bluuebunny/crossref_metadata_2025 huggingface dataset:
Sialic‐Acid Content of Low‐Density Lipoproteins Controls Their Binding and Uptake by Cultured Cells
Authors: FILIPOVIC Ivan , SCHWARZMANN Günter , MRAZ Wilfried , WIEGANDT Herbert , BUDDECKE Eckhart
Published: March 2005

The (high‐affinity receptor)‐mediated uptake of homologous low‐density (low‐ϱ) lipoproteins by cultured human arterial smooth muscle cells or human skin fibroblasts is controlled by the sialic acid content of low‐ϱ lipoprotein particles. This conclusion is derived from the following results.

1. Gangliosides incubated with native low‐ϱ lipoproteins associate with low‐ϱ lipoprotein particles. Low‐ϱ lipoproteins modified by associated GLac1, GGtet1, and GGtet2b + GGtet3 gangliosides are internalized by arterial smooth muscle cells at a rate up to 80% lower than native low‐ϱ lipoproteins or those preincubated with desialized gangliosides.

2. The inhibitory effect of gangliosides is specific for high affinity uptake and not detectable on skin fibroblasts deficient in low‐ϱ lipoprotein receptor.

3. Desialyzed low‐ϱ lipoproteins are internalized by smooth muscle cells up to 100% faster than native low‐ϱ lipoproteins, the enhancement of uptake corresponding to the degree of desialization.
Yeast tRNALeuUAG. Purification, Properties and Determination of the Nucleotide Sequence by Radioactive Derivative Methods
Authors: RANDERATH Erika , GUPTA Ramesh C. , CHIA Li‐Li S. Y. , Simon H. CHANG  , RANDERATH Kurt
Published: March 2005

A second major species of leucine tRNA, tRNALeuUAG (formerly designated tRNALeuCUA) was purified from baker's yeast in a three‐step procedure entailing BD‐cellulose chromatography in the presence and absence of Mg2+ and Sephadex G‐100 gel filtration. Results of aminoacylation and partial RNase T1 digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNALeum5CAA (tRNALeu3).

The primary structure of baker's yeast tRNALeuUAG was elucidated by application of sensitive radioactive isotope derivative (‘postlabeling’) methods. Complete RNase T1 and A and partial RNase U2 fragments, prepared from non‐radioactive tRNA and 5′‐half and 3′‐half molecules, were separated by two‐dimensional polyethyleneimine‐cellulose anion‐exchange thin‐layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H‐labeled nucleoside trialcohols in combination with base analysis. 2′‐O‐Methylated guanosine was characterized using the [γ‐32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the RNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis.

A different sequencing approach, consisting of a combination of ‘readout sequencing’ with tritium sequencing of complete RNase T1 and A fragments, was applied to the 3′‐half molecule. The 3′‐half molecule was labeled with 32P at its 5′ terminus, partially degraded with RNases T1, U2, and Phy1 and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 A260 unit of RNA and the analysis of the complete fragments about 6 A260 units.

Interesting structural features of tRNALeuUAG are (a) the location of unique, leucine tRNA isoacceptor‐specific sequences next to U‐8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1‐methyladenosine in the T loop, a modification not present in the structurally related tRNALeum5CAA, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.
Fluctuations in Proline and Other Free Amino Acids during the Mitotic Cycle of the Myxomycete Physarum polycephalum
Authors: THRELFALL Richard J. , THOMAS Alan J.
Published: March 2005

1. During synchronous growth of the acellular slime mould Physarum polycephalum the free amino pool had two maxima, one of 650 units [nmol/plasmodium dry weight (mg)] at metaphase and the other of 780 units in mid G2 with minima of 550 units before and after mitosis.

2. Proline formed 20–25% of the total pool with aspartic acid, glutamic acid, threonine, valine, leucine, lysine and arginine making up 55% of the pool.

3. The fluctuation of proline during the mitotic cycle was quite different from that of the other amino acids and was transiently very low during telophase.
Location and Encapsidation of the Coat Protein Cistron of Tobacco Mosaic Virus
Authors: LOMONOSSOFF George P. , BUTLER P. Jonathan G.
Published: March 2005

The coat protein cistron of tobacco mosaic virus has been located on the viral RNA starting between 975 and 1050 nucleotides from the 3′‐hydroxyl end. This locates its 5′ end close to the origin for virus assembly, where the first protein disk interacts with the RNA. It also means that the coat protein mRNA must have a short 5′‐untranslated tail and a long (over 500 nucleotides) 3′ one.

