bluuebunny/crossref_metadata_2025 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.
Metabolism of Steroid Hormones, Sterols, and Bile Acids in Liver Microsomes from Male, Female, and Male‐Pseudohermaphroditic Rats
Authors: Einarsson Kurt , Gustafsson Jan‐Åke , Goldman Allen S.
Published: March 2005

The metabolism of 4‐[4‐14C]androstene‐3,17‐dione and 4‐[4‐14C]pregnene‐3,20‐dione in 100000×g microsomes from livers of male, female and male pseudohermaphroditic rats was studied by thin‐layer chromatography and gas chromatography‐mass spectrometry. Sexual differences characterized the 16α‐hydroxylation, 3α ‐ and 3β‐hydroxysteroid oxidoreduction and 5α‐reduction of 4‐androstene‐3,17‐dione and the 6β‐hydroxylation, 20α‐ and 20β‐hydroxysteroid oxidoreduction and 5β‐reduction of 4‐pregnene‐3,20‐dione. In all these cases male pseudohermaphroditic rats showed identical enzyme activities to female rats. Furthermore, the following reactions participating in the biosynthesis of bile acids were studied: 7α‐hydroxylation of cholesterol, 5α‐reduction of 7α‐hydroxy‐4‐cholesten‐3‐one, 12α‐hydroxylation of 7α‐hydroxy‐4‐cholesten‐3‐one and 6β‐hydroxylation of taurochenodeoxycholic acid. Sexual differences characterized the two first‐mentioned reactions; also in these respects male pseudohermaphroditic rats were identical to female rats. The female character of the hepatic metabolism of the male pseudohermaphroditic rat may be due to the lack in these rats of a neonatal testosterone “imprinting” on the liver because of end‐organ insensitivity to testosterone and/or a deficient testicular biosynthesis of androgens.
Transplantable Immunoglobulin‐Secreting Tumours in Rats
Authors: Querinjean Pierre , Bazin Hervé , Beckers Andrée , Deckers Christian , Heremans Joseph F. , Milstein Cesar
Published: March 2005

The purification and chemical characterization of three Bence‐Jones proteins and one light chain of an IgG myeloma protein of the rat strain LOU/Wsl are described. The amino acid composition of the four chains is very similar but not identical. Fingerprint analyses show a pattern of peptides common to all four proteins as well as unique features. The compositions of the common peptides are given. The data indicate that the four light chains are of the kappa type.
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 Parallel Nature of the Interchain Disulphide Bonds of Immunoglobulins
Authors: Svasti Jisnuson , Milstein Cesar
Published: March 2005

[35S]Cystine‐labelled MOPC 21 (IgGl:χ) myeloma protein was prepared by incubating myeloma cells, grown as a suspension culture, in medium containing [35S]cystine. The radioactive protein was used as a tracer in conjunction with carrier myeloma protein. A peptic‐tryptic peptide of 36 residues was prepared, which contained ten half‐cystine residues and included the heavy‐light and all the heavy‐heavy inter chain disulphide bridges of the molecule. The peptide was digested with thermolysin and subjected to partial acid hydrolysis. A large number of small peptides was isolated and, when possible, their amino acid compositions were determined. From 170 possible arrangements of the disulphide bonds a unique arrangement was deduced in which all the heavy‐heavy interchain bonds were parallel. This is the first case in which the arrangement of all the interchain bonds of any of the human or mouse IgG subclasses has been established.
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 Energy Metabolism of Human‐Blood Polymorphonuclear Cells
Authors: Marchand Jean‐Claude , Leroux Jean‐Paul , Cartier Pierre
Published: March 2005

