DNA.Comparative studies of mutation induction after methylation (by methyl methanesulfonate) and ethylation (by ethyl methanesulfonate) showed that methylation was five to ten times more effective than ethylation. This observation confirms chemical studies of the reactivities of the individual bases of DNA but conflicts sharply with observations that methyl methanesulfonate is much less mutagenic than ethyl methanesulfonate in bacteriophage and some higher organisms. In each case the number of mutational events caused in transforming DNA followed single‐hit kinetics. After termination of alkylation, the rates of decrease of mutants caused by methylation was twice that caused by ethylation, which fits chemical estimates of depurination rates.Reverse mutation studies with different mutagens and base analogs indicated that methyl methanesulfonate predominantly methylates guanine whereas ethyl methanesulfonate alkylates guanine and also adenine; in both cases the major product is an N7‐alkyl purine. Accordingly the cause of base‐pairing mistakes is unlikely to be related to steric effects of the alkyl group but rather due to ionization of the alkylated guanine.In addition, inactivation of transforming activity was observed to be linear with treatment time of DNA with methyl or ethyl methanesulfonate in a semilogarithmic plot. Since the observed inactivation rates cannot be solely explained as inactivating mutagenic events produced by base alkylation, it is likely that phosphate alkylation is inactivating.Lethal and mutagenic DNA alterations are shown to be dissociated processes by the following observations. (a) After termination of alkylation, the number of mutants caused by alkylation of the purine bases decreased linearly with time indicating that depurination is inactivating. (b) Under identical conditions, the number of total transformants decreased three times faster than the number of mutants, indicating that in addition to depurination triester breaks must be responsible for inactivation. Therefore, base alkylations are mutagenic and depurination and triester breaks are lethal DNA alterations.
Cooperative Ligand Binding by Ferrihemoglobin
Authors: Banerjee Ramaprasad , HENRY Yann , Cassoly Robert 
Published: March 2005

Reactions of human ferrihemoglobin with azide and fluoride ions have been reinvestigated, particularly with a view to compare the effect of pH on the Hill coefficient and to study its dependence on observation wavelength. The results at pH 6 (with initial high‐spin aquo form) show the absence of cooperativity for the fluoride reaction but the existence of significant co‐operativity for the azide reaction, in agreement with the conclusions drawn by some previous authors.At pH 9, where the ferrihemoglobin is an equilibrium mixture of high and low‐spin states, the same reactions give different Hill coefficients; cooperativity appears for fluoride binding and diminishes for the azide reaction. The possible significance of concomitant changes of iron spin state and the appearance of cooperativity in hemoglobin reactions is discussed.