bluuebunny/crossref_metadata_2025 huggingface dataset:
Visualisation in the Electron Microscope and Size of RNA from Animal Cells
Authors: Granboulan N. , Scherrer K.
Published: March 2005

Ribosomal, preribosomal (pre‐rRNA) and messenger like (mlRNA) RNA molecules from animal cells have been spread in extended form for visualisation in the electron microscope, in order to measure their length. It is shown that the giant nascent pre‐rRNA and mlRNA consists of linear molecules of homogeneous shape. Their length ranges up to 3.5 μ in the case of pre‐rRNA and up to 8.5 μ for mlRNA. The correlation of apparent lengths and molecular weight of RNA is discussed. A spacing of 2.45 Å, based on the length and the chemically determined molecular weight of E. coli rRNA, allows one to assign molecular weights to rRNA and pre‐rRNA that are in fairly good agreement with published data based on sedimentation analysis. However, the 28 S rRNA has twice the length of the 18 S species, which is in disagreement with the mass relation of 2.5–3.0 observed by sedimentation. In the case of mlRNA the spacing of 3.17 Å, derived from the length to molecular weight relation of R 17 phage RNA, is more likely to give correct values than that based on rRNA. This conclusion derives from a discussion of the relation of length and molecular weights in various viral RNA molecules in a range of up to 107 daltons.

Two general conclusions may be drawn from the work presented:

The high sedimentation rate of pre‐rRNA and mlRNA is not based on artificial agglomeration or particular configuration but on the real mass of molecules which contain, in the case of mlRNA, up to 30,000 bases (molecules of up to 50,000 bases are likely to exist also) in covalent linkage.

The electron microscope may be used to determine molecular weights. The precision of the values obtained may be equal to or better than those determined by sedimentation if correct spacing data are available from reference molecules of the same RNA class.
The Mechanism of Formation of Polyunsaturated Fatty Acids by Photosynthetic Tissue
Authors: Gurr M. I. , Robinson M. P. , James A. T.
Published: March 2005

Labelled oleic acid, which is used as a substrate in studying the conversion of oleate into linoleate by Chlorella chloroplasts, is rapidly incorporated into the phospholipids. The phosphatidyl choline fraction accounts for almost all of the phospholipid label. Desaturation to linoleate lags behind the incorporation into lipids, but the newly synthesized linoleate remains esterified to phosphatidyl choline.

[1‐14C]Oleoyl‐phosphatidyl choline, incubated with chloroplasts, is converted into [1‐14C]‐linoleoyl‐phosphatidyl choline, but considerable difficulties arise in presenting the lipid to the enzyme in an acceptable form. Oleoyl‐phosphatidyl choline formed in situ, however, does not present these difficulties and is desaturated at a faster rate than the CoA derivative under conditions in which unesterified fatty acid cannot be “activated”, and hence cannot be desaturated. We conclude that the precursor fatty acid can be transferred directly from the lipid to the desaturase enzyme and the product immediately accepted by a lipid to form a “desaturation‐acylation” cycle. Our data neither prove, nor exclude the possibility that the fatty acid is transformed while still attached to the lipid in ester linkage.
Interaction of the RNA Polymerase Inhibitor Chromomycin with DNA*
Authors: Behr W. , Honikel K. , Hartmann G.
Published: March 2005

Formation of a complex between chromomycin and DNA has been shown to depend on the guanine content and on base pairing of the polydeoxynucleotides. A limit of association is reached when one chromomycin molecule is bound per four nucleotide base pairs. The rate of complex formation is independent of the size of the sugar side chains of the antibiotic and of the nature and base composition of DNA. In contrast the rate of complex dissociation increases with a decrease in size of the sugar side chains. The inhibition by chromomycin and its derivatives of DNA and RNA polymerase reactions exhibits similar features. Probably the dissociation rate of the complex determines the inhibitory activity. Chromomycin by complex formation protects guanine rich segments of DNA very efficiently against the attack of nucleases. After enzymatic hydrolysis chromomycin containing base paired oligonucleotide fragments remain in the reaction mixture and can be isolated by electrophoresis. The antibiotic is not covalently bound to these fragments and can be removed by thorough extraction with ether.
Lichtinduzierte, reversible Aktivitätssteigerung der NADP‐abhängigen Glycerinaldehyd‐3‐phosphat‐Dehydrogenase in Chloroplasten
Authors: Müller B. , Ziegler I. , Ziegler H.
Published: March 2005

Irradiation of fully developed green leaves as well as irradiation of isolated chloroplasts causes a rapid, reversible increase in the activity of NADP‐linked phosphoglyceraldehyde dehydrogenase. This increase is closely connected with the functioning of the non‐cyclic electron flow of photosynthesis. It is accompanied by a decrease in the activity of the NAD‐linked enzyme.

The experiments presented here show that it is not the kinase but the dehydrogenase which is the rate limiting factor in the over‐all reaction measured up to date.

The activation of the “dark‐enzyme” (= basic enzyme) resulting in the “light‐enzyme” (= activated enzyme) is not due to an unspecific reduction of S‐S‐groups in the reactive center. Dithiothreitol only restores the activity lost during the isolation procedure of the chloroplasts.

NADPH rather than dithiothreitol causes a specific activation of the basic enzyme. However the basic enzyme must be present in the completely reduced form to undergo maximal activation. Incubation with dithiothreitol and NADPH thus results in an activation rate as high as that caused by light in vivo.

Activation of the NADP‐linked dehydrogenase could also be attained by NADPH after its partial purification by ammonium sulfate fractionation.

The activation rate by incubation with different concentrations of NADPH shows a sigmoidal type of curve.