Pure and viable human granulocytes were prepared from peripheral blood, and incubated in a buffered saline medium. Glycolytic intermediates, adenine‐nucleotides, glycogen, glucose and oxygen consumptions, and lactate production were measured under standard (aerobiosis, pH 7.4) and modified conditions. Standard incubations allow the determination of mass‐action ratios, which localize regulatory steps at hexokinase, phosphofructokinase and glyceraldehyde‐phosphate dehydrogenase: phosphoglycerate kinase. Anaerobiosis results in an increased glucose consumption while lactate production remains normal, presumably because of an increased glycerolipid synthesis induced by the elevated NADH/NAD+ ratio. A positive crossover at phosphofructokinase level derives from ATP diminished content. 0.5 mM 2,4‐dinitrophenol induces an increased oxygen consumption, an ATP fall and a positive crossover at phosphofructokinase level with an augmented overall glycolysis. pH‐changes modify phosphofructokinase activity, as revealed by the positive and negative cross‐overs respectively observed at pH 7.80 and 6.85. 0.1 mM and 0.2 mM monoiodacetate inhibits overall glycolysis without increasing oxygen consumption; the NAD+/NADH ratio is notably decreased; an apparent positive crossover again appears at the phosphofructokinase step, but the decreased hexose‐monophosphate level may result from the diminished ATP, whose concentration is not far from the Michaelis constant of hexokinase. 2 mM to 20 mM sodium fluoride protects glycogen, inhibits glycolysis and dramatically increases oxygen consumption. 0.2 mM to 0.5 mM NaF‐activates glycolysis and glycogen breakdown, and inhibits oxygen consumption. 3 mM dibutyryl adenosine 3′:5′‐monophosphate increases glycogenolysis and induces two cross‐overs: a positive one at phosphofructokinase and a negative one at glyceraldehyde‐phosphate‐dehydrogenase: phosphoglycerate kinase level. The lipolytic action of 3′:5′‐AMP may explain those effects, by decreasing ATP/ADP and NAD+/NADH ratios. A comparison between over‐all glycolytic activities and cross‐over plots clearly indicates that granulocyte glycolysis is regulated at the hexokinase step by ATP availability as compared with Michaelis constant, at the phosphofructokinase level through an allosteric ATP inhibition, and at the glyceraldehyde‐phosphate‐dehydrogenase step by mass‐action of the NAD+/NADH ratio.
The Number of Catalytically Essential Carboxyl Groups in Pepsin
Authors: Paterson Ann K. , Knowles Jeremy R.
Published: March 2005

Pepsin has been modified by reaction with [14C]trimethyloxonium fluoroborate at pH 5.0. The loss of catalytic activity consequent upon modification can be ascribed to the esterification of carboxyl groups in the protein. The relationship between the number of methyl groups incorporated and the remaining catalytic activity provides clear evidence that at least two carboxyl groups are essential to the activity of pepsin. Mechanistic formulations that require two carboxyl groups now rest on firmer ground.
Action of Bleomycin on DNA and RNA
Authors: Müller Werner E. G. , Yamazaki Zen‐ichi , Breter Hans‐J. , Zahn Rudolf K.
Published: March 2005

Bleomycin, exclusively yet not necessarily, affects thymidine‐containing compounds such as DNA and polydeoxyribonucleotides by releasing free thymine and leaving aldehyde functions. Bleomycin action can be enhanced by adding reducing as well as oxidizing agents. Several radical scavangers were without influence. The guanidino moiety in the bleomycin could be traced as a functionally essential group. Molecular morphology and base sequence of the DNA strongly influence bleomycin activity. High bleomycin concentrations, besides modifying DNA into oligothyminic or athyminic nucleic acids, cause strand scissions. Possible reaction mechanisms are discussed.
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.
Conductimetry in Enzyme Studies
Authors: Lawrence Anthony J. , Moores Geoffrey R.
Published: March 2005

An improved multichannel conductivity apparatus is described. Techniques are reported for measuring single ion conductances and the quantitative nature of the method is demonstrated. The role of buffers is examined and the Good buffers are found to be particularly satisfactory. Reactions with metal chelates are examined critically and the possibility of artefacts is discussed. Assays are described for deaminases, amidases, oxidases, proteases and nucleases. The assay of decarboxylases is discussed. Kinetic data has been obtained for some of these enzymes and compared with published results. The enzymic hydrolysis of inorganic pyrophosphate is suggested as the basis of a coupled assay for a wide range of enzymes.
The Substrate Specificity of Exonuclease A5
Authors: Kozlov Igor A. , LVOVA Tatiana N. , Ledneva Rimma K. , TATARSKAYA Rachel I.
Published: March 2005

