bluuebunny/crossref_metadata_2025 huggingface dataset:
Study on the Mechanism of Lysosome Labilization by Inorganic Mercury in vitro
Authors: Lauwerys Robert , Buchet Jean‐Pierre
Published: March 2005

This investigation demonstrates that the labilization of rat liver lysosomes by Hg2+in vitro is the result of a complex series of events. The initial rapid binding of Hg2+ on lysosomal membranes is followed by an autocatalytic breakdown of lysosomes by the lysosomal hydrolytic enzymes. This secondary action is dependent on the concentration of Hg2+ which decreases the hydrolytic activity of the lysosomal enzymes. Furthermore an Hg2+ exchange reaction between solubilized lysosomal proteins, which have bound Hg2+, and lysosome membranes can induce lysosome labilization.
Peptidyl‐Donor Substrates for Ribosomal Peptidyl Transferase
Authors: Mercer Julian F. B. , Symons Robert H.
Published: March 2005

A study of the structural requirements for activity of low molecular weight compounds in the donor or peptidyl site of ribosomal peptidyl transferase requires methods for the chemical synthesis of potential donor substrates. Methods are described here for the preparation of cytidylyl‐(3′→ 5′)‐cytidylyl‐(3′→ 5′)‐2′(3′)‐O‐(N‐[3H]acetyl‐l‐leucyl)‐adenosine and cytidylyl‐(3′→ 5′)‐2′(3′)‐O‐(N‐[3H]acetyl‐l‐leucyl)‐adenosine and the corresponding l‐phenylalanine derivatives. A major difficulty overcome was the severe restriction placed on possible synthetic routes by the extreme alkali lability of the aminoacyl linkage. The synthetic route developed involved the stepwise synthesis of CpCpA and CpA appropriately protected with acid‐labile protecting groups. The free terminal 2′(3′)‐hydroxyls were aminoacylated, all protecting groups removed and the α‐amino groups acylated with [3H]acetic anhydride.

The chemically prepared N‐acetyl‐aminoacyl‐trinucleotide fragments had similar activity in the ribosome‐catalysed fragment reaction as reported for the corresponding pentanucleotide fragments obtained from biological sources. Further, the inactivity of the N‐acetyl‐aminoacyl‐dinucleotide fragments was confirmed under a wider range of conditions than those previously described.
Zur Biosynthese der Alkaloide vom Rhoeadin‐Typ
Authors: Rönsch Hasso
Published: March 2005

The biosynthesis of alkaloids with 2‐phenyl‐3‐benzazepine skeleton (rhoeadine type) starts from two molecules of tyrosine. Now it was shown, using Papaver bracteatum plants, that [N‐14CH3, 8‐14C]dl‐tetrahydropalmatine methiodide (Ia) is incorporated with high efficiency into alpinigenine (III). Apparently the incorporation of the N‐quaternary salt is a specific one.

A degradation sequence was applied which allows the selective determination of the radio‐activity in the non‐aromatic C‐atoms as well as the aromatic rings of alpinigenine.

The incorporation of a compound with tetrahydroprotoberberine structure is in accordance with current biogenetic views. But involvement of a N‐quaternary intermediate seems quite surprising. A novel mechanism of ring rearrangement may operate. Thus, ring splitting as a step for further transformation of tetrahydropalmatine should be initiated by N‐methylation followed by hydroxylation on one of the α‐C‐atoms (C‐14 or C‐8) and subsequent hydrolysis.

The significance of the feeding experiments in respect to biogenesis of benzazepine alkaloids is briefly discussed.
Ribonucleic Acid Polymerase from Eukaryotic Cells
Authors: Mondal Hrishikes , Ganguly Asok , Das Asis , Mandal Radha K. , Biswas Birendra B.
Published: March 2005

The role of protein factor B and C on RNA synthesis by RNA polymerase CI isolated from chromosomal non‐histone proteins of coconut nuclei has been studied further. Factor B has been implicated as the initiation factor on the experimental evidences that (a) in its absence, RNA polymerase CI shows only minimal activity; (b) it can bind with RNA polymerase and the enzyme · factor B complex then binds to DNA, but factor B alone can not bind to DNA; (c) it promotes the incorporation of [β,γ‐32P2]ATP into RNA and this stimulation reaches a plateau rather quickly while the incorporation of [14C]ATP in the interior of RNA chain continues; (d) it is active with native homologous DNA as template, but not with denatured or λDNA; (e) RNA molecules synthesized in its presence are of higher sedimentation value (10–20 S) than that synthesized in its absence (4 S); (f) it can completely counteract the inhibitory effect of rifampicin, which is known to inhibit RNA synthesis at the initiation step.

Factor C seems to facilitate the release of synthesized RNA from the DNA template since (a) it stimulates RNA synthesis by polymerase CI when added on top of factor B, but in absence of factor B, C alone is inactive; (b) it can reinitiate RNA synthesis after the reaction has reached a plateau in a system where DNA is limiting, an affect similar to that obtained at higher ionic strength. Factor C, however, does not influence the molecular size of RNA synthesized. Furthermore, the RNA polymerase CI is sensitive to α‐amanitin whereas the RNA polymerase CII is comparatively resistant. The former appears to synthesize the non‐ribosomal RNA whereas the latter synthesizes ribosomal RNA.
Hydrogen‐Ion‐Titration Studies of Pancreatic Phospholipase A and Its Zymogen
Authors: Janssen Lambert H. M. , de Bruin Simon H. , DE Haas Gerard H.
Published: March 2005

The titration curves of porcine pancreatic phospholipase A an its zymogen were measured in the pH region 2.0 to 9.5 at 25°C.

In the pH region 2 to 6 the titration curves were measured using an Ag‐AgCl electrode as reference electrode, at ionic strength 0.5. It was found that the titration of the carboxyl groups could be described with the Linderstrøm‐Lang model using an intrinsic pKof 4.5 and an electrostatic interaction factor ω of 0.073. The side‐chain carboxyl group of the second (viz. glutamyl) residue in prophospholipase appeared to have an abnormally low pK of 3.7. The maximum positive charge of the two proteins as determined by titration agreed well with the values predicted by the amino acid composition.

