bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymatic Catalysis in Solidified Media
Authors: Ceska Miroslav
Published: March 2005

Water insoluble blue starch polymer particles incorporated into agar medium were shown to be hydrolyzed by α‐amylase enzyme. When a drop of α‐amylase was placed on such a medium the enzyme diffused at the same time hydrolyzing the water insoluble blue starch polymer to form a clear area which was easily visible and easily measured. The size of the hydrolyzed area was studied with respect to the time of hydrolysis, temperature of hydrolysis, and enzyme concentration. The variance of repeated measurements of the diameter of 80 hydrolyzed areas, as well as one area measured ten times, was determined. The variance in diameter measurements of the hydrolyzed areas on different plates and at different distances from the center of the plate was determined.
Biological Activities of Chemically Modified Endotoxins
Authors: Rietschel Ernst Th. , Galanos Chris , Tanaka Atsushi , Ruschmann Ellen , Lüderitz Otto , Westphal Otto
Published: March 2005

The cell wall glycolipid (somatic antigen) of the Re mutant of Salmonella minnesota is comparable in its endotoxic activity to the complete wild type lipopolysaccharide. The glycolipid is composed mainly of lipid A and 2‐keto‐3‐deoxyoctonate (KDO); smaller amounts of ethanolamine and 4‐aminoarabinose, and traces of amino acids are also present. The serological specificity is determined by KDO residues. In order to determine if non‐lipid A constituents contribute to endotoxicity, amino groups and KDO units in the glycolipid were chemically modified by the introduction of substituents. When free amino groups were dinitrophenylated or succinylated the serological specificity was unaffected. By contrast, the original serological specificity was completely lost when the free hydroxyl groups of the KDO region were substituted with succinyl residues or the carboxyl groups converted to the methyl esters. In biological tests, however, the modified preparations exhibited full endotoxic activity. It is concluded that the polar groups, which determine the serological specificity of the KDO region of the glycolipid, do not play a specific role in endotoxicity.
Steroids in Meconium from Male and Female Newborn Infants
Authors: Gustafsson Jan‐Åke , Stenberg Åke
Published: March 2005

The steroids in meconium from normal male and female newborn infants have been studied by gas chromatography‐mass spectrometry. Practically all steroids were excreted as mono‐ and disulphates. The approximate concentrations of steroid mono‐ and disulphates were 430 and 470 μg per g of meconium. Twenty unsaturated and fourteen saturated steroids were completely identified. Most of the unsaturated steroids had a 3β‐hydroxy‐Δ5 structure. In addition, seven saturated steroids were tentatively identified. No significant qualitative differences were found in the steroid composition of meconium from male and female newborns. The results are discussed in relation to present knowledge of steroid metabolism in the foeto‐placental unit.
Functional Properties of Human Hemoglobin Treated with 5,5′‐Dithiobis, 3,3′‐Nitrobenzoic Acid
Authors: Amiconi Gino , Antonini Eraldo , Brunori Maurizio , Wyman Jeffries , Nason Alvin
Published: March 2005

The reaction of 5,5′‐dithiobis, 3,3′‐nitrobenzoic acid with hemoglobin and isolated chains has been studied and the functional properties of the derivatives have been determined. The oxygen affinity of treated hemoglobin, both at low and high salt concentration is higher than that of normal hemoglobin under the same conditions with no substantial change in heme‐heme interaction; the Bohr effect is somewhat reduced in the alkaline range, but is increased in the acid range. The treated β‐chains show enchanced oxygen affinity with respect to normal chains. Treated hemoglobin shows also, in comparison with normal hemoglobin, significant changes in the kinetics of CO binding and O2dissociation.
Functional Properties of Human Hemoglobin and Isolated Hemoglobin Chains Treated with Organic Mercurials
Authors: Giardina Bruno , Binotti Ines , Amiconi Gino , Antonini Eraldo , Brunori Maurizio , Mcmurray Cecil H.
Published: March 2005

The functional properties of human hemoglobin and of isolated α‐ and β‐chains with free SH groups blocked by 2‐chloromercuri‐4‐nitrophenol, 4‐chloromercuri‐2‐nitrophenol and p‐chloromercuribenzoate have been determined. The oxygen affinity of all the hemoglobin derivatives is slightly higher than that of the untreated protein; the shape of the oxygen equilibrium curve is pH dependent (except for p‐chloromercuribenzoate reacted hemoglobin). The alkaline Bohr effect is similar to that of normal hemoglobin, but in the acid range it appears to be modified to a different extent in the various cases. Thus the properties of the modified hemoglobins appear to depend on the details of structure of the organic mercurial reacted with the β93‐SH group. The results with isolated β‐chains show that the presence of the organic mercurials on F9(93), but not on G14(112) is associated with a decrease in oxygen affinity and a large increase in the oxygen dissociation velocity constant.
Acetylcholinesterase Interaction with a Lipoprotein Matrix
Authors: Grafius Melba A. , Bond Howard E. , Millar David B.
Published: March 2005

