bluuebunny/crossref_metadata_2025 huggingface dataset:
Nascent Subribosomal Particles in Tetrahymena pyriformis
Authors: Leick Vagn , Engberg Jan , Emmersen Julius
Published: March 2005

The metabolism and physical‐chemical properties of nascent (precursor) 40 and 60 S subribosomal particles have been studied in exponentially growing Tetrahymena pyriformis GL.

Analysis of formaldehyde‐fixed 40 and 60 S particles in isopycnic CsCl gradients reveals that they have a lower buoyant density than the corresponding 30 and 50 S ribosomal sub‐units. Moreover, overall labeling of the cells with [3H]amino acid and [32P]phosphate shows that the precursor particles attain larger 3H/32P ratios than the corresponding sub‐units, indicating that the precursor particles contain extra (accessory) protein that splits off when they are converted into ribosomal sub‐units. The extra protein constitutes 10 to 20% of the total structural protein.

Experiments in pulse‐labeling with radioactive amino acids indicate that 40 and 60 S particles are very rapidly labeled. This suggests that, apart from the RNA moiety, at least part of the structural ribosomal protein moiety associated with the nascent particles is newly synthesized.

The possible involvement of 40 and 60 S particles in sub‐unit exchange between rounds of translation was investigated by comparing their kinetics of labeling with that of the ribosomal sub‐units. The results of the comparison indicate that the rate of recycling of ribosomal sub‐units through the pool of 40 and 60 S particles is too slow to account for sub‐unit exchange between each round of translation.
The Nature of Inhibition of Mitochondrial Malate Dehydrogenase by Thyroxine, Iodine Cyanide and Molecular Iodine
Authors: Varrone Stelio , Consiglio Eduardo , Covelli Italo
Published: March 2005

The mechanism of inhibition of the enzyme activity of mitochondrial malate dehydrogenase by thyroxine, iodine cyanide and molecular iodine has been further investigated. The results obtained allow us to exclude formation of a protein/inhibitor stable complex, iodination of the enzyme or disaggregation of the protein molecule into inactive subunits as explanations for the enzyme inactivation induced by the iodine‐containing compounds. On the other hand, it has been demonstrated that in all cases the enzyme inhibition is dependent on the oxidation of certain—SH groups necessary for the activity.

Direct oxidation of the thiol groups of the protein by molecular iodine and iodine cyanide can be easily assumed, whereas the chemical mechanism of the thyroxine‐induced enzyme inactivation is harder to explain. However, since neither hormone deiodination nor metal chelation occur, while thyroxine by itself is also able to oxidize the thiol group of reduced glutathione, a direct effect of thyroxine on the sulfhydryl groups of the enzyme is postulated.
Evolution of the Polyribosome Distribution during in vivo Reticulocyte Maturation
Authors: Marbaix G. , Burny A. , Huez G. , Lebleu B. , Temmerman J.
Published: March 2005

Maturation of rabbit reticulocytes in vivo is accompanied by a shift in the polyribosome distribution from heavy to small clusters.
Inhibition of Protein Synthesis in the Reticulocytes by Aminoacyl Adenosine
Authors: Harbon Simone , Chapeville François
Published: March 2005

Leucyl adenosine and phenylalanyl adenosine inhibited protein synthesis in the reticulocytes, inhibition being accompanied by the release of growing peptide chains from the ribosomes. The action of aminoacyl adenosines was compared to that of puromycin.

Although leucyl adenosine was rapidly hydrolyzed in the incubation medium, inhibition was maintained within the reticulocytes for several hours, indicating a stabilization of the compound inside the cell. Reversibility of the inhibitory effect was temperature dependent.

Protein synthesis was inhibited less efficiently in the lysate than in intact cells; an enhanced hydrolysis of leucyl adenosine was noticed in the lysate.

No specificity of the inhibition reaction was observed concerning the amino acid moiety (leucine or phenylalanine), whereas in the nucleoside part, adenosine was required.
Markers of the Variable and Constant Region of Human Lambda Immunoglobulin Chains
Authors: Tischendorf Frank W. , Tischendorf Margrit M.
Published: March 2005

52 human lambda immunoglobulin chains could be divided into two antigenic subtypes, St(+) and St(–).

St(+) type chains represent a homogeneous structural group differing from St(−) type proteins by characteristic subclass specific positions in the N‐terminal sequence of amino acids. St(+) proteins follow the “Ha” type sequence. St(+) type molecules are present in normal L‐chain preparations. 19% of normal lambda chains appear to consist of St(+) type molecules.

St(−) type chains include at least two isotypic variants, following the “Bo” and “X” type sequence of amino acids respectively.

The results point in the direction that 3 germ‐line genes, one st(+) and two st(–) genes, encode the specificity region of lambda polypeptide chains.

No correlation between N‐terminal St markers and the lysine‐arginine interchange at position 190 of the C‐terminal half was found; thus evidence of the unigenic control of the lambda molecule was not provided.

190Arg and 190Lys proteins do occur in the three major antigenic lambda chain subclasses.