From an analysis of the titration curves covering the region 4.5 to 9.5, which were measured with a calomel electrode at ionic strength 0.1, the following could be concluded. The intrinsic pK and ω of the carboxyl groups appeared to be at this ionic strength 4.7 and 0.114, respectively. In both phospholipase and prophospholipase the number of titratable carboxyl groups was found to be 3 higher than given by the amino acid analysis. All 3 histidines present were titratable with an intrinsic pK of 6.6 and ω near zero. The terminal amino group which is free only in phospholipase appeared to have an apparent pK of 8.3.
Nature and Linkages of the Fatty Acids Present in the Lipid‐A Component of Salmonella Lipopolysaccharides
Authors: Rietschel Ernst Th. , Gotter Horst , LÜDERITZ Otto , Westphal Otto
Published: March 2005

Laurie, myristic, palmitic, and 3‐d‐(–)‐myristic acid in a ratio of about 1:1:1:3 are present in the lipid A part of the glycolipid derived from the Re mutant Salmonella minnesota R595. An additional fatty acid, Δ2‐tetradecenoic acid found in larger amounts after alkaline treatment was recognized as an artifact formed by a β‐elimination reaction from ester‐linked 3‐myristoxy‐myristic acid. Various methods for the stepwise liberation of fatty acids were used to identify their distribution on the backbone of lipid A which consists of β‐1,6‐linked glucosamine disaccharide units substituted at position 3′ with 2‐keto‐3‐eoxyoctonate and at positions 1 and 4′ with phosphate. It was found that the amino groups of the glucosamine residues are substituted by 3‐D‐(–)‐hydroxymyristic acid whose hydroxyl group is free. The three available hydroxyl groups at positions 3, 4, and 6′ of the glucosamine dissaccharide are substituted by equal amounts of lauric, palmitic, and 3‐D‐myristoxymyristic acid and, possibly, by smaller amounts of myristic and unsubstituted 3‐D‐hydroxymyristic acid. A similar structure of lipid A seems to exist in lipopolysaccharides of other Salmonella species.
Animal DNA‐Dependent RNA Polymerases
Authors: Gissinger Francis , Chambon Pierre
Published: March 2005

Calf thymus AI DNA‐dependent RNA polymerase was purified by chromatography on phosphocellulose, Sepharose 6B and DEAE‐Sephadex and by glycerol density‐gradient centrifugation. The polymerase was more than 90% pure and free of contaminating nucleases. Addition of 30% glycerol to all buffers increased the stability of the enzyme activity throughout the purification, but the final yield of activity was nevertheless low. This does not seem to be due to the loss of a stimulating factor during the purification. No evidence was found for an enzyme resembling rat liver AII DNA‐dependent RNA polymerase in calf thymus.
The Metabolism of Benzoate and Methylbenzoates via the meta‐Cleavage Pathway by Pseudomonas arvilla mt‐2
Authors: Murray Keith , Duggleby Clive J. , Williams Peter A. , Sala‐Trepat José M.
Published: March 2005

Pseudomonas arvilla mt‐2 grows at the expense of benzoate, m‐toluate (3‐methylbenzoate) and p‐toluate (4‐methylbenzoate), but not o‐toluate (2‐methylbenzoate). Under various conditions compounds with spectra characteristic of muconic semialdehydes can be made to accumulate in the media. These spectra indicate that benzoate is metabolised through catechol and 2‐hydroxymuconic semialdehyde (2‐hydroxy‐6‐oxohexa‐2,4‐dienoate), m‐toluate through 3‐methylcatechol and 2‐hydroxy‐6‐oxohepta‐2,4‐dienoate and p‐toluate through 4‐methylcatechol and 2‐hydroxy‐5‐methylmuconic semialdehyde (2‐hydroxy‐5‐methyl‐6‐oxohexa‐2,4‐dienoate).

Freshly harvested cells grown on all three substrates only consume oxygen at a significant rate when presented with the growth substrates themselves and catechol and the methylcatechols, but not with salicylate, protocatechuate or phenol or the cresols.

Extracts of cells grown on these substrates contain high induced levels of the suite of meta cleavage enzymes, including both the hydrolytic branch and the 4‐oxalocrotonate branch.

The substrate specificity of the early enzymes of the pathway suggests that only one non specific enzyme expresses each activity in cell‐free extracts and that it is nonspecifically induced during growth on all three carbon sources.

It is suggested that the metabolic role of the hydrolytic branch of the pathway is the assimilation of 3‐methylcatechol and its precursor, and that of the 4‐oxalocrotonate branch is to assimilate catechol and 4‐methylcatechol and their precursors.

o‐Toluate is not metabolised because it is neither a substrate for the benzoate oxidase system nor an inducer of any of the meta pathway enzymes.

It appears that benzoate and m‐and p‐‐toluates are the substrate inducers of the meta pathway enzymes in this organism. The ortho pathway is induced on incubating cells with catechol and the first inducer appears to be cis,cis‐muconate or possibly catechol itself.
A Glycopeptide from the Posterior Lobe of Pig Pituitaries
Authors: Holwerda Dirk A.
Published: March 2005

An extract from the posterior lobes of pig pituitaries contains a glycopeptide with a molecular mass of 3200 (± 5%). The glycopeptide is present in amounts of the same order as the hormones oxytocin and vasopressin.