The kinetic parameters, Km(app) and V, determined for the digestion of the substrates of type pNpN by exonuclease A5 have proved similar for different substrates, regardless of their nucleotide composition and base sequence. When dinucleotides, in whose bases the amino groups were acylated, were used, it was shown that V does not change but Km(app) decreases. The decrease in the value of Km(app) correlates with a decrease in the hyperchromicity, which results from the modification of the bases. In the series of oligonucleotides differing in length, i.e., d(pT)2, d(pT)3, d(pT)4, Km(app) decreases 60‐fold when a trinucleotide is used instead of a dinucleotide and four‐fold when a tetranucleotide is used instead of a trinucleotide, V changing comparatively little. Specially synthesized, the new model compounds of type pRpN, RpRpN and RpRpR, where N is a nucleoside and R is a substance of non‐nucleotide nature, were used. It was established that model substrates can be digested by exonuclease A5 at a rate close to that of hydrolysis of oligonucleotides of pNpN and NpNpN type, only when the terminus to be attacked contains N. The nature of R, adjacent to the nucleoside, is apparently inessential; we have shown that it may be an aliphatic diol or resorcinol. All these data give grounds for believing that for the enzyme‐substrate interaction the latter should possess at least two phosphate residues specifically oriented and one nucleoside at the 3′‐terminus bound with the remaining portion of the molecule by a phosphodiester bond across its 5′‐OH group. The structure of the heterocyclic base in these nucleosides is of no prime importance.
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.
The Effect of Bifunctional and Monofunctional Azo‐Dye Reagents upon the Oxygen‐Binding Mechanism of Human Hemoglobin
Authors: Fasold Hugo , MEYER Christa , Steinkopff Gudrun
Published: March 2005

Both p‐bis‐iodoacetamido‐2,2′‐dicarboxyazobenzene and 4‐iodoacetamido‐2,3′‐dicarboxyazo‐benzene were reacted with the accessible SH‐groups of human hemoglobin tetramers. The mono‐functional azo dye produced the expected, and uniformly modified hemoglobin, in which both cysteines‐β93 were alkylated. The bifunctional dye gave rise mainly to a symmetrically modified hemoglobin, forming cross‐links within the two β‐chains between cysteine‐93 and lysine‐82. A small amount of a second, asymmetrical hemoglobin derivative could be isolated, carrying the same cross‐link in one β‐chain, while the second cross‐link ran from one cysteine‐β93 to the histidine in position 45 of the neighbouring α, chain. Homotropic cooperativity of oxygen binding was reduced in all these derivatives of hemoglobin. Both cross‐linked hemoglobins possessed an oxygen‐binding curve with a Hill coefficient of 1.0–1.2. For the hemoglobin reacted with mono‐functional dye this coefficient varied between 1.65 and 1.72. Its Bohr effect amounted to 0.3, while the cross‐linked hemoglobins showed no Bohr effect.
The Influence of Steric and Electronic Parameters on the Substrate Behaviour of α‐Oxo Acids to Yeast Pyruvate Decarboxylase
Authors: Lehmann Harald , FISCHER Gunter , Hübner Gerhard , KOHNEBT Klaus‐Dieter , Schellenberger Alfred
Published: March 2005

The Michaelis constants of yeast pyruvate decarboxylase at full substrate activation and the maximum decarboxylation rates were determined from a total of 21 aliphatic and aromatic α‐oxo acids in the absence of buffer substances. By means of a pyruvamide‐activated enzyme it was possible to confirm experimentally the Km‐values obtained by extrapolation. The substrate binding rate of pyruvate decarboxylase increases linearly with increasing H+ concentration, which indicates that only the undissociated pyruvic acid acts as the substrate of the enzyme. Whilst the substrate binding rate can be correlated linearly with the steric substituent constants of aliphatic α‐oxo acids, it was not possible to find a corresponding relation between the aromatic α‐oxo acids and the Hammett σ‐constants, which indicates steric peculiarities in the active centre of pyruvate decarboxylase. On the other hand, a linear relation between the maximum velocities and the σ+‐values for direct conjugation was established, leading to some conclusions concerning the mechanism of the splitting‐off of aldehyde.
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.
New Form of A‐, B‐, and H‐Blood‐Group‐Active Substances Extracted from Erythrocyte Membranes
Authors: Gardas Andrzej , KOŚCIELAK Jerzy
Published: March 2005

