bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and Properties of Structured Chromatin from Guerin Ascites Tumour and Rat Liver
Authors: YANEVA Mariana , DESSEV George
Published: March 2005

The method proposed by Hancock for isolation of structured chromatin from tissue culture cells is modified and used for isolation of chromatin from Guerin ascites tumour and rat liver. Micrococcal nuclease digestion patterns and thermal denaturation of these chromatins are studied and cornpared with those of chromatins prepared by precipitation and extraction with salts (salt chromatins). In contrast to the multiphasic melting profiles of salt chromatins, the structured chromatins exhibit relatively homogeneous denaturation patterns under a variety of conditions, suggesting that their DNA is uniformly stabilized by histones and there are no free independently melting DNA stretches. Digestion of structured chromatin with micrococcal nuclease yields a characteristic pattern of fragments, showing that the subunit structure of the deoxyribonucleoprotein is intact. No discrete fragments are formed upon digestion of salt chromatin.
Somatic Antigens of Shigella
Authors: DMITRIEV Boris A. , KNIREL Yuriy A. , KOCHETKOV Nikolay K. , HOFMAN Irihna L.
Published: March 2005

The O‐specific polysaccharide obtained from the lipopolysaccharide of Shigella dysenteriae type 1 (Shigella shiga) by mild acid hydrolysis followed by fractionation on Sephadex G‐50 was found to be identical to that described by Morgan's group and was composed of l‐rhamnose, d‐galactose and N‐acetyl‐d‐glucosamine in a ratio 2:1:1. On the basis of methylation analysis data the polysaccharide was proved to be a linear chain of monosaccharide residues in pyranose forms substituted at position 3, except for that of galactose substituted at position 2.

Selective cleavage, based on the N‐deacetylation reaction of the polymer, together with determination of linkage configurations by chromic anhydride oxidation showed that the O‐specific polysaccharide is built up of repeating tetrasaccharide units whose proposed structure is given below

‐3)‐α‐l‐Rhap(1–3)‐α‐l‐Rhap(1–2)‐α‐d‐Galp(1–3)‐α‐d‐GlcNAcp(1‐ where Rhap = rhamnopyranose, Galp = galactopyranose, and GlcNAcp =N‐acetyl‐glucosamine.

The present findings confirmed the considerations of Heidelberger on the substitution patterns of l‐rhamnose and d‐galactose residues from the results of serological studies.
The Binding of Glycoconjugates to Human‐Milk d‐Galactosyltransferase
Authors: PRIEELS Jean‐Paul , DOLMANS Marcel , SCHINDLER Melvin , SHARON Nathan
Published: March 2005

Through the use of affinity chromatography, a homogeneous preparation of human β(1 → 4)‐d‐galactosyltransferase (the A protein of lactose synthase) was obtained. The specificity of this protein for glycoconjugates was studied in the presence and absence of human α‐lactalbumin.

A kinetic analysis of the transfer of d‐galactose to N‐acetyl‐d‐glucosamine and to β(1 → 4)‐linked N‐acetylglucosamine oligomers, suggested that the active site region of the enzyme contains more than one binding site for acceptor molecules. Furthermore, experiments with N‐acetylglucosamine‐β(1 → 4)‐N‐acetylmuramic‐pentapeptide isolated from Micrococcus luteus indicated that the presence of a peptide chain does not enhance enzymic activity, as compared with the corresponding free disaccharide. Similar results were obtained using ovalbumin and the ovalbumin glycopeptide (which have similar apparent Km values for A protein) as galactose acceptors. In contrast to its ability to inhibit N‐acetyllactosamine production, α‐lactalbumin did not inhibit the transfer of d‐galactose to the N‐acetylglucosamine oligomers or the glycopeptides.

Although α‐lactalbumin can switch the specificity of A protein from N‐acetyl‐d‐glucosamine to d‐glucose resulting in the production of lactose, no transfer of galactose was observed to β(1 → 4)‐linked glucose oligomers or to a collagen glycopeptide, d‐glucopyranosyl‐α(1 → 2)‐d‐galactopyranosyloxy‐β(1 → 5)‐lysine. It therefore appears that α‐lactalhumin can only modify human A protein for monosaccharide acceptors.
Developmental Changes in Prolyl Hydroxylase Activity and Protein in Chick Embryo
Authors: TUDERMAN Leena
Published: March 2005

Changes in prolyl hydroxylase activity and immunoreactive protein were studied in various chick embryo tissues during the embryonic development. Both the enzyme activity and the amount of immunoreactive protein increased till the 16th day of development and declined thereafter in all tissues studied. Comparison of the enzyme activity to the content of the total immunoreactive protein indicated that there are distinct differences in the degree of enzyme activity between different chick embryo tissues, and in the same tissue betweenn different stages of embryonic development. The highest relative enzyme activities were found in cartilage and skin, in which about 60%, of the enzyme was active on the 16th day of development and only 20–30% was active on the 20th day of development; the lowest values were observed in spleen and large vessels, in which below 10% of the enzyme protein was in the active form on the 20th day of development. Gel filtration studies demonstrated that in cartilage of 16‐day‐old chick embryos about 60% of the total immunoreactive enzyme in the tissue was present in the form of active prolyl hydroxylase tetramer, whereas on the 20th day of development only 30% of the enzyme protein in cartilage was in the tetramer form. By contrast, in large vessels of the 16‐day‐old chick embryos, essentially all the enzyme was in the form of prolyl hydroxylase monomers.
Continuous Measurement and Rapid Kinetics of ATP Synthesis in Rat Liver Mitochondria, Mitoplasts and Inner Membrane Vesicles Determined by Firefly‐Luciferase Luminescence
Authors: LEMASTERS John J. , HACKENBROCK Charles R.
Published: March 2005

Oxidative phosphorylation in rat liver mitochondria, the mitoplast or inner membrane‐matrix fraction, and inverted inner membrane vesicles was studied with firefly luciferase luminescence. All preparations showed relatively high ATP/O ratios under initial reaction conditions in the presence of Mg2+, but only intact mitochondria demonstrated significant respiratory control. Mitochondria and mitoplasts, but not vesicles, catalyzed nearly complete conversion of ADP to ATP. Vesicles converted between 50% and 70% of added ADP to ATP establishing a phosphate potential of 9.8 to 10.5 kcal/mol (43.9 kJ/mol). In “loosely coupled” submitochondrial systems which do not display respiratory control, the phosphate potential appears to be a useful parameter of the integrity of the phosphorylating membrane, while ATP/O ratios may be dependent on the relative concentrations of ADP and ATP in the reaction medium.

