bluuebunny/crossref_metadata_2025 huggingface dataset:
Histones from the Fruit Fly Ceratitis capitata
Authors: FRANCO Luis , MONTERO Francisco , NAVLET Jacinto M. , PERERA Julián , ROJO M. Carmen
Published: March 2005

Histones from the fruit fly Ceratitis capitata have been obtained and characterized by amino acid analysis and polyacrylamide gel electrophoresis. It has been found that the five major histone groups are present in this insect.

The fractionation of histones has been achieved by using acid or organic selective extractions. Ceratitis capitata F1 exhibited a reduced electrophoretic mobility when compared with the mammalian corresponding histone. On the contrary, fly F2A1, mobility was similar to that of mammalian homologous histone. Some differences were found, however, in amino acid composition. It has been found that cysteine does not occur in fly F2A1, but this amino acid seemed to be present in the F3 histone, which aggregated under oxidizing conditions.

A strongly basic protein was isolated together with histones when pharate adult chromatin was used as starting material. Amino acid analysis of this protein revealed the presence of hydroxyproline (19%), as well as high amounts of threonine (30%) and arginine (23%). It was found that this specific protein was not a nuclear component and it seemed to be located at the pharate adult envelope; the name of purarium‐specific protein is proposed for this protein. Its strongly basic nature is judged to be the cause by which it became attached to chromatin during the isolation procedure.
Separation of Isoenzymes by Partition in Aqueous Polymeric Two‐Phase Systems
Authors: BLOMQUIST Göran , HARTMAN Annika , SHANBHAG Vasant , JOHANSSON Göte
Published: March 2005

A method for the separation of isoenzymes by counter‐current distribution in aqueous biphasic systems is described. The phase systems used contained dextran and trimethylamino‐poly(ethyleneglycol). The method has been tested with three isoenzyme systems, namely, lactic dehydrogenase, malate dehydrogenase and enolase. Separation of isoenzymes in extracts of tissue or cells as well as in a mixture of commercial isoenzyme preparations were successfully achieved by this technique.
Biological Properties of Lipid A from Shigella sonnei
Authors: ŁUOWSKI Czesław , ROMANOWSKA Elżbieta
Published: March 2005

Lipid A preparations isolated from Shigella sonnei lipoplysacharides after mild acid hydrolysis were used as immunogens in rabbits. Titers of antisera to lipid A amounted to about 1:640 in passive hemolysis test. Antibodies to lipid A were purified on the column packed with lipid A coupled to Sepharose 4 B. Pure antibodies to lipid A examined in immunoelectrophoresis proved to be immunoglobulins G.

The specificity of antisera to lipid A was confirmed in passive hemolysis inhibition test elaborated in this work. From the results of the examination of various products of lipid A degradation in inhibition studies of lipid A—anti‐(lipid A) system, it can be concluded that amino groups of glucosaminyl units substituted by 3‐hydroxymyristoyl residues are immunodominants of lipid A.

Lipid A preparations from Shigella sonnei showed distinct anticomplementary activities which disappeared from the examined materials after removal of ester‐linked fatty acids. Shigella sonnei lipopolysaccharides, at suitable concentrations, inhibited also complement fixation. Phase II lipopolysaccharide was the best inhibitor.
Glycosphingolipids and Glycoproteins in the Wild‐Type and in a Non‐Aggregating Mutant of Dictyostelium discoideum
Authors: WILHELMS Otto‐Henning , LÜDERITZ Otto , WESTPHAL Otto , GERISCH Günther
Published: March 2005

Fractionation of the phenol‐water extract from aggregation‐competent cells of Dictyosteiium discoideum resulted in the isolation of two main fractions containing two different antigen specificities, I and II, respectively. Antigen I was also obtained from growth‐phase cells of Dictyostelium discoideum. This antigen is a glycolipid, and antigen II is a glycoprotein, both containing the same sugar constituents but in different proportions: the ratio of N‐acetylglucosamine—fucose—mannose was 12:5:2 and 12:5:6 in antigens I and II, respectively. The lipid constituents of antigen I were identified as behenic acid and 4‐hydroxysphinanine (C18‐phytosphingosine) present in a ratio of 1:1. In addition, phosphate and ethanolamine were found. Under various conditions, antigen I dissociated into subfractions.

The protein component of antigen II was rich in acidic and hydroxy amino acids. The carbohydrate moiety was linked through glucosamine units glycosidically to the hydroxy groups of the hydroxy amino acids. Antigen II strongly reacted with concanavalin A.

Cells of a non‐aggregating mutant showed markedly increased agglutinability with wheat germ agglutinin, and changed serospecificity and electrophoretic mobility of antigen I. Extracts of cells from this mutant, harvested at a stage analogous to aggregation competence, yielded fractions similar in composition to antigens I and II. However, the N‐acetylglucosamine—fucose—mannose ratio in this glycolipid was 12:1:3, i.e. the content of fucose was markedly reduced. With N ‐acetyl‐β‐hexosaminidase part of the N‐acetylglucosamine was liberated from this glycolipid indicating that N‐acetylglucosamine is a terminal sugar in the mutant antigen. Various results suggest that fucose is the terminal sugar in the wild‐type glycolipid.
Compartmentation of the Tryptophan‐Synthase‐Proteolyzing System in Saccharomyces cerevisiae
Authors: HASILIK Andrej , MÜLLER Hannelore , HOLZER Helmut
Published: March 2005

