bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycopeptides des immunoglobulines
Authors: Jouanneau Jacqueline , Razafimahaleo Edmond , Bourrillon Roland
Published: March 2005

An immunoglobulin M from Waldenström's disease (IgM Ga) was hydrolysed by papain and a glycopeptide fraction was isolated by gel filtration on Sephadex G‐100. This fraction was then successively resolved into fourteen glycopeptides by gel filtration on Sephadex G‐50 and by preparative paper electrophoresis at pH 1.9.

The amino acid and carbohydrate content of these glycopeptides was determined by ion‐exchange chromatography in an autoanalyzer.

All of them contain aspartic acid and serine, but they are quite different from one another, in amino acid composition and sequence about the carbohydrate‐peptide linkage, especially with respect to histidine, tyrosine, threonine, valine, glutamic acid, lysine and proline.

Furthermore, a number of glycopeptides contain only mannose and glucosamine, while others contain in addition, galactose, fucose, and N‐acetylneuraminic acid, in variable amounts.

These results suggest the presence of several linkage sites of the oligosaccharide chains on the H‐chain and the true heterogeneity of these oligosaccharide chains.

The peptide sequence of a few glycopeptides was reported, especially the following: Tyr‐Asn‐(carbohydrates)‐Ser‐Thr, also present in the Fc fragment of Immunoglobulin G.
Tryptophan Fluorescence Quenching in Horse Liver Alcohol Dehydrogenase
Authors: Luisi Pier Luigi , Favilla Roberto
Published: March 2005

The tryptophan fluorescence quenching by NADH or NAD in horse liver alcohol dehydro‐genase is investigated. Evidence is given that only one of the two tryptophan residues present in each subunit chain of the enzyme undergoes the quenching process. Such a quenching allows one to titrate exactly the enzyme active sites and to evaluate coenzyme binding constants. This quenching is then ascribed to a specific interaction between coenzyme and enzyme active site. The possible nature of this interaction is discussed.
Quantitative Measurement of Uptake of Alloantibody on Mouse Lymphocytes
Authors: Hämmerling Ulrich , Eggers Hans Joachim
Published: March 2005

Tritiated mouse alloantibodies were prepared by reaction of the gamma globulin fractions of antisera with DL‐[3‐3H]alanine‐N‐carboxy anhydride. The uptake of [3H]alanylated allo‐antibody against H‐2, TL or θ on mouse cells proved specific. On the basis of the known specific radioactivity the number of alloantibody molecules taken up per mouse lymphocyte was calculated for the three alloantigenic systems. In comparisons of cell populations from lymph nodes and thymus it was shown that there is a striking difference in alloantigen representation, qualitative as well as quantitative, implying extensive changes in alloantigenic surface patterns during the maturation of thymocytes into lymphocytes.
The Trapping of Uridine Phosphates byd‐Galactosamine,d‐Glucosamine, and 2‐Deoxy‐d‐galactose
Authors: Keppler Dietrich O. R. , Rudigier Jürgen F. M. , Bischoff Erwin , Deckker Karl F. A.
Published: March 2005

The effects ofd‐galactosamine,d‐glucosamine, and 2‐deoxy‐d‐galactose on rat liver uracil nucleotides were studiedin vivo. Enzymic and isotope dilution analyses of the UDP‐sugars and of uridine phosphates revealed three major, related changes: an accumulation of the respective UDP‐sugar derivatives, a marked decrease of UTP, UDP, and UMP, and a subsequent increase of the sum of hepatic uracil nucleotides. The decrease of uridine phosphates was accompanied by diminished contents of UDPG and UDP‐galactose. UMP of total liver RNA was not altered significantly. Inhibition of uridylate synthesis by use of 6‐azauridine resulted in a suppression of thed‐galactosamine‐induced stimulation of uridine phosphate synthesis and of the increase in total acid soluble uracil nucleotides. The trapping of uridine phosphates by formation of UDP‐sugar derivatives was most pronounced and most prolonged after administration ofd‐galactosamine. The uridine phosphate content was lowered to less than 10% of normal within three hours, while the sum of uracil nucleotides increased by 0.35 μmole x g liver−1x hour−1, from an initial value of 1.24 μmole/g. The quantitative analysis of the time dependent changes of UDP‐hexosamines, UDP‐N‐acetylhexosamines, UDPG, and UDP‐galactose revealed a pronounced alteration byd‐galactosamine of the UDP‐sugar pattern.

Corresponding changes in the distribution of liver uracil nucleotides were obtained after administration ofd‐glucosamine and 2‐deoxy‐d‐galactose. Both, however, are ineffective in provoking hepatitis. In contrast tod‐galactosamine,d‐glucosamine and 2‐deoxy‐d‐galactose do not lead to the formation of UDP‐hexosamines; furthermore they are less efficient in trapping uridine phosphatesin vivo. These observations contribute to an understanding of the orotate‐mediated prevention of the galactosamine‐induced liver damage, and of the role of pyrimidine nucleotides in this experimental hepatitis.
The Cleavage of Prolyl Peptides by Kidney Peptidases
Authors: Dehm Peter , Nordwig Arnold
Published: March 2005

Carboxypeptidase activity was detected in swine kidney microsomes. The enzyme was solubilised by treatment with trypsin and toluene. Approximately 330‐fold purification was achieved by subsequent chromatography on DEAE‐cellulose and hydroxylapatite.

As shown by polyacrylamide gel investigation, the carboxypeptidase preparation thus obtained was practically pure. It still contains trace impurities of aminopeptidase M which is quite similar in physical properties. The molecular weight of the carboxypeptidase was 240000 as determined by Sephadex gel filtration. It is activated by manganese ions and exhibits a pH‐optimum at 7.75.