The rapid kinetics of oxidative synthesis of ATP by inverted inner membrane vesicles was investigated in stopped‐flow rapid‐mixing experiments. After an oxygen pulse to reduced vesicles, ATP synthesis commenced within 20 ms. In antimycin‐inhibited vesicles, ATP synthesis resulting from the rapid oxidation of the terminal portion of the respiratory chain commenced within 20 ms and took approximately 100 ms for half completion. Since half oxidation of the terminal portion of the respiratory chain occurs within a few milliseconds, ATP synthesis resulting from such redox reactions must occur over a significantly longer time course than the redox reactions themselves.
Geometry of the Protein S4 from Escherichia coli Ribosomes
Authors: PARADIES H. Hasko , FRANZ Alfred
Published: March 2005

The shape of protein S4 from Escherichia coli ribosomes in solution was determined by hydrodynamic methods and low‐angle X‐ray scattering. The molecular weight of 24000 determined by low‐angle X‐ray scattering is within 3% of that found by sedimentation equilibrium analysis and 8% of that determined by amino acid sequence work. The radius of gyration of 3.36 nm. the radius of gyration of the cross section of 0.41 nm and the hydrodynamic studies revealed that protein S4 is not spherical, but rather has a markedly extended shape. Calculations of different conformations, e.g. random coil, based on the parameters evaluated from hydrodynamic methods, revealed a rod‐like structure of S4 with a length of 14 nm and a diameter of 1 nm. This is supported by a model of an equivalent scattering particle of uniform density based on all parameters obtained in this study.
Purification, Subunit Structure and Partial Amino‐Acid Sequence of Anthranilate‐5‐phosphoribosylpyrophosphate Phosphoribosyltransferase from the Enteric Bacterium Serratia marcescens
Authors: LARGEN Michael , MILLS Stanley Ely , ROWE Joan , YANOFSKY Charles
Published: March 2005

The enzyme anthranilate‐5‐phosphoribosylpyrophosphate phosphoribosyltransferase from Serratia marcescens was purified to apparent homogeneity. The purification procedure included ammonium sulfate precipitation, DEAE‐cellulose chromatography, Sephadex gel filtration and hydroxyapatite chromatography.

The molecular weight of the native protein as determined on a calibrated Sephadex G‐200 column was 45000. Dodecylsulfate‐polyacrylamide gel electrophoresis in the presence of reducing agent revealed a subunit molecular weight of 43000 ± 900, suggesting that the enzyme exists as a monomer.

The sequence of the amino‐terminal 38 residues revealed that three amino acids, glutamine (six residues), glutamic acid (five residues) and serine (five residues) comprised 42% of the sequence composition.
The Role of Escherichia coli dnaG Function in Coliphage M 13 DNA Synthesis
Authors: DASGUPTA Santanu , MITRA Saukar
Published: March 2005

Examination of the role of Excherichia coli dnaG function in different stages of M 13 phage DNA synthesis by ultracenuifugal analysis of intracellular phage DNA in a thermosensitive dnaG mutant shows that: (a) the formation of parental double‐strand replicative‐form DNA (rfDNA) from the infecting virus is independent of dnaG function; (b) the synthesis of progeny rlDNA requires dnaG product: (c) after a pool of rfDNA is made up, dnaG function is not required for the progeny single strand DNA (ssDNA) synthesis. The ssDNAs produced under nonpermissive condition are mostly circular and biologically functional.
The O9 Antigen of Escherichia coli
Authors: PREHM Peter , JANN Barbara , JANN Klaus
Published: March 2005

The lipopolysaccharide from Escherichia coli O9: K30 was isolated in about 2% yield with aqueous 45% phenol at 65 C, followed by ultracentrifugation. The polysaccharide moiety was obtained by graded hydrolysis and gel permeation chromatography. It consisted of a mannan which carried oil its reducing end the core oligosaccharide of the R1 type. The mannan contained 1 → 2 and 1 → 3 linkages in a ratio of 3:2 as determined by methylation analysis and mass spectrometry. On periodate oxidation, 58% of the mannose residues were destroyed.

Degradation of oligosaccharide mixtures with α‐mannosidase from jack bean meal, as well as a specific rotation of [α]25D=+ 89° indicated that all mannosyl linkages have the α‐configuration.

Smith degradation resulted in the liberation of mannosyl(1 → 3)‐mannose (bound to glyceraldehyde), as established by methylation analysis.

From these results we conclude that the O9 polysaccharide of E. coli has a pentasaccharide repeating unit of α‐mannosyl(1 → 3)‐α‐mannosyl‐(1 → 2)‐α‐mannosyl‐(1 → 2)‐α‐mannosyl‐(1 → 2)‐mannose, which are joined in the polysaccharide through α‐(1 → 3)‐mannosyl linkages.
The Role of Interchain Disulphide Bridges in the Conformational Stability of Human Immunoglobulin G1 Subclass
Authors: VENYAMINOV Sergei Yu. , RAJNAVÖLGYI Éva , MFDGYESI György A. , GERGELY János , ZÁVODSZKY Péter
Published: March 2005

The hydrogen‐deuterium exchange data of human immunoglobulin G1 (IgG 1) are interpreted by assuming fast fluctuations of the protein conformation, through which the peptide groups become exposed to the solvent. The probability of solvent exposure of peptide hydrogens reflects a rather loose conformation for native IgG in comparison with other globular proteins. The probability of solvent exposure is greater than 10‐3 for half of the peptide groups, which shows that the conformational transitions by which these groups are exposed to the solvent are accompanied by changes in standard free energy less than 17 kJ/mol (4 kcal/mol).