The isolation and purification of new group A‐, B‐ and H‐specific substances from human erythrocyte membranes is described. These substances contain galactose, fucose, glucosamine, sialic acid and amino acid residues. The A‐specific substance also contains galactosamine. They contain at most about 2% fatty acids or sphingosine. The substances are serologically active; however, both required combination with phospholipids for development of maximum activity ine haemagglutination inhibition tests. Addition of phospholipids was not required in serological precipitation tests. These blood‐group substances may be used to coat group‐O or Bombay‐Oh red cells, thereby conferring on them A‐, B‐, or H‐activity. They form high‐molecular‐weight aggregates in aqueous solution but disaggregate into smaller units in the presence of sodium dodecylsulphate.
Studies on an Inhibitor of Embryonic Differentiation
Authors: Niebel Jörg , TIEDEMANN Hildegard , Tiedemann Heinz
Published: March 2005

An inducing factor isolated from 9‐ to 11‐days‐old chicken embryos causes differentiation of the amphibian gastrula ectoderm into mesodermal (trunk and tail) structures. This inducing activity is prevented by an inhibitory factor. The inhibitor is found after gel filtration on Sephadex G‐200 in the void volume. After precipitation with hexadecylpyridinium chloride the inhibitor redissolves at ionic strengths I= 0.4–1.0. In isoelectric focusing experiments the factor bands near pH 3. The inhibitor is tentatively characterized as an acidic glycoprotein.
Biosynthesis of N‐Acetylneuraminic Acid in Morris Hepatomas
Authors: Harms Erik , KREISEL Wolfgang , Reutter Werner , MORRIS Harold P.
Published: March 2005

The biosynthesis of N‐acetylneuraminic acid was studied in liver, neonatal liver, regenerating liver and Morris hepatomas 7777, 5123 TC, 5123 C, 5123 D, 7800, 7795, 9121 and 9618 A. The following three parameters were investigated: the biosynthesis of N‐acetylneuraminic acid from d‐[1‐14C]glucosamine and d‐N‐acetyl‐[U‐14C]mannosamine; the content of free and protein bound N‐acetylneuraminic acid and of CMP‐N‐acetylneuraminic acid; the activity of the key enzyme (UDP‐N‐acetylglucosamine‐2′‐epimerase).

The biosynthesis of N‐acetylneuraminic acid was investigated in vivo by labelling experiments using [1‐14C]glucosamine and N‐acetyl‐[U‐14C]mannosamine as precursors revealing the same metabolic route in liver and hepatoma. The biosynthesis of N‐acetylneuraminic acid deriving from [1‐14C]glucosamine in vivo is diminished to about 15% in hepatoma compared to liver, whereas the synthesis of UDP‐N‐acetylglucosamine is not altered. The uptake of N‐acetyl‐[U‐14C]mannosamine and its conversion to N‐acetylneuraminic acid in liver and hepatoma is demonstrated. The synthesis of free N‐acetylneuraminic acid deriving from N‐acetyl‐[U‐14C]‐mannosamine is not changed in hepatoma. The incorporation of this precursor into protein‐bound N‐acetylneuraminic acid of hepatoma is four‐times higher.

The content of free and protein bound N‐acetylneuraminic acid and CMP‐N‐acetylneuraminic acid have been measured in livers and hepatomas by isotope dilution technique. About 90% of total N acid content in both livers and hepatomas is protein‐bound. In Morris hepatomas the content of protein‐bound N‐acetylneuraminic acid is elevated (between 5.93 and 8.38 nmol/mg protein) compared to liver (4.50 ± 0.52 nmol/mg protein). However the content of free. N‐acetylneuraminic acid including CMP‐N‐acetylneuraminic acid is diminished in Morris hepatomas (between 108 and 164 nmol/g hepatoma in comparison to 196 ± 28 nmol/g liver), whereas the content of CMP‐N‐acetylneuraminic acid alone is not greatly changed (31.4 ± 8.5 nmol/g Morris hepatoma 9121 in comparison to 40.8 ± 10.1 nmol/g liver).