There are characteristic differences in sensitivity between the activity of the basic enzyme and its ability to be activated at different temperatures between 52°–60°.

The results indicate that the action of NADPH may be an allosteric one, causing a transition from a NAD‐ (or possibly a NAD + NADP)‐dependent to a NADP‐dependent form of the enzyme. A diagram for a feed‐forward activation of the NADP‐linked dehydrogenase which possibly takes part in regulation of CO2‐fixation is presented.
Circular DNA from Shigella paradysenteriae
Authors: Jansz H. S. , Zandberg J. , van de Pol J. , van Bruggen E. F. J.
Published: March 2005

The strain of Shigella paradysenteriae Y6R contains super twisted circular DNA which comprises a minor fraction of the total DNA. The purified circular DNA was shown by sedimentation analysis to consist of two major components of 21 S and 14 S respectively which gave rise to 17 S and 11 S components after digestion with minute amounts of deoxyribonuclease. It was shown by electron microscopy that this DNA consists of super twisted circular form before and open circular forms after treatment with deoxyribonuclease. Two major size classes of open circular forms with contour lengths of 1.72 μ and 0.52 μ. were found in addition to a minor fraction of circular molecules of 1.13, 2.20 and 4.37 μ. The mean molecular weight as calculated from the contour lengths and sedimentation coefficients is 3.4 × 106 and 1.0 × 106 for the 1.72 μ (21 S) and 0.52 μ (14 S) DNA respectively.
Spectral Studies with Histidyl‐Tryptophan Peptides
Authors: Shinitzky M. , Fridkin M.
Published: March 2005

A series of linear and cyclic derivatives of histidyl‐tryptophan was synthesized for the study of intramolecular indole‐imidazolium interaction. The absorption spectrum and the fluorescence intensity of each of the peptides were examined in aqueous solution when the imidazole side chain was protonated (pH < 4) and uncharged (pH > 9). The absorption spectra of the protonated peptides show hypochromism around 280 mμ, whereas at shorter wavelengths hyperchromism appears. The hyperchromism is most intense around 240 mμ and it is attributed to the charge‐transfer band of the intramolecular indole‐imidazolium complex. The fluorescence intensity of all tested histidyl‐tryptophan peptides drops markedly with protonation of the imidazole side chain. The change in fluorescence intensity with pH superimposes to the potentio‐metric titration curve of the imidazole residue. From the preceeding results it may be concluded that (a) the electron affinity of imidazolium is smaller than that of the positively charged nicotin‐amide by 1.2 eV and (b) the indole‐imidazolium complex is not of a sandwich type. It is suggested that the charge‐transfer bond of this complex is located between the C‐2–C‐3 bond of the indole and the plus charge center of the imidazolium.
The Role of Endogenous Lipid in Gluconeogenesis and Ketogenesis of Perfused Rat Liver
Authors: Menahan L. A. , Wieland O.
Published: March 2005

The oral antidiabetic glycodiazine (2‐benzene sulfonamido‐5‐β‐methoxyethoxy pyrimidine), an inhibitor of hepatic lipolysis, was used to investigate the metabolic role of liver lipid in gluco‐genesis in the perfused livers of fasted (24–30 h) rats. In a concentration of 10 mM, glycodiazine inhibited almost completely endogenous ketone body formation.

Perfusion with pyruvate as substrate, in the presence of glycodiazine, resulted in a higher rate of pyruvate oxidation and suppressed the rate of glucose synthesis in comparison to livers perfused with pyruvate alone. Thus, maximal rates of gluconeogenesis from pyruvate would seem to require an adequate supply and oxidation of fatty acids.

Oleate, even in the presence of glycodiazine, stimulates glucose synthesis from pyruvate. Uptake of and ketone body formation from oleate were also not affected by glycodiazine. These results allow the conclusion that glycodiazine specifically inhibits liver lipolysis.

Glucagon (57 nM), although having no significant effect on gluconeogenesis from pyruvate at this concentration, overcame the inhibitory effects of glycodiazine on glucose formation and suppressed the elevated pyruvate oxidation which results from the inhibition of endogenous lipid mobilization.

Glucagon, in the absence of added substrates, stimulated gluconeogenesis, urea production and ketogenesis. The increase in gluconeogenesis due to glucagon was not impaired by glycodiazine. This indicates that intermediates arising from fatty acid oxidation do not limit the rate of gluconeogenesis under these conditions.
Study on Metabolism of Phospholipids in Animal Tissues
Authors: Káš J. , Hladík J. , Šícho V.
Published: March 2005

Metabolism of phospholipids in post mortem tissues of liver, kidney, spleen, and heart of guinea pigs was studied. The per cent composition of the individual groups of phospholipids in the above tissues in the fresh state and after a certain period of the dying process taking place under the described conditions is shown. Generally, one can state that phosphatidyl choline is the most important component of phospholipids in these tissues, in liver and in heart representing about 70% of the total amount. They are metabolized relatively faster, while sphingomyelin and phosphatidyl serine are metabolized most slowly. On the basis of the lipid phosphorus determination it is possible to conclude that within 24 h about 20–30% of the phospholipids are decomposed. During the following phase of the dying process no further pronounced decrease of phospholipids takes place. After this period the enzyme system splitting phospholipids is practically inactive. During the dying process an apparent increase of inorganic phosphorus can be observed, which indicates the decomposition of other phosphorus compounds in the course of this process.