The possible allelic nature of the lysine‐arginine interchange at position 190 is discussed.
Calcium Efflux from a Heavy Sarcotubular Fraction
Authors: Fairhurst Alan S. , Hasselbach Wilhelm
Published: March 2005

Calcium efflux has been measured from two different fractions of rabbit skeletal muscle partially loaded with calcium oxalate, under conditions in which the inward calcium pump is inoperative. Efflux from the heavy sarcotubular fraction (2000–8000×g) is stimulated by ryanodine and caffeine, whereas calcium efflux from a light fraction (12000–35000×g) is much less sensitive to these drugs. Quinidine and chlorpromazine stimulate calcium efflux from both muscle fractions. These effects of ryanodine and caffeine are completely blocked, and those of quinidine and chlorpromazine partially blocked, by 5 mM magnesium, although magnesium does not affect calcium efflux in the absence of drugs. Zinc stimulates and uranyl ion depresses calcium efflux.
Nucleotide Sequences in a Protected Area of the 16 S RNA within 30 S Ribosomal Subunits from Escherichia coli
Authors: Fellner Peter , Ehresmann Chantal , Ebel Jean‐Pierre
Published: March 2005

The 16 S RNA is partly protected against attack by T1 ribonuclease when it is enclosed in the 30 S ribosomal subunit. It has therefore proved possible to isolate 3 large fragments of the 16 S RNA resulting from digestion of the molecule within the 30 S subunit. The nucleotide sequences present within these fragments have been studied by using fingerprinting methods. It was found that the 3 fragments were derived from the same area of the molecule. We postulate that this area, encompassing 35–40% of the 16 S RNA is especially inaccessible to nuclease action, due to some major feature of the architecture of the 30 S ribosomal subunit.
A Sequence of 50 Nucleotides from Coliphage R17 RNA
Authors: Rensing Ulrich F. E. , Schoenmakers John G. G.
Published: March 2005

A sequence of 50 nucleotides of the RNA from coliphage R17 has been determined. It can be read through in only one translational frame. The sequence is not part of the coat protein cistron nor does it come from the untranslated sequences described so far.

Two possible “hairpin” secondary structures have been constructed.

The two‐dimensional technique on thin‐layer plates has been used to fractionate intermediates in limited exonucleolytic digests of two dodekamers which could not be sequenced by conventional techniques. The potential general use of this method has been discussed.
Incorporation of Galactose into Blood‐Groups (ABH) Precursor Substance by Lactose Synthetase from Human Milk
Authors: Schenkel‐Brunner Helmut
Published: March 2005

It has been shown that an enzyme preparation from human milk having lactose synthetase activity is capable of incorporating galactose residues into water‐soluble blood‐group substance. A suitable acceptor substance for investigating the enzymatic galactose transfer was obtained from blood‐group H‐substance by sequential removal of fucose and terminal galactose residues. When galactose was incorporated into this acceptor substance with the help of a preparation of galactosyl transferase from human milk in the presence of UDP‐galactose, a remarkable increase in cross‐reactivity with anti‐type‐14 pneumococcus sera could be observed. Since a glycoprotein with “type‐14 specificity”, having galactose as the serologically determinant group, is the precursor of the water‐soluble blood‐group A, B, H and Le substances, this enzymatic galactose transfer would appear to be an essential step in the biosynthesis of the blood‐group‐active compounds. Evidence for the identity of the enzyme responsible for the synthesis of type‐14‐specific sites with lactose synthetase of human milk was obtained from competition experiments.
Affinity Labeling by m‐Nitrobenzenediazonium Fluoroborate of Porcine Anti‐Dinitrophenyl Antibodies
Authors: Franěk Františiek
Published: March 2005

From the tryptic digest of γ‐chains of porcine anti‐dinitrophenyl antibody which has been affinity‐labeled by m‐nitrobenzenediazonium fluoroborate, an azotyrosine‐containing peptide consisting of 28 amino acid residues was isolated using affinity chromatography on Sepharose‐bound antibodies. The peptide contained a half‐cystine residue and a tryptophan residue, The thermolysin digest of this peptide yielded several shorter pep tides. The amino acid sequence in the vicinity of the half‐cystine was revealed by the finding of the peptide Leu‐Ser‐Cys(O3H). Azotyrosine was found in a nonapeptide, the amino acid sequence of which was characterized by the presence of more than one amino acid in most of the steps of sequential degradation.

Comparison of the amino acid sequence in the vicinity of the half‐cystine residue and of the partial amino acid sequence of the azotyrosine‐containing nonapeptide with the known sequences of heavy chains of man, rabbit, mouse and guinea‐pig immunoglobulins showed that the labeled tyrosine of γ‐chains of porcine anti‐dinitrophenyl antibody is located most likely in the hyper‐variable section between half‐cystine 22 and tryptophan 36.
Interconversion of Pyruvate Dehydrogenase in the Isolated Perfused Rat Liver
Authors: Patzelt Christoph , Löffler Georg , Wieland Otto H.
Published: March 2005

Active form and total activity of pyruvate dehydrogenase were measured in homogenates prepared from tissue samples obtained from the isolated rat liver during perfusion.

In the absence of substrate, the active portion accounted for about 20% of total activity in livers from fed rats, whereas only about 10% were found to be active in livers from starved rats. These activities remained rather constant during a perfusion period of 120 min. Addition of 10 mM pyruvate to the perfusion medium resulted in an immediate, three‐fold increase of the active pyruvate dehydrogenase. A similar effect was exerted by 20 mM fructose, which is known to be rapidly converted to pyruvate in liver. 10 mM lactate led to a considerably smaller increase of pyruvate dehydrogenase activity as compared to the effects obtained with pyruvate or fructose. Total activities of the enzyme did not change significantly during perfusion.

If the activities of active pyruvate dehydrogenase from livers perfused with and without substrate were plotted against medium pyruvate concentrations an exponential correlation could be observed.

Addition of 2 mM oleate abolished the effects of fructose or pyruvate on the formation of active pyruvate dehydrogenase. The simultaneous addition of d‐(+)‐decanoylcarnitine inhibited the action of oleate, indicating that fatty acid oxidation is required for the inactivation of pyruvate dehydrogenase by interconversion of the active to the inactive form.