The nature of the components and some of the association forces in aggregated acetylcholinesterase, extracted from the electric organ ofElectrophorus electricus, were investigated. The aggregated acetylcholinesterase (rapidly sedimenting, 70 S, and referred to as fast acetylcholinesterase) and the dissociated or “monomer” acetylcholinesterase (11.5 S and referred to as slow acetylcholinesterase) were isolated by zonal centrifugation. The fast acetylcholinesterase was further processed by gel filtration, resulting in a fast acetylcholinesterase of low specific activity and a slow acetylcholinesterase of high specific activity and a lowered sedimentation value (10.8 S). The fast acetylcholinesterase was exposed to the hydrolytic action of various enzymes (neuraminidase, hyaluronidase, alkaline phosphatase, and phospholipase A) and examined by sucrose gradient centrifugation techniques with no effect observed on the aggregation. However, phospholipases C and D and pancreatic lipase dissociated the fast acetylcholinesterase to the slow acetylcholinesterase (11.5 S). With phospholipase C, but not pancreatic lipase, the acetylcholinesterase was released from a matrix of ultraviolet absorbing, non‐enzymatic material which remained in its aggregated form. With pancreatic lipase the matrix was also dissociated into a “monomer” species with a sedimentation value similar to that of the “monomer” acetylcholinesterase (resembling dissociation in high ionic strength). The hydrolytic action of the lipases did not alter the sedimentation value of the “monomer” nor its property of reversibly forming a gelatinous precipitate at pH 4.0. However, the slow acetylcholinesterase recovered from the gel column was not sensitive to the acid pH. The results indicate that the aggregated or fast acetylcholinesterase is a complex between “monomer” and a matrix of lipoproteins which, in itself, is an aggregation of molecular species similar in sedimentation value to the “monomer” acetylcholinesterase. A model is presented. Comparison between the matrix and the receptor protein is made.
Co‐operative Non‐Enzymic
Authors: Podder Sunil K.
Published: March 2005

The absorbance and the optical rotatory dispersion of solutions of GpGpGpC and GpCpCpC individually and of the mixture of these two were measured in 0.1 M phosphate buffer (pH 7.2) as a function of temperature and concentration. The data indicate that below room temperature GpGpGpC self‐associates into large aggregates which presumably involve both G · C and G · G base pairs. It interacts as well with GpGpCpC to form a 1:1 GpGpGpC: GpCpCpC complex containing 4 G · C base pairs. The kinetics of formation of the 1:1 complex, as well as of self‐associated complexes, were studied by temperature jump relaxation method. The transition temperature Tmof the 1:1 complex was determined from the variation of the relaxation amplitude with temperature. From the concentration dependence of Tm, the values of – 21.6 kcal/mole and –50 entropy units were calculated for ΔH and ΔS respectively for the formation of the 1:1 complex.

Temperature‐jump relaxation experiments on the individual components over the temperature range 20–40°C indicate a single process which was too rapid to follow (t0.5≪ 5 μsec), corresponding to the melting of a single stranded helix. In experiments with mixed solutions a slower measurable process was observed in the millisecond time range, which was attributed to 1:1 complex formation with a recombination rate constant kr= 5.4 106M−1× sec−1 and a dissociation rate constant kd= 40 sec−1 (at 21°C), ΔE‡r= 4.5 kcal/mole and ΔE‡d=+ 28.7 kcal/mole. Analysis of thermodynamic and kinetic data in terms of the “all or none” model suggests that the formation of the second base pair is the rate limiting step in the G · C helix formation. A single base pair adjacent to a pre‐existing helical sequence can be formed in 0.1 μsec. In contrast, the apparent formation and dissociation rate constants of GpGpGpC aggregates are smaller than those of the 1:1 complex by several orders of magnitude. The existence of a syn‐anti type equilibrium below room temperature is postulated to explain this observation.
An Efficient Procedure for the Isolation of Polyribosomes from Tissue Culture
Authors: Gielkens Arno L. J. , Berns Ton J. M. , Bloemendal Hans
Published: March 2005

In this paper an efficient procedure is described for the isolation of polysomes from tissue culture cells. Optimal yields, especially of the heavier classes of polysomes can only be obtained at a rather high KC1 concentration in the presence of nonionic detergent. Evidence is provided that the lower polysomal yield after extraction at low salt concentration is not due to breakdown of the polysomes by RNAase.

Furthermore, the yield of polysomes is pH dependent only at low salt concentration, while at high salt concentration no influence of the pH is observed. Refreshing of growth medium several hours before harvesting the cells, increases the ratio of polysomes to 80 S monomers.
Recherches sur les acides désoxyribonucléiques d'Acetabularia mediterranea
Authors: Heilporn Viviane , Limbosch Sylvie
Published: March 2005

The density of the DNA present in the cytoplasm of Acetabularia mediterranea has been determined by centrifugation in a CsCl gradient. Electron microscope examination has demonstrated that a unique DNA having a density of 1.704 g/cm3 is extracted from a pure fraction of chloro‐plasts. The method elaborated by Suyama and Bonner for the isolation of the mitochondria has enabled us to isolate a sub‐cellular fraction with cytochrome oxidase activity, from which we were able to extract a small amount of pure DNA which is probably mitochondrial DNA, having only one density peak at 1.715 g/cm3; unlike mitochondrial DNA, chloroplast DNA is sensitive to prolonged maintenance of the algae in the dark, as well as to treatment of a suspension of chloroplasts and mitochondria with deoxyribonuclease. Under our experimental conditions, centrifugation of cytoplasmic DNA in CsCl to which ethidium bromide is added seems to exclude the possibility that circular molecules of DNA occur whether of chloroplastic or of mitochondrial origin. Both cytoplasmic DNA readily undergo thermal denaturation. Renaturation of chloroplast DNA is incomplete, whereas mitochondrial DNA renatures readily completely. During the process of cyst formation and nuclear multiplication, it is possible to extract newly formed DNA which is probably nuclear: it has a density of 1.696 g/cm3. All three DNA, with a boyant density of 1.696, 1.704, 1.715 g/cm3, respectively, disappear after treatment with deoxyribonuclease, but remain intact after treatment with a mixture of α‐ and β‐amylases.