The relative representation of the individual groups of phospholipids in liver mitochondria is different from that of the whole tissue. Liver mitochondria contain a relatively higher amount of phosphatidyl ethanolamine, a lesser amount of phosphatidyl choline, lysophosphatidyl choline being totally absent.
Über multiple Formen der Hexokinase in Hefe
Authors: KopperschlÄGER G. , Hofmann E.
Published: March 2005

Multiple forms of hexokinase have been detected by agar gel and polyacrylamide gel electro‐phoresis in crude extracts obtained from yeast cells of different strains. Electrophoretic mobility and number of enzyme forms depend on the conditions of cell disruption and vary with the respective yeast strain. Extracts from commercial baker's yeast, prepared by short‐time mechanical disruption contain two or three isozymes of hexokinase. Cell‐lysis by proteolytic procedures (toluene, extraction of dried yeast) changed the electrophoretic pattern profoundly.

Between the exponential and the stationary phases of growth characteristic differences in the pattern of the two isozymes of hexokinase have been found.

Five different strains of yeast revealed different patterns of hexokinase isozymes.

Electrophoresis on polyacrylamide gels, using a concentration gradient of the polymer, indicated that the differences in the mobilities of these enzymes are presumably not caused by major differences in their molecular sizes. Trypsin caused an increase of electrophoretic mobility of one or both forms of the enzyme from baker's yeast.
Isolation of Three Acid Phosphatases from Wheat Germ
Authors: Verjee Z. H. M.
Published: March 2005

Three different forms of acid phosphatase have been isolated from commercial wheat germ by ion‐exchange chromatography on DEAE‐cellulose.

The three components could not be separated by gel‐filtration, suggesting that the enzyme was homogeneous with respect to molecular weight. A molecular weight of 55000 was obtained for all the three isoenzymes, as determined by gel‐filtration on Sephadex G‐100 and Sephadex G‐200.

The isoenzymic nature of the three forms was suggested by differences in their pH optima, Michaelis constants, response to activation and inhibition, substrate specificities and electrophoretic mobilities.

The isolation procedure was readily adapted to the preparation of large quantities of the isoenzymes. These partially‐purified forms were electrophoretically indistinguishable from the three components which could be demonstrated in the original wheat‐germ extract.
Biosynthesis of 6β‐ and 6α‐Hydroxyprogesterone in the Human Foetal Liver
Authors: Lisboa B. P. , Gustafsson J.‐Å.
Published: March 2005

After incubation of progesterone with 105000 Xg microsomes of human foetal liver two C21O3 steroids were isolated by ascending one‐dimensional thin‐layer chromatography. Gas chromato‐graphy‐mass spectrometry analysis showed that the compounds were identical with 6β‐ and 6α‐hydroxyprogesterone. The significance of this finding in relation to earlier reports of 6‐hy‐droxylated C21 steroids in human pregnancy urine is discussed.
Effects of Phosphate and other Ionic Compounds on the Activity of Crystalline Beef Liver Glutamate Dehydrogenase
Authors: di Prisco G. , Strecker H. J.
Published: March 2005

At pH 9.0 the activity of crystalline glutamate dehydrogenase was increased by the addition to the incubation mixture of various electrolytes including sodium chloride, potassium phosphate, Tris, 2‐amino‐2‐methyl‐1, 3‐propanediol and methionine. Potassium phosphate was most stimulatory and, at an optimum concentration, increased activity three to four‐fold. No stimulation was obtained at pH 7.6. Potassium phosphate and the other electrolytes abolished activation by high concentrations of NAD or NADP and altered the inhibitory effects obtained by compounds which presumably do not interact at the NAD binding site. These electrolytes also overcame the inhibition by GTP, a ligand which binds at the same site as NAD, but they did not suppress the reactivity with ADP. It is proposed that at pH 9.0 potassium phosphate and other electrolytes alter glutamate dehydrogenase so that the regulatory site which binds NAD or GTP is no longer available to these ligands.
La biosynthése des stérols dans les tissus de tabac cultivés in vitro
Authors: Benveniste P. , Hewlins M. J. E. , Fritig B.
Published: March 2005

Tobacco tissues grown in vitro were fed with labelled acetic acid during 5, 30 and 150 min, respectively. 4, 4‐Dimethylsterols, 4‐methylsterols and sterols were extracted and purified and their specific activities were determined by acetylation of biosynthetically labelled alcohols with [3H]acetic anhydride of known specific activity.

On the basis of the measured specific activities, a tentative scheme for plant sterol biosynthesis is proposed. In particular, cycloartenol appears to play a direct role in this biosynthesis. The conversion of cycloartenol into sterols appears to be a non‐specific process with several possible parallel routes. The linear scheme previously proposed, involving the following transformations : cycloartenol → methylene‐24 cycloartanol → cycloeucalenol → obtusifoliol →“lophenols”, etc., appears to be only one of the pathways followed in our material.
Steroids in Germfree and Conventional Rats
Authors: Eriksson H. , Gustafsson J.‐Å. , Sjövall J.
Published: March 2005

The metabolism of 3β,21‐dihydroxy‐5α‐pregnan‐20‐one in incubations with caecal microorganisms has been studied. Pregnane‐3,20‐diols and pregnane‐3,20,21‐triols were the major products. Relatively more pregnanediols were formed under aerobic than under anaerobic conditions. There was no formation of pregnanediols from 5α‐pregnane‐3β,20β(and 20α),21‐triol and no epimerization of the 20‐hydroxy group or loss of 20α(or 20β)‐2H in deuterium labelled pregnanetriols. The 3β‐hydroxy group underwent partial epimerization during the incubation. The results show the existence of a microbial 21‐dehydroxylation of 21‐hydroxy‐20‐oxo steroids and offer an explanation for the absence of 21‐hydroxylated steroids in faeces from conventional rats.
The Use of Procion Blue as a Molecular Probe in the Study of Ribosomal Structure
Authors: Hultin T.
Published: March 2005

The triazinyl dye, procion brilliant blue MR, has a selective initial action on the proteins of rat liver ribosomes in situ, and can be used for detecting shielding effects in normal and modified particles. Alterations in the shielding pattern can be analyzed by disc‐electrophoresis.