The carboxypeptidase preferentially attacks substrates with a prolyl residue in the last but one position. No chain length dependence was found. Peptides free of proline were also cleaved, but normally at a rather low rate. The designation carboxypeptidase P (indicating its preference of proline peptide bonds) is therefore suggested.

The physiological role of the new peptidase is discussed.
Hefe‐Phosphofructokinase
Authors: Freyer Renate , Liebe Stefan , Kopperschläger Gerhard , Hofmann Eberhard
Published: March 2005

The effect of trypsin treatment with respect to the allosteric properties of yeast phosphofructokinase as well as its molecular weight has been studied. In the presence of fructose 6‐phosphate trypsin desensitizes yeast phosphofructokinase to ATP inhibition. Under the same conditions, activation of this enzyme by the positive allosteric effector AMP, however, remains uneffected. Addition of MgATP instead of fructose 6‐phosphate to the incubation medium containing trypsin protects the enzyme against ATP‐desensitization. After prolonged incubation of yeast phosphofructokinase with trypsin the enzyme becomes inactivated both with fructose‐6‐phosphate and with MgATP.

Using density gradient centrifugation in sucrose media the effects of trypsin treatment on the sedimentation behaviour of yeast phosphofructokinase has also been investigated. With fructose 6‐phosphate, trypsin converts the 560000 form of yeast phosphofructokinase into two enzymatically active forms of 510000 and 350000 daltons respectively, which are both insensitive to ATP inhibition.

On the other hand, in the presence of MgATP the peak of enzymatic activity sediments with 160000 daltons. This molecular form has the same high sensitivity to ATP inhibition as the untreated enzyme of molecular weight 560000. After the addition of fructose 6‐phosphate, the 160000‐molecule seems to dimerize giving an ATP‐sensitive form with a molecular weight of 340000.

In the light of studies about the existence of several interconvertible forms of yeast phosphofructokinase published recently by our laboratory, the results described in this paper are discussed in terms of the existence of dimeric and trimeric states, regarding the 160000‐moiety as the trypsin modified monomer of the enzyme. Kinetic and allosteric properties of these states including conditions for their formation and stabilization are summarized and the peculiarities of the trypsin‐unmodified and trypsin‐modified enzyme are compared.
Studies on Nucleolar Liver DNA
Authors: Ittel Marie‐Elisabeth , Wintzerith Marguerite , Zahnd Jean‐Pierre , Mandel Paul
Published: March 2005

Rat liver nucleoli were isolated from nuclear rat liver preparations. The nucleolar DNA and extranucleolar DNA, i.e. chromosomal DNA were examined. After deproteinization, nucleolar and chromosomal DNA were separated from RNA on 6 M CsCl isopycnic gradient.

Nucleolar and chromosomal DNA were of the same density (1.703 g/cm3) in CsCl. The specific activity of nucleolar DNA from normal adult rat liver was significantly higher than chromosomal DNA after 32P incorporation in vivo, independent of the time after the label. Nucleolar DNA is metabolically distinct from chromosomal DNA. Results are discussed and a possible role of nucleolar DNA replication in ribosomal RNA synthesis is suggested.
Occurrence and Function of a High‐Km Hexokinase in Immature Red Blood Cells
Authors: Gerber Gerhard K. G. , Schultze Maria , Rapoport Samuel M.
Published: March 2005

The formation of 14C02 from [1‐14C]glucose and [6‐14C]glucose by reticulocytes of rabbit and rat is dependent on the concentration of glucose over a wide range, indicating reactions preceding the formation of glucose‐6‐phosphate to be dependent on glucose concentration.

In haemolysates of rabbit reticulocytes a high‐Km hexokinase was demonstrated by its kinetic properties. It corresponds to the glucokinase of liver. This enzyme does not occur in mature red blood cells of man and rabbit, nor in reticulocytes of newlyborns with erythro‐blastosis fetalis. The presence of glucokinase in reticulocytes introduces a metabolic path of glucose utilization which is not subject to regulation by the phosphofructokinase.
Antigenic Changes in Shigella flexneri Following Conjugation with Escherichia coli HfrC
Authors: Manson Ronald , Simmons D. Alastair R. , Petrovskaya Valentina G.
Published: March 2005

The biosynthesis of Shigella flexneri O‐antigens proceeds through the rough precursor antigens to the (group antigen) variant Y form under the influence of bacterial chromosomal genes. Type specificity is then conferred on the growing molecule with the incorporation of terminal α‐glucosyl secondary side chains by specific UDP‐glucose transferases under the control of the appropriate prophage(s) at the type specific or T‐locus. When the various Sh. flexneri serotypes are conjugated with Escherichia coli HfrC, the T‐locus with its phage attachment site(s) is lost so that antigen biosynthesis stops at the end of the chromosomal or variant Y stage. These findings offer an explanation for the observation that many Sh. flexneri x E. coli HfrC hybrids carry Y‐specificity.
Optical Rotatory Dispersion and Circular Dichroism of DNA from Various Sources at Alkaline pH
Authors: Luck Gerhard , Zimmer Christoph , Snatzke Günther , Söndgerath Gertrud
Published: March 2005

The effect of deprotonation and alkaline denaturation on the conformation of DNA from various sources has been investigated by optical rotatory dispersion and circular dichroism measurements.

Upon transition to alkaline pH, changes in the optical rotatory dispersion at the characteristic peaks around 228 and 290 nm are observed. The variation of the optical rotations in case of A. T‐rich DNA is more pronounced than in case of G . C‐rich DNA. For G . C‐rich DNA, changes of rotations are mainly observed at pH values beyond 11.5; the transition ranging between pH 11.6 and 11.7. The transition is accompanied by a shift of the “cross‐over” point towards longer wavelengths presumably due to deprotonation of guanine residues.