Ficoll‐gradient fractionation of a metabolic lysate from yeast spheroplasts was used to prepare vacuole, mitochondria and cytosol fractions. Tryptophan synthase activity was exclusively found in the soluble fraction. Proteinases A, B and C as well as the tryptophan‐synthase‐inactivating activity were mainly found in the vacuole fraction. The heat‐stable activities inhibiting pröteinase and tryptophan‐synthase‐inactivating enzyme appeared in the soluble fraction. The subcellular separation of tryptophan synthase and the tryptophan‐synthase‐inactivating activities explains why in crude extracts from stationary yeast cells, high tryptophan synthase activities are observed in spite of high tryptophan‐synthase‐inactivating activity. The proteinases of a freshly prepared vacuole fraction from yeast spheroplasts are unable to inactivate externally added tryptophan synthase unless the organelles are disrupted by sonication or osmotic shock.
Preparation of Soluble and Insoluble Polysaccharide Conjugates of Peptides with a Free α‐Amino Group
Authors: JOST Rolf , YARON Arieh
Published: March 2005

Synthetic peptides were covalently attached to Sepharose and water‐soluble dextran of high molecular weight. In these polysaccharide conjugates, the bound peptides bear a free α‐amino group. The interaction of leucine aminopeptidase with its peptide ligands Thr‐Phe‐Pro (substrate) or Thr‐(But)‐Phe‐Pro (competitive inhibitor) was strikingly different in soluble dextran conjugates and insoluble Sepharose conjugates of these ligands. The affinity of the enzyme to its inhibitor Thr(But)‐Phe‐Pro was decreased by more than 100‐fold upon insolubilization of the peptide on Sepharose. When the peptide was coupled to dextran of weight‐average molecular weight ∼ 500000, the potency of the inhibition was not impaired.

Leucine aminopeptidase failed to hydrolyze the Sepharose‐bound substrate Thr‐Phe‐Pro to a measurable extent, but it released N‐terminal threonine from the soluble dextran conjugate of this peptide to an extent of at least 38% of the theory.

The interference, caused by the insoluble matrix was hardly overcome, when a new type of spacer, consisting of > 80 dl‐alanyl residues, was inserted between matrix and ligand. The spacer which is several enzyme subunit diameters in length, gave rise to a small fraction (about 5%) of accessible ligand but did not improve specific interaction to a sufficient extent in order to enable specific binding of leucine aminopeptidase to inhibitor‐, or substrate‐substituted Sepharoses.

Nonspecific adsorption of the enzyme was observed in an inhibitor derivative of Sepharose with ɛ‐aminohexanoic acid inserted as spacer.
5‐S RNA: Biosynthesis and Association to Immature Ribosomal Particles in the Nucleus of Lymphocytic Mouse Leukemia Cells, L5178 Y
Authors: AUGER Marie‐Annick , TIOLLAIS Pierre
Published: March 2005

In the nucleus of L5178 Y cells, a lymphocytic mouse leukemia cell strain, newly synthesized 5‐S ribosomal RNA first appears mainly in the nucleoplasm and then diffuses into the nucleolus. Kinetic studies have revealed that the appearance rates of 5‐S RNA and “45‐S fraction” ribosomal precursor RNA are identical. During the first 30 min, 5‐S RNA binds to proteins and constitutes a nuclear pool of independent ribonucleoprotein particles which sediment at approximately 11 S. After this time, it begins to integrate with the nucleolar “80‐S nascent” particle. This ribonucleoprotein particle contains “45‐S fraction” RNA, the direct product of ribosomal DNA genes and has been shown to represent the first stage of ribosome formation. 5‐S RNA remains associated to preribosomal particles during the nuclear maturation process leading to the large ribosomal subunit, located in the cytoplasm. In labeling experiments, the molar ratio of labeled 5‐S RNA compared to high‐molecular‐weight ribosomal RNA in ribonucleoprotein particles, continues to increase until reaching a value of 1:1 for steady‐state labeling. The association of 5‐S RNA to preribosomal particles is more labile than it is to the mature ribosomal subunit. Chemical products such as heparin or sarkosyl used to extract nucleolar ribonucleoprotein particles, were shown to detach 5‐S RNA from these particles; but dithiothreitol keeps the particles intact.
Studies on Lysostaphin
Authors: GROV Arne , IVERSEN Ole‐Jan , ENDRESEN Curt
Published: March 2005

Immunological resemblance between the endopeptidase, endo‐β‐N‐acetylglucosaminidase and N‐acetylmuramyl‐L‐alanine amidase of lysostaphin has been demonstrated. Only the peptidase was found to give precipitin reaction. All three enzymes sensitized tanned sheep erythrocytes to agglutination in homologous and heterologous enzyme antisera. Absorption experiments revealed the presence of specific determinant(s) in all the enzymes, determinant(s) shared between two and two enzymes, and a possible determinant(s) common to all three enzymes.
Dinucleotide Sequences in the Regions of T7 DNA Coding for Termination of Early Transcription
Authors: PETERS Gordon G. , HAYWARD Richard S.
Published: March 2005

Highly purified RNA polymerase from Escherichia coli terminates RNA synthesis in vitro at defined sites of the DNA of coliphage T7. One such site maps at the end of the major “early” transcription region of the genome. A second, distal site is most readily studied in certain mutant strains from which the first site has been deleted. At both sites, termination in vitro is simple (independent of rho or other factors) and both appear to be functional in vitro. We have applied to these sites a technique designed to study base sequences at and beyond RNA termini. The RNA terminated at the first site has cytosine at its 3′ end, and the first base coded by the DNA template beyond the RNA terminus is guanine. The specificity of this sequence is at least 92%. At the second terminator, which is found to be less efficient in its action, the corresponding bases are cytosine and adenine.
The Mechanism of Inhibition of Pig‐Plasma Benzylamine Oxidase by the Copper‐Chelating Reagent Cuprizone
Authors: LINDSTRÖM Anders , PETTERSSON Gösta
Published: March 2005

Treatment of pig‐plasma benzylamine oxidase with the copper‐chelating reagent cuprizone leads to the reversible formation of an enzyme · cuprizone complex with a dissociation constant of about 3 mM, followed by an irreversible conversion of this complex into an enzyme species exhibiting a new absorption band centered around 350 nm. The latter enzyme species shows no reactivity towards substrate or carbonyl reagents such as phenylhydrazine. One mole cuprizone per mole protein is sufficient to complete the chromophoric changes and to inactive the enzyme completely.