The activity of UDP‐N‐acetylglucosamine‐2′‐epimerase of all Morris hepatomas investigated was diminished to between 6% and 13% compared to that of host liver. In other fast growing liver tissue (neonatal and regenerating liver) this enzyme activity was not diminished but slightly increased showing that the decreased enzyme activity is a characteristic feature of Morris hepatomas.
Mechanism of the Exchanges Catalysed by the Oxoglutarate Translocator of Rat‐Heart Mitochondria
Authors: Sluse Francis E. , LIÉBECQ Claude , Goffart Georges
Published: March 2005

The kinetics of the exchange reactions between internal l‐malate and external 2–oxoglutarate or malonate have been measured at 4°C in the presence of external l‐malate (external‐product inhibitor), using preparations of rat‐heart mitochondria under conditions where the oxoglutarate translocator is operating exclusively. Measurements of initial rates were made at three concentrations of the internal substrate (l‐malate), three concentrations of the external substrate (2‐oxoglutarate or malonate) and three concentrations of the external reaction product (l‐malate).

The Michaelis constant for the internal malate is unaffected by the presence of the external product whereas the apparent‐maximum rate for each concentration of external substrate decreases when the concentration of external product increases. On the other hand the Michaelis constant for the external substrate, oxoglutarate or malonate, increases when the concentration of external malate increases.

The external product inhibits the exchange in a competitive way when external substrate is varied and in a non‐competitive way when internal substrate is varied.

The inhibition constant determined for the external malate is equal to the previously determined dissociation constant of the corresponding external‐malate · translocator complex.

These results are in perfect agreement with the previously proposed mechanism (rapid‐equilibrium random bi‐bi) and a mixed dead‐end and product inhibition.
Influence of Bile Salts on Cholesterol Synthesis in the Isolated Perfused Rat Liver
Authors: Decker Karl F. A. , BARTH Christian A. , Hackenschmidt H. Jürgen , ULLMANN Harald L. , Liersch Michael E. A.
Published: March 2005

The effect of taurocholate and cholate on hepatic cholesterol synthesis has been studied with the isolated perfused rat liver. By the infusion of bile salts at a constant rate to the portal vein, conditions in the enterohepatic circulation of taurocholate and cholatein vivowere simulated.

A 1‐h infusion of sodium taurocholate, at a rate of 10mg × (100g body wt)−1× h−1, did not inhibit cholesterol synthesis, as measured by3H2O incorporation.

The same results were obtained, when livers with enhanced cholesterol synthesis were studied or when bile salt infusions were extended to 2 h before starting incorporation measurements. Stimulation was achieved by feeding a 2% cholestyramine diet during 7 days to the donor animals.

A 3‐h infusion of taurocholate, at the above rate, exerted no toxic effect on liver metabolism, as judged from hepatic enzyme leakage to the perfusion medium, from the rate of ketone body production and the 3‐hydroxybutyrate/acetoacetate ratio.

Cholic acid in amounts equivalent to those of taurocholate did not influence cholesterol synthesis under these conditions.

It is concluded, that inhibition of cholesterol synthesis by bile saltsin vivois not mediated by a short‐acting (e.g.allosteric) mechanism at the enzymatic level, but rather by a change of enzyme content.
Replication of the Single‐Stranded DNA Bacteriophage M 13
Authors: Jaenisch Rudolf , JACOB Elard , Hofschneider Peter Hans
Published: March 2005