The recovery of characteristic oligonucleotides in nuclease‐protected rods during the growth from RNA and a protein disk preparation shows that elongation of the nucleated rods proceeds independently in both directions though, on average, much more rapidly along the longer 5′ tail than the shorter 3′ tail. protected RNA of length equal to that in the complete virion is first seen within 6 min, showing that the most rapidly elongated particles are substantially complete by this time.
Degradation of Poly(inosinic acid) · poly(cytidylic acid) [(I)n· (Cn)] by Human Plasma
Authors: DE CLERCQ Erik
Published: March 2005

The double‐helical RNA interferon inducer, poly(inosinic acid) · poly(cytidylic acid) [(I)n· (C)n], is rapidly degraded by nucleases present in human plasma (serum). When incubated at 37°C with 50% human plasma, (I)n· (C)n (10 μg/ml) showed a half‐life of 6 min, [as assessed by residual acid insoluble radioactivity of an (I)n· (C)n preparation which was 3H‐labelled in the (C)n component]. Sucrose velocity analysis revealed that human plasma nucleases specifically hydrolyzed the (C)n strand of (I)n· (C)n, as assessed with (I)n· (C)n preparations that were 3H‐labelled in either (I)n or (C)n. The plasma nucleases that were responsible for the degradation of (I)n· (C)n resembled pancreatic ribonuclease A rather than T1 ribonuclease in substrate specificity. (I)n· (C)n became completely inactive as an inerferon inducer when it was incubated with 50% human plasma or serum. However, its interferon‐inducing capacity was fully restored if (C)n but not (I)n was added to the host cells, after these had been exposed to the inactivated (I)n· (C)n. That the inactivating effect of human plasma on the interferon‐inducing potency of (I)n· (C)n could solely be attributed to hydrolysis of the (C)n strand was further supported by the finding that two analogues of (I)n· (C)n, which had become more resistant to degradation by pancreatic ribonuclease by virtue of some substitutions in the (C)n strand, i.e. a bromine at C‐5, (I)n· (br5C)n, or a sulfur at C‐2, (I)n· (s2C)n, were not inactivated in the presence of 50% human plasma (or serum).
Messenger RNA of the Large Subunit of Ribulose‐1,5‐Bisphosphate Carboxylase from Chlamydomonas reinhardi
Authors: SANO Hiroshi , SPAETH Eberhard , BURTON William G.
Published: March 2005

Polysomes specifically synthesizing the large subunit of ribulose‐1,5‐bisphosphate carboxylase were isolated from Chlamydomanas reinhardi cells by the indirect immunoprecipitation method. Electrophoretic analysis showed that the immunoprecipitated polysomes were of chloroplast origin. The mRNA coding for the large subunit which was purified from immunoprecipitated polysomes migrated at the 19‐S position on sucrose density gradients, and its molecular weight was estimated to be 7.3 × 105 by acid‐urea/agarose gel electrophoresis. The mRNA was translated in vitro with a cell‐free protein‐synthesizing system derived from Escherichia coli to give full‐length large‐subunit polypeptides.
Distribution of the Messenger RNA Coding for the Common Precursor of Corticotropin and β‐Lipotropin within the Bovine Pituitary
Authors: TAII Shunzo , NAKANISHI Shigetada , NUMA Shosaku
Published: March 2005