Irreversible dissociation of the ribosomes in Mg2−‐deficient media makes a normally resistant protein (“protein 5”) available to the reagent. This unmasking occurs even in the cold.

When the particles are incubated at 35° in the presence of aminoacridines, or in media with increased ionic strength, another previously shielded protein (“protein 10”) is unmasked by a time and temperature dependent reaction.

The unmasking of protein 10 by incubation in media with increased ionic strength is partially reversible by a time and tempreature dependent reaction. Although some detachment occurs in connection with the unmasking, the reversibility is probably confined to non‐detached molecules. The activity of the particles of endogenous or poly U‐dependent amino acid incorporation is not seriously impaired under these conditions.

The experiments indicate that organic reagents can be used to provide information about protein location and structural alterations in ribosomes, which confirm and extend structural data obtained by use of proteolytic enzymes.
Structure de l'haptoglobine de lapin
Authors: Cheftel R. I. , Parnaudeau M. A. , Bourrillon R. , Moretti J.
Published: March 2005

Phylogenic research on the haptoglobin of different animal species led us to study the structure of rabbit haptoglobin.

Rabbit haptoglobin, whose homogeneity was checked according to several criteria, was submitted to hydrolysis by pronase (72 h, 39°, pH 8.2); the hydrolysate was then filtered through Sephadex G‐25, and the resulting glycopeptide fraction was separated on Dowex 50‐X2 (H+) into two parts, one (A) not retained by the resin, the other one (B) collected after elution with 0.5 M NaCl.

Amino‐acid and carbohydrate composition, nature of end groups, and timing of sialic acid release, were determined on fraction A; from the amount of aspartic acid, the molecular weight was estimated to be no less than 2860.

Fraction B, which amounted to one third of fraction A, could not be analysed on account of the insufficient quantity available.

Quantitative paper electrophoresis at pH 3.5 and 6.2 yielded 8 glycopeptides from fraction A and 2 from fraction B. These various glycopeptides showed notable differences in their amino‐acid composition, mainly with respect to glutamic acid, proline, glycine and histidine. On the contrary, no significant differences were found in the carbohydrate composition, with the exception of the oses N‐acetylneuraminic acid ratio, which varies from 2 to 10.

These results suggest 6 oligosaccharide units to be present in rabbit haptoglobin, as compared to 7 in human haptoglobin, bound to peptidic fractions varying in their amino‐acid composition in the neighbourhood of the carbohydrate‐peptide linkage. Although the precise nature of this bond has not been ascertained, it does not appear to be an O‐glycosidic one.
Desensitization of Yeast Phosphofructokinase to ATP‐Inhibition by a Protein Fraction from Yeast
Authors: Atzpodien W. , Gancedo J. M. , Hagmaier V. , Holzer H.
Published: March 2005

A heat‐labile fraction from yeast is described which converts the ATP‐sensitive from of phosphofructokinase (Form s), inhibited by high concentrations of ATP, into a desensitized form (Form d), not inhibited by excess ATP. This desensitization reaction is time‐dependent.

Forms s and d are different, relatively stable forms of phosphofructokinase. Retaining their characteristic sensitivity or insensitivity to ATP, they can be fractionated by ammonium sulfate and acetone.

The heat‐labile fraction, desensitizing Form s to Form d, is neither dialysable nor ultrafiltrable. It is inactivated by trypsin treatment of the crude extract. It can be fractionated by ammonium sulfate and acetone. All these facts strongly suggest that the active principle is a protein.

The protein fraction from crude yeast extract desensitizing Form s to Form d, was about 50‐fold purified by treatment with protamine sulfate and fractionation with ammonium sulfate and acetone.

Besides the desensitizing protein, the conversion reaction is dependent on fluoride, AMP, ATP‐Mg++, and fructose‐6‐phosphate. Ado‐3′:5′‐P has no effect.
The Participation of Ornithine and Citrulline in the Regulation of Arginine Metabolism in Saccharomyces cerevisiae
Authors: Ramos F. , Thuriaux P. , Wiame J. M. , Bechet J.
Published: March 2005

The pools of arginine, ornithine and citrulline have been measured under different conditions of growth in wild type and argR mutants (non‐repressible for ornithine transcarbamylase and four other enzymes of the arginine biosynthetic pathway). Comparing these pools with the level of ornithine transcarbamylase and the rate of growth one may conclude:

The arginine pool of a wild type strain growing on ammonium as nitrogen source is 18 mM; it increases to 72 mM by addition of arginine to the medium and causes 95% repression of ornithine transcarbamylase synthesis. In decreasing the pool to 3.7 mM by a bradytrophic mutation the differential rate of ornithine transcarbamylase synthesis increases 6 times.

When the pool of ornithine is increased by external addition of this amino acid or by using ornithine as the only nitrogen source a repression occurs which reaches 50 to 70% simulataneously with an intense reduction of the arginine pool (≤ 1.2 mM). This low pool may reach a growth limiting value. This shows the existence of a double control by arginine and ornithine. Lysine mimics ornithine.

The primary effect of argR mutation is not at the level of the arginine pool.

A double regulatory mutant, argR and cpaO (which confers non‐respressibility to carbamoyl‐phosphate synthetase) shows that the double control of arginine and ornithine also reaches this enzyme.

The strain bearing simultaneously the argR and cpaO mutations shows an intense distrurbance of metabolism leading to cessation of macromolecules biosyntheses and a loss of viability when basic amino acids such as lysine, ornithine, α‐γ‐diamino butyric acid or arginine are added to a culture growing on glutamate as nitrogen source.

