bluuebunny/crossref_metadata_2025 huggingface dataset:
Urate Oxidase and its Association with Peroxisomes in Acanthamoeba sp.
Authors: Müller M. , Møller K. M.
Published: March 2005

Urate oxidase is present in axenic Acanthamoeba sp. (Neff strain). The enzyme shows an absolute requirement for molecular oxygen as electron acceptor and forms hydrogen peroxide. Its optimum of activity in 0.1 M Tris‐glycine buffer is at pH 9.4 with an apparent Km value of 18.7 μM. Its activity is inhibited by oxypurines, trichloropurine, oxonate and potassium cyanide. In all these properties the enzyme strongly resembles mammalian urate oxidases. Isopycnic centrifugation of Acanthamoeba homogenates in sucrose gradients showed that the distribution pattern of urate oxidase is identical with that of catalase but is different from the distribution of succinate dehydrogenase (a marker enzyme for mitochondria) and of acid phosphatase (a marker enzyme for lysosomes). The results strongly suggest the existence of a particle population with characteristics of peroxisomes in Acanthamoeba.
Protein Synthesis in Nuclei Isolated from HeLa Cells
Authors: Gallwitz D. , Mueller G. C.
Published: March 2005

Labeled amino acids were incorporated into isolated HeLa cell nuclei in vitro in the presence of a cytoplasmic pH 5 fraction and an energy generating system. Autoradiographically, the label appeared evenly distributed over the nuclei. Comparison of the nuclear and microsomal systems in vitro revealed differences in the ionic requirements for optimum function and in the polyacryl‐amide gel electrophoretic pattern of the proteins being synthesized. The inhibition of the protein synthesizing activity following inhibition of RNA synthesis in HeLa cells was more pronounced in the nuclear than in the microsomal system.
The Dissociation of Rabbit Reticulocyte Ribosomes with EDTA and the Location of Messenger Ribonucleic Acid
Authors: Nolan R. D. , Arnstein H. R. V.
Published: March 2005

Rabbit reticulocyte ribosomes were incubated with polyuridylic acid at 37° for 1 h under conditions suitable for protein synthesis. Ribosomes containing bound polyuridylic acid were isolated and dissociated into subparticles by treatment with EDTA. The subparticles were separated by zone centrifugation in a sucrose density gradient and the distribution of polyuridylic acid was examined by measuring the ability of gradient fractions to stimulate the incorporation of [14C] phenylalanine into protein by a cell‐free system. It was observed that the peak of polyuridylic acid activity corresponded to a region of the subparticle gradient having a sedimentation coefficient of about 50 S. When [14C] polyuridylic acid was used in the initial binding reaction, subsequent analysis of subparticles produced by treatment with EDTA confirmed the presence of polyuridylic acid on or close to the large ribosomal subparticle.
On the Problem of an Enzymatic Regulation of DNA Synthesis in Chlorella
Authors: Wanka F. , Poels C. L. M.
Published: March 2005

Uridine kinase and thymidylate kinase were investigated in Chlorella cultures synchronized by alternating periods of 16 hours light and 8 hours darkness. The enzymes were activated and stabilized by mercaptoethanol and glutathione.

Both enzyme activities increased periodically between the 8th and 18th hour after beginning of the light period. The increase was blocked in the presence of 15 μM actidione. When the light was turned off at the 12th hour the enzyme accumulation ceased after another 90 min.

DNA synthesis took place between the 10th and 18th hour and subsequently ceased completely, although high dTMP kinase activities were still observed. The results do not support the hypothesis that DNA synthesis in Chlorella is regulated by thymidylate kinase.
The Hydrolysis of the Naturally Occurring α‐Glucosides by the Human Intestinal Mucosa
Authors: Eggermont E.
Published: March 2005

An extract of human intestinal mucosa can degrade glycogen, isomaltose, sucrose and maltose through the action of several hydrolytic enzymes, which have been identified by experiments of competition between substrates and by some other properties. Besides an α‐amylase, which is soluble and probably of pancreatic origin, three truly intestinal α‐glucosidases are bound to cellular structures: a glucoamylase forms glucose from glycogen (Km= 0.65 mg/ml) and maltose (Km= 2 mM), a maltase‐isomaltase, from maltose (Km= 1 mM) and isomaltose (Km= 7 mM) and a maltase‐sucrase, from maltose (Km= 7 mM) and sucrose (Km= 17 mM).
The Sedimentation Properties of the Intestinal α‐Glucosidases of Normal Human Subjects and of Patients with Sucrose Intolerance
Authors: Eggermont E. , Hers H. G.
Published: March 2005

The maltase‐isomaltase, maltase‐sucrase and glucoamylase of the human intestinal mucosa have been solubilized by Triton X‐100 and submitted to centrifugation in a mannitol gradient. Under these conditions, the three enzymes sediment together in a 10 S fraction; with one normal mucosa out of 10 which were investigated a supplementary 13 S peak of both glucoamylase and maltase‐isomaltase was observed.

Several experimental conditions, like treatment of the extracts at 45° or solubilization of the α‐glucosidases by papain in the absence of potassium ions, induced an important inactivation of the maltase‐isomaltase and deep alterations in the sedimentation profile of the 3 glucosidases: the glucoamylase and the residual maltase‐isomaltase were distributed in 3 fractions with 13 S, 10 S and 6–7 S sedimentation coefficient, whereas the maltase‐sucrase was mostly present as a 7 S structure. In sucrose intolerance, the maltase‐sucrase is absent and the activities of the maltase‐isomaltase and glucoamylase are significantly reduced. The residual enzymes were ‐recovered as 13 S and 6 S structures instead of the normal 10 S species.