The distribution of the mRNA coding for the common precursor of corticotropin and β‐lipotropin among different parts of the bovine pituitary has been investigated by quantifying the mRNA activity with the use of a cell‐free protein‐synthesizing system. The results obtained have demonstrated that this mRNA activity is located both in the anterior lobe and in the intermediate lobe, while it is essentially not detectable in the neural lobe nor in the stalk. The structural identity of the translation products of corticotropin/β‐lipotropin mRNA from the anterior and from the intermediate lobe has been indicated by their molecular weight as well as by the electrophoretic patterns of the peptide fragments formed from them upon partial enzymatic proteolysis or upon cyanogen bromide cleavage. The specific activity of corticotropin/β‐lipotropin mRNA in the intermediate lobe is about 20‐fold higher than that in the anterior lobe, and the total activity of this mRNA in the former is about 2‐fold higher than that in the latter. In the intermediate lobe, the translation product of corticotropin/β‐lipotropin mRNA amounts to almost one‐third of the products encoded by total translatable mRNA. These results indicate that corticotropin/β‐lipotropin mRNA represents a major mRNA species in the intermediate lobe of the pituitary, thus suggesting that this lobe may perform a highly specialized function in producing a large amount of the common precursor of corticotropin and β‐lipotropin.
Induction of Cyclic AMP Phosphodiesterase by Disaggregation of the Multicellular Complexes of Dictyostelium discoideum
Authors: OKAMOTO Koji
Published: March 2005

When the multicellular complexes (slugs) of Dictyostelium discoideum were disaggregated, the activity of phosphodiesterase for adenosine 3′:5′‐monophosphate increased. The increase occurred first with the enzyme in the particulate fraction of the cell. Cytoplasmic and extracellular phosphodiesterase, as well as the macromolecular inhibitor for this enzyme, also increased at later times. The increase in these activities was blocked by inhibitors of protein and RNA syntheses. This indicates that the increase is not due to the activation of the enzyme, but due to its synthesis de novo, and that the synthesis is induced at the transcriptional level by the loss of cell contact. In the case of cellular phosphodiesterase, inhibitors of protein synthesis (cycloheximide and emetine) not only blocked the increase, but also caused a decrease in activity. The mechanism of such decrease was further investigated.
Brain Pyridoxal Kinase. Mobility of the Substrate Pyridoxal and Binding of Inhibitors to the Nucleotide Site
Authors: KWOK Francis , CHURCHICH Jorge E.
Published: March 2005

Pyridoxal kinase has been purified 2000‐fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. The interactions of the substrate pyridoxal and the inhibitor N‐dansyl‐2‐oxopyrrolidine (dansyl = 5‐dimethylaminonaph‐thalene‐1‐sulfonyl) with the catalytic site were examined by means of fluorescence spectroscopy. The increase in emission anisotropy that follows the binding of pyridoxal to the kinase was used to determine the equilibrium dissociation constant. Pyridoxal kinase binds one molecule of substrate with a Kd= 11 μM at pH 6. The emission anisotropy spectrum of bound pyridoxal reveals that the substrate is not rigidly trapped by the protein matrix.

N‐Dansyl‐2‐oxopyrrolidine is a competitive inhibitor with respect to ATP at saturating concentrations of pyridoxal. It binds to the enzyme with a dissociation constant of 6 μM. N‐Dansyl‐2‐oxopyrrolidine is immobilized by strong interactions with the enzyme, but it is displaced from the catalytic site by ATP. The results are consistent with the hypothesis that N‐dansyl‐2‐oxopyrrolidine binds at the nucleotide binding site of pyridoxal kinase.
Mitochondrial GTP‐AMP Phosphotransferase.
Authors: TOMASSELLI Alfredo G. , NODA Lafayette H.
Published: March 2005

Kinetic and equilibrium dialysis substrate binding studies have been done to investigate the properties of mitochondrial GTP‐AMP phosphotransferase. The results show that the enzyme has a specific requirement for divalent metal ions, namely Mg2+, Mn2+ or Ca2+ (Ca2+ is active only in the forward direction, the direction of formation of ADP). The reaction rate depends upon the ratio [Mg2+]: [substrate] rather than on the metal ion concentration alone. The enzymatic activity is influenced by NaCl (or KCl) and optimum pH occurs at 11.5 and 9.5 for guanosine and inosine nucleotides respectively. Examination of binding of substrates to the enzyme showed that there is one binding site (GTP site) for MgGTP, GTP, MgGDP or GDP per molecule of enzyme, with dissociation constants of 4.5, 4.4, 3.0, 2.2 μM respectively and one binding site (AMP site) for AMP, ADP or ATP per molecule of enzyme with dissociation constants of 20.9, 33.4 and 33.4 μM respectively. Since, within the limitations of equilibrium dialysis used in the present studies, AMP binding to one site of the enzyme could be detected only when GDP or GTP is present, the mechanism of the forward reaction may be assumed to be nearly ordered. For the reverse reaction there is no requirement of order of binding of the two nucleotides and so the mechanism of reaction may be assumed to be random.
DNA Polymerases of Anucleated Cells
Authors: TARRAGO‐LITVAK Laura , DESGRANGES Claude , ARAYA Alejandro , LITVAK Simon
Published: March 2005