Cuprizone reacts with free pyridoxal phosphate under formation of a reaction product showing maximum absorption at 350 nm in the visible region, and competes with phenylhydrazine for the protein‐bound pyridoxal phosphate in benzylamine oxidase.

Cuprizone does not extract any significant amounts of copper from benzylamine oxidase, but affects electron paramagnetic resonance spectra of the enzyme and its phenylhydrazone derivative in a way suggesting a direct interaction with at least one of the two copper ions present in the protein. The latter interaction does not appear to be identical with those involved in the formation of the inactive enzyme species absorbing at 350 nm.

It is concluded that the inhibitory action of cuprizone cannot be attributed to its copper‐chelating properties, but reflects an interaction with enzyme‐bound pyridoxal phosphate through a mechanism analogous to that governing Schiff‐base or hydrazone formation between enzyme and substrates or hydrazine derivatives.
Effect of Azide on Some Spectral and Kinetic Properties of Pig‐Plasma Benzylamine Oxidase
Authors: LINDSTRÖM Anders , OLSSON Bengt , PETTERSSON Gösta
Published: March 2005

The effect of azide on optical absorption and electron paramagnetic resonance (EPR) spectra of pig‐plasma benzylamine oxidase has been examined and correlated with the effect of azide on the catalytic activity of the enzyme.

A strong reversible interaction between azide and copper in benzylamine oxidase, corresponding to complex‐formation with a dissociation constant less than 0.2 mM, was detected by EPR techniques. This interaction is competitive with the EPR‐detectable binding of cuprizone to copper in the enzyme, but is not affected by reactions involving the active‐site pyridoxal phosphate and has no evident effect on enzyme activity.

A weaker reversible interaction between azide and protein‐bound copper, corresponding to complex formation with a dissociation constant of 40 mM, was detected spectrophotometrically and by EPR techniques. An absorption band centered at 390 nm is associated with the latter complex, the apparent stability of which was found to be unaffected by a 20‐fold variation of the oxygen concentration as well as by reduction of the enzyme with substrate.

Azide acts as an uncompetitive inhibitor of benzylamine oxidase with an inhibition constant close to 40 mM. 100 mM azide has essentially no effect on the reactivity of the active‐site pyridoxal phosphate towards phenylhydrazine or benzylamine. It is concluded that formation of the enzyme · azide complex exhibiting the 390 nm absorption band precludes reoxidation of the substrate‐reduced form of the enzyme, indicating that at least one of the two copper ions in benzylamine oxidase may have a direct catalytic function in relation to the reoxidation process.
Cathepsin D: Rapid Isolation by Affinity Chromatography on Haemoglobin‐Agarose Resin
Authors: SMITH Ross , TURK Vito
Published: March 2005

The intracellular proteinase, cathepsin D, has been isolated from bovine spleen and thymus, in times as short as several hours, by affinity chromatography of partially purified and unpurified tissue extracts on haemoglobin‐agarose resin. After subsequent separation from an inactive higher‐molecular‐weight protein by gel permeation chromatography, the enzyme from both tissues shows three dominant proteolytically active bands on gel electrophoresis at pH 4.3 and 9.5: this proteolytic activity is completely inhibited by the acid‐proteinase inhibitor, pepstatin. These enzyme electrophoretic patterns were approximately constant with variation in isolation time and with various preliminary purification procedures. The enzyme shows only traces of polypeptides other than that with an apparent molecular weight of 42000 on dodecylsulphate electrophoresis, in contrast to the enzyme prepared by conventional methods, which contains considerable amounts of smaller polypeptides. This difference in polypeptide composition is shown to be the result of degradation of the enzyme in vitro during isolation by the previously published methods.
The Reversibility of the Isomerization of the Δ8 to Δ7 Bond in Cholesterol Biosynthesis
Authors: SCALA Antonio , GALLI‐KIENLE Marzia , ANASTASIA Mario , GALLI Giovanni
Published: March 2005

The reversibility of the isomerization of the Δ8 to Δ7 bond in the biosynthesis of cholesterol has been re‐examined. For this purpose [4‐14C, 9α‐3H1]cholest‐7‐en‐3β‐ol has been incubated with the 10000 ×g soluble fraction of rat liver homogenate both under anaerobic and aerobic conditions. Almost complete exchange of the 9α hydrogen with the medium has been observed under anaerobic conditions, whereas under aerobic conditions 34% exchange with the medium has been calculated from the 3H/14C ratio found in cholesterol isolated after incubation. The stereospecificity of the Δ7 to Δ8 isomerization has been established by incubating in analogous conditions [4‐14C,7‐3H1]cholest‐7‐en‐3β‐ol. The synthesis of each labelled substrate is described.
A Conductimetric Study of Erythrocyte Lysis by Lysolecithin and Linoleic Acid
Authors: LAWRENCE Anthony J. , MOORES Geoffrey R. , STEELE James
Published: March 2005

A linear six‐channel conductivity apparatus has been developed for rapid and accurate kinetic measurements and used to investigate the net efflux of electrolyte from erythrocytes. The pre‐lytic conductance change and a change directly associated with lysis have been measured for cells treated with lysolecithin and linoleic acid.