Native DNAs show characteristic differences in their circular dichroism spectra dependent on the G · C content. Increasing the temperature (below the melting region) reveals enhancement of the circular dichroism band of native DNA around 270 nm whereas the opposite effect is found in the denatured state. As a possible explanation an increase of the chirality in the native helical DNA structure or a structure of intermediary character is considered. Titration up to pH 12 or higher produces a drastic increase of the rotational strength around 228 nm as well as changes in the other two circular dichroism‐maxima which may be correlated to a diminuition of the chiral and helical structure.
The Effects of Magnesium and Manganese on the Purine Ratio of RNA Transcribed in vitro by Rat Liver Nuclei
Authors: Moulé Yvonne , Rousseau N.
Published: March 2005

It is currently accepted that the base composition of the RNA synthesized in vitro by RNA polymerase of rat liver nuclei, depends on both the nature of the cation added to the incubation medium and the ionic strength of this medium. However, up to the present time, the foregoing results have been obtained on nuclei isolated in sucrose solution containing Mg2+ (1 to 3 mM). The influence of the isolation medium of nuclei has so far never been considered.

The present results demonstrate that the ratio A/G of the RNA synthesized in vitro is much more determined by the cation added to the isolation medium than that present in the incubation assay. Nuclei isolated either in the presence of 1 mM Mn2+ or in the absence of cation added to sucrose solution synthesize (at low ionic strength) RNA molecules with a ratio A/G 1 regardless of the ion present in the standard assay. Thus, presence of Mg2+ during the isolation of nuclei appears to suppress their ability to transcribe in vitro DNA‐like RNA although allowing an optimal synthesis of ribosomal RNA.

The properties of liver nuclei isolated in various conditions have been studied in relation to their transcriptional ability, and the results have been discussed taking into account the different possibilities of action of the cations upon the nucleus.
Hydrocarbon‐Utilizing Microorganism
Authors: Fujii Katsuhiko , Fukui Saburo
Published: March 2005

Cellular fatty acids of Corynebacterium simplex ATCC 6946 were studied using various chain length of n‐alkanes as a sole source of carbon substrate. The organism contained appreciable amounts of odd‐numbered fatty acids even when grown on even‐numbered n‐alkane, suggesting the involvement of α‐oxidation of fatty acids. Moreover various homologues of tuberculostearic acid (10‐methylstearic acid) and iso branched acids were derived specifically depending on the chain length of n‐alkane.

The compositions of monounsaturated and 10‐methyl branched‐chain fatty acids were remarkably affected by the level of intracellular vitamin B12, while those of normal saturated ones remained essentially constant. Namely, B12‐sufficient cells contained more 10‐methyl fatty acids than Δ9‐monounsaturated ones, and vice versa.

From the study concerning the enzymatic synthesis of 10‐methyl fatty acids, these acids were found to be formed by methyl group transfer from S‐adenosylmethionine to unsaturated fatty acid moieties of certain lipids, and methyl‐B12 did not serve as a direct methyl donor in this reaction. In the test organism, the presence of B12‐dependent N5‐methyltetrahydrofolate‐homocysteine transmethylase was confirmed, the activity of which decreased significantly in the cells of low B12‐level. These results indicated that the reduced activity of methionine‐synthesizing system causes a lowering in the transformation of Δ9‐monounsaturated acids into 10‐methyl fatty acids in C. simplex.
3‐Phosphoglycerate Kinase from Rabbit Sceletal Muscle and Yeast
Authors: Krietsch Wolfgang K. G. , Bücher Theodor
Published: March 2005

Phosphoglycerate kinase, isolated from rabbit muscle in highly purified crystalline form, was compared in terms of chemical, physical and kinetic properties to yeast phosphoglycerate kinase. A summary of the data is presented.

Both proteins are composed of a single polypeptide chain, and in electrophoresis only one band was found. The N‐terminus is masked. The C‐terminal amino acid of the muscle enzyme is valine.

Muscle and yeast enzyme have nearly identical molecular weights, Michaelis‐Menten constants, optimal pH values and Vmax. On the other hand, they differ significantly in their amino acid composition, especially in the content of sulfur and aromatic amino acids, in electrophoretic mobility and in heat stability. The muscle enzyme has an essential SH group. The single SH group of the yeast enzyme is apparently without importance for enzyme activity.

Differences in the same direction as those observed with yeast and muscle phosphoglycerate kinase are discussed in connection with other glycolytic enzymes from the same organisms.
Staphylococcal Ornithine Carbamoyltransferase
Authors: Zaharia O. , Soru Eugenia
Published: March 2005

A procedure for the purification of an ornithine carbamoylphosphate transferase isolated from a Staphylococcus aureus strain is reported. The procedure consists of the following steps: water extraction of the crude enzyme by autolysis under a toluene layer of the acetone dried bacteria cells, lyophilization of the crude extract, molecular sieving chromatography on a Bio Gel P‐150 column and as a final step the electrophoresis on Sephadex G‐200 plates.

A 100‐fold purification with a 50% yield is realized. The purified preparation so obtained appeared to be pure on the basis of acrylamide gel electrophoresis, thin layer chromatography, immunodiffusion and immunoelectrophoresis.

The apparent molecular weight of the enzyme as determined by molecular sieve chromatography is 200 000 ± 30 000.

The Km value for l‐ornithine as substrate is 3 mM and for carbamoylphosphate 0.7 mM. Arrhenius activation energy is 13 775 cal/mol. NH2‐terminal amino acid is glycine: COOH‐terminal amino acid is glutamic acid.