The possible physiological significance of pyruvate and fatty acids in the regulation of pyruvate dehydrogenase interconversion is discussed.
Cell Walls, Lipids, and Lipopolysaccharides of Pseudomonas Species
Authors: Wilkinson Stephen G. , Galbraith Lesley , Lightfoot Gordon A.
Published: March 2005

Cell walls from two EDTA‐sensitive and three EDTA‐resistant pseudomonads grown in nutrient broth have been analysed. Each wall had a composition broadly characteristic of gram‐negative bacteria, although the walls of Pseudomonas pavonacea and P. syncyanea were relatively rich in glycosaminopeptide and loosely‐bound lipid, respectively. Phosphatidylglycerol was the only phospholipid common to all species, although phosphatidylethanolamine was absent only from P. diminuta. The latter species contained the distinctive range of glycolipids and a phosphoglucolipid identified previously in cells grown on nutrient agar. However, the glycolipids and an ornithine‐containing lipid present in some other species (notably P. rubescens) when grown on nutrient agar were not detected. Except in P. rubescens, the major fatty acids in loosely‐bound lipids were C16, C16:1, and c18:1 acids: in P. rubescens, the major components were C15, iso‐Cl5, and C17:1 acids. Cyclopropane acids occurred in the lipids from P. stutzeri.

Components of the lipopolysaccharides were identified and determined. The lipid A fractions contained fatty acids, phosphorus, and (except for P. diminuta) glucosamine. The fatty acids included both hydroxy and non‐hydroxy acids. Apart from 2‐hydroxydodecanoic acid (present in P. syncyanea), all the hydroxy acids were β‐hydroxy acids, and 3‐hydroxydodecanoic acid predominated in all species except P. rubescens. Odd‐numbered hydroxy acids were minor components in P. diminuta and P. pavonacea and major components (C13 acids) in P. rubescens. The non‐hydroxy acids consisted of saturated acids (C12 to C14) and αβ‐unsaturated acids (dehydration products from the β‐hydroxy acids).

Unusual features of the polysaccharide fractions from the lipopolysaccharides included the occurrence of non‐phosphorylated heptose residues (P. diminuta), the presence of phosphorylated glucose residues and of aspartic acid (P. pavonacea), the probable absence of 2‐keto‐3‐deoxyoctonic acid and the presence of acid‐labile galactose residues (P. rubescens), the virtual absence of glucose (P. stutzeri), and the presence of alanine (P. syncyanea). Amino sugars (glucosamine, galactosamine, and quinovosamine) occurred in the polysaccharides from P. stutzeri and P. syncyanea only. Following chromatography of the partly degraded polysaccharides on Sephadex, the distribution of components between possible side chain and core regions of the polysaccharides could be determined.

The possible significance of the results for the sensitivity of the organisms to EDTA and for their taxonomy is discussed.
A Model for the Initiation of Transcription by DNA‐Dependent RNA Polymerase from Escherichia coli
Authors: Schäfer Rolf , Zillig Wolfram , Zechel Kasper
Published: March 2005

The stable initiation complexes formed by interaction of DNA‐dependent RNA polymerase from Escherichia coli containing initiation factor σ with native DNAs of bacteriophages T4, T5 and T7 have been differentiated into a “labile‐starter” and a “stable‐starter” class. The former exhibits a half‐life of 1 min with T5‐DNA and of 10 min with T4‐DNA under standard conditions, the latter a half‐life of more than 100 min for the first‐order decay in the presence of the strong template‐competitor heparin. The labile class is competed out by core enzyme, the stable is not. The relative amounts of both classes of complexes depend on ionic strength.

The stable‐starter class has been subdivided into a “delayed‐starter” complex formed rapidly and already possessing high stability in the presence of heparin but unable to directly initiate and an “immediate‐starter” complex as a first‐order transition product of the delayed‐starter state. From the immediate‐starter complex initiation can proceed immediately. The time for transition of half of the former into the latter complex is 3.5 min with T5‐DNA. The temperature dependence of the equilibrium concentration of the stable‐starter complex reveals a sigmoid shape indicating a highly cooperative event, probably a conformational change in the transition from delayed to immediate starter.

From the length‐average chain length of the transcripts of T5‐DNA in vitro and the length of the transcribed fraction of the DNA an approximate number of 14 promoters per DNA molecule was estimated. For T4‐DNA, calculation yields about 6. In excess of RNA polymerase about 27 copies of transcript per T5‐ “promoter” were produced from the stable complex under conditions excluding consecutive initiation by free enzyme and reinitiation. The corresponding value for T4‐DNA is between 1 and 4.

On the basis of these results, a model for the initiation of transcription is postulated, in which an initiation site on the DNA (promoter) is subdivided into an clement for primary recognition, the entry site R, an element for the storage of enzyme for immediate initiation, termed storage stretch S, which furnishes space for the stable alignment of a certain number of polymerase molecules, and an element for actual initiation, termed initiator I.
Étude de l'action de sels de cations monovalents sur les particules ribonucléoprotéiques de cerveau de rat
Authors: Stévenin James , Zawislak Richard , Jacob Monique , de Hildwein Georges
Published: March 2005

The dissociation of the nuclear particles by salts has been studied in order to throw some light upon their structure.

The density of nuclear mono‐ and polyparticles in CsCl was progressively increased as a result of the action of increasing KCl concentrations (from 0.4 to 1.0 M). The results indicate that proteins are progressively released from the particles and are not consistent with the existence of an “informofer” stable at high salt concentration.