The pre‐lytic response to lysolecithin shows three distinct concentration‐dependent phases which correlate with reported changes in morphology. A mechanism is proposed whereby in low concentration lysolecithin is confined to the outer lamella of the membrane but above a threshold level it may translocate to the inner lamella giving a temporary state of high permeability. The response to linoleic acid is more complex, but basically shows constant or increasing permeability after the initial addition without the apparent formation of transient high permeability states. The kinetic form of the response is suitable for accurate kinetic measurements and the leakage response to unsaturated fatty acids is suggested as a method for examining changes in membrane structure.
Synthesis of ATP from ADP and Inorganic Phosphate at the Myosin‐Subfragment 1 Active Site
Authors: MANNHERZ Hans Georg , SCHENCK Helmut , GOODY Roger S.
Published: March 2005

Experiments on the synthesis of ATP from ADP and Pi bound at the active site of the myosin S1 fragment show that the rate limiting step in the ATPase mechanism at 23 °C is associated with a surprisingly low equilibrium constant (about 16 at pH 8.). This, together with the dissociation constant of Pi from the S1 · ADP complex (about 10 mM) obtained from these measurements, and the previously measured rate constants for this system indicate that the dissociation constant of ATP is extremely small, probably in the region of 0.10 pM.
Error Frequency in 5‐S RNA from Cells Grown in 5‐Bromodeoxyuridine
Authors: STAMBROOK Peter , WILLIAMSON Robert
Published: March 2005

Chinese hamster cells were grown in the presence of 5‐bromodeoxyuridine (BrdUrd) for 72 h under conditions where about 50% of the thymine residues were replaced by bromouracil. The substitution in the genes coding for 5‐S ribosomal RNA occurred to approximately the same extent as in the rest of the genome. Cell growth and viability were not affected. Ribosomal 5‐S RNA labelled with [32P]phosphate was isolated from BrdUrd‐treated and control cultures and the oligonucleotides from a T1 ribonuclease digest analysed by fingerprinting techniques. The fingerprints of the two samples were similar. The frequency of errors in the nucleotide sequence of 5‐S RNA from BrdUrd‐treated cells was very low compared with the reported frequency of errors in a transcription system in vitro. The sequence of Chinese hamster 5‐S RNA is the same as that reported for other mammalian species.
A High‐Molecular‐Weight Protein Component Required for Natural Messenger Translation in Ascites Tumor Cells
Authors: STRYCHARZ William A. , RANKI Marjut , DAHL Hans‐Henrik M.
Published: March 2005

An IF‐M3‐deficient ascites cell‐free system is described.

An IF‐M3 species of high molecular weight has been isolated and partially purified from a high‐KCl ascites ribosomal wash fluid.

Translation of encephalomyocarditis virus RNA and globin mRNA is completely dependent upon the presence of the ascites IF‐M3, while the factor is not required with either poly(A,U,G) or poly(U) as messengers.

The sparsomycin technique for the generation of short initiation peptides has been employed to demonstrate that IF‐M3 acts at the level of chain initiation.

Rabbit reticulocyte IF‐M3 and IF‐E3, as well as rabbit liver IF‐M3, can substitute for ascites IF‐M3 in EMC‐RNA and globin mRNA translation.

The translation of EMC‐RNA in the ascites cell‐free system is, therefore, dependent upon the presence of two discrete initiation factors, a high‐molecular‐weight species, IF‐M3, and a smaller component, IFEMC.
Pantothenoylcysteine‐4′‐phosphate Decarboxylase from Horse Liver
Authors: SCANDURRA Roberto , BARBONI Erminia , GRANATA Filippo , PENSA Bernardo , COSTA Mara
Published: March 2005

Pantothenoylcysteine‐4′‐phosphate decarboxylase has been purified 700‐fold from horse liver by by an improved method. Enzyme activity was determined by measuring the amount of labeled CO2 produced from the labeled cysteine moiety of the substrate molecule. The enzyme is not bound to any cellular structure since more than 80% of the total activity of the homogenate is recovered in the supernatant fraction. The enzyme does not seem to contain pyridoxal phosphate, is strongly inhibited by this latter and by carbonyl reagents, such as sodium borohydride, hydroxylamine and phenylhydrazine. These findings strongly support the presence in the enzyme molecule of a reactive carbonyl group. The enzymic reaction rate is increased over 20‐fold by sulfhydryl compounds, has an absolute specificity for pantothenoylcysteine 4′‐phosphate and is not inhibited either by pantothenoylcysteine or by the reaction product, pantetheine 4′‐phosphate.
Subunit Interactions in Enzyme Catalysis
Authors: MOUTTET Christian , FOUCHIER Francis , NARI Joannès , RICARD Jacques
Published: March 2005

By using the principles of structural kinetics previously formulated, the effects of subunit interactions and conformation changes on transient rate of catalysis have been investigated for one‐substrate dimeric enzymes. The analytical solutions of transient rate equations and the expression of the induction time, τ have been obtained for the following models of protomer interactions: simple sequential, partially‐concerted, fully‐concerted, and exclusive allosteric.

The progress curve of the reaction during the pre steady‐state phase, as well as the curve expressing the variation of τ−1 against substrate concentration, [S], have been simulated by computer analysis. When the protomers do not exhibit any interaction in the simple sequential mechanism, the plot τ−1versus [S] is a straight line and the induction time is always positive. If subunit interaction occurs but does not change during substrate binding, the plot of τ−1 against [S] is still linear but the length of the induction time depends on the value of the interaction coefficient. If this value is much lower than one, the enzyme, although Michaelian, behaves as “hysteretic” when rapidly mixed with its substrate. In the same way, the exclusive allosteric model also predicts positive z values, but the plot of τ−1 against [S] is a curve reaching a plateau for high concentrations of the substrate. The sequential models (simple, partially‐concerted, fully‐concerted) can give rise to either positive or negative induction time. Moreover, the asymptote of the curve τ−1 against [S] is oblique. It is thus possible, at least in theory, from pre‐steady‐state kinetics alone, to rule out either the exclusive allosteric or the sequential models.