These observations allow the conclusion that the three α‐glucosidases are naturally linked to each other in the normal mucosa and that the alteration of one of them induces a structural rearrangement of the 3 enzymes. The absence of maltase‐sucrase in patients with congenital intolerance to sucrose can be regarded as the result of a single mutation of the corresponding structural gene and as the cause of a reorganization of the two other α‐glucosidases; this reorganization can explain a partial loss of the enzymatic activity.
On the Redox Potentials of Ubiquinone and Cytochrome b in the Respiratory Chain
Authors: Urban P. F. , Klingenberg M.
Published: March 2005

The standard redox potential of ubiquinone in submitochondrial particles is measured by equilibration with the succinate/fumarate system to about 65 mV at pH 7, with the redox ratio, n= 2. The pH dependence is ‐60 mV/Δ pH. The lower value measured for bound ubiquinone is discussed in comparison with the value found for isolated ubiquinone (104 to 112 mV, pH 7) determined in ethanol‐HCl.

In parallel experiments the standard redox potential of cytochrome b was measured to 72.5mV at pH 7. The pH dependence is ΔE/Δ pH = 0 at pH < 6.8 and ‐ 60 mV ΔHp at pH > 6.8.
Einfluß des pH‐Wertes auf die Kinetik der Hefe‐Hexokinase
Authors: Bohnensack R. , Hofmann E.
Published: March 2005

The kinetics of the yeast hexokinase reaction has been studied in the range of pH 6.3‐9.9 at 25°. Initial velocities were determined by varying the concentrations of glucose and in dependence of ionic species of MgATP complexes, which were calculated from the total concentrations and the known equilibrium constants. Maximum velocity is decreased in the acid region due to formation of an inactive central enzyme‐glucose‐MgATP complex with a pK value of 6.8. In the same range the Michaelis constant for glucose decreases too, indicating that this group is protonated in the enzyme‐glucose complex but not in the state of the free enzyme. In the alkaline region the Michaelis constant for glucose increases. This is caused by dissociation of a group with pK 9.9 in the free enzyme but not in the enzyme‐glucose complex. The Michaelis constant for the MgATP is not altered in the acid and neutral pH regions, but is also increased in the alkaline region. This finding shows the presence of an additional site of the enzyme dissociating with a pK of 9.9. This must be in the nondissociated state for binding of MgATP. Free uncomplexed ATP acts as a competitive inhibitor of MgATP. Due to the fact, that both the uncompetitive and the noncompetitive inhibition of ATP is abolished with increasing pH, it is suggested that inhibition is caused by the ion ATPH3−. The uncompetitive part of the inhibition is also decreased in the acid region. It appears therefore that the enzyme‐glucose complex can combine with the free ATP only when the group with pK 6.8 is dissociated. The pH effects on the kinetics of the yeast hexokinase reaction are consistent with the assumption that the dissociated form of the group with pK 6.8 of the central complex favours as base catalyst the release of a proton from the sugar and increases therefore the nucleophilic attack of the hydroxyl group of the sugar to the phosphoryl group of ATP.
Conditions in vitro which Affect Respiratory Control and Capacity for Respiration‐Linked Phosphorylation in Brown Adipose Tissue Mitochondria
Authors: Grav H. J. , Pedersen J. I. , Christiansen E. N.
Published: March 2005

Oxidative phosphorylation in brown adipose tissue mitochondria is greatly influenced by variations in the in vitro environment. Factors found to be of particular importance were the concentrations of serum albumin, of magnesium and EDTA, and the osmolality and the pH of the medium. The maximal P/O ratios obtained in an isoosmolar medium at pH 6.3, containing 2% serum albumin, 7.5 mM MgCl2, and 7.5 mM EDTA with pyruvate‐malate as substrate, and using the glucose‐hexokinase trapping system, were 2.8 for newborn guinea‐pigs and 2.6 for unweaned rats.

Under otherwise equal conditions the P/O ratios obtained with 4‐week‐old guinea‐pigs were higher than those with the newborn.

EDTA was a requirement for demonstration of respiratory control in brown adipose tissue mitochondria. Respiration with pyruvate‐malate or succinate (+ rotenone), and stimulated by inorganic phosphate and ADP, was found to be sensitive to oligomycin and to carbonyl cyanide phenylhydrazone. However, the small extent of stimulation by the phosphate acceptor system, the incomplete inhibition by oligomycin, and the fact that the respiratory rate after exhaustion of added ADP was higher than that prior to the addition of ADP, suggest that brown adipose tissue mitochondria are loosely coupled.

The measured respiratory control ratios all increased from the newborn to the 3‐week‐old guinea‐pigs.

Mitochondria from newborn and 6‐day‐old guinea‐pigs exhibited the phenomenon of “reversed acceptor control”. Thus, in the absence of added inorganic phosphate, ADP inhibited the respiration with pyruvate‐malate or with succinate. The reversed ADP control was abolished by carbonyl cyanide phenylhydrazone, but was not suppressed by treatment of the mitochondria with oligomycin.

Inorganic phosphate affects respiration in a composite manner. With all of the preparations tested, inorganic phosphate stimulated respiration whether exogenous ADP was present or not. In the absence of added ADP, the respiration initially stimulated by inorganic phosphate entered a subsequent inhibitory phase.

The implications of these findings to the control of brown adipose tissue thermogenesis are discussed.
Mutations Affecting the Repressibility of Arginine Biosynthetic Enzymes in Sacchromyces cerevisiae
Authors: Bechet J. , Grenson M. , Wiame J. M.
Published: March 2005

A method is described for isolating mutants of Saccharomyces cerevisiae which have lost repressibility by exogenous arginine for ornithine transcarbamylase.