We would like to suggest that following many years of laboratory culture, Acetabularia mediterranea may have given rise to new genetically distinct species. Such an hypothesis would explain the apparent lack of agreement between our own findings and those of Gibor and of Schweiger.
Investigation of Xanthine Oxidase
Authors: Bayer Ernst , Bacher Adalbert , Krauss Peter , Voelter Wolfgang , Barth Günter , Bunnenberg Edward , Djerassi Carl
Published: March 2005

The magnetic circular dichroism and circular dichroism spectra of xanthine oxidase are reported from 200–700 nm. The magnetic circular dichroism spectrum exhibits weak bands at 520 and 375 nm which are assigned to the iron‐sulfur and molybdenum chromophores, respectively. The tryptophan chromophore gives a strong positive magnetic circular dichroism band at 292 nm and from the intensity of this band in xanthine oxidase, the presence of a total of 23–24 residues per molecule has been calculated.
Comparative Structural Studies of the Active Site of ATP: Guanidine Phosphotransferases
Authors: der Terrossian Elisabeth , Desvages Gisèle , Pradel Louise‐Anne , Kassab Rhida , van Thoai Nguyen
Published: March 2005

The active thiol group of lombricine kinase from Lumbricus terrestris muscle was labelled with N‐ethyl‐[1‐14C]maleimide. The resulting inactivated N‐ethyl‐[1‐14C]succinimido enzyme was then subjected to tryptic hydrolysis.

The peptide containing the labelled essential thiol group was isolated and found to contain: Leu‐Gly‐Tyr‐Ile‐Thr‐[14C]Cys‐Pro‐Gly‐Ser‐Asn‐Leu‐Gly‐Thr‐Leu‐Arg. The amino acid sequence around this thiol group was very similar with that of homologous ATP: guanidine phosphotransferases previously studied, arginine kinase from Homarus vulgaris muscle, creatine kinases from ox brain and ox muscle and from rabbit muscle.

In addition among the other enzymes of this group, lombricine kinase is of special interest since it is the only dimeric enzyme of molecular weight ≃ 80000 which possesses only one essential thiol group and one nucleotide binding site per two subunits.
Ribosomal Proteins
Authors: Hindennach Ingrid , StÖFFLER Georg , Wittmann Heinz‐Günter
Published: March 2005

All 21 ribosomal proteins from 30 S subunits of Escherichia coliwere isolated in a pure state by the following methods: separation of subunits by zonal centrifugation in B X V rotors, extraction of proteins, CM‐cellulose chromatography with pyridine formate gradients, gel filtration on Sephadex G‐100 and, in some cases, preparative polyacrylamide block electrophoresis. The purity of isolated proteins was 97–100% shown by disc electrophoresis at pH 4.5 in urea, by two‐dimensional polyacrylamide electrophoresis, by dodecylsulfate gel electrophoresis and by cellulose acetate gel electrophoresis. The yield of the isolated proteins was 7–110 mg depending on the protein and the number of purification steps.

Fractionation of total 30 S proteins with ammonium sulfate is recommended for quick isolation of proteins S15 and S20 in large quantities.
Studies on Adenosine Deaminase
Authors: Sim Meng K. , Maguire M. Helen
Published: March 2005

Adenosine deaminase was purified 525‐fold from the maternal component of early gestation bovine placenta by acetone fractionation, salt fractionation, ion exchange chromatography and gel filtration. The final enzyme preparation was homogeneous when subjected to sedimentation analysis in the ultracentrifuge and had an s20,w value of 4.3 S. 1 mg of enzyme protein deaminated 252 μmoles of adenosine per min at 30 °C, and there was no loss in activity on storage in the presence of 1 mM 2‐mercaptoethanol at 2–4 °C for 12 months. The molecular weight by gel filtration was 35480 and the energy of activation was 6.66 kcal/mole. The molecular properties of the placental enzyme are generally similar to those reported for adenosine deaminase purified from calf intestinal mucosa and calf serum, but differ in that only one protein band is observed on cellulose acetate electrophoresis at pH 5.9 and 8.6.
Studies on Adenosine Deaminase
Authors: Maguire M. Helen , Sim Meng K.
Published: March 2005

The substrate specificity of adenosine deaminase purified from the maternal component of the bovine placenta has been studied in detail, and the effects of different types of inhibitors have been examined. The enzyme catalysed the hydrolytic removal of amino, chloro, hydroxylamino, methoxy and methoxyamino substituents from the 6 position of purine ribonuclesides; 2‐substitution by amino, chloro or fluoro groups reduced the V of adenosine and 6‐chloropurineriboside but 2‐amino substitution increased the V of 6‐methoxypurineriboside. The results indicate that both a bond‐forming component dependent on steric factors, and a bond‐stretching component dependent on the electronegativity of the leaving group are involved in the rate‐determining formation of the transition complex. 2‐Alkylamino‐, 2‐alkylthio‐, and 2‐halogenoadenosines are competitive inhibitors with Ki values which conform the importance of the basicity of N1 of substrates and inhibitors in their binding to the active site of the enzyme.

The enzyme was shown to have dissociating groups with pKa values of 9.8, 8.9 and 5.7. Enzyme activity was inhibited by p‐chloromercuribenzote (PCMB), and titration of the native enzyme with this mercurial showed that there are two sulphydryl groups per mole of enzyme. There was no reaction of the enzyme with the sulphydryl reagent, 5,5′‐dithiobis‐(2‐nitrobenzoic acid). 2‐Mercaptoethanol and glutathione in high concentrations inhibited the enzyme; inhibition by glutathione was irreversible.
Biochemische Differenzierung der Pr1‐/Pr2‐determinierenden von der MN‐determinierenden N‐Acetyl‐Neuraminsäure durch Acetylierungsversuche mit Erythrocytenglykoproteinen
Authors: Merz Wolfgang , Roelcke Dieter
Published: March 2005

In contrast to MN receptors, Pr1 and Pr2 antigens of isolated glycoprotein of erythrocytes are not inactivated by acylation of free amino groups.

By introduction of easily leaving groups, e.g. trifluoroactic residues, inactivation of the MN activity can be made reversible.