The above theoretical developments have been applied to the early kinetics of l‐phenylalanine ammonia‐lyase reaction. The induction time, that can be calculated from the progress curve, is negative for a large range of substrate concentrations. This result allows one to rule out, for that enzyme, the allosteric transition as a possible explanation of cooperativity. Sequential binding of substrate and subunit interactions can thus generate a burst, that is a transient activation of the reaction in the absence of any activator. It is tempting to assume this transient activation to be of regulatory significance.
Enzyme Polymorphism in Rat‐Liver Microsomes and Plasma Membranes
Authors: BLOMBERG Fred , RAFTELL Marika
Published: March 2005

Detergent‐soluble antigens from rat liver microsomes and plasma membranes were separated by two‐dimensional immunoelectrophoresis. Enzyme activities in the immunoprecipitates were assayed by different staining reactions.

Nine different microsomal esterases were detected. Six of these, including one esterase shared with plasma membranes, were inhibited by 0.1 mM difluorophosphate, but not by 1 mM eserine.

Plasma membranes and microsomes each contained at least ten nucleoside diphosphatase and triphosphatase‐active antigens. Only three precipitates with these activities were detected when plasma membrane extracts were tested against anti‐microsomal sera or microsomal extracts against anti‐plasma membrane sera. Although the possible immunological identity of these antigens remains to be established these findings suggest that plasma membranes and microsomes have at most three of these nucleoside diphosphatases and triphosphatases in common.

Seven microsomal nucleoside diphosphatases and triphosphatases were intimately associated with both acid phosphatase and NADH‐neotetrazolium reductase activities. These complexes also contained phospholipids as shown by incorporation experiments either in vivo, or in vitro, with [14C]‐choline or [14C]ethanolamine. Three microsomal UDPase‐active antigens did not hydrolyze ATP or ADP and were not associated with acid phosphatase or NADH‐neotetrazolium reductase activities. Furthermore, these antigens did not contain any of the labelled phospholipids.

Lipid‐containing multienzyme complexes of different composition were also detected in the plasma membranes. The nucleoside diphosphatases and triphosphatases in plasma membrane extracts were associated with NADH‐neotetrazolium reductase activity and two of them also contained l‐leucyl‐β‐naphthylamidase activity.

p‐Chloromercuribenzoate (0.1 mM) inhibited all NADH‐neotetrazolium reductase activity in the plasma membrane and microsomal precipitates and 10 mM tartrate all acid phosphatase activity in the microsomal precipitates, leaving the other activities intact. This indicated that the various enzyme activities present in the multienzyme complexes depended on separate catalytic sites.

The plasma membrane extracts contained three and the microsomal extracts two l‐leucyl‐β‐naphthylamidase‐active antigens, respectively. The latter two and one of the plasma membrane antigens were not found to be associated with any other enzyme activities or phospholipids, in contrast to the two plasma membrane antigens mentioned above.
Enzyme Polymorphism in Rat‐Liver Microsomes and Plasma Membranes
Authors: RAFTELL Marika , BLOMBERG Fred
Published: March 2005

Extraction of rat liver microsomes and plasma membranes with the detergents Lubrol W and sodium deoxycholate rendered extracts containing a multiplicity of electrophoretically and immunologically different enzyme‐active antigens as detected in two‐dimensional immunoelectrophoresis. To establish that this enzyme polymorphism was not merely due to an interaction of enzyme molecules with detergent micelles but reflected membrane organization, antigens solubilized by other agents like Triton X‐100, papain, phospholipase A or C were compared with those extracted by Lubrol W and deoxycholate.

Different enzyme‐active antigens were differently affected by the various solubilizing agents. Thus phospholipases A and C released the same esterase‐active antigens as did Lubrol‐ deoxycholate. In contrast, phospholipid‐containing multienzyme complexes (expressing nucleoside diphosphatase and triphosphatase, NADH‐neotetrazolium reductase, acid phosphatase and L‐leucyl‐P‐naphthylamidase activities in different combinations) were not solubilized in an enzymatically active form by these agents. On the other hand, such multienzyme complexes were released in significant quantities from the microsomes but not from the plasma membranes, when a highly diluted papain solution was used for extraction. The results are assumed to reflect differences in the molecular organization of individual enzymes in the various membranes.

In order to investigate the molecular identities of the antigens solubilized from the membranes with the different agents, 125I‐labelled antigens, extracted with papain or phospholipase A, were mixed with unlabelled detergent‐solubilized antigens. Autoradiography of the immunodiffusion plates showed that the enzyme‐active antigens released by papain or phospholipase A were electro‐ phoretically and immunologically similar to those solubilized by detergents.
Presence of Cytochrome c1 in Cytoplasmic “Petite” Mutants of Saccharomyces cerevisiae
Authors: CLAISSE Maurice L. , PAJOT Patrick F.
Published: March 2005

To show the occurrence of cytochrome c1 in ϱ− petite mutants of Saccharomyces cerevisiae, in which its presence was questioned, low‐temperature absorption spectra of various ϱ+ϱ− strains have been compared. Some of these strains carry nuclear mutations producing different types of cytochrome deficiencies, thus lowering the spectral interferences on cytochrome c1 and making easier its observation. The comparison was made with whole cells and with mitochondrial preparations. All the ϱ−mutants examined present a shoulder or a peak at a wavelength corresponding to cytochrome c1 in the ϱ+ strains. This absorption band at 552 nm in ϱ− mutants was further identified by isolating a cytochrome with an α absorption maximum at this wavelength. The low‐temperature absorption spectrum of this cytochrome was compared to low‐temperature absorption spectra of cytochrome c1 isolated from a wild‐type strain, and of cytochrome c and cytochrome b2. It is similar, if not identical, to the spectrum of the wild‐type cytochrome c1, and quite distinct of that of the other cytochromes.