Citrulline is shown to be used as nitrogen source via arginine, under these conditions the arginine pool increases to 190 mM, but does not produce the expected high repression. Citrulline or a derivative attenine repression.
Stereochemistry of Hydrogen Transfer from Pyridine Nucleotides Catalyzed by Δ4‐3‐Oxosteroid 5β‐Reductase and 3α‐Hydroxysteroid Dehydrogenase from Rat Liver
Authors: Björkhem I. , Danielsson H.
Published: March 2005

The stereochemistry of hydrogen transfer from NADPH in the conversion of Δ4‐3‐oxosteroids into 3‐oxo‐5β‐steroids and from NADH and NADPH in the conversion of 3‐oxo‐5β‐and 3‐oxo‐5α‐steroids into 3α‐hydroxysteroids, catalyzed by rat liver Δ4‐3‐oxosteroid 5β‐reductase and 3α‐hydroxysteroid dehydrogenase, respectively, was studied with C19‐, C21‐, C24‐, and C27‐steroids. In all cases, the hydrogen transferred from the pyridine nucleotides was the 4A‐hydrogen.
Separation of Large Quantities of Ribosomal Subunits by Zonal Ultracentrifugation
Authors: Eikenberry E. F. , Bickle T. A. , Traut R. R. , Price C. A.
Published: March 2005

The maximum capacity of the B‐XV zonal rotor for the separation of Escherichia coli ribosomal subunits has been investigated. These experiments were performed using a class of sucrose density gradients (hyperbolic gradients) which present certain theoretical advantages. 2 g of subunits could be utilised in single centrifugations with a final yield of 0.5 g of 30 S and 1.0 g of 50 S subunits of greater than 99° purity.
Molecular Weight and Quaternary Structure of Yeast l‐Lactate Dehydrogenase (Cytochrome b2)
Authors: Jacq C. , Lederer F.
Published: March 2005

Amino acid analyses of l‐lactate dehydrogenase from baker's yeast show that the minimum molecular weight (53000 daltons) of the protein is much lower than found in the literature (80000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2.
The Nucleotide Sequence of Methionine Transfer RNAM
Authors: Cory Suzanne , Marcker K. A.
Published: March 2005

The species of methionine tRNA which places methionine into internal positions of growing polypeptide chains, methionine tRNAM, was purified from Escherichia coli strain CA265 labelled with 32P by column chromatography on DEAE‐Sephadex and benzoylated DEAE‐cellulose. Sequence analysis of the products of complete and partial digestion of tRNAM by ribonuclease T1 and by pancreatic ribonuclease permitted the derivation of the total primary structure of this molecule.

The sequence of methionine tRNAM is where U* is probably 4‐thio‐uridine, and , A* and X are unknown nucleotides.
Chain Length Effects of n‐Alkyl 1‐Oxygen‐and n‐Alkyl 1‐Thio‐β‐d‐Xylopyranosides on β‐d‐Xylosidases
Authors: Kersters‐Hilderson H. , Claeyssens M. , Loontiens F. G. , Krynski A. , De Bruyne C. K.
Published: March 2005

The effect of an homologous series of n‐alkyl β‐d‐xylopyranosides (from methyl to octyl) and n‐alkyl 1‐thio‐β‐d‐xylopyranosides (from methyl to hexyl) on the activity of β‐d‐xylosidases from different origins (Aspergillus niger, Aspergillus oryzae and Bacillus pumilus 12) was investigated. Hydrolysis of n‐alkyl β‐d‐xylopyranosides (oxygen derivatives) was only measurable with the A. nigerβ‐d‐xylosidase, where maximal hydrolysis was observed with the n‐butyl derivative. Furthermore the behaviour of the series of oxygen and sulfur derivatives was purely competitive with respect to the hydrolysis of p‐nitrophenyl β‐d‐xylopyranoside by all of the three enzymes. The inhibition constants decreased with increasing chain length for both series and both fungal enzymes. For B. pumilus 12 such a relationship was only found for the thiocompounds, whereas for the oxygen series a pronounced maximal inhibition was observed for the n‐butyl and n‐pentyl derivatives. The contribution of hydrophobic regions at or near the active site is discussed and speculative interpretations are presented.
Cooperative Binding to Linear Biopolymers
Authors: Schwarz Gerhard
Published: March 2005

A linear lattice of equivalent binding sites with nearest neighbor cooperativity is used as a basic model for a linear biopolymer displaying cooperative binding of small ligands. Complicating effects due to the dimerization of free ligands are taken into account. The theory implies two types of intrinsic binding processes: (1) the binding of an isolated ligand (nucleation), and (2) the binding of a ligand in the immediate neighborhood of an already bound one (aggregation). The ratio of the respective association constants determines the degree of cooperativity. It is generally shown how the equilibrium properties of the system can be derived by means of the matrix method. Some properties of special practical significance, such as the equilibrium concentrations of free and bound monomer ligands, are discussed in more detail. On the basis of a pertinent kinetic model chemical relaxation of strong cooperative binding is also examined. The paper gives particular emphasis to the formulation of quantitative relations which are most useful for an experimental application of the oretical results. Appropriate procedures to plot measurable data and to evaluate basic parameters are proposed. They are applied to specific experimental systems in a subsequent set of papers.
Glutamate Dehydrogenase of Nuclear and Extra‐Nuclear Compartments of Chang's Liver Cells
Authors: Di Prisco Guido , Strecker Harold J.
Published: March 2005