The peptide portion is composed of seventeen amino acid residues: aspartic acid or asparagine (3), threonine (1), serine (3), proline (1), glycine (2), alanine (3), leucine (3) and arginine (1). The carbohydrate portion, about 55% by weight, is composed of fucose (1), galactose (1), mannose (3) and glucosamine (4). The glycopeptide exhibits none of the known physiological activities of hypophysis except for a slight lipolytic activity.
The Metabolic Divergence in the meta Cleavage of Catechols by Pseudomonas putida NCIB 10015
Authors: Sala‐Trepat Jose M. , Murray Keith , Williams Peter A.
Published: March 2005

A reinvestigation of the catabolic pathway(s) used by Pseudomonas putida NCIB 10015 (Dagley's strain) for the degradation of phenol and the cresols has proved the existence of a metabolic divergence after meta cleavage of the catechols formed by hydroxylation of the primary substrates. The ring‐fission products of catechol and 4‐methylcatechol are shown to be simultaneously catabolized by two different enzymic activities, an NAD+‐dependent dehydrogenase and a cofactor‐independent hydrolase. The metabolizing activities of both ring‐fission products in extracts of cells grown on phenol and the cresols (o‐, m‐ and p‐cresol) were found to be non specific; thermal inactivation of extracts of phenol‐grown cells has shown that this nonspecificity is attributable to only one enzyme expressing each activity and that the two activities are located on separate proteins.

Extracts of cells grown on all four substrates contain high induced levels of the meta cleavage suite of enzymes functional in the dissimilation of catechol, including both the 4‐oxalocrotonate branch (NAD+‐dependent 2‐hydroxymuconic semialdehyde dehydrogenase, 4‐oxalocrotonate tautomerase and 4‐oxalocrotonate decarboxylase) and the hydrolytic branch (2‐hydroxymuconic semialdehyde hydrolase).

The hydroxylase, oxygenase, dehydrogenase and hydrolase activities are shown to be nonspecific and can also act upon the methyl derivatives of their respective substrates. A constant pattern of specificity was found for these enzymes, independent of the monophenolic substrate used for growth.

From studies with a mutant lacking phenol hydroxylase, the entire suite of meta cleavage enzymes are shown to be coincidently induced from the top by the primary substrate (phenol or the cresols).

The evolutionary and physiological implications of the divergent pathway are discussed.
A Study of the Quantitative Variation of Histones, and Their Relationship to RNA Synthesis, during Erythropoiesis in the Adult Chicken
Authors: Billett Michael A. , Hindley John
Published: March 2005

Defined cell populations representing limited periods in the erythropoietic sequence of adult chickens have been obtained by buoyant density centrifugation of bone marrow cells on bovine serum albumin gradients. The histones of these various populations and of circulating erythrocytes have been compared quantitatively by polyacrylamide gel electrophoresis. No change in the relative amounts of F1, F2a1, F2a2 (and possibly F3 + 2b), could be detected between the early polychromatic erythrocyte and the mature erythrocyte, although changes in the distribution of the F1 subfractions were observed. The amount of the erythrocyte specific histone (F2c), however, increases approximately 2.5‐fold during this period. These events were correlated with a parallel decrease in RNA synthesis, both in whole cells and when measured in isolated nuclei. The results are discussed in relation to possible mechanisms of chromatin condensation and genome repression.
The Thiol Groups of Human Hemoglobin Carrying Heme only on the α‐Chains
Authors: Birchmeier Walter , Glatthaar Beat E. , Bradshaw Ralph A. , Winterhalter Kaspar H.
Published: March 2005

Hemoglobin ICII is a derivative of human HbA, carrying the heme prosthetic group only on the α‐chains. Reaction of ICII in the cyanmet form with p‐mercuribenzoic acid or iodoacetamide yields one titratable thiol group per αβ dimer as in HbA. The kinetics of the thiol modification of HbA and ICII with iodoacetamide indicate that ICII reacts more than 10‐times faster than HbA under pseudo‐first‐order conditions. The site of modification was determined from the tryptic peptides following chain separation. The modified thiol group in both HbA and ICII was identified as cysteine β‐93, whereas cysteine α‐104 and β‐112 remained unmodified. These results indicate a different conformation of the heme‐free and heme‐bound β‐chains, which seems to be independent of the nature of the ligands of the α‐chains.
Tetrahydrobiopterin, a Cofactor in Mitochondrial Electron Transfer
Authors: Rembold Heinz , Buff Klaus
Published: March 2005

Biopterin concentration was determined by the microbiological Crithidia test in sonified rat liver mitochondria (0.03 mM, 0.4 nmol/mg protein) and in high‐speed supernatant of rat livers (0.015 mM, 0.15 nmol/mg protein). Respiration of intact mitochondria is not influenced by presence of 7,8‐dihydroneopterin. Tetrahydropterins (biopterin, neopterin, 6,7‐dimethylpterin), on the other hand, lead to an elevated net oxygen consumption in metabolic state 3 as well as in state 4 rat liver mitochondria, but ATP formation is diminished in the presence of tetrahydropterin. In state 3, high tetrahydropterin concentrations (> 0.4 mM) seem to inhibit respiration; preincubation with reduced pterin shifts the inhibitory concentration to about 0.25 mM. Used as unique substrate, tetrahydropterins considerably activate respiration under state 4 conditions; at concentrations of 0.5–0.6 mM, saturation is gained and no further activation is possible with still higher concentrations. Low temperature‐difference spectra demonstrate a reduction of cytochromes c and a/a3 by tetrahydropterins, and no reduction of cytochrome b. Reduction of cytochromes is time‐dependent. This lag phase is influenced by substrates, ADP, type and concentration of tetrahydropterin.
Investigation of the Structure of Angiotensin II Using 13C Nuclear‐Magnetic‐Resonance Spectra
Authors: Zimmior Stephen , Haar Wolfgang , Maurer Wolfgang , Rüterjans Heinrich , Fermandjian Serge , Fromageot Pierre
Published: March 2005