The cytochrome c1 content, in the various ϱ+and ϱ− strains examined, is estimated with the help of a calculation procedure taking into account the spectral cross interferences of the cytochromes.

In the absorption spectra of the ϱ− strains, a shoulder is recorded at about 557–558.5 nm. The nature of this shoulder is discussed.
Relations between Structure and Function in Cytoplasmic Membrane Vesicles Isolated from an Escherichia coli Fatty‐Acid Auxotroph
Authors: SHECHTER Emanuel , LETELLIER Lucienne , GULIK‐KRZYWICKI Tadeusz
Published: March 2005

The results presented in this paper establish relationships between structural, morphological and functional properties of cytoplasmic membrane vesicles isolated from an Escherichia coli unsaturated fatty acid auxotroph. The membranes were isolated from cells grown in the presence of either oleic, linoleic, linolenic or elaidic acids.

High‐angle X‐ray diffraction studies show that the order‐disorder transitions induced by temperature variations and associated with the paraffin chains of the lipids are a function of the fatty acid composition of the membranes. In some cases “cocrystallization” of various lipid species takes place within a single type of ordered domains. In other cases there is segregation of various lipid species into more than one distinct type of ordered domain.

The various order‐disorder transitions observed induce morphological changes in the hydrophobic core of the membranes which can be detected by freeze‐etch electron microscopy. A random distribution of particles on the fracture faces is observed when the paraffin chains of the lipids are disordered. Upon ordering of the paraffin chains, particles are excluded from the ordered domains and as a consequence, smooth areas and areas with densely packed particles are observed. The ratio of the smooth surface to particulated surface is proportional to the amount of ordered paraffin chains present. Moreover, the size of the smooth domains is a function of the fatty acid composition of the membranes.

Discontinuities in the rate of d‐lactate‐dependent proline uptake as a function of temperature correlate with the order‐disorder transitions observed. The high energies of activation at low temperatures are attributed to decreased mobility of the carrier proteins upon aggregation. In contrast, phosphoenolpyruvate‐dependent vectorial phosphorylation does not respond to the ordering of the paraffin chains.
Translation of Semliki‐Forest‐Virus 42‐S RNA in a Mouse Cell‐Free System to give Virus‐Coat Proteins
Authors: SMITH Alan E. , WHEELER Tricia , GLANVILLE Nial , KÄÄRIÄINEN Leevi
Published: March 2005

Semliki‐Forest‐virus 42‐S virion RNA is translated in a mouse ascites cell‐free system. One of the major polypeptides made in vitro has been identified as Semliki‐Forest‐virus capsid protein by co‐electrophoresis on dodecylsulfate–polyacrylamide gels and by tryptic peptide analysis on paper. Another major product of the cell‐free system has been characterised as envelope protein by peptide analysis but it does not co‐electrophorese on gels with envelope proteins from purified virions. It is possible that the nascent, non‐glycosylated polypeptide is partially degraded by proleolytic enzymes present in the cell‐free extract, or alternatively that fragments of envelope protein are produced by premature termination on the viral RNA. Several other polypeptides are synthesised in the cell‐free system in variable amounts but these have not been identified. The genetic content and the mechanism of translation of Semliki‐Forest‐virus 42‐S RNA are discussed.
The Effect of Inhibitors of Protein Synthesis on Cholesterol Side‐Chain Cleavage in the Mitochondria of Luteinized Rat Ovaries
Authors: ARTHUR John R. , BOYD George S.
Published: March 2005

Conversion of cholesterol to pregnenolone, termed cholesterol side‐chain cleavage, has been studied in isolated mitochondria from luteinized rat ovaries.

Cholesterol in these mitochondria in the presence of cyanoketone is converted to one product pregnenolone on addition of a suitable electron donor. The percentage conversion of total mitochondrial cholesterol to pregnenolone is comparable to that measured by the percentage conversion of [4‐14C]cholesterol to [4‐14C]pregnenolone when the label has been preincubated at 29°C for 10 min with the mitochondria.

In mitochondrial incubations the depletion of endogenous cholesterol follows the production of pregnenolone. The initial rate of production of pregnenolone (0.8 nmol × mg protein−1× min−1) was maintained in incubations for 5–6 min. Addition of free cholesterol to incubations allowed the production of pregnenolone to continue at the initial rate for at least 30 min.

Treatment of rats with cycloheximide 20 min prior to killing, caused a 58% inhibition of luteal mitochondrial cholesterol side‐chain cleavage and a small but significant rise in the cholesterol content of isolated mitochondria. The cycloheximide treatment did not affect the total luteal mitochondrial cytochrome P‐450 but caused a 56% decrease in the inverted type I pregnenolone‐binding spectrum, suggesting cholesterol binding to cytochrome P‐450 had been reduced.

Rats treated with chloramphenicol 3 and 10 h before killing exhibited no differences in total luteal mitochondrial cytochrome P‐450 or mitochondrial cholesterol metabolism when contrasted with saline‐injected animals.

Puromycin was shown to bind to the rat luteal mitochondrial cytochrome P‐450 to produce a type I1 difference spectrum and to inhibit luteal mitochondrial cholesterol side‐chain cleavage in vitro.