Two different DNA polymerases have been purified and characterized from human platelets. In the mitochondrial fraction a unique activity of the polymerase γ type has been found. The same enzyme is found in the extramitochondrial supernatant. A second DNA polymerase, called ‘cytoplasmic’ DNA polymerase has been found in the 10000 ×g supernatant of human platelets.

The following properties of the latter DNA polymerase from human platelets are identical to those of DNA polymerase α from normal cells: DEAE‐cellulose and phosphocellulose chromatography, size, thermal stability, phosphonoacetic acid and ethidium bromide inhibition. However, some of its properties, like high resistance to N‐ethylmaleimide and the lack of DNA polymerization using synthetic RNA primers, are those of DNA polymerase β.
Taurocyamine Kinase from Body‐Wall Musculature of the Lugworm Arenicola marina
Authors: SURHOLT Bernhard
Published: March 2005

ATP: taurocyamine phosphotransferase was extracted from body‐wall musculature of the lugworm Arenicola marina and purified by sequential ammonium sulfate precipitation, Sephadex G‐25, DEAE‐cellulose and hydroxylapatite column chromatography to polyacrylamide discelectrophoretic homogeneity. The enzyme was enriched 860‐fold to a specific activity of 1200–1300 μmol×min−1× mg protein−1.

For direct measurement of the enzyme activity of forward as well as of reverse reaction one spectrophotometrical assay was evolved.

The purified enzyme has a molecular weight of approximately 60000 (assayed by means of sucrose‐density‐gradient centrifugation and by gel‐sieve chromatography on a Sephadex G‐100 column). Dodecylsulfate/polyacrylamide gel electrophoresis revealed three bands: at 22000, 13000–14000, and 11000.

Temperature optimum of the taurocyamine kinase activity was found to be 33°C. The pH optimum of the reaction velocity of the forward reaction is at pH 8.0, that of the reverse reaction at pH 7.2.

K
m values for the substrates of the forward reaction are 1.0 × 10−4 M for taurocyamine and 3.3 × 10−3 M for ATP, and for those of the reverse reaction 8.3 × 10−4 M for phosphotaurocyamine and 1.2 × 10−3 M for ADP. V value of the forward reaction is 240 μmol×min−1× mg protein−1, and that of the reverse 1340 μmol×min−1× mg protein−1.

Phosphocreatine and phosphoarginine are inactive. To become active the taurocyamine kinase needs Mg2+ (10 mM).

Isoelectric focusing of the purified enzyme showed a pattern of about 8 bands between pH 6.1 and 7.8, the main band lying at pH 7.3.

Within the cells of the body‐wall musculature 92–94% of the taurocyamine kinase activity were found to be in the cytosol, the mitochondria, in all probability, have 6–8% of the total activity.
Kinetic and Magnetic Properties of Cobalt(III) Ion in the Active Site of Carbonic Anhydrase
Authors: SHINAR Hadassah , NAVON Gil
Published: March 2005

Cobal(III) bovine carbonic anhydrase B was prepared by the oxidation of the cobalt(II) enzyme with hydrogen peroxide and was purified by affinity chromatography. The oxidation reaction is inhibited by specific inhibitors of carbonic anhydrase. The inhibition is explained by the fact that the Co(II)‐enzyme · inhibitor complex cannot be directly oxidized by hydrogen peroxide, but has to dissociate to give free Co(II) enzyme which is then oxidized. The Co(III) ion in Co(III) carbonic anhydrase cannot be directly substituted by zinc ions. It can be reduced by either dithionite or BH−4 ions to give, first, their complexes with the Co(II) enzyme, and upon their removal, a fully active Co(II) enzyme. Cyanide and azide bind to cobalt(III) carbonic anhydrase with similar rate constants of 0.060 ± 0.005 and 0.070 ± 0.007 M−1 s−1 respectively. These rates are faster than those found for Co(III) inorganic complexes. The Co(III) ion in both Co(III) carbonic anhydrase and Co(III) carboxypeptidase A was found to be diamagnetic, indicating a near octahedral symmetry.
Purification and Characterization of the Hypoxanthine‐Guanine Phosphoribosyltransferase from Saccharomyces cerevisiae
Authors: SCHMIDT Rainer , WIEGAND Helga , REICHERT Uwe
Published: March 2005