No differences between Pr1 and Pr2 antigens could be found with the reactions described in this paper.

Acylation in aqueous phase (buffer/anhydrous acetic acid) and in organic phase (pyridine/anhydrous acetic acid) gave identical results qualitatively and quantitatively. It was not possible to split the N‐acetic binding neither under mild alkaline conditions nor with aminoacylase. 4–5 amino groups out of 11 could be acylated, whereas succinylation in aquous phase (buffer/anhydrous succinic acid) resulted in modification of only one free amino group, the calculations being based on a molecular weight of 30000. Following succinylation MN activity was also lost, whereas the antigenic activity of Pr1 and Pr2 remained unchanged. The appearance of a negative charge instead of the positive charge of the free amino group on the glycoprotein did not affect Pr1 or Pr2 activity in any way.

If glycoprotein was esterified under conditions which cause esterification of free sialic acid to a wide extent neither MN activity nor Pr1/Pr2 activity was altered.
Mechanism of Action of Specific Antiserum on Pasteurella septica
Authors: Griffiths Elwyn
Published: March 2005

Circumstantial evidence suggests that the ability of the host to prevent the uptake of iron by pathogenic bacteria may constitute an important means of defence. A detailed biochemical study of the way antisera exert their antibacterial effects and the possible involvement of iron has now been initiated.

Work with Pasteurella septica antiserum has revealed a totally unsuspected way in which antiserum and complement exert their antibacterial effects. The process is very quick and specific and results in the cessation of bacterial multiplication; cells in stasis then appear to be killed by the operation of a secondary event. The inhibitory process operates by affecting the biochemistry of the bacterial cell leading first to an inhibition of net RNA synthesis and then to an inhibition of all macromolecular synthesis.

The presence of free iron in the serum did not prevent the initiation of the inhibition but it did allow macromolecular synthesis, cell multiplication and rapid growth to be resumed later. In contrast, the presence of haematin in the serum allowed the cells to continue multiplying in the presence of antiserum without delay. Addition of haematin to bacteria which had been inhibited by antiserum restored net RNA synthesis immediately. Net protein synthesis and multiplication were restarted within 15–20 min and DNA synthesis in 40 min.
Dissociation of Pila Haemocyanin at Low Ionic Strength
Authors: Hoebeke Johan J. , Elliott Francis G.
Published: March 2005

The study of the haemocyanin of Pila leopoldivillensis by means of zone electrophoresis on cellulose acetate has shown that its dissociation is favoured by lowering of the ionic strength.

These results were confirmed and extended over a larger range of pH and ionic strength values by means of light scattering measurements. An interpretation of the results is given in terms of ionic equilibria. The type and the number of charged groups involved in the dissociation into halves and into tenths are proposed.
Apo‐ and Reconstituted Holoenzymes of Metapyrocatechase from Pseudomonas putida
Authors: Takemori Shigeki , Katagiri Masayuki , Komiyama Tadazumi
Published: March 2005

In order to ascertain the role of metal in the structure and function of the iron‐containing oxygenase, we have successfully obtained stable, crystalline apo‐ and fully reconstituted holoenzymes from metapyrocatechase of Pseudomonas putida. Ultracentrifugally and electrophoretically homogeneous apoenzyme is prepared by dialysis against o‐phenanthroline followed by chromatography on DEAE‐Sephadex. The apoenzyme is essentially free of iron and inactive. Fully active holoenzyme, which contains 3 g‐atoms of iron per mol of protein, can be reconstituted by incubating the apoenzyme with ferrous ion. Using these preparations, the close correlation between iron content and activity of the enzyme has been demonstrated. The apoenzyme is, in terms of its molecular weight and sensitivity to sulfhydryl group reagents, indistinguishable from the reconstituted holoenzyme, indicating that no gross change of the protein structure has occurred.
Substrate‐and Antibiotic‐Binding Sites at the Peptidyl‐Transferase Centre of Escherichia coli Ribosomes
Authors: Fernandez‐Muñoz Rafael , Monro Robin E. , Vazquez David , Torres‐Pinedo Ramon
Published: March 2005

In Escherichia coli there is one binding site per ribosome for lincomycin, chloramphenicol and erythromycin.

These antibiotics act at closely related sites on the 50‐S subunit. There are, however, some interesting differences in the properties of the three sites, such as: (a) the apparent possibility of obstructing the lincomycin site without closing the chloramphenicol site, (b) asymmetry of competition effects between chloramphenicol and erythromycin, (c) differential responses to ethanol and (d) differential responses to other antibiotics (puromycin, macrolides, streptogramins A and B and others also acting on the larger ribosome subunit).
Determination of the Stability Constant of the Megnesium‐Ion Complex of Isocitrate
Authors: Londesborough John C. , Dalziel Keith
Published: March 2005

The stability constant of the magnesium‐ion complex of isocitrate has been measured by spectrophotometric and fluorimetric titrations of 8‐hydroxyquinoline with magnesium chloride in the presence and absence of isocitrate. Values of 195 ± 15M−1 at 19 °C, and 25°C, and 217 ± 10M−1 at 37°C were obtained at pH 7.0 and ionic strengh 0.15.
Transcription of Mammalian Chromatin by Mammalian DNA‐Dependent RNA Polymerases
Authors: Butterworth Peter H. W. , Cox Ronald F. , Chesterton C. James
Published: March 2005