The results are discussed in terms of a protein which is rapidly turning over and which may be synthesised extra‐mitochondrially and in some way affect the binding of cholesterol to luteal mitochondrial cytochrome P‐450.
Kinetic Studies of the Reaction of Thiol Groups of Calf‐Thymus Histone F3 with 5‐5′‐Dithiobis(2‐nitrobenzoic acid)
Authors: PALAU Jaime , DABÁN Juan R.
Published: March 2005

The reactivity of 5‐5′‐dithiobis(2‐nitrobenzoic acid) with thiol groups of calf thymus histone F3 was studied by using the tripeptide glutathione and 2‐mercaptoethanol as models. The results found for glutathione suggest that the reaction is dependent on the nucleophile‐S− concentration. The reactivity of thiol groups of the histone in 2.4 mM sodium phosphate solutions at room temperature was found to be much higher than for glutathione and almost independent of pH between pH 5 to 7. The reaction follows second order kinetics, K2(app.) at pH 5 being 4000 times higher than K2 for glutathione. The pKa(app.) for thiol groups of histone F3 was estimated to be 4.2 indicating the predominance at low pH of ionic forms of the thiol groups. At 24 mM sodium phosphate, pH 6.6, 80% of thiol groups react at a similar high rate as in the case of 2.4 mM sodium phosphate, and the remaining 20% reacts very slowly, the kinetics being of first order. Local denaturation is not obtained by urea treatment and high temperatures (85°C), but guanidine hydrochloride completely unfolds the microenvironments containing cysteine. It is proposed that such reacting microenvironments are important in the biological role of the histone F3.
Effect of Reconstitution Conditions on the Structure of Escherichia coli 30‐S Ribosomal‐Subunit Components
Authors: LEMIEUX Gerald , LEFEVRE Jean‐François , DAUNE Michel
Published: March 2005

16‐S RNA and five pure ribosomal proteins (S3, S4, S6, S7 and S8) from Escherichia coli 30‐S subunits were studied by circular dichroism in regard to the reconstitution of biologically active subunits. A small structural variation upon heating could be measured for RNA. It cannot be interpreted in terms of stacking or disruption of hydrogen bonds. However, most proteins underwent important structural modifications when heated to 42°C in reconstitution solvent. This effect was not dependent on the presence of magnesium salt. The relative amounts of secondary structure of native and heat‐denatured ribosomal proteins were calculated by using three reference curves obtained from standard proteins, the exact secondary structure of which has been determined by X‐ray crystallography. In contrast with earlier results, about 18%β‐structure was detected in native proteins. This discrepancy can most probably be attributed to the calculation method used. Results are discussed in relation to reconstitution of ribosomes.
Nucleoside‐Diphosphate Derivatives of 2‐Deoxy‐d‐glucose in Animal Cells
Authors: SCHMIDT Michael F. G. , SCHWARZ Ralph T. , SCHOLTISSEK Christoph
Published: March 2005

After administration of 14C‐labelled 2‐deoxy‐d‐glucose or 3H‐labelled nucleosides to cells in tissue culture the following activated intermediates of the deoxysugar have been isolated and characterized: the 6‐phosphate derivative, the 1‐phosphate derivative, the 1,6‐bisphosphate derivative, UDP‐2‐deoxy‐d‐glucose, GDP‐2‐deoxy‐d‐glucose and 2‐deoxy‐d‐gluconic acid 6‐phosphate. After extraction with acid and organic solvents, 2‐deoxy‐d‐glucose could be recovered by acid hydrolysis from the cells. Labelled 2‐deoxy‐d‐galactose was not found either bound to UDP or to the cells, indicating that no significant epimerisation of 2‐deoxy‐d‐glucose at the UDP level occurred in these cells.
New Inhibitors of RNA Synthesis in vitro Isolated from Escherichia coli
Authors: KAGAWA Haruo , EGBERTS Egbert
Published: March 2005

Three inhibitor proteins (I1, I2, and I3) were isolated from Escherichia coli. They depress the transcription of E. coli and T3 phage DNA by DNA‐dependent RNA polymerase from E. coli and show the following characteristics.

The inhibitors I1 and I3 consist of subunits; I2, behaves as a single protein in dodecylsulfate‐polyacrylamide gel electrophoresis.

Neither the initiation of the transcription of E. coli DNA nor the size of the RNA synthesized is influenced by the inhibitors.

In an E. coli DNA‐dependent system reinitiation of RNA synthesis at high salt concentration seems to be blocked by the inhibitors.

When inactivated, the inhibitors stimulate RNA synthesis and behave like termination factors giving rise to the production of RNA molecules of discrete size, shorter than those of control experiments.

It is suggested by hybridization competition experiments that the inhibitors I1 and I3 preferentially inhibit ribosomal RNA synthesis.

The inhibitors also depress the transcription of T3 phage DNA by E. coli RNA polymerase resulting in the synthesis of discrete RNA species smaller than those of control experiments without KCl as well as with 0.16 M KCl.
Phospholipid Metabolism in Embryonic Rat Fibroblasts following Stimulation by a Combination of the Serum Proteins S1 and S2
Authors: HOFFMANN Rainer , RISTOW Hans‐Jürgen , PACHOWSKY Heinz , FRANK Werner
Published: March 2005

Phospholipid metabolism has been investigated in embryonic rat fibroblasts which have been incubated in serum‐free medium and subsequently stimulated by the growth‐stimulating serum proteins S1 and S2:

Incorporation of 32P into phosphatidylinositol iancreases rapidly within a few minutes of stimulation; after 30 min it is 7.4‐fold higher in stimulated cells when compared with the controls. Phosphatidylcholine synthesis and uptake of 32P into the acid‐soluble pool is not significantly enhanced during this time. Labelling of phosphatidylserine, phosphatidylethanolamine and sphingomyelin during this period is just above the background level.

Incorporation of 32P into phosphatidylinositol and phosphatidylethanolamine is strongly inhibited in the presence of dibutyryl‐adenosine 3′: 5′‐monophosphate and theophylline whereas the metabolism of phosphatidylcholine is not influenced.