Two unmasked SH‐groups and two S‐S interchain bridges per molecule were found.
Effects of Valine Deprivation on the Biosynthesis of Ribosomal and Messenger RNA in a Mammalian Cell Line (L 5178 Y)
Authors: Tiollais Pierre , Galibert Francis , Boiron Michel
Published: March 2005

It has been well demonstrated in bacteria that RNA syntheses are non‐coordinated. In order to study this problem in mammals, qualitative and quantitative studies of rRNA, polysomal mRNA, and RNA of heterogeneous ribonucleoproteic particles syntheses have been performed, during exponential growth and valine starvation. During the latter, the residual synthesis of these three classes of RNA were respectively calculated as 38, 70 and 90% of the control. Whatever is the physiological significance of these particles one can conclude that the RNA syntheses in mammals are not coordinated. This result resembles those observed in stringent bacteria strains.

In addition, the “45 S fraction” isolated from sucrose gradient appears to be heterogeneous in CsCl gradient. This could be interpreted by the existence of several classes of particles, heterogeneous ribonucleoproteic particles and small ribosomal subunits more or less mature.

Finally, some heterogeneous ribonucleoproteic particles with high density (ϱ= 1.60 g/cm 3) were found. This is not in complete accordance with the generally finding, that these particles are characterized by a low density.
Observations on the Biosynthesis of Ubiquinones by Animals
Authors: Whistance Geoffrey R. , Field Frederick E. , Threlfall David R.
Published: March 2005

Tissues of a number of avian and mammalian species have been examined for the presence of 2‐polyprenylphenols, 6‐methoxy‐2‐polyprenylphenols, 6‐methoxy‐2‐polyprenyl‐1,4‐benzoquinones and 5‐demethoxyubiquinones. In no instance was evidence found of the presence of any of these compounds, although most tissues contained at least two ubiquinones and one ubiquinol.

Tracer studies wiht p‐hydroxy[U‐14C]benzoic acid have been carried out to detect the precursor of ubiquinone‐9, “5‐demethoxyubiquinone‐9”, reported by Olson and co‐workers [14, 15] in rat liver. The results obtained provided good evidence that this compound is ubiquinol‐9.
The Sequence around the Active‐Center Tyrosyl of Porcine Carboxypeptidase B
Authors: Roholt Oliver A. , Pressman David
Published: March 2005

Iodination of porcine carboxypeptidase B causes a loss of both peptidase and esterase activity; the presence of the inhibitor β‐phenylpropionate during iodination prevents the loss.

A residue in the active site of the enzyme was labeled by a procedure in which one portion of the enzyme was iodinated with 125I‐labeled iodine and a second portion with 131I‐labeled iodine in the presence of the inhibitor β‐phenylpropionate. The two preparations were mixed, digested with elastase and the iodopeptides separated. Two iodopeptides which appear to have come from the active site were isolated; both had high 125I/131I radioactivity ratios relative to the unfractionated digest. The labeled residue is tyrosyl in both iodopeptides. The same results were obtained when the inhibitor ɛ‐aminocaproate was used instead of β‐phenylpropionate. The yield of the two iodopeptides approached a total of about 1 mole/mole of enzyme in each of several experiments. The sequences of the two iodopeptides were found to be Thr‐Ile‐(monoiodo)Tyr‐Pro‐Ala and Ile‐(monoiodo)Tyr‐Pro‐Ala, and both appear to represent the same tyrosyl residue in the site of porcine carboxypeptidase B.
Catalytic Activity of Trypsin with an Acylated “Active” Serine
Authors: Bresler Simon E. , Krutyakov Valerii M. , Vlasov Gennadii P.
Published: March 2005

The applicability of the acylenzyme hypothesis to the mechanism of tryptic hydrolysis of amide and peptide bonds was studied. “The active” serine of trypsin was acylated with radicals of different size. If the acylenzyme mechanism is valid the kcat of esterase, amidase and peptidase activities of acyltrypsin must always decrease to the same extent. To decrease the degree of deacylation of carboxylic derivatives of trypsin, the enzymatic activity was measured at 25°, pH 6.0 and average incubation time 1 min. As compared with kcat of the native trypsin the values of kcat of esterase and amidase residual activities were found, respectively: for tetraformyl‐trypsin 43 and 75%, for heptaformyl‐trypsin 22 and 55%, for monoacetyl‐trypsin 6 and 37%, for triacetyl‐trypsin 1.4 and 22%, for monopropionyl‐trypsin 9 and 23%, for mono(dimethylpropionyl)‐trypsin 12 and 12%, for mono‐(diethylphosphoryl)‐ or mono‐(dimethylphosphoryl)‐trypsin 1 and 1%. Monoacetyl‐trypsin demonstrated 6.4% of esterase and 58% of peptidase activity. On the extrapolation to complete acylation (respectively to the zero esterase activity) the residual amidase activity decreased in the series formly‐, acetyl‐, propionyl‐trypsin and was equal to zero for dimethylpropionyl‐trypsin. The latter acylating radicals was similar in its size and hydrophobic nature to the smallest dialkylphosphoryl radicals. It is suggested that the acylenzyme mechanism is at leat not a single pathway of the tryptic hydrolysis of amide and peptide bonds.
Excretion of Steroid Hormones in Adults
Authors: Eriksson Håkan , Gustafsson Jan‐Åke
Published: March 2005

The steroids in faeces from three healthy persons, one woman and two men, were analyzed by gas‐liquid chromatography and gas chromatography‐mass spectrometry. Both C19O2, C19O3, C21O2 and C21O3 steroids were found in the free steroid and in the steroid monosulphate fractions.