The transcription of rat‐liver chromatin has been studied using partially purified from AI and highly purified form B DNA‐dependent RNA polymerases isolated from rat liver. Chromatin contains endogenous RNA polymerase activity. This activity is evident only when the RNA polymerase assays are carried out at high ionic strength and its appearance as the ionic‐strenght increases is thought to be due to the removal of chromatin‐associated proteins which block further transcription by the bound enzyme. This activity is insensitive to the rifampicin derivative AF/0‐13 which is shown to inhibit initiation of RNA synthesis by mammalian RNA polymerases. From AI polymerase is virtually inactive in the transcription of chromatin whereas the from B RNA polymerase (α‐amanitin sensitive) actively transcibes chromatin. This activity has two salt concentration optima: (a) 0.01 M (NH4)2SO4 or 0.03 M KCI; (b) 0.16 M (NH4)2SO4 or 0.25 MKCI. Both these activities are inhibited by rifampicin AF/0‐13. A comparison of the activity of the rat‐liver from B polymerase and the Micrococcus lysodeikticus RNA polymerase demonstrates that chromatin is a more efficent template for the rat‐liver enzyme. Evidence is presented that the rat‐liver form B and the Micrococcus lysodeikticus RNA polymerases bind to and transcribe from different sites on the chromatin DNA.
Isolierung und partielle Charakterisierung eines 8Sα3‐Glykoproteins aus Humanserum
Authors: Haupt Heinz , Baudner Siegfried , Kranz Theodor , Heimburger Norbert
Published: March 2005

Isolation and partial characterization of a still‐unknown 8Sα3‐glycoprotein from human serum is described. Supernatant II is used as the starting material; it is a side fraction from the Rivanol®‐ammonium‐sulphate procedure of preparing albumin and gamma‐globulin on a large scale.

8Sα3‐glycoprotein proves to be homogeneous according to free‐boundary electrophoresis and to molecular‐sieve electrophoresis. During electrofocusing several bands can be observed in the rangs of pH 4.2–4.6. In the characterization of this protein by ultracentrifugation a slight inhomogeneity becomes evident which is not observed when gel filtration is applied. The sedimentation constant is 7.87 S and the molecular weight is calculated to be 220000. The sedimentation coefficient decreases by treatment with propionic acid or an acid solution of urea. A partial dissociation of the molecule is achieved by means of reducing agents. It is also possible to dissociate this protein completely with 2‐mercaptoethanol followed by alkylation and treatment with an acid‐urea solution.

8Sα3‐glycoprotein contains approximately 30% carbohydrate, from which 9.2% is sialic acid. The protein moiety accounts for 70% of the total molecule. The isolated protein is characterized by high solubility in solutions containing protein‐precipitating agents. The concentration in normal human serum is in the range of 3.1–4.5 mg/100 ml.
Differential Incorportion of 5‐Azapyrimidines into the RNA of Phage f2 and of Bacterial Host
Authors: DoskoČIL Jiří , ŠOrm František
Published: March 2005

Particles of phage f2 formed in the presence of 5‐azacytidine have nearly normal sedimentation coefficient and buoyant density, but their infectivity with respect to populations of normal phage is reduced. RNA extracted from these particles is partly degraded to small fragments and has very low template activity.

Incorporation studies in vivo indicate that phage RNA‐replicase can utilize both 5‐azacytidine and 5‐azauridine triphosphates for the synthesis of RNA. Phage RNA, however, contains much less 5‐azacytosine than either replicative‐from RNA or host RNA; apparently some secondary mechanism is operative, tending to eliminate plus‐strands of RNA with high content of of 5‐azacytosine from entering mature phage particles.

Analyses of replicative‐form RNA formed in cultures treated with 5‐azacytidine show that this analogue is incorported mostly instead of cytosine, but may also replace uracil quite frequently, apparently by incorrect base pairing.
Relationships between the 3α‐and 20β‐Hydroxysteroid
Authors: Gibb William , Jeffery Jonathan
Published: March 2005

Crystalline cortisone reductase prepared from Strephtomyces hydrogenans has 3α‐ and 20β‐hydroxysteroid: NAD oxidoreductase activity. The total rate of reaction for mixtures of 17 α, 21‐dihydroxypregn‐4‐ene‐3,11,20‐trione with 5α‐androstan‐3,16‐dione, and of 17β‐hydroxy‐1α‐methyl‐5α‐androstan‐3‐ one with either of these compounds indicated that these substrates do not react independently. 3β‐Hydroxy‐5α‐androstan‐16‐one inhibited the reduction of both 17α, 21‐dihydroxypregn‐4‐ene‐3,11,20‐trione and 5α‐androstane‐3,16‐dione.

20β‐Hydroxypregn‐4‐en‐3‐one also inhibited the reduction of both these substrates. The findings allow the view that 3α‐ and 20β‐activities may depend upon a single active centre with complex binding characteristics.
The Effect of Tetranitromethane on Apo‐Aspartate‐Aminotransferase from Pig Heart
Authors: Barra Donatella , Bossa Francesco , Ferraro Anna , Giartosio Anna , Turano Carlo
Published: March 2005

Both holo‐ and apo‐aspartate‐aminotransferase are sensitive to tetranitromethane; however, the inactivation which follows nitration is partial for the holoenzyme, and complete for the apoenzyme. Cysteinyl and tyrosyl residues are modified in both forms of the enzyme; in the apoenzyme a greater number of tyrosines are available to the reagent. The complete inactivation of the apoenzyme is correlated with the loss of only one tyrosyl residue, which is selectively modified under particular conditions. The loss of activity is caused by the failure of the nitrated apoenzyme to recombine with the coenzyme.
Metabolic Instability of Myelin Protein and Proteolipid Fractions
Authors: Marks Neville , Mela Paula , D'monte Brian
Published: March 2005

Turnover of protein components of myelin in vivo was shown to be comparable to that of other brain proteins. Myelin was purified, to remove soluble contamination, by water shock (fraction M2) and centrifugation on sucrose and CsCl gradients. These methods gave two fractions, one of which consisted of characteristic myelin membranes free of visible contamination (M3), and the second consisted of fragmented membranes and vesicles (M4).