Besides permeability mutants, three complementary classes of mutations were found: argRI, argRII and argRIII which are recessive and define three loci. No evidence for a linkage between any of these three loci or with the gene coding for ornithine transcarbamylase has been obtained.

Strains bearing mutations at either of these loci cannot be distinguished on a phenotype basis: after growth on minimal medium, the l‐ornithine carbamoyl transferase activity is twice that of the wild type strain; the mutations modify neither the growth rate nor the permeability to arginine.

The mutations might affect the structure of an hetero‐polypeptidic aporepressor.

The level of ornithine transcarbamylase in diploids is proportional to the number of argF+ genes in regulated as well as in non‐regulated cells.
The Subunit Structure of Phenylalanyl‐tRNA Synthetase of Escherichia coli
Authors: Kosakowski M. H. J. E. , Böck A.
Published: March 2005

Fingerprints of tryptic digests of phenylalanyl‐tRNA synthetase of Escherichia coli NP 3 suggest that the smallest identical subunit is one fourth of the size of the native protein (mol. wt. 181000). Determination of the subunit size by gel electrophoresis reveals a molecular weight of 43000 ± 2000. By the criteria employed the enzyme therefore seems to be composed of four identical subunits.

Phenylalanyl‐tRNA synthetase which has been denatured by treatment with 8 M urea is able to reform active enzyme indistinguishable by its size from the native protein. Again, a size of about 45000 was determined for the subunits active in the association reaction.
Phosphorolysis of Aminoacyl‐tRNA by Polynucleotide Phosphorylase from Escherichia coli
Authors: Kaufmann G. , Littauer U. Z.
Published: March 2005

Valyl‐transfer ribonucleic acid was degraded by polynucleotide phosphorylase from Escherichia coli in the presence of phosphate or arsenate. A compound that was identified as 2′(3′)‐O‐valyladenosine 5′‐pyrophosphate was isolated from the phosphorolytic digest of valyl‐tRNA while 2′(3′)‐O‐valyladenosine 5′‐monophosphate was isolated from the arsenolytic digest. It was concluded that valyl‐tRNA can be phosphorolyzed by polynucleotide phosphorylase. The rate of phosphorolysis of aminoacylated tRNA was lower than that of uncharged tRNA. N‐blocked derivatives of valyl‐tRNA and phenylalanyl‐tRNA were also phosphorolyzed by polynucleotide phosphorylase. Some possible uses of nucleoside diphosphates substituted in their sugar moiety are suggested.
The Determination of Molecular Weight of Bacterial Genome DNA from Renaturation Rates
Authors: Gillis M. , Ley J. De , Cleene M. De
Published: March 2005

A new method is described for the determination of the total molecular weight of haploid genome DNA. It is based on initial optical renaturation rate measurements of precisely known concentrations of fragmented DNA. The theoretical basis of the measurement is presented. The actual state of replication of the genome has little effect. The exact conditions and the effects of DNA concentration, renaturation time, size of DNA fragments, buffer concentration, optimal temperature and % (G + C) have been determined. Several types of bacterial DNA of known genome size were included as a control. As an application, the DNA genome size have been determined of 40 different bacteria. The method appears to offer advantages by its simplicity, rapidity and reproducibility.
Quantitative Studies on Cell Proteins in Suspension Cultures
Authors: Volpe P. , Eremenko‐Volpe T.
Published: March 2005

The protein per cell decreases during a 96‐hour growth of Chang's and HeLa cells cultivated in suspension. This decrease is very high for the cytoplasmic proteins, while the nuclear proteins appear to be more stable. The content of cell proteins is reestablished during a phase of intensive enrichment which occurs during the first 30–60 min of the newly inoculated culture. The rate of protein labelling in this phase appears to be greater in the nuclei than in the cytoplasm. Both the level of rapid accumulation and the decrease of the protein content with time appear to be a function of cell density. The rapid protein accumulation occurs when this density in the culture is lowest. With time and increase of the cell density in the culture, the protein content per cell goes down. In non‐synchronized cells this decrease is steady. In “naturally” synchronized cells it shows itself in an oscillatory fashion. The artificial thymidine synchronization of HeLa suggests that the fluctuating protein production in the “naturally” synchronized cultures of these cells is linked with the periodical doubling of the cell number during the growth cycle which includes, for HeLa, at least three cell cycles.
Chemical Structure of Tubular and Glomerular Basement Membranes of Human Kidney
Authors: Mahieu P. , Winand R. J.
Published: March 2005

A method involving the use of sieves and differential centrifugation for the preparation and separation of basement membranes from human renal tubules and glomeruli is described. Electron microscopic examination of the material obtained shows the glomerular membrane as a single dense structure, while the tubular membrane is composed of two dense layers separated by a space filled with loose fibrils exhibiting the same periodicity as collagen. The carbohydrate composition of both types of membranes is the same but tubular membranes have a lower content of some specific sugars. Differences also exist in amino acid composition. The content in hydroxylysine is higher in the glomerular membrane while the content in hydroxyproline is higher in the tubular membrane.