Incubation of various precursor steroids with intestinal contents recovered from the ileostoma of a colectomized patient demonstrated the presence of intestinal bacterial 16α‐ and 21‐dehydroxylating enzymes. Furthermore, it was shown that a Δ16‐C21 steroid was converted to a 17α‐pregnane derivative and that the side chain of 11β,21‐dihydroxy‐4‐pregnene‐3,20‐dione could be removed by the intestinal bacteria.
Bovine Erythrocyte Cupro‐Zinc Protein
Authors: WOOD Edward , Dalgleish Douglas , Bannister William
Published: March 2005

The physicochemical properties and circular dichroism of bovine erythrocyte cupro‐zinc protein were investigated. In sedimentation velocity experiments in the ultacentrifuge, the protein sedimented as a single peak with an s°20,w of 3.06 S. The diffusion coefficient was 8.27 F. the partial specific volume 0.736 ml/g, and the instrinsic viscosity 0.033 (g/100 ml)‐1. Combination of these hydrodynamic data indicated a molecular weight near 33 000, which was in good agreement with chemical and gel filtration data. The molecular weight was also determined by the sedimentation equilibrium method using interference optics. By this method M̄w was 33 500, and the protein appeared to be homogeneous. The circular dichroism spectrum of bovine erythrocyte cupro‐zinc protein was unique among copper proteins in having positive contributions at 610, 440 and 350 nm, which disappeared on removing the metal atoms. In the ultraviolet there were positive contributions near 260, 270 and 290 nm, and removal of the metal atoms changed these into negative contributions at 265 and 285 nm and a positive contribution at 295 nm. Possible assignments of these bands are discussedd. In the far ultraviolet, the circular dichroism spectra of the native protein and apoprotein showed few differences, indicating that the metal atoms do not play a dominant role in determining protein conformation. However, urea had a profound effect on the circular dichroism of the apoprotein but little effect on that of the native protein. Neither the native protein nor the apoprotein showed the duble minimum between 200 and 220 nm characteristic of α‐helix, but rather the existence of a minimum at 210 nm suggested that the proteins contained some β‐pleated sheet structure. Reduction and carboxymethylation of thiol groups greatly reduced the ultraviolet circular dichroism, indicating that the disulphide bridges have a strong influence in determining the tertiary structure of the protein.
The Interaction of Fungal Laccase with Hydrogen Peroxide and the Removal of Fluoride from the Inhibited Enzyme
Authors: Brändén Rolf , Malmström Bo G. , Vänngård Tore
Published: March 2005

Electron paramagnetic resonance analysis shows that H2O2 added to native fungal laccase binds to one specific Cu2+ of the four copper atoms in the enzyme (the so‐called Type 2 or “nonblue” Cu2+).

This binding of H2O2 is associated with a new absorption band in the visible and nearultraviolet spectrum of the enzyme with its maximum absorbance at 400 nm.

Fluoride bound to Type 2 Cu2+ of the enzyme, as commonly prepared, can easily be removed by dialysis of the reduced enzyme. Presence of H2O2 in this dialysis makes the removal of fluoride faster.
The Uptake of C4‐Dicarboxylic Acids by Escherichia coli
Authors: Kay William W. , Kornberg Hans L.
Published: March 2005

Escherichia coli inducibly form a system effecting the uptake of a number of C4‐dicarboxylic acids; glucose or inhibitors of protein synthesis prevent this induction. After growth on C4‐dicarboxylic acids; glucose or inhibitors of protein synthesis prevent this induction. After growth onC4‐dicarboxylic acids, washed cells take up such acids rapidly and, initially, linearly with time; the Km for this process is approx. 30 μM and the Vmax is approx. 25 nmoles X min−1 mg dry wt −1, irrespective of the identity of the acid transported. C4‐dicarboxylic acids other than fumarate, succinate, malate, aspartate, and maleate, are takep up poorly. Washed cells talking up 14C‐labelled C4‐dicasrboxylic acids incorporate isotope mainly into macromolecular cell components; no significant intracellular accumulation of C4‐acids is observed. Mutants blocked in component reactions of the tricarboxylic acid cycle are also greatly impaired in their ability to take up C4‐acids; mutants blocked in reaction ancillary to the cycle are not. It is suggested that E. coli effect the uptake of C4‐dicarboxylic acids througha common, inducible and highly specific transport system.
Isotope Discrimination in Steroid Hydroxylations
Authors: Bjökhem Ingemar
Published: March 2005

The possible occurrence of isotope effects in steroid hydroxylations by rat and guinea‐pig liver was studied in vitro and in vivo.

In vitro, the 7α‐hydroxylation of [24‐14C + 7α−3H]taurodexycholic acid was found to involve a moderate isotope effect, whereas 6β−hydroxylation of [4−14C + 6β−3H]taurochenodeoxycholic acid and 7α−hydroxylation of [4−14C + 6−3H, 7α−2H]cholesterol and [4−14C + 6−3H, 7α−2H]‐cholestanol did not involve significant isotope effects. An isotope effect was observed in the autoxidative 7α‐hydroxylation of [4−14C + 6−3H, 7α−2H]cholesterol.

In vivo, [4−14C + 7α−3H]cholesterol was converted into chenodeoxycholic acid and cholic acid and [4−14C + 6β−3H]chenodeoxycholic acid into α‐ and β−muricholic acid without the occurrence of isotope effects, whereas [24−14+ 7α−3H]deoxycholic acid was converted into cholic acid with the occurrence of a moderate isotope effect.

The results show that breaking of the C‐H bound is not rate limiting in the 7α−hydroxylation of cholesterol and cholestanol or in the 6β−hydroxylation of taurochenodeoxycholic acid but may be rate limiting in the 7α−hydroxylation of taurodeoxycholic acid.
On the Mechanism of Hemoglobin‐Haptoglobin Complex Formation
Authors: Pavlíček Zdeněk , Jaenicke Rainer
Published: March 2005

In order to elucidate the mechanism of complex formation between human haptoglobin, Hp II, and bovine hemoglobin, Hb, measurements of the peroxidase activity and histidine determinations, as well as spectrophtometric and optical rotatroy dispersion titration experiments were performed.