Using the fast Fourier transform technique it has been possible to get readable natural‐abundance carbon‐13 nuclear magnetic resonance spectra of some oligopeptides, in particular the octapeptide angiotensin II. An assignment of the carbon‐13 resonances to the distinct carbons of the peptides has been made by comparison of the resonances with the corresponding resonances of free amino acids. From the pH dependence of the chemical shifts of the carbon‐13 resonances of the octapeptide angiotensin II, pK values can be derived for titratable groups of this hormone. These pK values as well as the shape of the titration curves are interpreted in terms of intramolecular interactions.
Purification and Properties of DNA‐Dependent RNA Polymerases from Yeast
Authors: Ponta Helmut , Ponta Ulrike , Wintersberger Erhard
Published: March 2005

A procedure for the large‐scale purification of yeast RNA polymerases A and B is described. The final enzyme preparations obtained are fairly homogeneous, as revealed by electrophoresis in polyacrylamide gels containing sodium dodecylsulfate. Both enzymes contain two large subunits with molecular weights of about 190000 and 135000 for polymerase A and 175000 and 140000 for polymerase B in a 1:1 ratio. The third polymerase from yeast (enzyme C) has not yet been sufficiently purified to determine its subunit structure. All three RNA polymerases require four ribonucleoside triphosphates, DNA and Mn2+ for activity. Denatured DNA is preferred over native DNA as template. Ammonium sulfate in low concentrations (20 to 50 mM) stimulates the enzyme activities two to three‐fold; high concentrations are inhibitory. Only RNA polymerase 3 is sensitive towards α‐amanitin. This enzyme is almost totally inhibited by a concentration of 100 μg/ml of the drug, whereas polymerases A and C remain virtually unaffected even at high concentrations of α‐amanitin. The RNA polymerases from yeast have, in general, many properties in common with the corresponding enzymes of multicellular organisms.
On the Microenvironment of the Pyridoxamine 5‐Phosphate Residue in NaBH4‐Reduced Glycogen Phosphorylase b
Authors: Cortijo Manuel , Shaltiel Shmuel
Published: March 2005

The absorption and fluorescence properties of NaBH4‐reduced glycogen phosphorylase b were studied and used for probing the microenvironment of the pyridoxamine 5′‐phosphate residue and for monitoring structural transitions occurring in the enzyme. Free pyridoxamine 5′‐phosphate in a solvent mixture composed of about 80% dimethylformamide and 20% water was found to simulate the optical properties of the enzyme at neutral pH (absorption maxima, molar absorption, fluorescence excitation and emission maxima and quantum yield). Therefore it is suggested that at pH 7, where the enzyme exhibits maximal catalytic activity, the pyridoxamine 5′‐phosphate residue is “buried” in a hydrophobic microenvironment. Upon gradually lowering the pH of the medium to 4.8, there are structural changes in the enzyme which are reflected in an increase of the molar absorption around 330 nm and of the quantum yield of the cofactor fluorescence. These transitions can be simulated with free pyridoxamine 5′‐phosphate by increasing the water content of the dimethylformamide‐water mixture to 80–90%. It seems, therefore, that acidification of the medium causes structural changes in the enzyme which expose the cofactor site.
Covalently Bound Flavin ind‐6‐Hydroxynicotine Oxidase fromArthrobacter oxidans
Authors: Brühmüller Margarete , Möhler Hanns , Decker Karl
Published: March 2005

d‐6‐Hydroxynicotine oxidase fromArthrobacter oxidansgrown on citrate anddl‐nicotine was obtained as a homogeneous protein as judged by ultracentrifugation and disc electrophoresis. From the sedimentation and diffusion coefficients of 3.54 S and 6.69 F, respectively, and from sedimentation equilibrium measurements a molecular weight of 53000 was calculated.d‐6‐Hydroxynicotine oxidase consists of one polypeptide chain and contains one mol FAD per mol enzyme. The flavin was shown to be covalently bound to the protein.

In the presence of oxygen,d‐6‐hydroxynicotine oxidase convertsd‐6‐hydroxynicotine to [6‐hydroxypyridyl‐(3)]‐(4‐N‐methylaminopropyl)‐ketone. Molecular oxygen could be replaced as electron acceptor by methylene blue and 2,6‐dichlorophenolindophenol, but not by one‐electron acceptors. Anaerobic reduction of the enzyme with substrate did not reveal a semiquinoid flavin intermediate. The spectral changes obtained by the photochemical reduction ofd‐6‐hydroxynicotine oxidase in the presence of EDTA, however, indicated an anionic semiquinone form.

d‐6‐Hydroxynicotine oxidase proved to be highly specific for the structural and absolutely specific for the stereochemical configuration of its substrate. Among others,l‐6‐hydroxynicotine and the reaction product were found to be competitive inhibitors.
Covalently Bound Flavin in d‐6‐Hydroxynicotine Oxidase from Arthrobacter oxidans
Authors: Möhler Hanns , Brühmüller Margarete , Decker Karl
Published: March 2005

A substituted riboflavin (HNO‐flavin) was isolated from d‐6‐hydroxynicotine oxidase. It was obtained from flavin peptides by hydrolysis with 6 N HCl at 95°C or with aminopeptidase M. The riboflavin derivative had the spectral characteristics of 8α‐substituted flavins. It showed a pH dependence of fluorescence with a pK of 4.65 and 86%, quenching at pH 7. In thin‐layer chromatography it was identical with 8α‐(N‐3‐histidyl)‐riboflavin. Hydrolysis of HNO‐flavin in 6N HCl at 125°C liberated 1 mol histidine per mol flavin as shown by amino acid analysis. Since FAD is the coenzyme of d‐6‐hydroxynicotine oxidase, these results are taken as evidence that this enzyme contains 8α‐(N‐3‐histidyl)‐flavin‐adenine dinucleotide in the active center.
The Synthesis of 3‐Deoxy‐5‐O‐Methyloctulosonic Acid anti Its Behaviour in the Warren Reaction
Authors: Charon Daniel , Szabó Ladislas
Published: March 2005