In the range of pH 6.2–8.45, at 25 °C no gross conformational changes are reflected in the hydrogen‐deuterium exchange behaviour of the native, the reduced‐nonalkylated‐reassociated and the reduced‐S‐alkylated‐reassociated IgG 1.

No difference could be detected in the conformational stability of the native and reoxidised reassociated IgG1 proteins. The lack of inter‐subunit disulphide bridges in S‐alkylated‐reassociated molecules results in all increased conformational motility. This destabilization of protein conformation affects about 90% of the peptide groups covered by the measurements, and corresponds to changes in standard free energy of 8 kJ/mol on the average.
A Comparison of Some Kinetic Properties of Soluble and Bound Lactate Dehydrogenase Isoenzymes at Different Temperatures
Authors: NITISEWOJO Poedijono , HULTIN Herbert O.
Published: March 2005

A comparison was made of some kinetic properties of three chicken lactate dehydrogenase iso‐enzynmes (1, 3 and 5) at 4, 16, 23 and 40 °C. Assays were performed with an enzyme concentration of 0.01 μM at pH 6.0. Under the conditions of assay, lactate dehydrogenase 3 and 5 bound to the particular fraction of homogenized skeletal muscle and were evaluated in the soluble and particulate state.

Binding of isoenzymes 3 and 5 to the cellular particulate fraction decreased V. This decrease was much greater for lactate dehydrogenase 5 than 3. Values of V for lactate dehydrogenase isoenzymes 1 and 3 did not follow a simple Arrhenius relationship; there was a rapid change in activity between 16 and 23 °C.

The apparent Km (pyruvate) values of all isoenzymes (bound or soluble) increased with increasing temperature, changing 4—10‐fold. The apparent Km for lactate dehydrogenase 5 was greater than that for lactate dehydrogenase 3, which in turn was greater than that for lactate dehydrogenase 1.
Raffinose Metabolism in Escherichia coli K12
Authors: SCHMID Kurt , SCHMITT Rüdiger
Published: March 2005

The utilisation by Edcherichia coli K12 of raffinose as sole carbon source depends on a new raffinose transport system, an invertase and an α‐galactosidase specified by the Raf‐plasmid D1021. The α‐galactosidase was purified to homogeneity from a mutant strain with constitutive synthesis of the enzyme, α‐Galactosidase hydrolyzes p‐nitrophenyl‐α‐d‐galactoside (Km 0.14 mM), methyl‐α‐d‐galactoside (Km 30mM), melibiose (Km, 3.2 mM) and rallinose (Km 60 mM). The enzymatic activity is strongly inhibited by Ag+, p‐chloromercuriphenyl sulfonic acid and, to a lesser extent, by iodoacetamide. Isoelectric focusing indicates the existence of one form of α‐galactosidase with an isoelectric point of 5.1. The purified enzyme has an SW,2o value of 11.7 ± 0.3S and a molecular weight of 329000 ± 4000; this value is not reduced at high dilutions. When examined by dodecylsulphate gel electrophoresis, purified α‐galactosidase yields a single subunit band of molecular weight 82000 suggesting that the intact enzyme consists of four subunits. Amino acid analysis indicates the presence of approximately 712 amino acid residues per quarter molecule including 8 half‐cystine residues. No carbohydrate moiety has been detected. High resolution electron micrographs and Markham rotation of α‐galactosidase show enzyme molecules of approximately 11 × 11 nm containing four globular subunits in a tetragonal arrangement.

The plasmid‐coded α‐galactosidase differs from the homologous E. coli enzyme by substrate affinities. cofactor requirements, stability and toluene resistance. It can, therefore, be used as a marker enzyme suitable for the detection in vivo of Raf‐plasmids
Quantitative Study of Secondary Structure of Histones H1, H2A, and H4 in Solution by Infrared Spectroscopy
Authors: SHFSTOPALOV Boris V. , CHIRGADZI Yu. N.
Published: March 2005

The secondary structure of histories H1, H2A, and H4 (F1, F2a2, and F2a1) has been quantitatively studied in heavy water (2H2O) solutions in a wide range of histone concentration, p2H, and concentration of sodium chloride using an improved infrared spectroscopy method. Under all conditions there are about 5–10% of α helix. Conditions favourable for aggregation induce formation of antiparallel pleated sheet structure to an extent of about 15% in H1 and H2A and about 30% in H4. When the p2H and concentration of NaCl are in the physiological range, there is the sane content of this structure in H2A and H4 and none in HI.
The Structure of Pentasaccharides and Hexasaccharides from Blood Group Substance H
Authors: KOCHETKOV Nicolai K. , DEREVITSKAYA Varvara A. , ARBATSKY Nicolai P.
Published: March 2005

Six individual reduced pentasaccharides and hexasaccharides were isolated after sodium borohydride degradation of blood group substance H from pig stomach linings. Their structure was determined by means of periodate oxidation, methylation analysis, mass spectrometry, partial acid and enzymic hydrolysis and chromic anhydride oxidation. It has been found that all the oligosaccharides investigated contain the common tetrasaccharide core to which terminal α‐l fucose and N‐acetyl‐α‐D‐glucosamine residues are attached. Some problems of carbohydrate chain heterogeneity in blood group substances are discussed.
The Inhibition of Isocitrate Oxidation by Palmitoyl‐l‐carnitine and Palmitoyl‐CoA in Rat Liver Mitochondria
Authors: LENARTOWICZ Ewa , WINTER Christa , KUNZ Wolfgang , WOJTCZAK Anna B.
Published: March 2005

Palmitoyl‐l, carnitine decreases the oxidation of isocitrate in rat liver mitochondria in state 3 by 25–30% Palmitoyl‐l‐carnitine acts as an additional substrate raising the‐rate of oxidative phosphorylation, NAD reduction and ATP/ADP ratio in mitochondria.