Following the raaction by slowly mixing the constituents of the complex in small portinos, the geometry of the complex turns out to depend on the sequence of mixing of the reactants. Adding Hb to Hp, a final complex Hb2→ Hp is formed. Teh inverse sequence of mixing leads to a complex of identical composition (Hp → Hb2), but different arrangement of the constituents, as shown by differences in the accessibility of histidine residues in both types of the complex. As suggested by the comparison of the histidine balance of both complexes, Hb2→ Hp may be described by the combination of αβ‐dimers of Hb with Hp (Hp (αβ)4), while Hp → Hb2 corresponds to the complex with two Hb tetramers (Hp(α2β2)2).

Investigating the saturation process of complex formation in greater detail by spectrophtometric titration and optical rotatory dispersion, intermediary complexes are observed which prove the mechanism of complex formation to be a consecutive association process.

Both series of complexes, Hp → Hb and Hb → Hp, seem to generate closely related configurations characterized by similar spectral and functional properties.
On the Mechanism of Action of Ribonuclease T1
Authors: Rüterjans Heinz , Pongs Olaf
Published: March 2005

The chemical shifts of C‐2 protons of the three histidine resedues of ribonuclease T1 have been studied as a function of pH in 2H2. From the resulting titration curves pK values are obtined for single histidine residues. The pK values of two histidine reidues (A and B) ae unusually high (7.1 and 8). The third histidine reisdue (C) exhibits a very anomalous titration behaviour, so that a pK value could not be derived for this residue. The result are interpreted residues. The presence of two histidine residues (A and C) in the active site of the enzyme is indicated by change, which occur in their C‐2 proton magnetic resonance absorption region on the addition of guanosine 3′‐phosphate. Histidine A interacts with the phosphate group of guanosine 3′‐phosphate. Histidine C is located at the side, where the guanine base is bound by the enzyme. On carboxymethlation of glutamic acid 58 of ribonuclease T1 the pH dependece of the chemical shift of the C‐2 protons of all three histidine residues change. The C‐2 proton magnetic resonace signls of histidine A and B sift upfield, shereas the one of histidine C shifts downfield. From the data it is concluded that glutamac acid 58 is located at the binding site near istidne C of ribonuclease T1
A Study on the Motion Of Protenis in Excitable Membrane Fragment by Nanosecond Fluorescence Polarization Spectroscopy
Authors: Wahl Philippe , Kasai Michiki , Changeux Jean‐Pierre
Published: March 2005

The motion of two fluorescent dyes, 1‐anilino‐8‐naphthalene sulphonate, which binds reversibly, and 5‐dimethylamino‐1‐naphthalene sulphonyl chloride, which binds covalently to excitable membrane fragments in vitro, has been studied by nanosecond fluorescence polarization spectroscopy. Both dyes were strongly immobilized by their association wtih membrane proteins. Solubilization by Triton X‐100 of an important fractioon (60%) of the proteins from membrane fragments heavily labelled with 5‐dimethylamino‐1‐naphthalene sulphonyl chloride was accompanied by a dramatic increase of motion. It is concluded that proteins are strongly immobilized within the membrane phase.
Zur Biosynthese der 2‐n‐Alkyl‐4‐hydroxychinolinderivate (Pseudane) bei Pseudomonas aeruginosa
Authors: Ritter Christiane , Luckner Martin
Published: March 2005

Sixty strains of the bacterium Pseudomonas aeruginosa (Schroeter) Migula were proved to synthesis 2‐n‐alkyl‐4‐hydroxyquinolines (pseudans). The compounds were detexted in most of the strains, but only few of them showed high alkaloid production. The pseudans are located in the cells. Excretion into the medium after long cultivation seems to be due to autolytic processes.Thin‐layer chromatography of the purified alkaloid fraction and gas chromatography of the acids producd by oxydative degradation of the alkaloids hve shown that all strains form pseudan VII (2‐n‐heptyl‐4‐hydroxyquinoline) and pseudan IX (2‐n‐nonyl‐4‐hydroxyquinoline) only.

Experiments on the physiology and biosynthese of the pseudans were carried out using strain 2977, which produces these compounds in good yields. The formation takes place during the idiophase (Fig. 1). After feeding anthranilic acid and acetate the production rate was considerably increased (Table 1). Strain 2977 degrades tryptophan by the “aromatic pathway” forming anthranilic acid as an interrmediate (Fig. 2). The production of higher amounts of this compound seems to be essential for a good rate of pseudan formation.

To study the biosynthesis, isotope labelled preparations of anthranilic acid, tryptophan, acetate, malonate and β‐ketodecanoic acid methyl ester were fed (Table 2). For localization of the incoroprated radioactivity, procedures for the chemical degradation of the pseudan molecule were developed. They permit the serate estimation of the C‐atoms 4–8a and the nitrogen as anthranilic acid, C‐atoms 2 and 3 each as CO2, the two terminal C‐atoms of the side chain as CO2 and methylamine respectively and the side chain as a whole. The results obtained have shown that the pseudan molecule is formed from anthranilic acid and acetate. Feeding malonic acid, it was demonstrated that the side chain is built starting from the terminal C‐atoms (Fig. 4). The high sepcific incorporation of β‐keto[3H]decanoic acid methyl ester indicates that a 3‐keto acid is an intermidate. Thus the pathway shown in Fig.5 for the biosynthesis of the pseudans may be assumed to be valid. The incorporation of [2‐14C]acetate into the pseudans was twice that of [1‐14C]acetate. This effect may be due to an equilibrium between malonyl CoA and free malonic acid, which enables the formation of [1,3‐14C2]malonyl‐CoA from [1‐14C]malonyl‐CoA. During the formation of the β‐keto acid half of the radioactivity is lost from this compound.
On the Mechanism of Accelaration of Carboxypeptidase A Activity
Authors: Schechter Isreal , Zazepizki Etty
Published: March 2005