Polyparticles of high sedimentation coefficient were more resistant to the action of salts than smaller ones. Respectively 69, 35 and 22% of 60‐S, 110‐S and 170‐S particles (density in CsCl: 1.40 g/ml) were dissociated with 1 M KCl into complexes of density (in CsCl) higher than 1.60 g/ml. A further increase in salt concentration or in time of contact, substitution of K+ by Na+ or Li+ ions or the replacement of Mg2+ by EDTA had little effect on the dissociation of 110–170‐S polyparticles. Polyparticles were more easily dissociated by salts when they were first treated with RNAase at very low concentrations, leading to only a few cuts of the polynucleotide chains but not to the hydrolysis of poly‐ into monoparticles. Together the results suggest a compact configuration for the polyparticles.
Inhibition on the Reducing Side of Photosystem II by Carbonylcyanidem‐Chlorophenylhydrazone and Lithium 3,5‐Diiodosalicylate
Authors: Homann Peter H.
Published: March 2005

Several investigators have shown that carbonylcyanidem‐chlorophenylhydrazone (CCCP) inhibits photosystem II of chloroplasts by acting on a site close to the oxygen‐evolving center. Evidence is presented in this paper which suggests that CCCP interferes also with the electron transfer between the primary acceptorQand the secondary acceptor pool,A.A similar action in this electron acceptor complex is described for the photosynthesis inhibitor salicylaldoxime, and other phenolic compounds like salicylate and, in particular, lithium 3,5‐diiodosalicylate. The latter two agents are known for their ability to increase the solubility of hydrophobic peptides and proteins in aqueous solutions. Hence, our findings may be of help in attempts to understand the structure‐activity relationship of the large class of structurally diverse herbicides which inhibit photosynthetic electron transport betweenQandA.
Initiation of Aerobic Oxidation of Sulfite by Illuminated Spinach Chloroplasts
Authors: Asada Kozi , Kiso Kuniaki
Published: March 2005

Spinach chloroplasts induced aerobic oxidation of sulfite on illumination. The oxidation of sulfite was not found in KCl‐ and Tris‐treated chloroplasts and was inhibited by 3(3,4‐dichloro‐phenyl)‐1,1′‐dimethylurea, suggesting that the reaction is induced through the electron transport system in chloroplasts. The oxidation rate of sulfite was much higher than the rate of the Hill reaction. In addition, inhibition by several sugars indicates that the oxidation of sulfite proceeds as a chain reaction. Although catalase did not affect the reaction, ferredoxin plus NADP, cytochrome c and superoxide dismutase prevented the oxidation of sulfite. We thus propose that a superoxide anion formed by the univalent reduction of oxygen by illuminated chloroplasts is the initiator of sulfite oxidation.
Conformational Studies on Lipoamide Dehydrogenase from Pig Heart
Authors: van Muiswinkel‐Voetberg H. , Veeger Cornelis
Published: March 2005

NAD+ forms two pairs of spectrally distinguishable complexes upon binding to oxidized lipoamide dehydrogenase. The shape and magnitude of the spectra is dependent on temperature and pH. The flavin‐absorption band at 450 nm is not shifted uniformly. Intrinsic dissociation constants only could be calculated at 430 nm. In 30 mM phosphate buffer pH 7.2 at 25 °C K1d= 50–60 μM, while at 0 °C K1d= 40 μM for the high‐affinity pair; for the low‐affinity pair these values are 200–250 μM and 110–130 μM respectively. The high‐affinity pair is also present at pH 5.6 at I= 0.1 and I= 0.2, the values at 25 °C are respectively 50 μM and 35 μM.

Computer simulation suggests that the activating effect of NAD+ on the NADH : lipoate reductase activity can be explained by assuming that binding of NAD+ shifts the pK‐value of a protonated group at the active centre from 6.2 to 4.9–5.0. This suggestion is supported by the observation that binding of NAD+ at the high‐affinity site is accompanied by liberation of 0.9 ± 0.07 equiv. H+/mol flavin. From titration data it was calculated that for NAD+ binding K1d= 65 μM.

The results indicate that binding of NAD+ at the high‐affinity site, even at partial saturation, shifts the monomer‐dimer equilibrium towards the dimer. Furthermore the difference in reduction state upon reducing the enzyme with NADH at 25 °C and 0 °C is not due to differences in affinity for NAD+, but to conformational changes of the protein around the flavin moiety. It has to be considered that the two active centres of the dimer are not completely independent.
Binding of Ligands to the Inorganic Pyrophosphatase of Bakers' Yeast
Authors: Rapoport Torn A. , Höhne Wolfgang E. , Heitmann Peter , Rapoport Samuel
Published: March 2005

Binding studies of ligands to the inorganic pyrophosphatase of bakers' yeast were performed by means of the following techniques: flow dialysis, an electrode sensitive to divalent metal ions, difference spectroscopy and kinetic measurements. The effects of tyrosine modification on binding (and vice versa) were also studied.

The results were:

There exist per enzyme molecule four binding sites for the divalent metal ions, presumably two per subunit. Two of the four binding sites are identical with the activator sites postulated from previous kinetic experiments. Binding to these sites induces spectral changes which are similar for all the metal ions. The association constants fall in the order Mn2+ > Co2+ > Mg2+. The role of the other binding sites is still unknown.

Calcium pyrophosphate is bound at two sites per enzyme molecule. It is a good analogue to the actual substrates, i.e. pyrophosphate complexes of Mg, Mn, Co, Zn, since it is bound tightly but is not split by the pyrophosphatase. It protects the enzyme effectively against inactivation by nitration of tyrosines with tetranitromethane.

PPi in the absence of divalent metal ions is weakly bound and is therefore a bad substrate analogue. Pi is also only weakly bound. It is concluded that a stable covalent phosphoryl‐enzyme is not formed under the experimental conditions.

Nitration of an active tyrosine residue in the substrate‐binding site weakens the binding of Co2+ to the activator site by a factor of about 6 while the affinity of calcium pyrophosphate to the substrate site probably completely vanishes.
The Denaturation by Urea and Guanidinium Chloride of Trypsin and N‐Acetylated‐Trypsin Derivatives Bound to Sephadex and Agarose
Authors: Gabel Detlef
Published: March 2005

Coupling of trypsin to cyanogen‐bromide‐activated Sephadex, but not to agarose, results in a stabilization of the enzyme towards denaturation by urea.