Palmitoyl‐CoA added to mitochondria oxidizing isocitrate in state 3 causes a strong inhibition of isocitrate oxidation and of oxidative phosphorylation and a considerable elevation of intramitochondrial NADH/NAD and ATP/ADP ratios. The effect of palmitoyl‐CoA is dependent on its concentration and is competitive with ADP. Carnitine restores only oxidative phosphorylation, but the oxidation of isocitrate remains inhibited. Evidence is presented that the transport of isocitrate is not affected by palmitoyl‐CoA in this case. From these results it can be concluded that the action of palmitoyl‐CoA is due to the inhibition of adenine nucleotide translocation.

The kinetic studies of NAD‐dependent isocitrate dehydrogenase in the soluble fraction of sonicated mitochondria revealed that the enzyme is very sensitive towards the inhibition by NADH and only very slightly affected by ATP (Ki for NADH and ATP are 0.017 and 3.6 mM respectively). On the basis of the kinetic data the relative contribution of NADH and ATP in the inhibition of isocitrate oxidation by fatty acids was calculated.

It is concluded that the inhibition of isocitrate oxidation caused by palmitoyl‐l‐carnitine and palmitoyl‐CoA is primarily due to the increased reduction of NAD, whereas the increase of ATP/ADP ratio is much less important.
Effects of Antibodies to Various MÒlecular Forms of a Mutationally Altered Escherichia coli Alkaline Phosphatase on Its Activation by Zinc
Authors: PAGES Jean‐Marie , VARENNE Stanislas , LAZDUNSKI Claude
Published: March 2005

Immunogenic and antigenic properties of a Zn2+‐deficient alkaline phosphatase produced in a mutant (U‐47) of Escherichia coli have been studied. The native U‐47 enzyme, that exists in monomerdimer equilibrium, was used as immunogen. From the antisera obtained, four antibody populations directed to the various molecular forms of U‐47 enzyme have been purified by affinity chromatography using specific antigens coupled toglutaraldehyde‐activated beads of indubiose. 70% of the total antibody obtained was directed both to the monomeric and the dimeric forms. 9% was directed to the dimer but showed a low affinity for the monomer; 10% and 11% were specifically directed respectively to the monomer and the dimer. Each antibody population purified had a specific effect on the catalytic activity of the Zn2+‐activated U‐47 enzyme. The anti‐monomer‐dimer and the anti‐dimer‐monomer inhibit to the same extent whereas the specific anti‐monomer does not alter the activity significantly and the specific anti‐dimer causes a 30% activation. The catalytic activity of the alkaline phosphatase produced in wild‐type strains was also reduced by these anti‐U‐47 enzyme antibodies. However, whereas the anti‐monomer had again very little effect, the anti‐dimer‐monomer and the anti‐monomer‐dimer inhibited this enzyme to different extents. The specific anti‐dimer also inhibited this wild‐type alkaline phosphate. Antibodies of high affinity to the dimeric form of U‐47 enzyme, i.e. specific anti‐dimer or anti‐dimer‐monomer, caused a 30% activation when they were added prior to the reactivation process by Zn2+. Specific anti‐monomer strongly inhibited this reactivation process. The Fab fragment of the anti‐wild‐type phosphatase antibody, under the same conditions, caused a 300% activation. The extents of interactions of the various molecular forms of U‐47 enzyme and of the wild‐type enzyme with the anti‐monomer‐dimer and with the anti‐dimer have been determined. U‐47 enzyme monomeric form has three determinants exposed and the dimeric form has five determinants exposed for interacting with the anti‐monomer‐dimer antibody, the free wild‐type enzyme has only two determinants exposed to this antibody. These determinants might be close to the active site or in another critical location since this antibody can reduce the catalytic activity of the wild‐type enzyme to half the original value. The anti‐dimer antibodies can interact with three determinants exposed at the surface of the free Zn2+‐reactivated U‐47 enzyme and the non‐covalent binding of one mole of inorganic phosphate results in the exposure of one more antigenic determinant.
l‐Phenylalanine: tRNA Ligase of Escherichia coli K10
Authors: PIMMER Jutta , HOLLER Eggehard , ECKSTEIN Fritz
Published: March 2005

The Km and V values of the tRNA‐charging reaction have been measured for l‐phenylalanine: tRNA ligase and the geometric isomers of adenosine 5′‐O‐(1‐thio)triphosphate, adenosine 5′‐O‐(2‐thio)triphosphate and for 5′‐O‐(3‐thio)triphosphate. All ATP analogs were found to be substrates with values of Km similar or (up to 10‐fold) higher, and with values of V in the range of 10–30% compared with the natural substrate.

The dissociation constants of the binary enzyme · nucleotide and ternary enzyme · nucleotide ·l‐phenylalaninol complexes were analysed as a function of the position of the sulfur atom indicating those phosphate groups which are involved in an enzyme‐triphosphate interaction. The results are consistent with a participation of the β and γ‐phosphate in the binary complex formation and an additional interaction at the positions of the α and β‐phosphate groups in the ternary complexes.
Affinity Chromatography of Collagen Glycosyltransferases on Collagen Linked to Agarose
Authors: RISTELI Leila , MYLLYLÄ Raili , KIVIRIKKO Kari I.
Published: March 2005

Denatured citrate‐soluble collagen was coupled to agarose by the cyanogen bromide activation technique, and columns prepared from this material were studied for affinity chromatography of collagen glycosyltransferases. Both collagen glycosyltransferases became bound to the column, the degree of binding and the capacity of the column being higher with the glucosyltransferase than with the galactosyltransferase. The addition of Mn2+ enhanced the binding, especially with the glucosyltransferase. The enzymes were eluted from the column with small peptides prepared from collagen, and they were separated from the peptides by gel filtration. With this procedure a collagen glucosyltransferase purification of about 5000‐fold and a collagen galactosyltransferase purification of about 1000‐fold was obtained from chick embryo extract by relatively simple steps.
Synthesis of the Brain‐Specific S‐100 Protein in a Cell‐Free System from Wheat Embryo Programmed with Poly(A)‐Containing RNA from Rabbit Brain
Authors: MAHONY James , BROWN Ian , LABOURDETTE Gerard , MARKS Alexander
Published: March 2005