A number of peptides derivatives were found to increase the peptides activity of caboxypeptidase A towards benzyloxycarbonyl‐depeptides. The accelerators are either completely resistant to cleavage by carboxypeptidase A or degraded at a very slow rate. To evaluate the mode of action of the activators (A), the kinetics of hydrolysis of the substrate (S) by the enzyme (E) was followed by measuring the effect of a constant concentration of A on varying concentrations of S and vice versa. These studies indicate that an EA complex is formed, and that EA reacts with S to form an active ternary complex, EAS. Acceleration is observed because EA has a higher affnity for S than the free enzyme. Ultracentrifuge studies show that The activators do not induce aggregation of carboxypeptidase A.
Purification from Crude Extract by Affinity Chromatography of the Inhibitor of Deoxyribonuclease I
Authors: Lindberg Uno , Eriksson Sigbritt
Published: March 2005

A one step procedure, consisting of affinity chromatgography, was worked out for the purification from crude extracts of calf thymus of the protein inhibitor specific for pancreatic deoxyribonuclease (DNAase I).

For this purpose DNAase I was immobilized on agaros. This solid phase enzyme conjugate was shown to have the ability to adsorb from crude extracts one major protein, which could be eluted again with 3 M guanidine‐HCl in 1.0 M soudium acetate and 30% glycerol. This protein preparation had only a little DNAase inhibiting activity, but could be idnetified by gel electrophoresis and peptide mapping as the inhibitor protein described recently. The low recovery of inhibitor activity was due to poor stability of the inhibitor protein in the solvent system found necessary to effect its elution from the column material.
Amino Acid Sequence of Lima Bean Protease Inhibitor Component IV
Authors: Tan Celline G. L. , Stevens Frits C.
Published: March 2005

Commmerical lima bean inhibitor was fractionated into four apparently homogeneous components as previously described by Jones, Moore and Stein in 1963.

Reduced and alkylated component IV was hydrolyzed with tryspin and the resulting peptides were separated by ion exchange charomatography on Dowexs 50 X2 or gel filtration on Bio‐Gel P‐6. Where necessary, further purification of the peptides was carried out by paper chromatography, paper electrophoresis or a combination of both.

Seven peptides were obtained in pure form and their compositions are reported here. The amino acid sequenceof six of these peptides was determined, using classical methods. When allowance is made for the occurence of two homologous peptides, presumably resulting from heterogeneity in the original protein preparation, the tryptic peptides isolated, account for the complete composition of the protein.

In an attempt to obtain overlapping sequences the tryptic digest was also performed on the reduced, alkylated and guanidinated protein. Five tryptic peptides, including one containing homoarginine as the carboxy‐terminal residue, were isolated in pure form from the digest; their amino acid compositions are reported.
Amino Acid Sequence of Lima Bean Protease Inhibitor Component IV
Authors: Tan Celine G. L. , Stevens Frits C.
Published: March 2005

Using ion exchange chromatography, gel filtration and paper electrophoresis eight peptides were isolated in pure form from a chymotryptic digest of lima bean inhibitor component IV. They account for 76 out of the 84 resiodues of the protein. Complete or partial sequnces of these peptides were determined using classical methods. This information, together with inforation obtained on the tryptic peptides and reported in the preceding paper allowed the deduction of the complete amino acid sequence of lima bean inhibitor IV. The results show that the original protein preparation was heterogeneous as reflected by the occurrence of conservative substitutions at positions 26, 37 and 39. A detailed examination of the sequence revealed the presence of a repetitive sequence with residues 50 through 61 clearly homologous to residues 23 through 34. Of added interest is the fact that the anti‐chymotrypsin site of lima bean inhibitor, as recently determined by Krahn and Stevns, was found to be located in one of the homologous regions. This has led us to speculated that the anti‐trypsin site could be located in the other homologous region and that this double‐headed inhibitor, with indpendent sites for trypsin and chymotrypsin, may have evolved by gene duplication.
Isolation and Properties of Crystalline Initiation Factor F1 from Escherichia coli Ribosomes
Authors: Lee‐Huang Sylvia , Sillero María Antonia G. , Ochoa Severo
Published: March 2005

Initiation factro F1 was isolated from Escherichia coli ribosomes as a crystalline basic protein of molecular weight 9400 ± 100 daltons. It contains all of the common twenty amino acids, with only one residue eachy of histidien, proline, cryteine, methionine, and tryptophan, and has alanine and lysine, respectively, at the amino‐and carboxy‐terminal ends.
Thyroid Hormone Induced Changes in the Enzyme Activity Pattern of Energy‐Supplying Metabolism of Fast (Whit) Slow (Red), and Heart Muscle of the Rat
Authors: Kubišta Václav , Kubištová Jarmila , Pette Dirk
Published: March 2005

Influeces of thyroid hormones onthe activity levels of glyucogen phosphorylase, hexokinase, trisoephosphate dehydrogenease, critrate synthase, 3‐hydroxyacyl‐CoA dehydrogenease, mitochondrial glycerolphosphate dehydrogenase and the contnt of cytochrome a3 were examined in a “white” (m. rectus femoris), “red” (m. soleus) and heart muscle of male rats. Of the enzymes examined, glycerolphosphate dehydrogenase and hexokinase, revaled most significant increases, with comparable extent and simultaneous time of onset. The pattern o reaction was, however, different in the three types of muscle. In white muscle, an increase of hexokinase, but not of glycerolphosphate dehydrogenase was found. In red muscle, the two enzymes increased in parallel and in heart muscle the increase in glycerolphosphate dehydrogenase was the largest and exceeded by far that of hexokinse. In skeletal muscles it could be shown that the increase of hexokinase activity was limited to hexokinase‐isoenzyme II, hexokinase I being unaffected. Histochemical staining for glycerolphosphate dehydrogenase revealed that the activity of this enzyme is predominalty demonstrable in the subsarcolemmal mitochondria. The increased activities of hexokinase in the skeletal muscles and of glycerophosphate dehydrogenase in red and heart muscle are interpreted as a coordinate process of a “long‐term metabolic regulation” which is related to an elvelatd aerobic carbohydrate catabolism of muscle tissue and to an enhanced rate of basal metabolism.
X‐Ray Small‐Angle Scattering of Yeast Glyceraldehyde‐3‐phosphate Dehydrogenase as a Function of Saturation with Nicotinamide‐Adenine‐Dinucleotide
Authors: Durchschlag Helmut , Puchwein Gerd , Kratky Otto , Schuster Inge , Kirschner Kasper
Published: March 2005