Selectively N‐acetylated trypsin derivatives were prepared and coupled to Sephadex.

When a single amino group, which is required for the immobilization reaction, was available for coupling, the insoluble derivative was denatured as the free one.

The availability of two or three amino groups for coupling diminished the rate of denaturation considerably. When more than approximately four amino groups were available for coupling, the insoluble derivatives were fully active in 8 M urea.

Neither the binding to Sephadex nor that to agarose influenced the denaturation of the enzyme by guanidinium chloride.

Differences between the denaturation mechanisms of urea and guanidinium chloride are discussed.
The Bacterial Degradation of 5‐Amino‐4‐chloro‐2‐phenyl‐3(2H)‐pyridazinone
Authors: de Frenne Eberhard , Eberspächer Jürgen , Lingens Franz
Published: March 2005

From the culture medium of bacteria, which utilize 5‐amino‐4‐chloro‐2‐phenyl‐3(2H)‐pyridazinone (pyrazon) as sole source of carbon four metabolites could be isolated. These metabolites were identified as 5‐amino‐4‐chloro‐2‐(2,3‐cis‐dihydroxycyclohexa‐4, 6‐diene‐1‐yl)‐3(2H)‐pyridazinone (compound I), as 2‐(5‐amino‐4‐chloro‐3‐oxo‐2,3‐dihydro‐2‐pyridazino)‐cis,cis‐muconic acid (compound II), as 2‐pyrone‐6‐carboxylic acid (compound III) and as 5‐amino‐4‐chloro‐3(2H)‐pyridazinone (compound IV).

Cell‐free extracts of the pyrazon‐degrading bacteria were shown to metabolize 2‐hydroxy‐muconic acid with a stoichiometric release of CO2.

A pathway for the bacterial degradation of 5‐amino‐4‐chloro‐2‐phenyl‐3(2H)‐pyridazinone is proposed.
Biological Role of Xanthine Oxidase and Tetrazolium‐Reductase Inhibitor
Authors: Fried Rainer , Fried Lygia W. , Babin Donald R.
Published: March 2005

The biochemical properties of tetrazolium reductase inhibitor (“reductase inhibitor”) from beef brain and liver were compared with superoxide dismutase (erythrocuprein) from beef erythrocytes, using xanthine oxidase and xanthine as a model system.

In all assays the behavior of erythrocuprein and the reductase inhibitor was identical; it is concluded that the tetrazolium reductase inhibitor is a member of the superoxide dismutase class of enzymes. Its possible identity with other proteins of this group remains to be established.

Xanthine dehydrogenase is activated by natural or synthetic detergents. These compounds eliminate the enzymatic action of both erythrocuprein and reductase inhibitor. Low levels of cyanide, which do not inhibit xanthine dehydrogenase block superoxide dismutase.

The biological role of xanthine oxidase is discussed. We propose that an important function of xanthine oxidase is to provide an ubiquitous source of hydrogen peroxide and superoxide radicals which serve as oxidants for coupled biological oxidations.
Conditions Affecting the Dissociation Reaction in vitro of Rat‐Liver Ribosomes
Authors: von der Decken Alexandra
Published: March 2005

Subunits were obtained by incubation of ribosomes with cell sap, puromycin, GTP, phospho‐enolpyruvate, pyruvate kinase, subsequent exposure to 0.15 M KCl and 1 mM MgSO4 followed by sucrose density‐gradient separation.

Addition of ATP reduced the yield of the subunits but had no influence on their purity as judged by polyacrylamide‐gel electrophoretic analysis of their RNA. Reassociation of the sub‐units to form 80‐S ribosomes and restoration of polyuridylic‐acid‐dependent synthesis of polyphenylalanine was highest when the subunits were prepared by incubation of ribosomes in the absence of ATP with or without puromycin.

In the absence of puromycin, the dissociation rate per minute was 8% and 2% of the total at 35° and 25°C, respectively.

Dietary protein restriction excerted no influence on the dissociation of the ribosomes incubated with cell sap of liver from rats fed a normal protein diet, while cell sap from protein‐restricted rats had a decreased capacity to dissociate ribosomes of normal rats. These differences were significant per wet weight of liver but not per mg cell‐sap protein, due to a simultaneous decrease in the protein content of the cell.
Structure and Function of the Genome of Coliphage T5
Authors: Bujard Hermann , Hendrickson Herbert E.
Published: March 2005

Separated strands of ligated DNA from coliphage T5+ and single‐stranded fragments purified from alkaline‐denatured DNA were used in DNA‐DNA annealing experiments. The products of such experiments were analyzed by analytical ultracentrifugation to equilibrium in a CsCl density gradient as well as by electron microscopy. Pycnometric analyses showed that the heavy strand is the uninterrupted strand, whereas the light strand is the ligated form of the nicked strand. All fragments of length shorter than one half of the complete DNA molecule are derived from the light strand. A new arrangement of the single‐stranded fragments of the T5+ chromosome has been deduced from electron microscopic analysis and has resulted in a new model of the physical structure of the DNA of T5+.
Structure and Function of the Genome of Coliphage T5
Authors: Hendrickson Herbert E. , Bujard Hermann
Published: March 2005