Gel electrophoresis after solubilization of the material demonstrates the presence of two, may be three, major proteins.
Pyruvate Dehydrogenase of Escherichia coli K 12
Authors: Dennert G. , Höglund S.
Published: March 2005

Purification of the pyruvate dehydrogenase complex from E. coli K 12 YMel is described. The pure enzyme has a specific activity 3–5 times higher than the enzyme originally isolated from E. coli Crookes. The electrophoretic mobility was determined to be 2.4 × 10−5 volt−1× cm2× sec−1. Ultracentrifugal analyses indicated a sedimentation constant of s°20,w= 53.5 × 10–13 sec. Light scattering showed a molecular weight of 3 × 106 and the frictional ratio was calculated to be f/f0= 1.28. The preparation of microcrystals in 30–35% ammonium sulphate is described and electron microscopic pictures of crystalline monolayers are presented.
Studies on Deoxyribonucleic Acid Polymerases from Yeast
Authors: Wintersberger Ulrike , Wintersberger Erhard
Published: March 2005

Mitochondrial DNA polymerase from Saccharomyces cerevisiae was purified 80‐to 100‐fold with good yield by DEAE‐cellulose chromatography of the extract prepared by disruption of highly purified yeast mitochondria. The enzyme requires all four deoxyribonucleoside triphosphates, Mg++ and DNA for activity. The optimal Mg++ concentration is 50 mM. Different DNAs, in native as well as denatured or “activated” form, serve as template and primer. The preference for denatured over native DNA and the effective promotion of DNA synthesis by mitochondrial DNA is of special interest. Mitochondria from a cytoplasmic “petite” mutant also contain a DNA polymerase which can be solubilized and purified like the enzyme from wild type yeast. Identical properties were found for the polymerases from normal and mutant mitochondria. On the other hand, characteristic differences between the mitochondrial enzyme and the polymerases A and B obtained from mitochondria‐free cell extracts were noted in the behaviour on DEAE‐cellulose columns, the optimal Mg++ requirements and the effectiveness of different DNA templates. DNA synthesis with all three enzymes is inhibited in a similar fashion by the intercalating dye acriflavin. The molecular weight of the mitochondrial DNA polymerase from yeast is about 150000.
Degradation of Phenols by Intact Cells and Cell‐Free Preparations of Trichosporon cutaneum
Authors: Neujahr Halina Y. , Varga Janos M.
Published: March 2005

A yeast strain, enriched and isolated from soil using phenol as major carbon source, was identified as Trichosporon cutaneum. After growth on phenol washed cell suspensions of the organism oxidized phenol and some phenol derivatives without delay, others after varying lag periods.

Cell‐free preparations from the yeast strain were capable of hydroxylating various phenols as judged by a substrate‐ and NADPH‐dependent oxygen uptake. Certain properties and the cofactor requirement of this hydroxylating system were investigated. Diphenols were hydroxylated at rates considerably higher than monophenols. Mono‐halogen substituted phenols were hydroxylated at rates comparable to that of phenol hydroxylation. The relative hydroxylating activities with respect to different phenol derivatives were markedly changed after 3–5 weeks' storage, thus indicating the presence of several hydroxylating enzymes. The cell‐free preparations also contained the enzyme catechol 1,2‐oxygenase.

Results of studies with intact cells and cell‐free preparations indicated that not only phenol but also resorcinol, quinol and catechol were further hydroxylated prior to ring fission.

Catechol when supplied as substrate of catechol 1,2‐oxygenase preparations, was oxidized by the “ortho type” of ring fission. The enzyme involved has broad substrate specificity.
Expression du génome chez les hybrides interspécifiques
Authors: Denis Herman , Brachet Jean
Published: March 2005

The interspecific hybrid Arbacia lixula♂×Paracentrotus lividus♀ which stops developing at the onset of gastrulation, was previously found to contain much more DNA of maternal origin (P. lividus) than DNA of paternal origin. In spite of this, the RNA synthesized by the blocked gastrula hybridizes better with DNA of paternal type than with DNA of maternal type. An explanation of this observation was sought in the present paper by means of saturation and competition experiments. The higher affinity of RNA from the hybrid gastrula for paternal DNA could be due to the fact that RNA of paternal type is complementary to a longer portion of the corresponding DNA than RNA of maternal type. This explanation was not satisfactory since the number of genes of paternal type which are active in the hybrid is not very different from the number of genes of maternal type.

In normal embryos of A. lixula and P. lividus, RNA from unfertilized eggs and from plutei was found to be less competitive against labeled RNA from gastrulae than gastrula RNA itself. Transcription of the genome is therefore partially stage‐specific. This stage‐specificity is maintained in the hybrid as far as transcription of the maternal genome is concerned, but is lost as far as transcription of the paternal genome is concerned. The hybrid gastrula was shown to synthesize more RNA molecules of A. lixula type than does the normal gastrula of this species, whereas RNA of P. lividus type is produced in equal amounts in normal and in hybrid embryos. These observations explain the hybridization properties of the RNA synthesized by the blocked gastrula.

The results suggest that the genes of paternal type that are very active in the hybrid are those genes which in normal embryos are transcribed at all stages of development and probably also in the adult. This class of genes is much more active in a foreign cytoplasm than in the cytoplasm of its own species. On the other hand, the genes whose activity is restricted to the gastrula stage in normal embryos are not active in the hybrid. This class of genes does not seem to function in a foreign cytoplasm.
Steric Course of the Succinate Dehydrogenase Catalysed Exchange of Hydrogen between (S)‐Chlorosuccinate and Water
Authors: Rétey János , Seibl Josef , Arigoni Duilio G. , Cornforth John W. , Ryback George , Zeylemaker Willem P. , Veeger Cees
Published: March 2005

The exchange of hydrogen between (S)‐chlorosuccinate and water in the presence of succinate dehydrogenase has been studied by spectrometry (mass, nuclear magnetic resonance, and optical rotatory dispersion). It is concluded that (a) the two hydrogen atoms exchanged are the methine hydrogen and HR of the methylene group; (b) processes exchanging one and two atoms proceed simultaneously; (c) the methine hydrogen is exchanged faster than HR; (d) all molecules in which HR has been exchanged have also exchanged the methine hydrogen.
Acylglucoses of the Corynebacteria and Mycobacteria
Authors: Brennan P. J. , Lehane D. P. , Thomas D. W.
Published: March 2005