Polyadenylated polysonial RNA was prepared from rabbit cerebral hemispheres using phenol extraction and chromatography on oligo(dT)‐cellulose. This RNA directed the synthesis of the brain‐specific S‐100 protein in cell‐free extracts front wheat embryo. S‐100 protein was absent from the products of endogenous incorporation and from a reaction programmed with kidney mRNA. These results suggest that S‐100 protein mRNA contains a poly(adenylic acid) sequence and rule out the necessity of a brain‐specific factor for translation of S‐100 protein mRNA.
Structural Determination of Three Glycoasparagines Isolated from the Urine of a Patient with Aspartylglycosaminuria
Authors: LUNDBLAD Arne , MASSON Parvesh K. , NORDÉN Nils E. , SVENSSON Sigfrid , ÖCKERMAN Per‐Arne , PALO Jorma
Published: March 2005

Three different glycoasparagines have been isolated from the urine of a patient with aspartylglycosaminuria and their structures determined using sugar, amino acid and methylation analysis, enzymic degradation and measurements of the optical rotations. The structures were 2‐acetamido‐1‐N‐(4′‐L‐aspartyl)‐2‐deoxy‐β‐d‐glucopyranosylamine(yield 135 mg/l) β‐d‐galactopyranosyl‐(1 → 4)‐2‐acetamido‐1‐N‐(4′‐L‐aspartyl)‐2‐deoxy‐β‐D‐glucopyranosylamine (yield 35 mg/l), and α‐D‐mannopyranosyl‐(1 → 6)‐β‐d‐mannopyranosyl‐(1 → 4)‐2‐deoxy‐β‐d‐glucopyranosyl‐(1 → 4)‐2‐acetamido‐1‐N‐(4′‐L‐aspartyl)‐2‐deoxy‐β‐d‐glucopyranosylamine (yield 30 mg/l). The first two compounds have previously been described, whereas the third compound is different from any of the glycoasparagines isolated before.
Glutathione Reductase from Human Erythrocytes
Authors: WORTHINGTON David J. , ROSEMEYER Michael A.
Published: March 2005

The catalytic properties of glutathione reductase from human erythrocytes have been studied over a range of buffer conditions and substrate concentrations. This study provides cptimal conditions for determinime the basic kinetic parameters of the enzyme.

The catalytic behaviour of glutathione reductase is consistent with spatially separated binding sites for its substrates.

In certain assays anomalies were observed which are correlated with an inactivation of the enzyme by NADPH. Concurrent sedimentation experiments showed that NADPH promoted aggregation of the enzyme. Both inactivation and aggregation could be connected with oxidation of thiols at the active site.

The relation of the properties of glutathione reductase to cellular conditions is discussed.
Polypeptide‐Chain Elongation Promoted by Guanyl‐5′‐yl Imidodiphosphate
Authors: GIRBES Tomás , VAZQUEZ David , MODOLELL Juan
Published: March 2005

In a purified system from Escherichia coli containing ribosomes complexed with poly(uridylic acid) and N‐acetyl‐phenylalanyl‐tRNA, the nonhydrolyzable analog of GTP, guanyl‐5′‐yl imidodiphosphate (Guo‐5′‐P2‐NH‐P), promotes polypeptide synthesis at a rate several times slower than GTP. The activity is completely dependent on elongation factors EF‐T (i.e. EF‐Ts + EF‐Tu) and EF‐G. Examination of individual steps of the elongation cycle in partial reactions shows that Guo‐5′‐P2‐NH‐P is as efficient as GTP in promoting the EF‐T‐dependent binding of phenylalanyl‐tRNA to the ribosomal A site. In contrast, Guo‐5′‐P2‐NH‐P promotes the translocation‐dependent binding of phenylalanyl‐tRNA to a ribosome complexed with A‐site‐bound N‐acetyl‐phenylalanyl‐tRNA much more slowly than GTP. This slow rate of binding is due to the presence of EF‐G on the ribosome, and not to sluggish translocation, since (a) the rate remains slow even after translocation of N‐acetylphenylalanyl‐tRNA is completed, (b) it is greatly speeded up by removal of EF‐G from the reaction mixture (after translocation has occurred), and (c) it is slowed down again by readdition of the factor. Moreover, with post‐translocated ribosomes and in the absence of EF‐G, formation of dipeptide subsequent to the EF‐T‐dependent binding of phenylalanyl‐tRNA is much slower when binding of this substrate has been promoted by Guo‐5′‐P2‐NH‐P than it is when promoted by GTP. The results suggest that, during polymerization with Guo‐5′‐P2‐NH‐P, EF‐G and EF‐Tu are slowly released from the ribosome and, consequently, the steps of the elongation cycle subsequent to translocation and aminoacyl‐tRNA binding (aminoacyl‐tRNA binding and peptide bond formation, respectively) are delayed. Thus, during the elongation cycle, GTP hydrolysis is probably essential for fast release of the factors from the ribosome.
The Keratin Chains of Avian Scale Tissue
Authors: WALKER Ian D. , BRIDGEN John
Published: March 2005

The three major proteins of chick scale keratin were isolated as their S‐carboxymethylated derivatives and shown to be similar or identical in molecular weight by gel filtration but to he distinct by amino acid analysis and gel electrophoresis. The major amino‐terminal sequence of scale keratin chains was determined and shown to be highly homologous to the corresponding region of feather keratin chains. The carboxyl‐terminal peptides of the three scale keratin fractions differed in sequence but were all homologous to the carboxyl‐terminal segment of feather keratin chains. The pronounced concentration of cysteine residues at the amino‐terminal and carboxyl‐terminal segments suggested a similar role for these regions in both scale and feather keratin chains, namely to provide a disulphidelinked matrix to maintain the organisation of fibrils which arise from the internal hydrophobic segments of both types of chain. Analysis of a large hydrophobic segment from each of the three isolated protein fractions revealed that each was composed largely of repeating tripeptide units of the type Gly‐Gly‐X (where X = Phe, Leu or Tyr). At a few positions in each hydrophobic peptide, microheterogeneity was apparent in the sequences indicating that each isolated protein fraction was composed of at least three different chains each encoded by a different gene. A minimum of nine keratin genes are therefore expressed in scale tissue.
Localization and Possible Role of an Adenosine Triphosphatase in Chlorobium thiosulfatophilum
Authors: BURNS David D. , MIDGLEY Melvin
Published: March 2005

Evidence is presented that the ATPase activity detected in cell extracts of Chlorobium thiosulfatophilum is bound to the cytoplasmic membrane rather than to the chlorobium vesicles.