1. Hypoxanthine‐guanine phosphoribosyltransferase (EC 2.4.2.8) from Saccharomyces cerevisiae was purified 9400‐fold by affinity chromatography giving rise to an electrophoretically homogeneous preparation.

2. The molecular weight of the enzyme was determined by gel filtration with Sephadex G‐100 and by sodium dodecylsulfate gel electrophoresis. Both methods reveal a molecular weight of 51000.

3. The enzyme requires Mg2+ and has its pH optimum at 8.5.

4. Isoelectric focussing as well gel electrophoresis of the purified extract reveals a single band which exhibits enzyme activity. The isoelectric point of the enzyme is 5.1.

5. The enzyme displays Michaelis‐Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine and phosphoribosylpyrophosphate of 23 μM, 18 μM, and 50 μM respectively.
Site of Interaction between Phenazine Methosulphate and the Respiratory Chain of Bacillus subtilis
Authors: BISSCHOP Arnold , BERGSMA Jack , KONINGS Wil N.
Published: March 2005

Membrane vesicles from Bacillus subtilis W23 and from the menaquinone‐deficient Bacillus subtilis aroD were incubated with phenazine methosulphate and excess soluble phenazine methosulphate was subsequently removed by washing (preincubated vesicles). Phenazine methosulphate which remains attached to the membrane is accessible from the outer surface of the membrane because it can be reduced chemically by NADH or ascorbate and the reduced forms can mediate electrons to horse heart cytochrome c in the external medium of the membrane vesicles.

The oxidation rates of ascorbate in preincubated vesicles of B. subtilis W23 and B. subtilis aroD and of NADH in preincubated vesicles of B. subtilis aroD increased with increasing amounts of phenazine methosulphate attached per mg membrane protein. Electrons from membrane‐attached reduced phenazine methosulphate are essentially all mediated to the terminal part of the respiratory chain before cytochrome a601 because the oxidation of reduced phenazine methosulphate is almost completely inhibited by cyanide and only to a small extent by 2‐heptyl‐4‐hydroxy‐quinoline‐N‐oxide while NADH oxidase is strongly inhibited by both compounds. Furthermore, reduced phenazine methosulphate reduces completely cytochrome a601 and not all cytochromes b560 and c553.

Active transport of amino acids is at least ten‐fold more effectively stimulated by NADH or ascorbate oxidation via phenazine methosulphate than by NADH oxidation via NADH dehydrogenase. Possible explanations are discussed.
A 13C Nuclear‐Magnetic‐Resonance Study of CO2‐HCO3− Exchange Catalyzed by Human Carbonic Anhydrase C at Chemical Equilibrium
Authors: SIMONSSON Ingvar , JONSSON Bengt‐Harald , LINDSKOG Sven
Published: March 2005

The effects of human carbonic anhydrase C on the 13C nuclear magnetic resonance spectra of equilibrium mixtures of 13CO2 and NaH13CO3 were measured at 67.89 MHz. Enzyme‐catalyzed CO2‐HCO3− exchange rates were estimated from the linewidths of the resonances. The results show that: (a) the maximal exchange rates are larger than the maximal turnover rates; (b) the exchange is equally rapid with 1H2O or with 2H2O as solvents; (c) the exchange is equally rapid in the presence or in the absence of added buffers; (d) the apparent substrate binding is weaker than predicted if steady‐state Km values are assumed to represent substrate dissociation constants.