Acylglucoses have been identified as major components of the soluble lipids of Corynebacterium diphtheriae, Mycobacterium smegmatis and BCG, grown in the presence of glucose. These have been purified and the structures determined. All contain one fatty acid per mole of glucose. Mass spectrometry of the methylated lipids and other methylation studies and periodate oxidation indicate that the fatty acids are esterified to position 6 of the glucose. The major fatty acid of the acylglucose from C. diphtheriae and M. smegmatis is corynomycolic acid .
Synthese der Dihydrofolat‐Reduktase in synchronisierter Hefe
Authors: Jaenicke Lothar , Scholz Konrad , Donike Manfred
Published: March 2005

The course of synthesis of dihydrofolate reductase (5,6,7,8‐tetrahydrofolate:NADP oxidoreductase, EC 1.5.1.3) was followed in synchronized yeast cells in the presence of 2,3,5‐trisethyleneimino‐1,4‐benzoquinone (Trenimon®) as an inhibitor of DNA replication. The following dependency was found between stages of DNA‐synthesis and the time course of enzyme formation: whereas in the absence of the inhibitor, enzyme synthesis takes place in a discontinuous, stepwise fashion during one generation cycle [7], after inhibition of DNA replication by 0.01 mg/ml Trenimon, synthesis sets in at the normal interval. However, its increase is continuous and almost linear; or exponential, following the pattern of the synthesis of RNA and total protein. A regulatory mechanism is envisaged in which the synthesis of dihydrofolacte reductase is influenced solely by the replication of DNA and independent on metabolites of the enzyme. On the other hand, the level of free thymine‐derivatives increases two‐fold in the Trenimon‐inhibited cells, although the overall synthesis of thymine is greatly retarded. This is probably due to regulation of thymine synthesis by the end‐product of the reaction chain.
The Controlled Dissociation of Proteins from Rat Liver Ribosomes by Potassium Chloride
Authors: Clegg J. C. S. , Arnstein H. R. V.
Published: March 2005

Up to 50% of the protein was dissociated from rat liver ribosomes when they were zonally centrifuged through a solution containing KCl in concentrations up to 1.0 M. Sucrose gradient analysis of the resulting particles showed subunits whose sedimentation rate decreased in a characteristic manner as the KCl concentration used to produce them increased. The large subunit in particular showed apparently discontinuous changes in sedimentation behaviour, suggestive of transitions from one discrete state to another. Further evidence for the existence of well‐defined protein‐deficient states was obtained by isopyenic centrifugation of the subunits in CsCl gradients after formaldehyde fixation. These showed that a spectrum of protein‐deficient particles with characteristic buoyant densities could be obtained from each subunit: treatment with KCl resulted in a mixture of these whose composition was a function of the KCl concentration. Electrophoretic analysis on polyacrylamide gels of the proteins released and those remaining on the subunits showed that the removal of proteins was selective and the quantitative increase in proteins removed with increasing KCl concentration was accompanied by greater heterogeneity. It is suggested that removal of protein from rat liver ribosomes is an ordered, stepwise phenomenon similar to that observed in bacterial ribosomes treated with CsCl or LiCl.
Energy Production in Rat Parotid Gland
Authors: Feinstein Haim , Schramm Michael
Published: March 2005

Rat parotid gland slices showed a very slow rate of glucose utilization and lactic acid production, both in the presence and in the absence of oxygen. Epinephrine, which caused rapid enzyme secretion by the slices did not affect the low rate of glycolysis.

A rough calculation indicates that in the parotid gland ATP is produced by oxidative phosphorylation 50 times faster than by glycolysis. The mitochondria, when isolated and tested in presence of EDTA or EGTA showed respiratory activity and respiratory control comparable to those of liver mitochondria. Adenosine 3′ : 5′‐cyclic phosphate, which is an intermediate in induction of enzyme secretion, showed no clear‐cut effect on the functions of the parotid mitochondria. Isolation of the mitochondria in the absence of the above chelators yielded a preparation which was almost depleted of NAD, respired feebly on NAD linked substrates and showed no respiratory control. Addition of NAD+ restored respiration. The damage caused to these mitochondria during isolation was attributed to the high calcium concentrations found in the gland. Addition of small amounts of free calcium to functional parotid mitochondria indeed caused the loss of NAD and the decline of respiration. Tested in parallel experiments, liver mitochondria were much more resistant to added calcium. Their initial calcium content was one tenth that of the parotid mitochondria. The control of calcium distribution in the parotid gland and the possible functions of calcium are discussed.
Sulfenyl Halides as Modifying Reagents for Polypeptides and Proteins
Authors: Boccù Enrico , Veronese Francesco M. , Fontana Angelo , Benassi Carlo A.
Published: March 2005

The determination of tryptophan and cysteine content in proteins by means of the selective reaction of sulfenyl halides with these amino acids has been carefully investigated. By reaction with 2‐ or 4‐nitrophenylsulfenyl chloride in aqueous acetic or formic acid, the nitrophenyl chromophore is covalently bound to the tryptophan and the cysteine residues of the protein, giving a thioether and a mixed disulfide respectively. The cysteine content has been determined spectrophotometrically at 450 nm from the 4‐nitrothiophenol released from the mixed disulfides of the cysteine residues after exposure of the 4‐nitrophenylsulfenyl chloride‐labelled protein in 0.1 N NaOH (at this wavelength the corresponding chromophore covalently bound to tryptophan does not absorb). Tryptophan has been estimated in the sulfenylated proteins by spectrophotometric determinations at 365 or 328 nm of the 2‐ and 4‐nitrophenylsulfenyl chromophores (ɛM 4000 and 11700 respectively), following removal of the yellow label from the cysteine residues, by dissolving the modified protein in 0.1 N NaOH. The procedure has been shown to be accurate for both amino acids with a wide variety of proteins.
Accumulation of Squalene during Hepatic Cholesterol Synthesis in vitro
Authors: Onajobi Funmilayo D. , Boyd G. S.
Published: March 2005