The activity of this ATPase is inhibited in vitro by various carbodiimides, phloridzin and sodium azide.

The apparent Km for ATP is ∼ 0.2 mM and the enzyme shows product inhibition by ADP.

Photophosphorylation, characterized in vivo, is inhibited by many of the compounds that in hibit the ATPase.
Synthesis of a Class of DNA‐Binding Proteins in Synchronized Untransformed and Virus‐Transformed Cells
Authors: MELERO José A. , SALAS Maria L. , SALAS José
Published: March 2005

The synthesis of a class of proteins with affinity for denatured DNA has been studied in synchronized cultures of the established hamster fibroblast line NIL‐1 and its virus‐transformed derivative NIL‐1‐hamster sarcoma virus. It has been found that the synthesis of a DNA‐binding protein (P6, molecular weight 50000) in synchronized untransformed NIL‐1 cells follows a pattern different from that observed in the transformed cells. The protein is low in stationary cultures of NIL‐1 and NIL‐1‐hamster sarcoma virus but increases after the cells are stimulated to grow, although the time of maximal P6 synthesis relative to cellular DNA synthesis is different in NIL‐1 and NIL‐1‐hamster sarcoma virus. In contrast, the pattern of synthesis of two other DNA‐binding proteins (P7 and P8′) is essentially identical in synchronized untransformed and transformed cells. P7 is very low in resting cultures of both types of cells, but greatly increases early after the cells are stimulated to divide, while P8′ is high in stationary cultures and decreases slightly after the initiation of cellular DNA synthesis has occurred.
Use of a Sequence‐Specific DNA‐Binding Ligand to Probe the Environments of EcoRI Restriction Endonuclease Cleavage Sites
Authors: KANIA Jürgen , FANNING Thomas G.
Published: March 2005

The DNAs of bacteriophage lambda and adenovirus were incubated with the sequence‐specific DNA‐binding ligand 6,4′‐diamidino‐2‐phenylindole. Digestion of the ligand DNA complexes with EcoRI nuclease and subsequent agarose gel electrophoresis demonstrated that the ligand inhibited nuclease activity at some sites, but not at others. The results suggest that.diamidino‐2‐phenylindole can be used to probe the immediate environments of the EcoRI cleavage sites.
Binding of Aminoacyl‐tRNA to Reconstituted Subparticles of Escherichia coli Large Ribosomal Subunits
Authors: KAZEMIE Mirabotalib
Published: March 2005

The activity of 50‐S subunits to stimulate the binding of aminoacyl‐tRNA to the 30‐S subunits was lost when the particles were washed with LiCl concentrations higher than 1.0 M [Kazemie, M. (1975) Eur. J. Biochem. 58, 501–510]. The particles could regain this activity when they were incubated with the corresponding LiCl washes. This effect of LiCI washes was used as an assay for identification of 50‐S subunit proteins which are essential for binding of aminoacyl‐tRNA.

A protein mixture containing mainly L1, L11 and L16 can reactivate 50‐S ‘core’ particles, consisting of 10, proteins and 23‐S RNA, to bind aminoacyl‐tRNAs in the presence of 30‐S subunits. Besides 5‐S RNA, protein L24 has a stimulatory effect on the binding of aminoacyl‐tRNA. Proteins L2, L20 and L23 are possibly required for maintaining the 50‐S subunits in the correct conformation for binding of aminoacyl‐tRNAs.
Structural Studies of Chicken Erythrocyte Histone H5
Authors: CRANE‐ROBINSON Colyn , DANBY Shirley E. , BRADBURY E. Morton , GAREL Annie , KOVACS Anne‐Marie , CHAMPAGNE Madeleine , DAUNE Michel
Published: March 2005

Spectroscopic studies (nuclear magnetic resonance, circular dichroism and infrared) have been carried out on chicken erythrocyte histone H5 and on three peptides cleaved therefrom: 1‐31, 32‐197 and 58‐197. It is shown that at ionic strengths above 0.1 M part of the H5 molecule takes up a globular conformation containing 14% a helix but no β sheet structure. Several details of the circular dichroism and nuclear magnetic resonance spectra indicate that the globular region is located in the N‐terminal half of the molecule and this proposal is supported by the observation that the peptide 32–197 is largely incapable of folding and the peptide 59–197 is completely incapable of folding. Structural similarities and differences between histone H5 and histone H1 are discussed.
Calcium Regulation in Heart Cells
Authors: AFFOLTER Hubert , CHIESI Michele , DABROWSKA Renata , CARAFOLI Ernesto
Published: March 2005

A study has been carried out of the interaction of dog heart mitochondria and sarcoplasmic reticulum with Ca2+‐bound to troponin or to its Ca2+‐binding component (troponin‐C). Both organelles are able to release more than 80% of the Ca2+ bound to the two proteins. However, sarcopiasmic reticulum is only able to do so in the presence of oxalate, whereas mitochondria are active also in the absence of permeant Ca‐complexing anions. On the other hand, calcium uptake in heart mitochondria is severely inhibited by Mg2+. The results are discussed with reference to the problem of the control of Ca2+ in heart sarcoplasm.
Acetyl‐CoA Hydrolase; Activity, Regulation and Physiological, Significance of the Enzyme in Brown Adipose Tissue from Hamster
Authors: BERNSON Vibeke S. M.
Published: March 2005

Acetate production in hamster brown adipose tissue is a consequence of the existence of an acetyl‐CoA hydrolase. The enzyme is soluble and is localised to the mitochondrial matrix.