Incorporation following intracisternal injection of [14C]lysine occurred at later time periods in myelin compared to mitochondria, probably as a result of different rates for penetration of precursor. The half‐lives for the different categories of protein and proteolipid components prepared by Triton X‐100 acetate procedures were markedly heterogeneous. In myelin, the highest incorporation occurred in the soluble basic proteins with half‐lives of 14‐21 days (fraction III), followed by the interface (Folch‐Lees, fraction II) material with half‐lives of 12–24 days, and the insoluble proteins (Wolfgram, fraction III) with half‐lives of 28–32 days. The half‐lives varied with the pulse period used (15–360 min) and give evidence of both short‐lived and long‐lived proteins. Heterogeneity of turnover was supported by the complex disc‐gel patterns found with electrophoresis on acrylamide using a phenol‐urea solvent system for solubilization. In comparable mitochondrial fractions, incorporation was highest in the interface material (half‐life 6–4 days), followed by insoluble proteins (half‐life 3–6 days), and lowest in the soluble (brain) fraction (half‐life 6–26 days).

Considerable hydrolase activity was observed in crude fractions and in hypotonic myelin fractions, particularly acid and neutral proteinases, aminopeptidase, and monoacyl and dipeptidyl arylamidases. It appears that hydrolases associated with the sheath could be involved in turnover, but in most cases are not associated with the highly purified membranes prepared in sucrose and CsCl gradients. Purification resulted in an increased specific activity of an aminopeptidase hydrolysing Leu‐Gly‐Gly as the substrate, and of ribonucleoside‐2′ : 3′‐cyclic‐phosphate esterase. It is concluded that these two hydrolases are intrinsic components of the myelin sheath membranes.
Regulation of δ‐Aminolevulinic Acid Synthetase by Drugs and Steroids in vivo and in Isolated Perfused Rat Liver
Authors: Bock Karl W. , Krauss Eva , FrÖHLING Wolfgang
Published: March 2005

δ‐Aminolevulinic‐acid‐synthetase induction by drugs depends upon the nutritional state of the animal. In allylisopropylacetamide‐treated animals the enzyme induction is reduced in fed compared with starved rats whereas after phenobarbital treatment an induction of the enzyme seen in starved animals is not observed in fed rats. δ‐Aminolevulinic‐acid‐synthetase induction is studied in relation to the synthesis of cytochrome P450.

In the isolated rat liver perfused with either rat blood or washed bovine erythrocytes and Eagle minimal essential medium, δ‐aminolevulinic acid synthetase decreases sharply. This decrease can be prevented by the continuous addition of dexamethasone (9α‐fluoro‐16α‐methyl‐pregna‐1,4‐diene‐11β,17α,21‐triol‐3,20‐dione) or hydrocortisone (pregn‐4‐ene‐11β,17α,21‐triol‐3,20‐dione) but not by testosterone (androst‐4‐ene‐17β‐ol‐3‐one) or etiocholanolone (5β‐androstan‐3α‐ol‐17‐one). δ‐Aminolevulinic acid synthetase cannot be significantly induced by allylisopropyl‐acetamide unless corticoids are added to the perfusion medium. The results indicate that the synthesis of heme and, consequently, hemoproteins is controlled by corticoids or their metabolites in adult rat liver.
Electron Flow and Cholesterol‐Side‐Chain Cleavage in Ovarian Mitochondria
Authors: Robinson John , Stevenson Patricia M.
Published: March 2005

The supply of electrons for the hydroxylation reactions associated with cholesterol side‐chain cleavage has been studied in intact porcine luteal mitochondria. The work was based on an assay of the NADPH‐dependent side‐chain‐cleavage reaction, the activity of the mitochondrial enzyme complex being expressed as the percentage of [4‐14C]cholesterol converted to the products, [4‐14C]pregnenolone and [4‐14C]progesterone, in 1 h. The reaction could best be supported by intermediates of the tricarboxylic acid cycle, although NADPH, and to a lesser degree NADH, would also support some activity.

Investigations of the ADP:O ratios of the substrates which could support cholesterol side‐chain cleavage showed that they were lower than the ADP:O ratios found for the same substrates in liver mitochondria from the same animal; respiratory‐control ratios were also lower in the ovarian preparation. In the adrenal cortex, another steroid‐hydroxylating tissue, similar results obtained by other workers have been interpreted as indicating that the two oxidative chains, the first terminating in cytochrome oxidase and the second in cytochrome P450, are connected by an energy‐dependent transhydrogenase. In the ovary this explanation may be true for succinate, but it does not satisfy the results obtained with substrates which reduce NAD(P)+. Although spectrophotometric assays showed that there was some energy‐linked transhydrogenase, as well as non‐energy‐linked transhydrogenase activity in ovarian mitochondria, all the evidence obtained by investigating cholesterol side‐chain cleavage supported by NADH and the NAD+‐specific 2‐oxoglutarate dehydrogenase indicated that in ovary hydrogen transfer from NADH to the cholesterol‐hydroxylation site was not dependent on energy. In contrast, inhibitors of NADH oxidase markedly stimulated NADH‐supported side‐chain cleavage. Energy was required for the transfer of electrons from succinate to the site of cholesterol side‐chain cleavage.
Pyridine‐Nucleotide Transhydrogenase
Authors: van den Broek Hendrikus W. J. , Veeger Cornelis
Published: March 2005

Anaerobic titration experiments of transhydrogenase from Azotobacter vinelandii with NADPH and NADH in the presence of NADase from Neurospora crassa show that two moles of reductant are required to reduce one mole of enzyme‐bound flavin. These observations have been confirmed by titrations with sodium dithionite. No stable 2‐equivalent reduced intermediate can be detected.