Glutamate dehydrogenase activity is present in both the nuclear and mitochondrial fractions of Chang's liver cells. After solubilization, the two activities were shown to differ from each other in response to variation of pH, in the stimulating effect of anions, especially inorganic phosphate, in response to variation of the concentration of NAD+, and in the ratio of velocities obtained with saturating concentrations of NADH and NADPH. During the 4‐day growth cycle of Chang's liver cells, glutamate dehydrogenase activity in nuclei attained a maximal activity of 4‐ to 7‐fold the starting value, within 12–16 hours after incubation in fresh growth medium. Glutamate dehydrogenase in the extra‐nuclear fraction required 24 hours to reach its maximal activity level of about 150% of the initial value. Incubation of cells in a medium lacking an essential amino acid resulted in a significant decrease of glutamate dehydrogenase activity of the extra‐nuclear compartment, but no change in the nuclear activity. It is concluded that two glutamate dehydrogenases are present in Chang's liver cells and that they may have different functions.
Regulation of Pyruvate Carboxylase in Rat Liver Mitochondria by Adenine Nucleotides and Short Chain Fatty Acids
Authors: Walter P. , Stucki J. W.
Published: March 2005

The activity of pyruvate carboxylase of intact rat liver mitochondria was measured by improved methods under various experimental conditions. In agreement with previous work, elevated ADP concentrations in the medium caused a strong inhibition of the enzyme activity. A partial reversal of this inhibition was observed upon addition of octanoate or decanoate, but not with octanoyl‐l‐carnitine. Furthermore, atractylate completely reversed this inhibition whereas dinitrophenol or oligomycin increased it. In these experiments, no clear correlation between pyruvate carboxylase activity and the levels of adenine nucleotides of the whole medium was apparent. However, the measurement of the intramitochondrial adenine nucleotide levels revealed a close inverse relationship between ADP levels and pyruvate carboxylase activity, as fatty acids and atractylate lowered whereas oliogomycin elevated the intramitochondrial ADP levels. Other evidence indicates that the intramitochondrial ATP to ADP ratio may also be of importance in regulating the pyruvate carboxylase activity. In the experiments with octanoate, the intramitochondrial AMP was strongly elevated and the ADP plus ATP pool was smaller. In agreement with this finding, the amounts of rapidly exchanging adenine nucleotides were found to be smaller in the presence of octanoate than in its absence, as measured by the incorporation of added [14C]ATP. The addition of octanoyl‐l‐carnitine caused only small changes in the intramitochondrial adenine nucleotide pattern and had also no significant effect on the incorporation of added [14C]ATP into the mitochondria.

The solubilized mitochondrial pyruvate carboxylase could also be inhibited by ADP but this inhibition could not be reversed by octanoate nor with high amounts of acetyl‐CoA.

Oxygen uptake measurements with intact mitochondria showed a strong increase in the rate of respiration upon addition of octanoate or octanoyl‐l‐carnitine to the ADP supplemented system. This increase could be inhibited by oligomycin indicating that the stimulated respiration is coupled to an increased phosphorylation of ADP.

It is concluded that the pyruvate carboxylase activity is regulated mainly by the level of intramitochondrial ADP. The lowering of the intramitochondrial ADP level and the resulting stimulation of pyruvate carboxylase by fatty acids is related on one hand to the production of intramitochondrial AMP by fatty acid activation resulting in a lowering of the ADP plus ATP pool, and on the other hand to the increased utilization of ADP for oxidative phosphorylation.

The possible physiological implications of these findings are discussed.
The Stability of a Codon Transfer RNA Complex
Authors: Högenauer Gregor
Published: March 2005

Equilibrium dialysis experiments indicate complex formation between the trinucleotide ApUpGp and tRNAfMet, the dissociation constant of the complex being approximately 150 μM. Unfractionated tRNA containing only minute quantities of tRNAMet fails to form complexes with ApUpGp to a detectable extent thus suggesting that the binding ability for this triplet is displayed solely by methionine accepting tRNAs.

Deacylation of tRNA appears not to interfere with its complex forming ability. The strength of a trinucleotide—tRNA interaction is not altered measurably if the terminal phosphate group is detached from the triplets.

The observed binding phenomenon is interpreted as a codon—anticodon interaction in the absence of ribosomes.
Amino‐acylation du tRNA1Val de Escherichia coli par la phéenylalanyl‐tRNA synthétase de levure
Authors: Taglang R. , Waller J. P. , Befort N. , Fasiolo F.
Published: March 2005

Comparison of the known primary structures of several tRNAs reveals striking similarities between the nucleotide sequences of yeast tRNAphe and Escherichia coli tRNA1Val. On the assumption that such structural analogy might lead to aminoacylation of both tRNAs by each of the corresponding aminoacyl‐tRNA synthetases, a detailed study of this system was undertaken.

The results indicate that under optimal conditions, E. coli tRNA1Val can be completely aminoacylated by yeast phenylalanyl‐tRNA synthetase, while the reciprocal (i.e. valyl‐tRNAPhe (yeast) formation by E. coli valyl‐tRNA synthetase) could not be demonstrated under any of a variety of conditions tested.

Conditions for Phe‐tRNA1Val (E. coli) formation by yeast phenylalanyl‐tRNA synthetase differ radically from those leading to aminoacylation of tRNA in homologous systems. In particular, the final yield of aminoacyl‐tRNA varies with enzyme concentration and is markedly affected by the nature of the buffer and the pH, as well as by the presence of monovalent cations and organic solvents such as ethanol and dimethyl sulfoxide. Similar observations have already been reported in the study of the aminoacylation of tRNA in several heterologous systems.