The chemical synthesis of 3‐deoxy‐5‐O‐methyloctulosonic acid (configuration on C‐4 and C‐5 unknown) from 4‐O‐formyl‐2‐O‐methyl‐d‐arabinose and oxaloacetic acid is described. When treated with periodate and thiobarbiturate, the compound exhibits a strongly positive Warren reaction (millimolar absorption coefficient: 13 mM−1 cm−1); a mechanism for this reaction is proposed. Structural features necessary for the production of a positive reaction from 3‐deoxyaldulosonic acid derivatives in this test are outlined. Some limitations of the Warren reaction as used for the quantitative estimation of this class of compounds are pointed out.
An Analysis of DNA‐Containing Particles Recovered from the Cytoplasm of Differentiating Chick‐Muscle Cells
Authors: Bell Eugene , Merrill Charlotte , Lawrence Charles B.
Published: March 2005

Labeled DNA‐containing particles from the cytoplasm of embryonic chick muscle can be dissociated in 0.5 M KCl to a basic particle of buoyant density 1.57 g/ml in CsCl. DNA‐containing particles of density 1.43 g/ml are found in a large distribution of distinct sizes whose sedimentation values in sucrose range from 16 S to greater than 100 S. DNA‐containing particles in a range of densities centering about a peak of 1.52 g/ml are found cosedimenting with polyribosomes in sucrose. The latter particles contain at least two subunits which are dissociable by treatment with EDTA; the DNA‐containing subparticle has a density of 1.43 g/ml.

Newly made RNA is found in association with DNA‐containing particles banding at 1.43 g/ml which are about 40 S and larger but not with particles of density 1.57 g/ml.

Virtually all of the rapidly labeling cytoplasmic RNA taken from the polyribosomal region of a sucrose gradient is associated with particles of densities 1.43 g/ml and 1.55 g/ml; a minor portion may be associated with particles of density 1.52 g/ml which are found cosedimenting uniquely with polyribosomes.

Practically all nascent chain protein is associated with particles of density 1.55 g/ml, which is the density of ribosomes or polyribosomes.

Addition of labeled DNA to cytoplasmic extracts does not yield any of the foregoing particles, and no binding of label to polyribosomes or cosedimenting structures is observed. DNA‐containing particles from the nucleus band at densities different from particles recovered from the cytoplasm.

We propose that the particles recovered from the cytoplasm are naturally occurring and not artifacts of fractionation and processing, and we present a model for their utilization in the transfer and use of genetic information.
Alkaline Denaturation of Nucleohistone
Authors: Ohba Yoshiki , Hayashi Masako
Published: March 2005

The ultraviolet spectral change at 245 nm of tyrosine residues in histones in alkaline solution was studied. The difference spectrum of histones between alkaline and neutral solution yields an absorption maximum at 242 nm. However, the influence of configurational changes of backbone structure of histones in alkaline solution, results in the true peak position being located near 245 nm. This was established from artificial difference spectra between histones and N‐acetyl‐l‐tyrosine ethyl ester in 0.5 N NaOH, conditions under which ionization of phenolic hydrogens of histones is immediately complete. This ease of ionization permits the quantitative estimation of histones in nucleohistone by measuring the ratio of absorbance at 244 nm to that at 260 nm, so long as we can prepare the nucleohistone from calf thymus which does not contain non‐histone protein.

Alkaline spectrophotometric titration of nucleohistone revealed that conformational changes of histones occur when the double helical structure of DNA is in the process of disruption in a nucleohistone molecule. This conformational change occurred without the dissociation of histones and DNA. The dissociation of histones and DNA in alkaline pH region was discussed.
Localization of Protein A in the Bacteria
Authors: Sjöquist John , Movitz Jan , Johansson Inga‐Britt , Hjelm Hans
Published: March 2005

Staphylococcus aureus, strain Cowan I, contains 1.7% protein A and its isolated cell walls contain 6.7%.

Removal of contaminating cytoplasmic membrane fragments from the cell walls does not decrease the content of protein A. Extraction of cell walls with trichloroacetic acid releases teichoic acid but not protein A. Extractions with high concentrations of LiCl do not remove protein A. Only bacteriolytic enzymes can release the protein.

Protein A isolated from bacteria after lysozyme digestion contains peptidoglycan constituents. More effective bacteriolytic enzymes such as lysostaphin release peptidoglycan fragments from this protein A preparation.

We conclude from these results that protein A is a cell wall component of S. aureus and is covalently linked to the peptidoglycan structure.
Isolation and Characterization of Thaumatin I and II, the Sweet‐Tasting Proteins from Thaumatococcus daniellii Benth
Authors: van der Wel Henrik , Loeve Kees
Published: March 2005

From an aqueous extract of the fruit of the tropical plant Thaumatococcus daniellii Benth, two sweet‐tasting basic proteins, here named thaumatin I and thaumatin II, were isolated by ultrafiltration, gel nitration on Sephadex G‐50 and ion‐exchange chromatography on SE‐Sephadex C‐25, using a sodium chloride concentration gradient.

The proteinaceous character of the two sweet‐tasting compounds was proven by the characteristic ultraviolet absorption spectrum, the presence of almost 100% polypeptide material as determined by the biuret method, the yield of 100% amino acids on hydrolysis with hydrochlorid acid, the positive reaction with amido black colouring agent (as used in the analysis of the fractions by polyacrylamide gel and starch gel electrophoresis) and by the disappearance of the sweet taste of the compounds after incubation with trypsin.

The basic character of the two proteins appears from their isoelectric points of almost 12, as estimated by starch gel electrophoresis at different pH values, and from the precipitation of the proteins at pH 12.

The molecules of thaumatin I and II are very similar as shown from their amino acid compositions, which are practically equal; they also have the same N‐terminal amino acid alanine and molecular weights of 21000 ± 600 and 20400 ± 600, respectively (calculated from ultracentrifugal data).

The proteins lose their sweetness on heating, on splitting of the disulphide bridges in the molecule and at pH values below 2.5.