The synthesis in vitro of cholesterol from dl‐[2‐14C]mevalonic acid by the rat liver 18000 ×g supernatant was studied. It was shown that in the presence of air and necessary cofactors, the 18000 ×g supernatant incorporated dl‐[2‐14C]mevalonic acid mainly into cholesterol plus cholesterol esters while a small percentage of the radioactivity was associated with squalene, squalene oxide, lanosterol and some unidentified products. However, when serum (rat or human) was added to the incubation mixture, the incorporation of dl‐[2‐14C]mevalonic acid into cholesterol was decreased, with a concomitant accumulation of radioactive squalene. There was a stoichiometric relationship between the concentration of serum in the incubation mixture and the decrease in cholesterol synthesis or the accumulation of squalene.

Pretreatment of the serum by dialysis, heat, or reaction with p‐chloromercuribezoate (PCMB), and N‐ethylmaleimide, all failed to annul the squalene accumulating effect of serum.

Fractionation of rat serum showed that an α1‐globulin possessed the squalene accumulating activity. Examination of Cohn fractions of human serum showed that the squalene accumulating activity was present in Fraction III‐0, Fraction IV‐1 and Fraction IV‐4. Delipidization of Fraction IV‐1 led to an increase in the squalene accumulating activity of this fraction.

Purification of delipidized Fraction IV‐1 by a combination of ammonium sulphate precipitation, ion‐exchange chromatography and perchloric acid precipitation gave a fraction, active in accumulating squalene, with the mobility of an α1‐globulin (α1‐protein). Ultracentrifugal analysis showed that the α‐protein was composed of a major component whose sedimentation velocity was 2.1 Svedberg units and a minor component of higher sedimentation velocity. Incubation of α1‐protein with [14C]cholesterol led to the binding of these lipids to the protein. Comparison of α1‐protein with an apo‐protein of α1‐liporprotein obtained from Dr. A. Scanu (Chicago) showed that both had similar physical, chemical and biological properties, indicating that α1‐protein, the squalene accumulating factor of delipidized Fraction IV‐1, is the apo‐protein of α1‐lipoprotein.

The present evidence indicates that the squalene accumulating factor of serum acts by binding squalene.
The NAD Kinases of Saccharomyces cerevisiae
Authors: Apps D. K.
Published: March 2005

Subcellular fractions of a strain of Saccharomyces cerevisiae have been prepared by enzymic digestion of the cell walls, osmotic lysis of the protoplasts and sorbitol density gradient centrifugation of the lysate. The mitochondrial fraction has both NAD‐ and NADH‐phosphorylating enzymes, whilst the cytoplasm contains a single NAD‐specific kinase. These three enzymes differ in their kinetic properties and in their stability to heat and to an alkylating agent, 3‐(bromoacetyl) pyridine. The properties of the cytoplasmic NAD kinase resemble those of NAD kinase partially purified from autolysates of pressed bakers' yeast.
Localization of the Glycerol‐Phosphate Dehydrogenase in the Outer Phase of the Mitochondrial Inner Membrane
Authors: Klingenberg Martin
Published: March 2005

Evidence for the localization of the mitochondrial glycerol‐phosphate dehydrogenase in the outer phase of the inner membrane is presented.

Glycerol‐phosphate does not permeate through the inner membrane to the matrix space in a number of mitochondria which actively oxidize glycerol‐phosphate.

The impermeability of the inner membrane to ferricyanide is demonstrated and this forms a basis for elucidating membrane localization of carriers.

In mitochondria from various tissues ferricyanide can interact directly with glycerolphosphate dehydrogenase but not with succinate dehydrogenase.

The oxidation of glycerol‐phosphate, in contrast to that of succinate, is insensitive to substrate depletion brought about by uncouplers.

The results are interpreted to show that both the substrate and the acceptor‐site of glycerol‐phosphate dehydrogenase are exposed to the outer side of the inner membrane. Thus, for the operation of the glycerol‐phosphate dehydrogenase, mitochondria do not require translocases for glycerol‐phosphate and dihydroxyacetone‐phosphate.
Light‐Induced Alkylation and Dealkylation of the Flavin Nucleus
Authors: Walker Wolfram. H. , Hemmerich Peter , Massey Vincent
Published: March 2005

The spectral course of irreversible photoreduction of the flavin nucleus by arylacetates and related compounds has been investigated in detail. The resulting benzylated dihydroflavins and their reoxidation products have been characterized spectrophotometrically, among them 5‐benzyl‐flavosemiquinone radical and 5‐benzylflavoquinonium cation. Reversible and irreversible flavin redution corresponding to hydride‐ or group‐transfer from substrate to flavin are discussed with respect to their scope and limitations and possible biological implications in the action mechanism of flavin dependent dehydrogenases.
Maturation et intégration du RNA 5 S au cours de la biosynthèse de la particule ribosomale 50 S
Authors: Galibert F. , Eladari M. E. , Hampe A. , Boiron M.
Published: March 2005

It is well known that protein synthesis is stopped either by chloromycetin or by starvation of an essential amino acid for a RCrel strain, ribosomal RNA synthesis continues but products do not appear in 50 S and 30 S ribosomes. It has also been shown that 5 S RNA synthesis is altered by the addition of chloromycetin but in an unknown manner. In this paper we report that:

(a) during amino‐acid starvation of a RCrel strain of Escherichia coli, 5 S RNA is transcribed as 5 S relax RNA located outside the ribosomes, in the supernatant fraction;