The results are discussed in relation to the structure of the two tRNAs.
A Steady‐State Kinetic Investigation of the Reaction Mechanism of the Tryptophan Synthetase of Escherichia coli
Authors: Creighton Thomas E.
Published: March 2005

The initial rate of glyceraldehyde‐P formation catalyzed by the tryptophan synthetase of Escherichia coli from indole‐3‐glycerol‐P has been measured as a function of the concentrations of indole‐glycerol‐P, serine, tryptophan, and indole. The conversion of indole‐glycerol‐P to indole and glyceraldehyde‐P is proposed to proceed by the ordered sequential release from the enzyme of indole followed by glyceraldehyde‐P. Tryptophan does not appreciably inhibit this reaction.

Tryptophan formation is proposed to occur by the random sequential addition of serine and indole‐glycerol‐P to the enzyme. Tryptophan is a product inhibitor of the reaction, apparently combining with the free enzyme and also forming an enzyme‐(indole‐glycerol‐P)‐tryptophan deadend complex.

Indole inhibits non‐competitively with respect to both indole‐glycerol‐P and serine, and is proposed to combine with the free enzyme and with the enzyme having bound indole‐glycerol‐P, serine, or both substrates.

The implications of the results for the catalytic center and subunit structure of the enzyme are discussed.
Studies on Deoxyribonucleic Acid Polymerases from Yeast
Authors: Wintersberger Ulrike , Wintersberger Erhard
Published: March 2005

A mitochondria‐free cell extract from Saccharomyces cerevisiae was used as source of yeast DNA polymerase for the partial purification and characterization of the enzyme. Upon DEAE‐cellulose chromatography, the total DNA polymerase activity separates into a major fraction, DNA polymerase A, and a minor fraction, DNA polymerase B. The final purification achieved was 150‐to 200‐fold for DNA polymerase A and 60‐to 80‐fold for DNA polymerase B. The two polymerases A and B differ from each other in some properties. Both purified enzyme preparations contain only traces of nucleases and very little, if any, terminal transferase. Activity is totally dependent on the presence of DNA, all four deoxyribonucleoside triphosphates and Mg++. With “activated” DNA or with poly d(A‐T) as template the DNA polymerases A and B exhibit an optimal activity. Native DNA preparations promote DNA synthesis to a low degree only, while denatured DNAs are even less active as templates. The enzymes have a molecular weight around 150 000; however, larger aggregates have been observed under certain conditions.
Glycannes liés à la fraction insoluble de la cornée de roussette (Scyliorhinus canicula L.)
Authors: Moczar Elemer , Moczar Madeleine
Published: March 2005

The distribution of the heteropolysaccharide chains in the insoluble tissular network of the dogfish cornea was investigated. For this purpose, the corneas were extracted with a buffer solution composed of 1 M CaCl2, 0.05 M tris and 0.02 M citric acid to remove soluble proteins. The residual insoluble stroma was successively hydrolysed by collagenase and pronase. The glycopeptides obtained were separated by gel filtration and high voltage electrophoresis. Two types of sugar chains were detected:

(a) polyosides composed of galactose, glucose, mannose, glucosamine, fucose, xylose and sialic acid, some of them at least linked to aspartic acid (type 1);

(b) galactose or glucosidogalactose linked to hydroxylysine (type 2).

For one heteropolysaccharide chain of type 1, 6 galactosidohydroxylysines and 4 glucosidogalactosidohydroxylysines were found.

The insoluble stroma was submitted to a fractionated extraction procedure by trichloroacetic acid and water in order to remove all collagenous material. The residual structural glycoprotein exempt from hydroxyproline was hydrolysed by pronase and the glycopeptide fraction was isolated by exclusion chromatography and electrophoresis. A single major glycopeptide fraction of the type 1 containing galactose, glucose, mannose, xylose and glucosamine was obtained.

This clear‐cut separation of the two types of glycopeptides by the tricholoroacetic acid treatment indicates that they are bound to different peptide chains: glycane of type 1 is bound to the peptide chain of structural glycoproteins (insoluble in hot trichloroacetic acid) and the glycane of type 2 is bound to the peptide chains of collagen (soluble in hot trichloroacetic acid).
Röntgenkleinwinkel‐Untersuchungen an der Fettsäuresynthetase aus Hefe
Authors: Pilz I. , Herbst M. , Kratky O. , Oesterhelt D. , Lynen F.
Published: March 2005

Fatty acid synthetase, a multienzyme complex from yeast, was analysed by small angle x‐ray scattering. The molecular weight determined by this method (2.2 × 106) was in close agreement with the value determined earlier by ultracentrifugation.

The experimental data indicate that the complex is contained in a ellipsoid volume with axial ratios 1:1:1.2. The relationship of radius of gryation to volume and the height of the second maximum in the scattering curve suggest that the complex proteins enclose a hollow space.

A model is proposed where the theoretical scattering curve closely aggrees with the curve measured for the fatty acid synthetase. The model consists of a spherical shell, composed of spheres, having an average wall thickness of 20 Å and divided in half by a planar wall also 20 Å thick. The outer radius is 125 Å. On the basis of this model the catalysis of fatty acid synthesis by the synthestase is discussed.
Existence and Functions of Two Enzymes with β‐Ketoadipate: Succinyl‐CoA Transferase Activity in Pseudomonas fluorescens
Authors: Hoet P. P. , Stainer R. Y.
Published: March 2005