Evidence is presented that in M 13 infected Escherichia coli cells five different messenger RNA species can be detected which upon analysis by polyacrylamide‐gel electrophoresis have molecular weights of 1.3 × 106, 8 × 105, 5 × 105, 3 × 105, and 1 × 105, respectively. One of them corresponds to an M‐13‐specific messenger RNA species reported earlier to correspond to one round of transcription on the DNA template. The five RNA species are found early and late after infection though a shift in their relative amounts can be observed. Between them no clearcut precursor relationship exists. Upon examination in dimethylsulfoxide gradients a different size distribution of M 13 mRNA is observed which has an upper limit at a molecular weight of 4–5 × 105 and a maximum at 9 × 104. From the results in dimethylsulfoxide gradients which are confirmed by an independent method it may be concluded that by gel electrophoresis aggregates of M 13 mRNA have been examined. The size distribution observed in dimethylsulfoxide gradients leads to the conclusion that M 13 mRNA is synthesized in the form of oligo or monocistronic RNA molecules. No polycistronic M‐13‐specific mRNA corresponding to one round of transcription on the DNA template can be detected. As revealed by a pulse‐chase experiment the half‐life time of M 13 mRNA is unusually long, being of the order to 20 min. This result together with the finding on the M 13 mRNA sizes is discussed under the aspect of the physiology of M 13 infection.
A Mutant of Escherichia coli Defective in Ribonucleosidediphosphate Reductase
Authors: Fuchs James A. , NEUHARD Jan
Published: March 2005

A procedure to isolate Escherichia coli mutants defective in ribonucleotide reduction is described. Using a parental strain defective in dCTP deaminase (paxA) and thus unable to synthesize dUMP from deoxycytidine nucleotides, we selected for cells unable to reduce UDP at a rate sufficient to satisfy the cell's need for thymidine nucleotides. This was accomplished by selecting for cells resistant to 2,4‐diammo‐5‐(3,4,5‐trimethoxybenzyl)pyrimidine (trimethoprim) in the presence of thymidine and screening these cells for a thymidine requirement that could be satisfied by deoxyuridine. Genetic analysis as well as enzyme assays of four such mutants chosen for study indicated that three had a defective thymidylate synthetase. One, LD195, which had normal thymidylate synthetase activity, was found to have a conditional deoxyuridine requirement that is only expressed under favourable growth conditions. Since restoration of a functional dCTP deaminase to derivatives of LD195 eliminated the deoxyuridine requirement, it was concluded that a combination of two mutations is responsible for the expression of this phenotype. Analysis of nucleotide pools of LD195 grown exponentially under permissive conditions revealed that both the dTMP and dTTP pools were 6‐fold lower than in the parent indicating a deficiency in the production of a thymidine nucleotide precursor. Enzyme assays of crude extracts of LD195 indicated that this deficiency was due to a defective ribonucleosidediphosphate reductase. This conclusion is proven by an extensive enzymatic analysis presented in an accompanying paper.
Properties of the Elongation Factors from Escherichia coli
Authors: Chinali Gianni , PARMEGGIANI Andrea
Published: March 2005

The interaction of elongation factor G (EF‐G) with ribosomes has been studied during or in the absence of elongation of the polypeptide chain in a system from Escherichia coli in vitro.

Mutual saturation curves of ribosomes and EF‐G suggest a rapid exchange of the factor from ribosome to ribosome. In the GTPase reaction, V/2 was reached at [ribosomes]/[EF‐G] ∼ 14 and at [EF‐G]/[ribosomes] ∼ 33. In the poly(U)‐directed poly(phenylalanine) synthesis, V/2 was obtained at [ribosomes]/[EF‐G] ∼ 12 and at [EF‐G]/[ribosomes] ∼ 6.

Kinetics of the exchange of EF‐G between two pure acetyl‐aminoacyl‐tRNA ·mRNA · ribosome complexes containing synthetic homo‐ or heteropolymeric mRNAs were investigated by following their respective EF‐G‐dependent incorporation of differently labelled amino acids into the corresponding polypeptides in experimental conditions in which EF‐G was either added free or already prebound to one of the complexes prior to addition of the second one.

In all experiments the rates of incorporation showed that the exchange of EF‐G between the two complexes was immediate and complete. These observations indicate that EF‐G behaves as a virtually free component which interacts catalytically with ribosomes during each round of elongation of the polypeptide chain.