Differences found in the titration of the flavoprotein with dithionite, NADPH in the absence and presence of NADP+, and NADH in the absence and presence of NADP+ and NAD+ indicate that NADP+ and NAD+ interact in a different manner with the enzyme. From the results obtained it is concluded that NADP+ has a higher affinity for the oxidised enzyme, while NAD+ has a higher affinity for the reduced form of the enzyme. The results also indicate that the level of reduction of the enzyme‐bound flavin is determined by the ratio of reduced and oxidised pyridine nucleotides.

Assuming an unknown reducible group X will exist next to the flavin, a reduction scheme is proposed in which the enzyme is reduced to the 2‐equivalent reduced state, followed by dissociation of the oxidised pyridine nucleotide and internal transfer of electrons to the reducible group X. Reduction to the 4‐equivalent reduced state is supposed to be the last step which is not followed by dissociation of the product. Titration experiments of the dithionite‐, NADH‐ or NADPH‐reduced enzyme with NADP+ and photoreduction experiments support the proposed reductive mechanism.
The Identification of 3‐O‐Methyl‐D‐xylose and 3‐O‐Methyl‐L‐xylose as Constituents of the Lipopolysaccharides of Myxococcus fulvus and Rhodopseudomonas viridis, respectively
Authors: Weckesser Jürgen , Rosenfelder Günter , Mayer Hubert , Lüderitz Otto
Published: March 2005

Lipophilic sugar constituents with identical chromatographic properties were identified in the lipopolysaccharides of the two gram‐negative bacteria, Myxococcus fulvus (Myxococcaceae) and Rhodopseudomonas viridis (Rhodospirillaceae). Both sugars were characterized as 3‐O‐methylpentoses by mass spectrometry, and xylose was obtained after demethylation. The optical rotation of the O‐methyl sugars and the reactivity of the demethylated sugars with D‐glucose oxidase revealed that Myxococcus fulvus contains 3‐O‐methyl‐D‐xylose whereas Rhodopseudomonas viridis contains 3‐O‐methyl‐L‐xylose.
Detection and Specificity of Lipid A Antibodies Using Liposomes Sensitized with Lipid A and Bacterial Lipopolysaccharides
Authors: Kataoka Tateshi , Inoue Keizo , Kinsky Stephen C.
Published: March 2005

Previous reports have shown that liposomal model membranes are appropriate objects for investigating the molecular basis of complement‐dependent membrane damage. The present study describes a different application for liposomes: their usefulness in screening sera suspected of containing antibodies against potential lipid antigens. Liposomes, actively sensitized with untreated or alkali‐treated lipid A derived from Salmonella minnesota, were found to release trapped glucose marker when incubated with a source of unheated complement and the antilipid A serum whose preparation is described in the preceding paper [1]. In the presence of complement, the anti‐lipid A serum could also promote loss of glucose from liposomes which had been actively sensitized with various untreated and alkali‐treated S or R form S. minnesota lipopolysaccharides. In contrast, several different anti‐R‐form sera had little effect on liposomes sensitized with either untreated or alkali‐treated lipid A. These results are in agreement with observations on the immunological specificity of lipid‐A antibodies using complement‐dependent hemolysis of passively sensitized human erythrocytes as test system. Alkali‐treated, but not untreated, lipid A could also be employed for passive sensitization of liposomes; in this regard, lipid A behaves similarly to S and R form lipopolysaccharides.
Liver Acetyl‐Coenzyme‐A Carboxylase
Authors: Hashimoto Takashi , Isano Hiroshi , Iritani Nobuko , Numa Shosaku
Published: March 2005

The mechanism of the liver acetyl‐coenzyme‐A carboxylase reaction was studied by kinetic experiments. Studies on inhibition by kynurenate, a novel dead‐end inhibitor binding to the uncarboxylated form of the enzyme, as well as malonyl‐CoA‐[14C]acetyl‐CoA exchange studies further substaintiated our previous conclusions that the reaction proceeds through the ordered bi‐bi uni‐uni ping‐pong mechanism, and that the site of action of citrate, an allosteric activator, lies predominantly on the carboxylated form of the enzyme. The complex patterns produced by malonyl‐CoA inhibition were accounted for by mixed product and dead‐end inhibition; malonyl‐CoA was found to form a dead‐end complex with the inactive (or less active) species of the carboxylated form of the enzyme.

On the other hand, the dissociation constant of the uncarboxylated enzyme · citrate complex was estimated to be 3–14 μM in terms of [14C]citrate binding, protection against avidin inactivation and shift in the protomer‐polymer equilibrium. This value is about 103‐fold smaller than the dissociation constant of the carboxylated enzyme · citrate complex found by kinetic studies, i.e. 3–6 μM, so that the effect of citrate on the uncarboxylated form of the enzyme does not manifest itself upon kinetic analysis of the carboxylase‐catalyzed reactions. These findings indicate that carboxylation of the biotinyl prosthetic group gives rise to a conformational change at the citrate site, resulting in a marked decrease in the affinity of the enzyme for the activator. This further supports our previously proposed concept that the main role of citrate in the carboxylase catalysis is to keep the carboxylated form of the enzyme in active conformation by shifting the equilibrium between the active and inactive (or less active) species of this enzyme form.
Stereochemistry of the re‐Citrate‐Synthase Reaction
Authors: Wunderwald Peter , Buckel Wolfgang , Lenz Helmut , Buschmeier Verena , Eggerer Hermann , Gottschalk Gerhard , Cornforth John W. , Redmond John W. , Mallaby Richard
Published: March 2005

R‐Acetate and S‐acetate, as the coenzyme‐A esters, were converted into citrates on recitrate synthase.

The citrate were assayed for chirality at the 2‐position (a) by incubation with aconitate hydratase (aconitase) and (b) by cleavage to malate by citrate lyase combined with malate dehydrogenase, followed by incubation of the isolated malate with fumarate hydratase (fumarase).