Acetyl‐CoA hydrolase has an apparent Km for acetyl‐CoA of 51 μM, and a specific activity at 30 °C of 870 nmol of acetate formed/min per mg 100000 × g supernatant protein.

The enzyme is noncompetitively activated by ADP and inhibited by NADH and the effect of these nucleotides may well serve to regulate the enzyme activity in vivo.

A strong product inhibition by CoA is observed. The inhibition is of S‐linear‐I‐hyperbolic noncompetitive nature.

The hydrolase has a Q10 of 2.0, which represents a 7.3% change in the rate of acetate production per °C. The energy of activation is 12200 cal/mol (53 905 J/mol).

The regulatory role of acetyl‐CoA hydrolase for fatty acid oxidation in brown adipose tissue of the hamster (a hibernator) at low as well as at normal body temperature is discussed.
Solubilization of the Chromatin‐Bound Estrogen Receptor from Chicken Liver and Fractionation on Hydroxylapatite
Authors: GSCHWENDT Michael
Published: March 2005

High‐affinity estrogen‐binding sites can be solubilized from the liver chromatin of estrogenized chickens by treatment of the chromatin with 2 M KCl/5 M urea and fractionation on hydroxylapatite. Two estrogen‐binding proteins are eluted from hydroxylapatite columns by 20 mM phosphate (binding protein I) and 200 mM phosphate (binding protein II), respectively.

The binding protein I is part of a non‐histone protein fraction containing acid‐soluble and insoluble proteins, whereas the binding protein II elutes together with high molecular weight nonhistone proteins containing acid insoluble proteins only. Both binding proteins exhibit the same affinity for estradiol (Kd∼ 10 ‐9 M).

From chromatin of untreated chickens very small amounts of binding protein I (0.1 pmol/mg protein compared to 1.9 pmol/mg protein from estrogenized chickens) with the same affinity for estradiol as that from estrogenized animals can be solubilized. Binding protein II is not detectable.

The “soluble nuclear estrogen receptor” extracted from crude liver nuclei of estrogenized chickens by 0.5 M KCl behaves on hydroxylapatite very similarly to salt/urea‐dissociated chromatin with respect to the binding protein I. No binding protein II, however, can be demonstrated.

Chromatography of various preparations on Bio‐Gel A‐1.5 m indicates that the binding protein II is a residual chromatin fragment containing an unseparated binding protein‐DNA complex, whereas the binding protein I represents the solubilized nucleic‐acid‐free chromosomal estrogen receptor. The “soluble nuclear receptor” and the binding protein I, however, are not identical with respect to their chromatographic behaviour on Bio‐Gel A‐1.5 m, even though their estrogen binding entity remaining after trypsin treatment seems to be very similar.
Préparation et caractérisation physico‐chimique partielle de la transferrine sérique ovine
Authors: GUÉRIN Gérard , VREEMAN Hendrik J. , NGUYEN Thanh Cac
Published: March 2005

Preparation and Partial Physico‐Chemical Characterization of Sheep‐Serum Transferrin

Sheep‐serum transferrin shows marked polymorphism and more than 20 alleles have been identified although only 4 or 5 of these have a frequency higher than 1%. Each of the alleles has two bands in starch‐gel electrophoresis, corresponding to a major and a minor fraction.

This paper describes the isolation and partial characterisation of the two fractions from the transferrin of sheep homozygous for Tf B. The purification consisted of: (a) precipitation by ammonium sulphate, (b) chromatography on CM‐cellulose and, finally (c) chromatography on DEAE‐Sephadex. The purification procedure had no effect on the electrophoretic mobility of the two fractions and they appeared homogeneous by starch‐gel and polyamide‐gel electrophoresis and by immuno‐electrophoresis.

The amino‐acid compositions of both fractions were very similar and the sequences of the first eight amino‐acid residues: Ser‐Pro‐Glu‐Lys‐Thr‐Val‐Arg‐Trp‐ were identical for both bands. These results, and the fact that the two fractions are found in all genetic variants and always have the same relative mobility strongly suggest that the differerences do not lie in the polypeptide chain.

Froth the results of hydrolysis by neuraminidase and assay of sialic acid, the major and minor fractions most probably contain two and three sialic acid residues (exclusively N‐acetylneuraminic acid) respectively, thus explaining the different electrophoretic mobilities. The sialic‐acid content has been calculated on the basis of a molecular weight of 77500 as determined both by low‐speed equilibrium ultracentrifugation and by Archibald's method. The following physico‐chemical constants have been established: absorption coefficient at 280 nm, A11cmmg/cm3= 1.25 ± 0.01 cm2· mg−1; partial specific volume, v̄= 0.734 cm3· g−1; sedimentation coefficient s°20,w= 5.15 S; refractiveindex increment, dn/dc = 0.173 cm3· g−1.
Equilibrium Studies on the Refolding and Reactivation of Rabbit‐Muscle Aldolase after Acid Dissociation
Authors: ENGELHARD Martin , RUDOLPH Rainer , JAENICKE Rainer
Published: March 2005

Dissociation, denaturation, and deactivation of aldolase from rabbit muscle in the acid pH range have been investigated using sedimentation analysis, fluorescence, circular dichroism, and activity tests. Under comparable experimental conditions the pH‐dependent profiles of deactivation and denaturation parallel the dissociation of the enzyme. In the range of dissociation at pH 4‐ 5 tetramers and monomers are in equilibrium. Intrinsic chromophores and far‐ultraviolet circular dichroism suggest the transition to be a complex multistep process.