The main conclusion concerning the catalytic mechanism of the enzyme is that the proton‐transfer processes which limit turnover rates in the steady state are not directly involved in CO2‐HCO3− exchange. In addition, the results suggest that CO2‐HCO3− interconversion takes place by a nucleophilic mechanism, such as a reversible reaction of zinc‐coordinated OH− with CO2.
Determination of Leb‐Active Oligosaccharides in Urine of Pregnant and Lactating Women by Radioimmunoassay
Authors: ZOPF David A. , GINSBURG Victor , HALLGREN Peter , JONSSON Anne‐Charlotte , LINDBERG Bo S. , LUNDBLAD Arne
Published: March 2005

Antiserum obtained by immunizing a goat with lacto‐N‐difucohexaose I, a Lebblood‐group hapten, coupled to poly‐L‐lysine was used in a radioimmunoassay to detect Leb‐active oligosaccharides (chiefly lacto‐N‐difucohexaose I) in urine of 138 pregnant and lactating women of different ABO and Lewis blood groups. Specificity of the method was determined by comparing inhibitory activities of 18 oligosaccharides. Only women who belonged to the Lebblood group excreted Leb‐active oligosaccharides in urine. Leb‐active oligosaccharides increase during pregnancy, reaching levels up to approximately 70 μmol lacto‐N‐difucohexaose I equivalents per pmol creatinine in the third trimester and early post‐partum period. Excretion varies considerably but tends to be highest in those individuals who strongly express the Lebantigen on their red blood cells or who belong to blood group O. Leb‐active oligosaccharides were detected in plasma of a few individuals but at concentrations 1000‐fold lower than in urine.
Molecular Transformations in Sarcolasmic Reticulum of Fast‐Twitch Muscle by Electro‐Stimulation
Authors: HEILMANN Claus , PETTE Dirk
Published: March 2005

Chronic electro‐stimulation of fast‐twitch rabbit muscle with the frequency pattern received by a slow‐twitch muscle induces a progressive transformation of the sarcoplasmic reticulum. After 2 days stimulation activities of Ca2+‐dependent ATPase and of Ca2+ transport begin to decrease, and are paralleled by a progressive decrease in Ca2+‐dependent and Ca2+, Mg2+‐dependent phosphoprotein formation, reduced rate of dephosphorylation and a rearrangement of the electrophoretic polypeptide and phosphoprotein patterns. These findings suggest a transformation of the sarcoplasmic reticulum to resemble that of a slow‐twitch muscle. This transformation is paralleled by increase in time‐to‐peak of twitch contraction and half relaxation time and occurs before conversion of the myosin light chain pattern is observed. The parallel time course of changes in contractile properties of stimulated muscle and the molecular and functional properties of the sarcoplasmic reticulum emphasizes the definitive role of the latter in determining the twitch characteristics of fast and slow twitch muscles.
Modification of DNA in Chromatin with Methyltransferase from Haemophilus influenzae Rd
Authors: DOENECKE Detlef
Published: March 2005

The accessibility of DNA in nucleosome dimers (as a model of the chromosomal chain of nucleosomes) was determined by means of modification methylases from Haemophilus influenzae Rd. Using these enzymes, the rate of modification of nucleosome dimers is about one fifth the rate observed with protein‐free DNA from chromatin subunit dimers. Methylated DNA sites in nucleosome dimers are readily accessible to micrococcal nuclease. The analysis of the fragment pattern of nucleosomes after methylation and mild nuclease treatment reveals that the methylated sites are predominantly located in the internucleosomal linker DNA.

Polylysine binding experiments further support this interpretation. This compound preferentially interacts with the nucleosomal core DNA and protects it against internal cleavage. It neither affects the degradation of methylated sites drastically nor does it inhibit the methylation of nucleosome dimers.

Thus, a combination of protection, cleavage and modification is proposed as a useful tool for the analysis of the structure of chromatin.
Yeast Killer Toxin: Purification and Characterisation of the Protein Toxin from Saccharomyces cerevisiae
Authors: PALFREE Roger G. E. , BUSSEY Howard
Published: March 2005