Structural changes of yeast glyceraldehyde‐3‐phosphate dehydrogenase as a function of saturation with NAD have been measured with the X‐ray small‐angle scattering technique. Samples of yeast glyceraldehyde‐3‐phosphate dehydrogenase were investigated at pH 8.5 and 40° and five different degrees of saturation with the coenzyme NAD (degree of saturation Y = 0, 0.23, 0.46, 0.72, 0.99). Because of the temperature‐sensitivity of the sample, the use of a special flow device became necessary to keep the time of exposure of the samples to 40° and to X‐ray irradiation below 1 h.

Apo‐enzyme revealed a contraction in volume depending upon the degree of saturation. Fully saturated the contraction amounts to 7%. As the observed degree of contraction is not linearly proportional to the degree of saturation, the simple sequential mechanism cannot explain the observations. To the extent that they coincide with the quantitative predictions of the allosteric model for this particular enzyme, based on calculations involving the equilibrium constants obtained from chemical relaxation experiments under identical conditions, they strongly support the latter mechanism.

Comparison of the experimental scattering curves with theoretical scattering curves of compact full cylinders did not bring about satisfactory agreement in the range of the first submaximum. Hollow circular cylinders with h:2r1= 0.6:1 (apo‐enzyme) or h:2r1= 0.58:1 (holo‐enzyme) may be regarded as sufficiently equivalent in scattering as far as the form of the main maximum is concerned as well as the position and relative intensity of the submaximum. Even if the molecule consists of four subunits they consequently must be arranged in such a way that the entire particle is very similar to a hollow cylinder.

The experimental determination of the radius of gyration (R= 32.1 Å in the case of the apo‐enzyme, or 31.7 Å in the case of the holo‐enzyme) leads to the calculation of the absolute particle dimensions: Apo‐enzyme: h= 48.5 Å, r1= 40.4 Å, r2= 6.1 Å, Holo‐enzyme: h= 46.4 Å, r1= 40.2 Å, r2= 6.0 Å.

The nature of the structural changes occuring in the allosteric transition can now be described in terms of an increase of the inherent anistropy and of a considerable decrease in the solvent included in the tetrameric assembly.

Exposure of the enzyme to 40° over a longer period of time leads to significant alterations in the scattering curves. These can be interpreted on the basis of growing aggregates in polydisperse solutions. This process is accompanied by a considerable decrease of enzyme activity.
Effects of Hydroxyurea and 1‐β‐D‐Arabinofuranosyl‐cytosine on Deoxyribonucleotide Pools in Mouse Embryo Cells
Authors: Skoog Lambert , NordenskjÖLD Bo
Published: March 2005

Mouse embryo cells incubated in medium with 0.5% serum contain only few cells which synthesize DNA. When the serum concentration was raised, DNA synthesis was induced and the pools of the deoxyribonucleoside triphosphates expanded. In such cultures the pool of dGTP was the smallest of the deoxyribonucleoside triphosphate pools and its size corresponded to only approximately 30 sec of DNA synthesis. The dCTP pool was 10–20 times larger than the dGTP pool.

Addition of hydroxyurea resulted in an almost instantaneous loss of the pool of dGTP and also in a decrease in the size of the dATP pool. In contrast the pools of dCTP and dTTP increased. However, the turnover of the dTTP pool was greatly diminished.

Removal of hydroxyurea from the medium resulted in a rapid normalization of the triphosphate pools but only in a more gradual restoration of DNA synthesis.

During incubation in 1‐β‐D‐arabinofuranosyl‐cytosine (arabinosyl‐cytosine), the pools of dATP, dGTP and dTTP expanded, while the pool of dCTP showed an initial decrease. The turnover of the dTTP pool was not markedly affected in spite of the fact that DNA synthesis was greatly reduced. When arabinosyl‐cytosine was removed the pools were normalized and DNA synthesis was slowly restored.

Our results thus show that the inhibition of DNA synthesis by hydroxyurea is correlated with diminishing pools of dGTP and dATP and a decreased turnover of dTTP, indicating a generally decreased deoxyribonucleotide formation presumably due to the inhibition of the enzyme ribonucleoside‐diphosphate reductase. The inhibition of DNA synthesis by arabinosyl‐cytosine is not correlated with a similar depletion of deoxyribonucleoside triphosphate pools.
Temperature‐Jump Relaxation Studies on Transfer Ribonucleic Acids
Authors: Dourlent Maurice , Hélène Claude , Yaniv Moshe
Published: March 2005

Equilibrium and kinetic measurements of the thermal denaturation of tRNA1Val and tRNA2Tyr from Escherichia coli have been obtained by absorption melting curves and temperature‐jump experiments. Besides a very fast effect attributed to changes in stacking equilibria, several relaxation times in the millisecond range are observed at 260 nm. The incomplete separation of the different relaxation times suggests a complex process, in agreement with the equilibrium data. At 335 nm, where the absorption is due only to 4‐thiouridine, the melting profile is monophasic and for both tRNAs only one relaxation time is observed in the millisecond range. The thermodynamic parameters derived from the results obtained at this wavelength indicate a cooperative melting of short double helical sequences. The results are discussed on the basis of the clover‐leaf model.