Thaumatin I and II have an intensely sweet taste, the degree of sweetness being about 1600 times higher than that of sucrose on a Weight basis or 105 times on a molar basis. The threshold values are near 10−4% or 48 nM.
Regulation of Pyruvate Dehydrogenase in Mitochondria of Rat Liver
Authors: Portenhauser Rudolf , Wieland Otto
Published: March 2005

Active (dephospho) and inactive (phospho) forms of pyruvate dehydrogenase are extracted from freshly prepared rat liver mitochondria in the same proportion as from freeze‐clamped samples of whole rat liver. The active form increases within a few minutes on incubation of mitochondria with pyruvate (state 4) in a concentration‐dependent manner, the maximal effect (80% active form) being obtained at 2 mM pyruvate. Half‐maximal activation occurs between 0.3 and 0.4 mM pyruvate. ADP, in the absence of exogenous substrate, also stimulates the formation of active pyruvate dehydrogenase. Palmitoyl‐l(–)‐carnitine counteracts the effects of pyruvate or ADP on pyruvate dehydrogenase interconversion. The lowest values of the active form of the enzyme were observed during state 4 respiration with palmitoyl‐l(–)‐carnitine, succinate, 2‐oxoglutarate, and 3‐hydroxybutyrate. These studies lend support to the view that, in liver, the state of the pyruvate dehydrogenase system is under metabolic control and that ADP may thereby play an essential role.
Extension of RNA Molecules with DNA An
Authors: Peters Gordon G. , Hayward Richard S.
Published: March 2005

Sequences in DNA, which act as signals for the termination of transcription, may lie wholly or partially beyond the distal end of the DNA region actually transcribed into RNA. In a hybrid formed between an intact RNA molecule and its DNA complement, any such untranscribed signal should lie in the region of single‐stranded DNA close to the 3′‐hydroxyl terminus of the RNA. We show here that hybrids of this type, formed on circular bacteriophage DNA, act as efficient primers for Escherichia coli DNA polymerase I and T4‐induced DNA polymerase, under the normal conditions for DNA synthesis in vitro. The DNA product is covalently linked to the 3′‐end of the RNA, and co‐purifies with it in CsCl equilibrium centrifugation. Priming by RNA is readily detectable even when DNA primers are also present. These and other features of the reaction suggest that it might form the basis of a general method for the study of DNA sequences beyond the termini of RNA chains.
Post‐Replication Repair of DNA in Ultraviolet‐Irradiated Mammalian Cells
Authors: Lehmann Alan R. , Kirk‐Bell Susan
Published: March 2005

Ultraviolet light produces pyrimidine dimers in cellular DNA. In mouse cell lines the great majority of these pyrimidine dimers are not excised, but remain in high molecular weight DNA. Hence the DNA used as template for DNA synthesis in the first generation after ultraviolet irradiation must contain such dimers. In two mouse cell lines (L5178Y and 3T3), alkaline sucrose sedimentation studies showed that the DNA pulse‐labelled shortly after irradiation contained gaps, which by analogy with the situation in Escherichia coli, are presumed to be opposite the pyrimidine dimers. In contrast such gaps could not be detected in DNA pulse‐labelled several hours after irradiation, despite the fact that dimers must have been present on the template strands. A possible explanation is that gaps were still being formed, but were filled in rapidly and were therefore not detected. This hypothesis was tested as follows. Either 1 or 8 h after ultraviolet irradiation cells were pulse‐labelled in the presence of theophylline. This purine inhibits the filling in of gaps presumably by binding to the DNA in the gap. Even in the presence of theophylline, the late‐synthesized DNA was considerably larger than the early‐synthesized DNA. This suggests that at late times after irradiation, gaps were either not formed at all opposite the dimers, or were so transient that theophylline could not bind and inhibit their filling in. A model for synthesis of DNA on templates containing pyrimidine dimers in mammalian cells, based on these findings and other recent results, is discussed.
Conformational Isomers of Nitrosyl‐haemoglobin
Authors: Trittelvitz Eberhard , Sick Hinrieh , Gersonde Klaus
Published: March 2005

The electron spin resonance spectra of the nitric oxide derivatives of horse and human haemoglobin indicate the existence of two Conformational states, the distribution of which depends on pH. The pK‐value of this allosteric transformation is 5.35. This pK is shifted by 0.3 units to 5.65 in the presence of a 50‐fold molar excess of 2,3‐bisphosphoglycerate. The ESR spectrum of nitrosyl‐myoglobin corresponds exactly to that of the alkaline species of nitrosyl‐haemoglobin, but no pH dependent change was observed. These findings might suggest a relation between the observed Conformational transition and the acid Bohr effect.

At 77 K the spectrum of the acid as well as that of the alkaline species of NO‐haemoglobin indicates rhombic symmetry of the inner molecular electric field. Raising the temperature to 298 K the ESR spectrum of the alkaline species shows a transition to axial symmetry. The spectrum of the acid species exhibits hyperfine structures in the direction of the x‐ and z‐axis, that of the alkaline species in the direction of the z‐axis only. Substitution of 14NO by 15NO reveals that these hyperfine lines are caused by an interaction of the unpaired electron with the N‐nucleus of the 6th ligand. The hyperfine coupling tensor of the acid species (15N: ΔH3, 3= 23 G; 14 N: ΔH3,3,3= 17 G) is smaller than in case of the alkaline species (15N:Δ H3,3= 32 G; 14N:ΔH3,3,3= 23 G).
Investigation of the Active Center of Porcine‐Pancreatic Amylase
Authors: Elödi Pal , Móra Susan , Krysteva Milka
Published: March 2005

The effect of maltose was studied in porcine pancreatic amylase. At neutral pH 1% (29 mM) maltose produced with amylase a difference spectrum characteristic of the perturbation of tryptophan. The molar absorption difference at the maximum wavelength was Δ290= 1200.

The difference spectrum appeared to be specific for maltose. Perturbation difference spectra measurements in 20% polyethylene glycol indicated that one tryptophyl side chain per mol amylase was involved in the interaction with maltose.