This work shows that Pseudomonas fluorescens can synthesize two different inducible enzymes which possess β‐ketoadipate: succinyl‐CoA transferase activity. Transferase I is operative in the activation of free β‐ketoadipic acid and is specifically induced in cells grown either with this acid, or with an aromatic precursor (benzoate) oxidized through the β‐ketoadipate pathway. Transferase II is specifically induced in cells grown with saturated dicarboxylic acids of chain length C6 through C10, and its physiological function is that of an adipate: succinyl‐CoA transferase; its ability to activate β‐ketoadipic acid reflects non‐specificity with regard to the CoA acceptor.
Effect of Ethanol Metabolism on Redox State of Steroid Sulphates in Man
Authors: Cronholm Tomas , Sjövall Jan
Published: March 2005

Ethanol intake has been shown to cause a rapid increase of the concentration of androst‐5‐ene‐3β,17β‐diol 3‐sulphate and a simultaneous decrease of the concentration of dehydroepiandrosterone sulphate in plasma. The ratio between the 17β‐hydroxysteroid and the 17‐ketosteroid increased with the ethanol concentration up to 30–50 mg ethanol/100 ml blood when a 6 to 10‐fold increase was obtained. The ratios between the 3‐sulphates of 5α‐androstane‐3α, 17β‐diol and androsterone and between the monosulphates of 5α‐androstane‐3β,17β‐diol and 3β‐hydroxy‐5α‐androstan‐17‐one also increased during ethanol metabolism. Deuterium was rapidly incorporated into the D‐ring of the three C19 steroid diols, reaching about 20 atom per cent excess 10 to 20 min after oral administration of 0.1 g [1‐2H2]ethanol per kg body weight. The concentrations of steroids with a sulphate group at C‐17 did not change and deuterium was not incorporated into these steroids or into the 17‐ketosteroid sulphates. The ratio between the 3‐sulphates of 5‐pregnene‐3β,20α‐diol and pregnenolone increased only slightly and incorporation of deuterium into the side chain of the C21 diol reached a maximum of about 4 atom per cent excess about 1 hour after the ethanol administration.

The results indicate that the ratio between 3‐sulphates of 17β‐hydroxysteroids and the corresponding 17‐ketosteroids in plasma is determined by the NADH/NAD+ ratio in the liver cell cytoplasm. They also indicate that the 3‐sulphate of 5‐pregnene‐3β,20α‐diol is not formed from pregnenolone sulphate at the same site or with the use of the same coenzyme molecules as the 17β‐hydroxysteroids.
Der Testosteronstoffwechsel in der isoliert perfundierten Rattenleber
Authors: Staib R. , Sonnenschein R. , Staib W.
Published: March 2005

The metabolism of testosterone was studied in perfused livers of normal male and female rats and of male rats, four days and four weeks after castration. The rats were treated twenty days with cyproterone acetate and cyproterone and four days with phenobarbital (Luminal). Livers were perfused with [4‐14C]testosterone (7.2 mg, 9 μC) in 250 ml Krebs‐Ringer‐bicarbonate solution. In some experiments the metabolism of testosterone was measured under perfusion conditions without oxygen and with addition of albumin in the perfusion medium.

The isolated perfused rat liver shows a sex difference in the metabolism of testerone. The liver of females produces more 5α‐androstanediols than the liver of male rats. The quotient 5α‐/5β‐dihydrotesterosterone in females has a value of 2.63 and that in male rats is equal to 0.68. The production of 5β‐dihydrotestosterone is significantly smaller in female than in male rats. The quotient of the highly polar (H) and remaining metabolites (R) in male animals is 1.2 and that in female rats is only 0.5. The small quotient in the female rats is explained by the significant elevation of the remaining metabolites through the more active 5α‐reductase.

Four days after castration of male rats, we can see a very significant decrease of the hydroxymetabolites (H) and a significant increase of the remaining metabolites (R). So we find a quotient H/R of 0.33 in the males as compared to that in the females (0.5). The elevation of the remaining metabolites, based on a significant increase in the production of 5α‐androstanediol is caused through an increase in the activity of 5α‐reductase after castration.

We could observe a similar change in the metabolism in rat liver after four weeks castration, but the differences are not so marked as in four day castrated animals.

After a twenty day application of cyproterone acetate, the metabolism of testosterone showed no important changes, except the absence of androstenedione. Application of cyproterone indicated a significant increase of the hydroxymetabolites. The reason for this will probably be found in an induction of the microsomal oxydases.

After a four day treatment with phenobarbital (Luminal) the hydroxymetabolites show a significant increase, which can be explained by an induction of the microsomal oxydases.

Under conditions without oxygen, the metabolism of testosterone significantly declines. As expected we find less highly polar derivatives.

On addition of albumin to the perfusion medium, we observe a significant three fold increase in the metabolism of testosterone.
Formation and Metabolism of 7α‐Hydroxy‐5α‐cholestan‐3‐one and 7α,12α‐Dihydroxy‐5α‐cholestan‐3‐one in Rat Liver
Authors: Björkhem I. , Einarsson K.
Published: March 2005

The synthesis of 7α‐hydroxy‐5α‐cholestan‐3‐one and 7α,12α‐dihydroxy‐5α‐cholestan‐3‐one is described. These compounds were shown to be formed from the corresponding Δ4‐3‐oxo‐steriods in the presence of rat liver microsomes fortified with NADPH or an NADPH‐generating system. Liver microsomes from female rats were three to four times more active than those from male rats. In the presence of microsomes fortified with NADH or NADPH, 7α‐hydroxy‐5α‐cholestan‐3‐one was converted into 5α‐cholestane‐3β,7α‐diol. NADH was the preferred cofactor. 7α‐Hydroxy‐5α‐cholestan‐3‐one and 7α,12α‐dihydroxy‐5α‐cholestan‐3‐one were converted mainly into allocholic acid in bile fistula rats. The role of microsomal Δ4‐3‐oxosteroid 5α‐reductase in the formation of 5α‐bile acids is discussed.