As demonstrated with [3H] inositol, the inhibition of phosphatidylinositol synthesis is restricted to dibutyryladenosine 3′:5′‐monophosphate; adenosine 3′:5′‐monophosphate; guanosine 3′:5′‐monophosphate and dibutyryl guanosine 3′:5′‐monophosphate are ineffective.

The growth‐stimulating serum factors must be present continuously for activation of phosphatidylinositol synthesis; within 1 h after serum deprivation the incorporation of 32P into phosphatidylinositol decreases to about 20% of the original value; in contrast, phosphatidylcholine biosynthesis is not influenced significantly during this time period.
Influence of Calf Serum on the Transport of 2‐Aminoisobutyric Acid in Embryonic Rat Fibroblasts
Authors: FRANK Werner , RISTOW Hans‐Jürgen
Published: March 2005

Transport of 2‐aminoisobutyric acid in embryonic rat fibroblasts is mediated by a carrier system, Km, app.= 8 × 10−3 M. It was also shown by competition experiments with naturally occurring amino acids that the alanine carrier is responsible for 2‐aminoisobutyric transportation.

With cells incubated for 24 h in serum‐free medium, a final Km, app. cannot be demonstrated indicating that under these conditions uptake occurs only by diffusion. After stimulation of the cells by the growth‐stimulating serum factors S1 and S2 no increase in accumulation of 2‐aminoisobutyric acid was found during the first hour, but a Km, app. value can be measured after about 2 h.

Efflux of 2‐aminoisobutyric acid is also brought about by an active mechanism which is strongly dependent on the temperature and potassium concentration of the culture medium, and which is also influenced by ouabain.

In the permanently growing transformed rat cell lines which have been examined, transport of 2‐aminoisobutyric acid appears to be independent of the presence of serum factors, nearly identical Km, app. values being found within cells grown with and without serum.
Polyol Metabolism in Monkey‐Kidney Epithelial‐Cell Cultures
Authors: HUTTON John C. , WILLIAMS John F. , SCHOFIELD Philip J. , HOLLOWS Frederick C.
Published: March 2005

An investigation of polyol synthesis by monolayer cultures of monkey kidney epithelium cells was undertaken as a consequence of the observation that these cells accumulated high concentrations of acyclic hexitols (200–600 μmol/g protein) when incubated in media containing elevated glucose [590 mg‐% (w/v)] concentrations.

It was established that the acyclic hexitol which accumulated when monkey kidney epithelial cell cultures were incubated in high glucose medium was sorbitol. The involvement of the polyol pathway of metabolism in this phenomenon was implicated from radioactivity incorporation experiments using [U‐14C]glucose substrate (the specific radioactivity of the sorbitol product was 92 % of the specific radioactivity of the added glucose) and from studies involving the use of the aldose reductase inhibitor 3,3′‐tetramethylene glutaric acid, which caused marked inhibition of polyol synthesis.

The intracellular glucose concentration was found to be an important factor regulating the steady state concentrations of the pathway intermediates sorbitol and fructose in monkey kidney epithelial cell cultures. Changes in the oxygen tension in the incubation medium or the presence of the hormones vasopressin, insulin or epinephrine were shown not to affect the concentrations of sorbitol or fructose in monkey kidney epithelial cell cultures incubated in media containing a high glucose concentration.

Cultures of HeLa Cells, goldfish testes fibroblasts, or ascites tumor cells did not exhibit the same capacity as monkey kidney epithelial cell cultures for sorbitol synthesis. To our knowledge, the present findings constitute the first reported example of polyol pathway activity in cells in monolayer culture.
Deuterium‐Labelling of sn‐Glycerol 3‐Phosphate during Metabolism of [1‐2H2]Ethanol in Rats
Authors: CURSTEDT Tore
Published: March 2005

The incorporation of deuterium into different positions of sn‐glycerol 3‐phosphate in liver and into glucose in plasma of rats given [1‐2H2] ethanol has been determined. The labelling of sn‐glycerol 3‐phosphate reached a steady state after about 1 min. The deuterium excess was about 5 atoms% at C‐1, 15 atoms% at C‐2, and 5 atoms% at C‐3. Plasma glucose contained less than 2 atoms% deuterium. Administration of glucose or fructose did not influence the labelling of sn‐glycerol 3‐phosphate. The results are compared with those obtained for the glycerol moiety of phosphatidyl cholines. It is concluded that there is more than one pool of sn‐glycerol 3‐phosphate in the liver, and that the major part has a glycolytic origin.
Molecular Morphology of Ribosomes
Authors: MICHALSKI Chester J. , SELLS Bruce H.
Published: March 2005

Lactoperoxidase‐catalyzed iodination of 30‐S and 50‐S subunits of Escherichia coli ribosomes maintained at 0°C revealed that proteins L2, L5, L10, L11, L15, L26, L27, L29 and L30 of the large subunit and S2, S3, S7, S9, S10, S11, S12, S14, S19 and S21 of the small subunit were the most susceptible to iodination. Unfolding of the 50‐S subunit by the removal of Mg2+ greatly increased iodination of the majority of the large subunit proteins while comparatively few of the 30‐S proteins became more susceptible to iodination under identical conditions. Less extensive conformational changes were observed when the iodination reaction was allowed to proceed at higher temperatures. At 37°C, significant increases in the iodination of proteins S6, S13, S15, S16, L3, L8, L9, L18 and L19 were noted suggesting that a loosening of the ribosome structure occurs at this temperature. Enzymatic iodination had little effect on the activity of the subunits in vitro with respect to G‐factor‐dependent GTPase and poly(U)‐directed poly(phenylalanine)‐synthesizing activities. The lactoperoxidase‐catalyzed iodination system is evaluated as a probe for detecting conformational changes and for determining the topographical nature of the ribosome.