The dissociation constant of the amylase · maltose complex calculated from the concentration dependence of the absorption difference at 290 nm was Ks= 13 mM. Maltose inhibited amylase activity competitively and an inhibition constant of Ki= 25 mM was obtained, a similar value to that found spectrophotometrically. It is assumed that the tryptophyl side chain interacting with maltose may be involved in the binding of substrate by pancreatic amylase.
Metabolism of Insulin and Glucagon
Authors: Ansorge Siegfried , BOHLEY Peter , Kirschke Heidrun , LANGNER Jürgen , Wiederanders Bernd , HANSON Horst
Published: March 2005

An enzyme catalyzing the reductive breakdown of insulin was isolated and purified from microsomes of rat liver. Its properties resemble strongly those of glutathione‐insulin transhydrogenase (thiol‐protein disulfide oxidoreductase) from bovine liver, human liver, bovine pancreas, and human kidney.

The enzyme was separated from microsomes by deoxycholate and purified by gel filtration on Sephadex G‐75 and subsequent chromatography on DEAE‐Sephadex A‐50.

Disc electrophoreses showed that highly purified, fresh enzyme preparations consist of only two enzymatically active proteins, forming one main band and a faint band, without any further visible contaminants.

The content of glutathione‐insulin transhydrogenase in rat liver amounts to about 0.5–1% of total liver proteins as calculated from the purification factor.

The molecular weight of the microsomal transhydrogenase determined by gel filtration on Sephadex G‐75 is 50000 to 55000. Using disc electrophoresis in dodecylsulfate‐containing gels molecular weights of 60000 and 121000 were determined for the main component and the second, slower‐migrating band of the enzyme, respectively.

The Michaelis‐Menten constants for insulin and glutathione (in the presence of EDTA) are 31 μM (mol. wt 6000) and 1.43 mM, respectively. Maximum breakdown of 125I‐labelled insulin was found at pH 7.3.

It seems very probable that our transhydrogenase from rat liver microsomes is identical or very similar to the enzyme catalyzing the reactivation of “randomly‐oxidized” ribonuclease (Anfinsen).
The Primary Structure of the Major Parvalbumin from Hake Muscle
Authors: Capony Jean‐Paul , PECHÉRE Jean‐François
Published: March 2005

With the aim of establishing a starting point for the determination of the complete amino acid sequence of the major parvalbumin from hake muscle, the peptides resulting from the tryptic digestion of the S‐sulfo‐derivative and of the performic‐acid‐oxidized protein were examined.

A total of 20 peptides were thus obtained in sufficient quantity and in a state of purity adequate for further studies. Their complete or partial sequence is reported.

When due account is taken of obvious overlaps, the total of these peptides fully accounts for the composition of the whole molecule with the exception of one alanine residue, and for the number of peptides expected from the presence of 12 lysines and 1 arginine in this parvalbumin.
A New Class of Heptose‐Defective Mutant of Salmonella typhimurium
Authors: Lehmann Volker , HÄMMERLING Günter , Nurminen Marjatta , MINNER Inge , Ruschmann Ellen , LÜDERITZ Otto , Kuo Tseng‐Tong , STOCKER Bruce A. D.
Published: March 2005

Salmonella typhimurium rfa‐657 strains, including both mutants and transductants, were shown to contain in their lipopolysaccharides both the (usual) l and the d‐isomer of glycero‐d‐manno‐heptose. By the application, in sequence, of two different extraction procedures S and R form lipopolysaccharides could be isolated separately from a representative rfa‐657 strain. The R lipopolysaccharide was predominantly of the Re chemotype, only a few 2‐keto‐3‐deoxyoctonate (KDO) residues being substituted by heptose (Rd2 chemotype) or by longer core chains. The d‐heptose was enriched in the R lipopolysaccharide, while the S lipopolysaccharide contained mainly the natural l‐isomer. It was found that d‐heptose can replace l‐heptoses I to III of the wild‐type core. However, an elongation to completeness of the core stubs containing d‐heptose seldom occurs.

The heterogeneity in structure of the lipopolysaccharide produced by this class of (leaky) mutants is assumed to be due to an altered 6‐epimerase which, in the wild form, catalyses the conversion of NDP‐d‐glycero‐d‐manno‐heptose into NDP‐l‐glycero‐d‐manno‐heptose and which is determined by the gene rfa‐657 situated between cysE and pyrE. The symbol rfaD is proposed for this gene.
Fractionation of Tyrosine‐Rich Proteins from Oxidized Wool by Ion‐Exchange Chromatography and Preparative Electrophoresis
Authors: Brunner Helmut , BRUNNER Anneraarie
Published: March 2005

On oxidizing wool by performic acid a soluble protein fraction is obtained containing high amounts of tyrosine, phenylalanine and glycine. This fraction, termed “δ‐keratose”, is extremely heterogeneous as shown by solubility characteristics, electrophoresis and end‐group determination. Anion‐exchange chromatography allows isolation of subfractions with different amino‐acid compositions. This process, however, involves a marked loss of tyrosine‐rich proteins absorbed irreversibly. Repeated preparative electrophoresis on polyvinylchloride layers permits isolation of many subfractions on a preparative scale without loss of aromatic amino acids. The amino‐acid composition of these subfractions is investigated. In spite of the wide differences among them some common features for all fractions can be established. It is shown that possible contamination by other proteins is not responsible for the heterogeneity of δ‐keratose but rather due to a wide variation in the amino‐acid composition among the demonstrated tyrosine‐rich proteins.
Thermal Stability of Poly(U) · tRNA: Ribosome Complexes with Phe‐tRNAPhe and Peptidyl‐tRNAPhe
Authors: Hamburger Albert David , LAPIDOT Yehuda , de Groot Nathan
Published: March 2005

The stability of ribosomal complexes, carrying [14C]Phe‐tRNAPhe, Gly‐Gly‐[14C]Phe‐tRNAPhe or Ac[14C]Phe‐tRNAPhe at the A site (aminoacyl site) or at the P site (peptidyl site) has been studied with thermal dissociation techniques. Gly‐Gly‐Phe‐tRNAPhe forms a more stable P‐site complex but a less stable A‐site complex than Phe‐tRNAPhe. The P‐site complex with Gry‐Gly‐Phe‐tRNAPhe is more stable than the A‐site complex with the same substrate. Phe‐tRNAPhe, bound enzymatically at the A site, forms a more stable complex than Phe‐tRNAPhe bound non‐enzymatically at the P site. The complex with Phe‐tRNAPhe, formed enzymatically in the presence of GTP, is considerably more thermostable than the complex formed in the presence of Guo‐5′‐P2‐CH2‐P.