Separated strands of ligated DNA of coliphage T5+ and purified specific single‐stranded fragments were used in DNA‐RNA hybridization studies with RNA extracted from cells at various times after infection by T5+. The ratio of the binding of pre‐early RNA to the heavy (uninterrupted) and light (nicked) strands is about 2 to 1, whereas for early RNA the ratio is about 3 to 1. Most, if not all, late RNA is transcribed from the heavy strand. Pre‐early RNA binds appreciably to a newly discovered small (Mr= 1.5 × 106) fragment of DNA derived from the light strand as well as to other, larger, fragments. The regions of transcription of the various classes of RNA are discussed in relation to a new model of the chromosome and the genetic and functional maps of T5.
Synthesis of Lens Protein in vitro
Authors: Berns Ton J.M. , Schbeurs Victor V.A.M. , van Kraaikamp Marjolijn W.G. , Bloemendal Hans
Published: March 2005

Messenger ribonucleic acid was isolated from calf lens polyribosomes and fractionated. Previous identifications of the newly synthesized products have been extended in that the fidelity of the translation of the 14‐S mRNA was assayed by fingerprinting analysis. The results suggest that lens messenger is translated with a high degree of fidelity in the cross systems even to the extent that the newly synthesized αA2 chains contain an acetylated methionine residue in the N‐terminal position like the native chains. There seems to be no requirement for cell‐specific protein factors in order to obtain accurate translation. Evidence is accumulating that the acetylation is not involved in the initiation mechanism.
Hydrolysis of [3H,4C] Phosphatidylethanolamines by Acid Phospholipases A of Subcellular Fractions of Rat Spleen
Authors: Lloveras Jacques , Douste Blazy Louis
Published: March 2005

Rat spleen exhibits maximal phospholipase A activity at pH 4.5. This activity is due to a phospholipase A1 and a phospholipase A2. After injection of Triton WR 1339, rat spleens were fractionated by differential centrifugation. The mitochondria + lysosomes fraction was further fractionated on a sucrose gradient. Three lysosomal populations were individualized of which the heavier one was sedimented with mitochondria.

With phosphatidylethanolamines as substrate, phospholipase A1 and A2 are present in similar ratio in all the subcellular fractions and in the three lysosomal populations.

Rat spleen is about 1.3‐fold richer in phospholipase A1 than in phospholipase A2. The fraction from centrifugation at 1000×g for 10 min contains respectively 40% and 44% of the total phospholipases A1 and A2 activities, the mitochondria + lysosomes fraction only 28% and 26%. Free ribonucleoprotein particles have low phospholipases A1 and A2 activities.
Luminescence Quenching in Azurin
Authors: Finazzi‐Agrò Alessandro , Giovagnoli Carlo , Avigliano Luciana , Rotilio Giuseppe , MondovÍ Bruno
Published: March 2005

The luminescence of oxidized or reduced azurin, an intensely blue copper protein, has been observed. The addition of Cu2+ or Hg2+ reduced the luminescence of the apoprotein to one third of its value while Ag+ reduced it to two thirds. This quenching is explained in terms of tryptophan distorsion produced by the different coordination of these metal ions. A singlet‐singlet internal conversion is postulated as the mechanism for luminescence quenching in all azurin derivatives. Copper is able to bind both the silver‐ and mercury‐treated protein, as shown by electron paramagnetic resonance experiments, without restoring the native blue color. Moreover addition of copper reduced the luminescence in the silver‐treated protein to that of the native protein; if NaCl is now added, the blue color is restored. This NaCl effect is absent in the mercury‐treated apoprotein.

These results suggest that the blue color is related to the binding of copper to at least one of the ligands involved in the mercury or silver complexing, most probably a sulfur atom.
Identification of Unconjugated Androst‐5‐ene‐diol in Human Peripheral Plasma
Authors: Demisch Klaus , Schöffling Karl , Seiler Nikolaus
Published: March 2005

In 2000 ml of pooled human male plasma unconjugated 3β, 17β‐dihydroxyandrost‐5‐ene as diacetate was identified. The identification was based on paper chromatography, acetylation followed by thin‐layer chromatography and final mass spectrometry. Furthermore aliquot parts of the extract after paper‐chromatography were subjected to a protein‐binding assay to assure the 17β‐hydroxyl configuration.
Spin‐Labelled Phosphofructokinase and Its Interactions with ATP and Metal·ATP Complexes as Studied by Magnetic‐Resonance Methods
Authors: Jones Robert , Dwek Raymond A. , Walker Ian O.
Published: March 2005

Rabbit skeletal muscle phosphofructokinase has been specifically modified at its most reactive thiol group with the nitroxide spin label, 4‐(2‐iodoacetamido)‐2,2,6,6–tetramethyl piperidinooxyl. The specific activity and allosteric kinetics are only slightly altered in the modified enzyme.

The electron spin resonance spectrum of the labelled enzyme at pH 7.5, in the absence of stabilising ligands, indicates that the label has moderate mobility, with a rotational correlation time, τR, of 3.3 ns, which is greater than the mobility of the smallest fully active oligomer of 340000 molecular weight (τR= 90 ns).

The spectrum is sensitive to pH and to the binding of ATP and its magnesium and manganous complexes. The binding of the metal complexes produces an immobilisation of the label (τR= 4.7 ns). In addition to this, MnATP causes a 74% quenching of the signal height as a result of dipolar spin‐spin interaction.

At pH 7.5, MnATP and ATP bind competitively to the labelled enzyme with dissociation constants of 540 μM and 1.3 mM respectively. The binding of MgATP is sigmoidal with [S]0.5= 400 μM and a Hill interaction coefficient of 2.0.

A two‐state model is proposed to account for the electron spin resonance spectral changes, in which the cooperative interactions are due to metal‐ATP binding at catalytic sites, and the dipolar interactions are due to metal‐ATP binding at a separate regulatory site closer to the spin label.