(b) this 5 S RNA which is slightly longer than the normal product is convertible into a true 5 S RNA when protein synthesis is restored after blockage of RNA synthesis by actionomycin;

(c) part of the 23 S RNA from a 25 S particle appears after resumption of protein synthesis in a 40 S particle which contains normal 5 S RNA;

(d) a pool of 5 S precursor RNA exists outside the 50 S ribosome. This 5 S precursor RNA is isolated;

(e) this precursor which is isolated from the 105000 ×g supernatant fraction after pulse labelling, migrates on gel electrophoresis between 5 S relax RNA and true 5 S RNA.
Postnatal Differentiation of the Enzyme Activity Pattern of Energy‐Supplying Metabolism in Slow (Red) and Fast (White) Muscles of Chicken
Authors: Bass A. , Lusch G. , Pette D.
Published: March 2005

Activity patterns of key enzymes in energy‐supplying metabolism were studied in a typical fast (white) muscle (pars posterior of m. latissimus dorsi) and in a typical slow (red) muscle (pars anterior of m. latissimus dorsi) of the chicken during postnatal development. The differentiation of these enzyme activity patterns occurs postnatally, and is practically completed after 3 weeks. After hatching, the enzyme activity patterns of the two muscles resemble each other, and correspond to that of a slow muscle, with predominantly aerobic metabolism. The slow muscle does not change its metabolic type markedly during development. The postnatal differentiation of the fast muscle, however, implies a pronounced change of its metabolic type. It consists of a parallel increase of absolute and specific activities of enzymes representing the following metabolic systems: glycogenolysis (glycogen phosphorylase), glycolysis (triosephosphate dehydrogenase), lactic fermentation (lactate dehydrogenase), and glycerolphosphate metabolism (glycerol‐3‐phosphate dehydrogenase). These concomitant changes suggest a coordinate synthesis of the respective enzymes. This conclusion applies also to a second group of enzymes which, however, decrease in absolute and specific activities. This group consists of enzymes representing the citric‐acid cycle (citrate synthase), fatty‐acid oxidation (3‐hydroxyacyl‐CoA dehydrogenase) and glucose phosphophorylation (hexokinase). Thus, enzymes belonging to constant proportion groups change in parallel during postnatal development of the muscle, and metabolic differentiation is reflected mainly by inverse shifts of constant proportion enzyme groups representing aerobic and anaerobic systems of energy‐supplying metabolism.
The Phosphodiesterases in the Rat Pancreas
Authors: van Venrooij W. J. W. , Poort C.
Published: March 2005

The purification of two phosphodiesterases found in the rat pancreas is described. The phosphodiesterase I, an enzyme of the snake venom type, has been purified about 500 times, the phosphodiesterase II, an enzyme of the spleen type, about 60 times. The phosphodiesterase I activity, optimal at a pH of about 9.0, is completely dependent on the presence of divalent metal ions. The phosphodiesterase II, being optimally active at a pH of around 6.0, does not show this dependency.

RNA and oligonucleotides are degraded only to mononucleotides. The phosphodiesterase I liberates 5′‐mononucleotides, the phosphodiesterase II only 3′‐mononucleotides. Oligonucleotides ending on a 5′‐phosphate group and on a 2′‐phosphate group are preferentially degraded by the phosphodiesterase I; those ending on a 3′‐phosphate group by the phosphodiesterase II. Both enzymes readily degrade oligonucleotides ending on a cyclic 2′:3′‐phosphate group. The possible significance of the latter finding is discussed.
Chicken Liver Mitochondrial Phosphoenolpyruvate Carboxykinase
Authors: Felicioli R. A. , Barsacchi R. , Ipata P. L.
Published: March 2005

Kinetic studies on the phosphoenolpyruvate carboxykinase (EC 4.1.1.32) catalyzed reactions (i. e. the phosphoenolpyruvate synthesis and the oxaloacetate HCO3− exchange) have been carried out with the use of a purified enzyme preparation. Intersecting initial velocity plots were obtained conforming to a sequential mechanism for both reactions.

Competitive inhibition patterns were observed for both reactions with ITP as variable substrate and IDP as inhibitor. For oxaloacetate exchange reaction noncompetitive and mixed P‐enolpyruvate inhibition patterns were observed with respectively oxaloacetate and ITP as variable substrates.

These results are consistent with an ordered kinetic mechanism where ITP is added first, followed by oxaloacetate, and the products are released in the order CO2, P‐enolpyruvate and IDP.

A reaction mechanism involving the synthesis of a ternary transitory complex, common to both reactions, is postulated.
Isolement et propriétés des chaînes du DNA de bactériophage 2 C
Authors: Truffaut Nicole
Published: March 2005

Bacillus subtilis phage 2 C DNA is double‐stranded, with a high molecular weight (about 100 million), a low Tm (77.8%), and a high buoyant density (1.742 g/cm3) in a CsCl density gradient. Denatured DNA centrifuged in a CsCl gradient reveals two bands (1.752 and 1.762 g/cm3) corresponding to the complementary strands.

In this communication, we report preparative separation of two strands from denatured DNA, either by chromatography on a methylated albumin‐kieselguhr column, or by density‐gradient centrifugation in the presence of poly G. The melting curves of native and denatured DNA, and of isolated fractions before and after mixing and annealing, are compared. As shown by spectrophotometry, and by buoyant density, a good renaturation was obtained by mixing and annealing the “light” and “heavy” fractions; each isolated fraction cannot be renatured alone.

Poly G strongly binds to the heavy fraction, increasing its density in a CsCl gradient by about 48 mg/cm3; there is also some binding to the light fraction (density increase under the same conditions, 6 mg/cm3). The resulting density difference between the two fractions is more pronounced than the natural one: 52 mg/cm3 instead of 10 mg/cm3. Poly C only interacts with the light fraction and host ribosomal RNA with the heavy one.