At pH ∼ 2.3 the enzyme is split into its fully inactive monomers which still contain some residual secondary structure. After reassociation under optimum conditions (0.2 M phosphate buffer pH 7.6, 1 mM EDTA, 0.1 mM dithiothreitol, 0 °C, enzyme concentration 0.4–50 μg/ml)up to 95 % enzymic activity is recovered which belongs to a renatured tetramerie species indistinguishable from the native enzyme by all available biochemical and physicochemical criteria.
Kinetics of Refolding and Reactivation of Rabbit‐Muscle Aldolase after Acid Dissociation
Authors: RUDOLPH Rainer , ENGELHARD Martin , JAENICKE Rainer
Published: March 2005

Tetrameric rabbit muscle aldolase has been dissociated to the monomer at pH 2.3 and fully reassociated and reactivated at pH 7.6.

Kinetics of reactivation and refolding were followed by slow and fast kinetic techniques after neutralization of the acidic enzyme solutions by dilution or rapid mixing. Rate constants (k), reaction orders (n), and activation energies (E) were calculated from measurements on the time, concentration, and temperature dependence of the reactions. The experimental results prove reactivation at high enzyme concentration (c > 4 μg/ml) to obey first‐order kinetics; at lower concentrations a transition to a higher reaction order is observed. Because of concentration‐dependent deactivation at low enzyme concentrations reactivation measurements could not be extended below c ∼ 0.5 μg/ml. In the accessible concentration range incomplete separation of reassociation and transconformation processes as well as intrinsic residual activity of the isolated subunits lead to an average value of n = 1.40 ∼ 0.18.

Renaturation as monitored by protein fluorescence is a multi‐step process composed of a fast increase in fluorescence emission (first‐order rate constant k ∼ 15 s−1) and a slow concentration‐dependent decrease which parallels the recovery of enzyme activity (n = 1.46 ± 0.12). The activation energy of both processes is of the order of E = 12–16 kcal/mol (50–67 kJ/mol). Reassociation is a prerequisite of full catalytic function and native fluorescence.
Membrane‐Bound F1 ATPase from Micrococcus Sp. ATCC 398E
Authors: HULLA Franz W. , HÖCKEL Michael , RISI Sergio , DOSE Klaus
Published: March 2005

A chemically reactive ATP analogue, 6‐[(3‐carboxy‐4‐nitrophenyl)thio]‐9‐β‐D‐ribofuranosylpurine 5′‐triphosphate (Nbs6ITP) has been synthesized. It has the ability to form stable thioether bonds between the 6‐position of the purine ring and aliphatic mercapto groups. The nucleotide moiety of the reagent has been covalently bound to agarose, via iminobispropylamine and N‐acetyl‐homocysteine as spacer with the purpose of producing an affinity chromatography material. The affinity matrix binds solubilized F1 ATPase from a crude extract of Micrococcus sp. membranes. Afterwards the enzyme can be selectively eluted from the column at a defined ATP concentration. This method is superior to the conventional purification with respect to speed and convenience of the preparation. The affinity chromatography leads in a one‐step process to the same purity of enzyme, substituting several steps of the conventional method.

In addition, the affinity matrix was used for binding studies. Although the presence of Mg2+ ions is a prerequisite for the hydrolysis of nucleoside 5′‐triphosphates, evidence is presented indicating that the binding of the nucleoside triphosphates to highly purified F1 ATPase from Micrococcus sp. appears not to be influenced by Mg2+ ion concentrations so far examined.
The Kinetics in vivo of the Synthesis of Albumin‐Like Protein and Albumin in Rats
Authors: URBAN Jöre , CHELLADURAI Mohanathasan , MILLERSHIP Anne , SCHREIBER Gerhard
Published: March 2005

The incorporation of radioactivity into total protein, albumin‐like protein and albumin in liver, and into total protein and albumin in blood plasma, was studied for a time period of 2 h after intra‐portal injection of l‐[l‐14C]leucine.

In the liver, radioactivity began to increase in albumin‐like protein before 2.5 min and in albumin at about 10 min after injection. In the plasma, radioactive albumin appeared at about 15 min. No albumin‐like protein could be detected in the plasma.

Maximum radioactivity was reached first in albumin‐like protein, then in hepatic albumin and finally in albumin in the blood‐stream. The maximum specific radioactivity of albumin‐like protein was 15 tines higher than that of extravascular hepatic albumin which, in turn, was 6 times higher than that of plasma albumin.

The increase of radioactivity in albumin in the blood corresponded almost quantitatively to the decrease of radioactivity in albumin‐like protein.

It is concluded that the albumin‐like protein is the precursor of albumin in vivo. It is converted into albumin 5–6 min before the appearance of newly synthesized albumin in the blood‐stream.
Hamster Brown‐Adipose‐Tissue Mitochondria
Authors: HEATON Gillian M. , NICHOLLS David G.
Published: March 2005

The specific ability of fatty acids to increase the proton conductance of the inner membrane of mitochondria from the liver and brown adipose tissue of cold‐adapted hamsters was compared. The liver and brown‐adipose‐tissue mitochondria had their effective proton conductances increased by respectively 0.028 and 0.94 nmol H+· min−1.(mV of proton electrochemical gradient)−1 for each nmol of palmitate bound. No difference could be detected between the abilities of liver and brown‐adipose‐tissue mitochondria to bind fatty acids. Purine nucleotides did not displace fatty acids from the brown‐adipose‐tissue mitochondria. The endogenous fatty acid content of hamster brown‐adipose‐tissue mitochondria prepared in the absence of albumin was found to be equivalent to 17 ± 7 nmol of palmitate/mg protein. The fatty acid content was reduced to 1 nmol/mg after preincubation of the mitochondria with CoA, ATP and carnitine. No inert pool of fatty acids could be detected. The endogenous fatty acids of hamster liver mitochondria were less than 4 nmol of palmitate equivalent/mg protein. Some of the fatty acid associated with the brown‐adipose‐tissue mitochondria originates during preparation of the mitochondria. In the light of these results, the physiological role of fatty acids in controlling the proton conductance of the brown‐adipose‐tissue mitochondrial inner membrane, and hence non‐shivering thermogenesis, is re‐evaluated.