The longitudinal relaxation rate of water protons at 35 MHz is not detectably altered by the presence of the spin label on the enzyme. An analysis of enhancement data obtained in the presence of manganous ions and ATP indicates that the enhancement of the ternary complex, ɛt, is unchanged compared with the native enzyme, but that the dissociation constant for Mn ATP increases from 260 μM in the native enzyme to 480 μM in the labelled enzyme.

The quenching of the electron spin resonance spectrum and the enhancement in the ternary complex of enzyme with MnATP have together been used to estimate the manganese‐nitroxide distance.

The longitudinal and transverse relaxation rates of the H‐2, H‐8, and H‐1′ protons in MgATP bound to the spin‐labelled enzyme are increased in comparison with the native enzyme due to the dipolar spin‐spin interactions. This allows the proton‐nitroxide distances to be estimated.

The distances calculated from the dipolar interactions have been used to obtain a self‐consistent three‐dimensional model of the regulatory site in relation to the spin label.
The Primary Structure of a Protein, Component 0.62, Rich in Glycine and Aromatic Residues, Obtained from Wool Keratin
Authors: Dopheide Theo A. A.
Published: March 2005

The amino acid sequence of component 0.62, a protein derived from wool keratin, with molecular weight of 6950, rich in glycine and aromatic residues, has been determined. The protein contains no lysine, histidine, glutamic acid, isoleucine or methionine. Of the total residues, more than 50% are glycine and aromatic amino acids. The sequence contains two sections rich in glycine, (Gly‐X)3 and (Gly‐X)4, which comprise 22% of the sequence.
Enzymatic Conversion of Human Blood‐Group‐O Erythrocytes into A2 and A1 Cells by α‐N‐Acetyl‐d‐galactosaminyl Transferases of Blood‐Group‐A Individuals
Authors: Schenkel‐Brunner Helmut , Tuppy Hans
Published: March 2005

Human O‐erythrocytes, when incubated with UDP‐N‐acetyl‐d‐galactosamine and α‐N‐acetyl‐d‐galactosaminyl transferase prepared from gastric mucosa of blood‐group A1 individuals, first acquired A2‐specificity and, after continued incubation, became agglutinable by anti‐A1 reagents. Group‐H specificity gradually disappeared. When A2‐erythrocytes were subjected to the same treatment, they required a shorter time of incubation than O‐cells to develop A1‐properties. These findings support the hypothesis of low and high densities of A‐specific sites on the cell surfaces of A2‐ and A1‐erythrocytes, respectively.

Acetylgalactosaminyl transferase prepared from the gastric mucosa of blood‐group‐A2 individuals was similar to the corresponding enzyme from A1‐individuals in being capable of transferring acetylgalactosamine from UDP‐acetylgalactosamine to water‐soluble H‐substance and of converting O‐erythrocytes into A2‐cells. With H‐substance as the substrate the blood‐group‐A2 enzyme was considerably less active than the A1‐enzyme based on equal weights of mucosal material extracted. When preparations of A1‐enzyme were diluted so as to show a transferase activity towards blood‐group‐H substance equal to that of the A2‐enzyme preparation, there was still a marked difference between the enzymes with regard to O‐erythrocytes, the speed of their conversion into A2‐cells being much less with the blood‐group‐A2 enzyme as compared with blood‐group‐A1 enzyme. These experiments clearly indicate that blood‐group‐A1 and A2 enzymes have different substrate specificities.
The Influence of Glycosaminoglycans on the Formation of Fibers from Monomeric Tropocollagen in vitro
Authors: ÖBrink Björn
Published: March 2005

Well‐characterized, monomeric, lathyritic tropocollagen and a variety of well‐characterized glycosaminoglycan preparations have been used in turbidimetric studies on collagen fiber formation in vitro. The experiments have been performed under physiological ionic conditions and at 37°C. It has been shown by others that the fibrillogenetic process consists of two stages, a nucleation phase and a fiber growth phase. By addition of polysaccharides before and after the nucleation phase it was possible to differentiate between the effects of the polysaccharides on the nucleation and on the fiber‐growth, respectively. Chondroitin sulphate, dermatan sulphate, heparan sulphate, heparin and proteoglycans of chondroitin sulphate and dermatan sulphate, which at 4°C form complexes with tropocollagen, in most cases accelerated the fiber formation, when they were added before the nucleation phase. This phenomenon was ascribed to an effect on the nucleation process. When added after the nucleation phase chondroitin sulphate, a heparan sulphate fraction and especially the proteoglycans delayed the fiber formation. This observation was ascribed to an inhibition of fiber growth from the nuclei.

Hyaluronic acid, which increases the activity of the tropocollagen molecules by steric exclusion, accelerated both the nucleation and the fiber growth processes. Keratan sulphate, which does not form complexes with collagen, had no or a slightly decelerating effect, either when added before or after the nucleation.

It could be demonstrated by light‐scattering technique that certain glycosaminoglycans influenced the properties of the collagen fibrils formed during the early fiber formation process.

The observation that certain polysaccharides had different effects on collagen fiber formation, when added before or after the nucleation, shows that the state of aggregation (nucleation) of collagen is of prime importance in determining the effects exerted by the polysaccharides. This finding may explain some of the contradictory data in the literature. Chemical and physical heterogeneity of the glycosaminoglycans also influenced their mode of interaction with collagen during fiber formation.
The Subunit Structure of the Aliphatic Amidase from Pseudomonas aeruginosa
Authors: Brown Paul R. , Smyth Maurice J. , Clarke Patricia H. , Rosemeyer Michael A.
Published: March 2005

The molecular weight of Pseudomonas aeruginosa amidase was found to be 200000 by sedimentation equilibrium analysis; the sedimentation coefficient was 10.6 S. The subunit molecular weight determined by electrophoresis in dodecylsulphate‐acrylamide gels and gel filtration on Sephadex G‐200 in dodecylsulphate‐p‐chloromercuribenzoate solution was 33000—35000. The amino acid composition of the enzyme was determined.