In each case, the citrate (or malate) derived from R‐acetate retained most of its tritium, and the citrate (or malate) derived from S‐acetate lost most of its tritium, on incubation with the hydratase.

It was concluded that if a normal intramolecular deuterium‐isotope effect is operative, the condensation of oxaloacetate with acetyl‐coenzyme A on re‐citrate synthase proceeds with inversion of configuration at the methyl group.
Studies of the Biosynthesis of the O9 Antigen from Escherichia coli O9:K30(A):H12
Authors: Fitzgerald‐Chandler Donna K. , Jann Klaus
Published: March 2005

The O9 antigen from Escherichia coli O9:K30 (A):H12 contains mannose (90%) and glucose (10%). Incorporation of [14C]mannose from guanosine diphosphate [14C]mannose (GDP‐[14C]Man) into acetic‐acid precipitates has been studied with a preparation from cells ground with alumina. The time‐course of mannose incorporation was biphasic (rapid reaction for 2–5 min followed by a slower, linear reaction for 5–20 min) and the incorporation was stimulated by uridine diphosphate glucose (UDP‐Glc) during the early part of the reaction. The reactions (mannose incorporation after 2 min and 15 min, ± UDP‐Glc) were nevertheless proportional to protein concentration. [14C]Glucose was incorporated into acetic‐acid precipitates from UDP‐[14C]Glc and this reaction was slightly stimulated by GDP‐Man. Radioactivity from GDP‐[14C]Man was incorporated into butanol extracts and the early part of the reaction (2–5 min) was markedly stimulated by UDP‐Glc. Bacitracin did not inhibit the incorporation of mannose into acetic‐acid precipitates in the early part of the reaction, but did inhibit incorporation about 20% between 5 and 30 min.

Larger‐scale incubation mixtures were extracted with 45% aqueous phenol. The product was of high molecular weight as indicated by Sephadex G‐100 chromatography and yielded a single radioactive peak corresponding to mannose after total hydrolysis and paper chromatography. This high‐molecular‐weight product precipitated with anti‐O9 antiserum in an antigen‐binding assay, indicating that it was serologically active. Thus, it is likely that the O9 antigen of E. coli O9: K30(A):H12 is synthesized from GDP‐Man and UDP‐Glc through a butanol‐extractable (lipid‐linked) intermediate. Reactions appear to be analogous to those described for lipopolysaccharide biosynthesis in Salmonella.
Nature de l'hétérogénéité interspécifique des protéines des ribosomes de mammifères
Authors: Houssais Jean François
Published: March 2005

The nature of the differences between ribosomal proteins of mammalian species (man, mouse, rat, hamster), separated by polyacrylamide gel electrophoresis, has been investigated in cell lines and inter‐specific somatic hybrids cultured in vitro, and in livers of the corresponding animals. The proteins of the large and small ribosomal subunits have only a few bands with the same electrophoretic mobilities. Comparing, for each kind of subunit, the ribosomal proteins in the four species, two categories of proteins are described: (a) proteins which are always present, qualitatively and quantitatively constant; (b) proteins which exhibit qualitative or quantitative differences. One qualitative difference, possibly due to a mutation slightly altering the electrophoretic mobility of one protein of the large subunit of the syrian hamster has been observed. The mouse‐hamster hybrid cells possess on their large ribosomal subunits, the proteins from each “parent”. Several quantitative differences, affecting only certain proteins of the subunits, are described. They seem to be the main source of the heterogeneity of ribosomal proteins observed between mammalian species. These findings are discussed in relation to the expression of the genome, and to the structure and function of the ribosomes during the process of speciation and differentiation.
Sterol Biosynthesis in Euglena gracilis Z.
Authors: Anding Claude , Brandt Roger D. , Ourisson Guy
Published: March 2005

Squalene, squalene‐2,3‐oxide, triterpenes and 4α‐methylsterols of Euglena gracilis Z. have been characterised by thin‐layer chromatography, gas chromatography and mass spectrometry. Their distribution in dark‐grown and light‐grown Euglena cells is quantitatively and qualitatively different, reflecting differences in the sterol compositions. However the sterol precursor is found to be in each case cycloartenol, whereas lanosterol is not detected. A new 4α‐methylsterol is isolated and the structure of 4α‐24‐dimethyl‐5α‐cholesta‐8(9)‐en‐3ß‐ol proposed. 24‐Methylene lanostenol is formed in light‐grown Euglena. None of the compounds described have been found in water‐soluble fractions nor in the form of fatty‐acid esters. The following compounds have also been isolated: 24‐methylene cycloartanol, 24‐methylene agnostenol (?), ß‐amyrin, obtusifoliol, 24‐methylene lophenol and 4α‐methylzymosterol.
Métabolisme phospholipidique des hématies nucléées
Authors: Soula Georges , Souillard Cécilia , Gard Catherine , Douste‐Blazy Louis
Published: March 2005

The in vitro incorporation of [32P]orthophosphate into chicken‐erythrocyte phospholipids has been studied by means of one‐ and two‐dimensional thin‐layer chromatography as a time‐related function: from 10 min to 6 h.

Labelled phosphatidic acid appears after 10 min and represents about 90% of the total labelled phospholipids. Sphingomyelin turnover is as low as in mammal erythrocytes whereas glycero‐phospholipids' turnover is high. The specific activity of phosphatidylserine is ten times higher than that of phosphatidylethanolamine; phosphatidylserine decarboxylase seems to be absent. In contrast with the metabolism of non‐nucleated erythrocytes, the [32P]orthophosphate is incorporated into phosphatidylglycerol, the turnover of which is important.

These data should agree with a de novo biosynthesis of phospholipids in nucleated erythrocytes, but many studies on mammal erythrocytes have demonstrated that there are essentially exchange reactions of phospholipids with plasma phospholipids and eventual acylation of plasma lysophospholpids.