Native DNA, and both fractions isolated by centrifugation in the presence of poly G, were analysed for base composition using 32P‐labelled DNA. Native DNA had a (G + C) content of 39.1%. As for the isolated fractions, there are more pyrimidines (cytosine, and hydroxymethyluracil replacing thymine) in the “heavier” fraction, which preferentially interacts with poly G.

Both strands from infectious DNA were assayed for infectivity with negative results. The “renatured” DNA also gave negative results, probably owing to some natural or accidental breakage.
Détermination de la structure d'un hexaose isolé du lait de femme, le dilactaminyl‐lacto‐N‐tétraose
Authors: Grimmonprez L. , Bouquelet S. , Bayard B. , Spik G. , Monsigny M. , Montreuil J.
Published: March 2005

The authors have determined the complete structure of a hexasaccharide isolated from human milk: the dilactaminyl (or di‐N‐acetylneuraminyl)‐lacto‐N‐tetraose. This new sugar results from the substitution onto the lacto‐N‐tetraose (β‐d‐galactopyrannoside‐1,3‐β‐d‐N‐acetylglucosaminopyrannosido‐1,3‐β‐d‐galactopyrannosido‐1,4‐d‐glucopyrannose) of two residues of N‐acetylneuraminic acid. The first is grafted in the 3‐position of the terminal galactose residue and the second in the 6‐position of the N‐acetylglucosamine residue.
A Mechanism of the Irreversible Inactivation of Bovine Pancreatic Ribonuclease by Diethylpyrocarbonate
Authors: Wolf Barry , Lesnaw Judith A. , Reichmann Manfred E.
Published: March 2005

The irreversible inactivation of ribonuclease with diethylpyrocarbonate was studied. The products of the reaction were electrophoresed on polyacrylamide gels in sodium dodecyl sulfate and found to contain polymers of ribonuclease. Other proteins reacted similarly with diethylpyrocarbonate. Ribonuclease treated with maleic anhydride was unable to polymerize when treated with diethylpyrocarbonate, but when the N‐maleyl group was removed by incubation at low pH, the capacity of the ribonuclease to polymerize was restored. It was concluded that the diethylpyrocarbonate reaction forms amide bonds between ɛ‐amino groups of lysine and carboxylic groups of aspartic or glutamic both intermolecularly or intramolecularly. In either case the enzyme was inactivated. A mechanism based on the known chemical reactivities of diethylpyrocarbonate is proposed. The conclusions are further substantiated by studies of tryptic peptides and microbiuret positive products obtained from a reaction of diethylpyrocarbonate with assorted amino acids.
Identification of C19O2 and C21O2 Steroids in the Mono‐ and Disulphate Fractions of Human Faeces
Authors: Laatikainen T. , Vihko R.
Published: March 2005

Steroid monosulphate and disulphate fractions were obtained from human faeces by chromatography on Sephadex LH‐20 and solvolysed. The steroids liberated were fractionated on silicic acid. Fractions containing C19O2 and C21O2 steroids were studied by thin‐layer chromatography, gas‐liquid chromatography and gas chromatography‐mass spectrometry.

Dehydroepiandrosterone, 5α‐androstane‐3α,17α‐diol, 5α‐androstane‐3α,17β‐diol, 5α‐androstane‐3β,17α‐diol, 5α‐androstane‐3β,17β‐diol, 5β‐androstane‐3α,17β‐diol, 5‐androstene‐3α,17β‐diol, 5‐androstene‐3β,17α‐diol, 5‐androstene‐3β,17β‐diol, 5α‐pregnane‐3α,20α‐diol, 5α‐pregnane‐3β,20α‐diol, 5β‐pregnane‐3α,20α‐diol, 5β‐pregnane‐3β,20α‐diol,pregnenolone, 5‐pregnene‐3α,20α‐diol and 5‐pregnene‐3β,20α‐diol were found in the monosulphate fraction and 5‐androstene‐3β,17α‐diol as well as 5‐androstene‐3β,17β‐diol in the disulphate fraction of human faeces.

Several steroids identified earlier in the disulphate fraction of human bile were found in the monosulphate fraction of human faeces, indicating partial hydrolysis of diconjugates in the intestine. The androstanediols and pregnanediols found in the faeces were primarily of a 3β‐hydroxy‐5α‐ or 3β‐hydroxy‐5β‐structure, whereas those in the bile were of a 3α‐hydroxy‐5α‐ or 3α‐hydroxy‐5β‐structure. This suggests conversion of a 3α‐hydroxyl to a 3β‐hydroxyl in the intestine in vivo.
Studies on the Metabolism of C19 Steroids in Rat Testis
Authors: Gustafsson Jan‐Åke , Lisboa Belisário P.
Published: March 2005

The presence of testicular 6α‐ and 6β‐hydroxylases has been shown by the isolation of 6α‐ and 6β‐hydroxytestosterone after incubations of testosterone with testicular preparations of adult rats. The steroids were isolated by thin‐layer chromatography and identified by gas chromatography‐mass spectrometry.
Polyribonucleotides Containing a Phosphorothioate Backbone
Authors: Eckstein Fritz , Gindl Hans
Published: March 2005

The enzymatic synthesis of two polyribonucleotides, poly r(s̄As̄U) and poly r(s̄U) containing a sugar‐phosphorothioate instead of a sugar‐phosphate backbone, is described. The formation and thermal stability of double‐stranded polymers is not impaired by this modification. They can also serve as templates for RNA‐polymerase. Single stranded polymers can serve as messengers. Both double and single‐standed polymer exhibit some degree of resistance to nucleases.