These results provide additional evidence that aminoacyl‐tRNA and N‐blocked aminoacyl‐tRNA interact with different stabilities with the ribosomal binding sites. It is suggested that the observed differences in thermal stabilities reflect the substrate specificity of the peptidyl transferase.
Dihydropteridine Reductase
Authors: Lind Kirsten E.
Published: March 2005

Binding of NADH and quinoid dihydropteridine to dihydropteridine reductase from rat liver has been studied by the techniques of protein fluorescence quenching and ligand protection.

The dissociation constans obtained for the binary complexes by the two methods were in agreement.

For NADH, the constant was 0.07 μM and for quinoid dihydropteridine, 2–3 μM (30 °C, pH 6.8). The constants have been compared with the apparent Michaelis constants.
Variantes de mycosides caractérisées par des résidus glycosidiques substitués par des chaînes acyles
Authors: Gastambide‐Odier Mireille
Published: March 2005

The hydrogenolysis of peracetylated mycoside G′ cleaves this mycoside between the aromatic nucleus and the acylated sugars. The mass spectrum of one of these sugars is typical of an ethyl 2‐O‐mycolyl‐3‐O‐methyl‐4‐O‐acetyl‐6‐deoxypyanoside (I): The highes peaks (m/e 1243, 1215 and 1187) are due to splitting on both sides of the methyl branching of the mycolic chain. Other important peaks prove that the sugar cycle is pyranic, that the mycolic chain is bound to the hydroxyl carried by C‐2 and that the methoxyl group is on C‐3. The acyl chain (IV) fragments (m/e 995, 967 and homologues) are typical of a mycolic acid with a methoxyl group (m/e 253, 281, 325) and a C22 side chain, as indicated by the +CH=6;Ċ–C22H45 peak.

The structure of mycosides A, B and G comprising polar sugars bound through an aromatic nucleus to an apolar aglycone suggests that these substances might function as a carrier or cofactor in the vicinity of the bacterial membrane or close to the bacterial cell wall. That mycolic acids which esterify hydroxyl groups of mycoside sugars are found to exist favors this conclusion, the more so as mycolic substances are constituants of the cell walls (arabinogalactane mycolate) and of the bacterial outer layer (cord factor).
Metabolism of Ethanol and Fructose in the Isolated Perfused Pig Liver
Authors: Damgaard Stig E. , Lundquist Frank , Sestoft Leif , Tønnesen Klavs , Hansen Finn Vallø
Published: March 2005

Ethanol at concentrations below 10 mM was oxidized at a rate of 0.7 μmol×g−1× min−lby the perfused pig liver. Infusion of fructose caused a stimulation (up to 120%) of ethanol oxidation, which was proportional to the rate of fructose elimination.

Stimulation was observed also whend‐glyceraldehyde (a fructose metabolite) was used. Even at highd‐glyceraldehyde concentrations, the increase in ethanol metabolism was less than with fructose.

Hepatic acetate metabolism was not affected by ethanol but was stimulated up to 100% by fructose in the presence of ethanol.

The concentration ratios of sorbitol/fructose and lactate/pyruvate in the perfusion medium were increased in experimental periods with ethanol and fructose compared to periods with fructose alone. The data indicate equilibrium between these metabolites and the free NADH/NAD in the cytosol.

The steady‐state concentrations of D‐glyceraldehyde and glycerol were increased when ethanol was infused together with fructose, while the production ofd‐glycerate was reduced to zero.

The rate of fructose elimination was not influenced by ethanol, although the output of lactate and glucose were diminished.

Accumulation of the reduced metabolites (sorbitol, glycerol,l‐glycerol 3‐phosphate) accounted for only a small part of the reducing equivalents generated during fructose‐stimulated ethanol oxidation. Complete oxidation of the ethanol metabolized would require 3.6 μmol O2×g−1×min−lcompared to a measured uptake of 1.8 μmol× g−l×min−1. As ketone bodies and acetaldehyde were not produced in significant amounts, the retained acetate must be used in syntheses,e.g.of fatty acids.

The experimental results are interpreted in terms of a model, the malate shuttle. Data from experiments with pyrazole and dinitrophenol support the interpretation.
Introduction of “Nicks” and “Chops” into Human Mitochondrial DNA in vivo and in vitro
Authors: Koch Jürgen
Published: March 2005

After prolonged exposure of HeLa cells (ATCC: CCL2) to ethidium bromide (1μg× ml−l in the growth medium) the bulk of the mitochondrial DNA of these cells sediments with a sedimentation coefficient of 21 S. This DNA species is a linear duplex DNA molecule with the length of the circumference of the closed circular mitochondrial DNA. This linear DNA contains single‐strand nicks or gaps as shown by sedimentation after denaturation or by sedimentation through alkaline sucrose gradients. The number of fragments obtained after denaturation increased with decreasing molecular weight reaching a peak at the fragment size of 1/10 of the single‐stranded mitochondrial DNA.

Most of the covalently closed phosphodiester backbones of the supercoiled mitochondrial DNA contain three sites which are sensitive to cleavage by alkali and at least one site which is sensitive to the action of RNAase.