Only one N‐terminal amino acid, methionine, and one C‐terminal amino acid, alanine, were found indicating that the subunits were identical. Further evidence for a structure comprising 6 identical subunits came from both tryptic fingerprints and reaction of the enzyme with the cross‐linking reagent dimethyl suberimidate.
Duck‐Haemoglobin Synthesis in Frog Cells
Authors: Lane Charles‐Daniel , Gregory Caroline M. , Morel Carlos
Published: March 2005

9‐S RNA was prepared from duck reticulocytes by dodecylsulfate treatment of the ribonucleoprotein particle derived from EDTA‐treated reticulocyte polyribosomes. The messenger content of this RNA was assayed by microinjection into oocytes of the frog Xenopus laevis. The 9‐S RNA was found to direct the synthesis of a molecular species that eluted from Sephadex G‐100 with added duck reticulocyte haemoglobin. When analysed on a cation‐exchange resin this material was found to contain the three globin‐chain species characteristic of duck reticulocyte haemoglobin. Moreover, paper electrophoresis at pH 6.5 and pH 3.5, and paper chromatography showed that these globin‐like species made in the oocyte were virtually indistinguishable, in terms of their histidine‐containing tryptic peptides, from the corresponding globin chains derived from duck reticulocyte haemoglobin.

A comparison between the products made following the injection into oocytes of reticulocyte 9‐S RNA from rabbits, mice or ducks proved that it is the exogenous messenger that gives rise to the information content of the proteins whose synthesis is elicited by the injected RNA.

The results also showed that the oocyte contains translational systems that are neither cell‐type nor species specific, and that duck globin messenger RNAs require no reticulocyte‐specific factors for their translation in oocytes.

The construction of a saturation curve showed that there is a linear relationship between the amount of messenger injected and the amount of haemoglobin synthesized, up to the point at which haemoglobin synthesis constitutes over 35% of the total protein being made. Consequently the frog oocyte provides a convenient assay system for small quantities of duck globin mRNAs, and the messenger activity of as little as 1 ng of 9‐S RNA can be detected very simply. Thus a combination of the duck reticulocyte and frog oocyte may provide a useful system for studying post‐transcriptional control, and the formation of mRNA, in a eukaryotic cell.
Preparation in vitro of a 2‐Thiocytidine‐Containing Yeast tRNAPhe‐A73‐C74‐s2C75‐A76 and Its Interaction with p‐Hydroxymercuribenzoate
Authors: Sprinzl Mathias , Scheit Karl‐Heinz , Cramer Friedrich
Published: March 2005

2‐Thiocytidine (s2C) is incorporated into yeast tRNAphe‐A73‐C74 by tRNA nucleotidyl transferase. This leads to the formation of tRNAPhe‐A73‐C74‐s2C75, which can be further converted to tRNAPhe‐A73‐C74‐s2C75‐A76. tRNAPhe with the s2CMP‐containing 3′‐end can be aminoacylated with the corresponding synthetase, exhibiting the same Km‐values and slightly lowered V compared with tRNAPhe‐A73‐C74‐C75‐A76. The s2CMP incorporated is a specific site for binding p‐hydroxymercuribenzoate; formation of 1:1 complex with a dissociation constant Kd= 8.7 μM was observed. The modified tRNA can be crystallized.
Presence of Ergosterol in Light‐Grown and Dark‐Grown Euglena gracilis Z
Authors: Anding Claude , Ourisson Guy
Published: March 2005

The results show, in addition to the previously reported sterols, the presence of ergosterol and 24S‐Δ5,7,22‐stigmastatrienol, two Δ5,7‐sterols. Ergosterol represents respectively 30% and 73% of the total sterols in green and white Euglena gracilis Z.
Studies on the Induction and Repression of Enzymes in Rat Liver
Authors: Iwasaki Yoshifumi , Lamar Carlos , Danenberg Kathleen , Pitot Henry C.
Published: March 2005

Tyrosine aminotransferase may be separated into four isolatable forms on hydroxyapatite columns. Form I is found in kidney, heart, brain and other tissues while forms II, III and IV appear to be unique to the liver in the rat. Forms II, III and IV were purified more than 1000‐fold from liver while form I was purified 30‐fold from kidney and heart.

The several forms have been compared kinetically and physicochemically. The Km for tyrosine of form I is significantly higher than that of the other three forms. On the other hand, in the presence of glutamate, V for all substrates studied is significantly lower for form II than for the other three forms. In addition, Kis for 2‐oxoglutarate in the presence of glutamate is somewhat lower and in the presence of pyridoxal 5′‐phosphate is significantly higher than for forms III and IV. The moledular weights and absorption spectra of forms II, III and IV could not be distinguished one from another. In contrast to forms II, III and IV the activity of form I was destroyed by brief heating at 60°C in the presence or absence of cofactors. For I also had a higher pH optimum than the other three forms.

In the fetus, glucagon, adenosine 3′:5′‐monophosphate and guanosine 3′:5′‐monophosphate induced predominantly form II. In the neonatal animal, hydrocortisone induced both forms II and III, while hydrocortisone in the presence of insulin induced form IV “prematurely”. This latter induction was insensitive to the action of actinomycin D. The rates in vivo of degradation of forms II, III and IV after hydrocortisone induction were indistinguishable. In the isolated perfused liver, hydrocortisone induced forms II, III and IV as had previously been shown in vivo, while adenosine 3′: 5′‐monophosphate induction was limited to forms II and III and insulin stimulated the level of form IV only. The significance of the differences in form I as compared to the hepatic forms, as well as the implications of selective metabolic regulation of the other three hepatic forms, are discussed.