Killer toxin from killer strains of Saccharomyces cerevisiae was isolated from concentrates of extracellular medium by precipitation in poly(ethylene glycol) and chromatography through glyceryl‐controlled‐pore glass. The toxin migrated as a single protein band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A molecular weight of 11470 was determined for the toxin protein from its electrophoretic mobility and amino acid composition. Gel filtration of the active toxin indicated that the 11470‐Mr monomer was the active unit. Electrophoretic comparison of extracellular concentrates from a killer strain and an isogenic non‐killer showed the presence of the toxin protein only in the killer‐derived material. The activity of the toxin was most stable between pH 4.2 and 4.6 At 30°C toxin from a superkiller strain was more stable than that from a normal killer.
Peptidyl Transferase of Bacterial Ribosome: Resistance to Proteinase K
Authors: BERNABEU Carmelo , CONDE Pedro , VÅZQUEZ David , BALLESTA Juan P. G.
Published: March 2005

70‐S ribosomes and 50‐S ribosomal subunits from Escherichia coli D10 were treated with proteinase K for increasing periods of time. Peptidyl transferase activity and sparsomycin‐induced binding of (U)C‐A‐C‐C‐A‐[3H]Leu‐Ac were tested in the treated particles, the binding of the substrate being more sensitive to the protease than peptide bond formation. Comparison of the amounts of proteins present in the treated particles with the residual activity indicates that only proteins L3 and L14 are released at a similar rate to that at which peptidyl transferase activity is lost. Proteins related to this ribosomal activity by other techniques are lost at a faster rate than the activity itself. In addition, the results indicate that sparsomycin stimulates the binding of the substrate by a different mechanism from that which inhibits peptide bond formation.
Poly(adenylic acid)‐Containing RNA of Euglena gracilis during Chloroplast Development
Authors: VERDIER Gérard
Published: March 2005

The transcriptional origin of the polyadenylated RNA has been studied in dark‐grown cultures of Euglena gracilis and during light‐induced chloroplast development. Complementary DNA (cDNA) to poly(A)‐containing RNA from different preparations (isolated chloroplasts and total cell polyribosomes from the wild‐type Z strain or from the aplastidic mutant W3BUL) were synthesized and used for hybridization experiments.

a) In Euglena cells illuminated for 0, 1 or 24 h, 7–10% of the total cell poly(A)‐containing RNA are transcribed from chloroplast DNA. However, when RNA is extracted from isolated chloroplasts, this proportion of poly(A)‐containing RNA is about two times higher (20%); in this case, the poly(A)‐containing RNA could belong to a specific class and be transcribed from 18–20% of the double‐strand chloroplast DNA.

b) In the dark‐grown cultures or in the greening cultures, the major part of the total cell poly(A)‐containing RNA is transcribed from the non‐repetitive sequences of nuclear DNA. The isolated chloroplast preparations contain also a high proportion of poly(A)‐containing RNA from nuclear origin.

c) About 10% of the total cell poly(A)‐containing RNA of the wild‐type strain, absent in the poly(A)‐containing RNA of the aplastidic mutant, appear hybridizable to the nuclear genome of this mutant. Thus the transcription of some of the RNA encoded by nuclear DNA could be controlled by the presence of the chloroplast. Moreover, the fact that isolated chloroplast preparations appear to be enriched in such poly(A)‐containing RNA from nuclear origin, suggests that these RNA are located at the level of the chloroplast.
Molecular Organization of the Outer Membrane of Salmonella typhimurium
Authors: GMEINER Jobst , SCHLECHT Siegfried
Published: March 2005

Analysis of whole cells of Salmonella typhimurium wild type and of various lipopolysaccharide mutants for their molar lipopolysaccharide content per mg dry weight revealed increasing lipopolysaccharide concentrations concomitant with the degree of lipopolysaccharide sugar deficiency. Cell walls were prepared from all strains by mechanical breakage of cells in the presence of 0.4% sodium dodecylsulfate. The use of the detergent for selective removal of cytoplasmic membrane was justified since it was shown that only minor amounts of lipopolysaccharide were extracted from whole cells during sodium dodecylsulfate treatment. Cell walls were analyzed for peptidoglycan, lipopolysaccharide, phospholipids, proteins and lipoprotein covalently linked to peptidoglycan. The results obtained on a molar basis indicate strongly that rough mutants incorporated increasing amounts of lipopolysaccharide into their cell walls. At the same time, the protein composition changed in some of the mutants. In cell walls of deep rough mutants the lipopolysaccharide concentration increased to about four times that found in the wild type, accompanied by increasing amounts of phospholipids, whereas the protein content decreased. Based on these findings different molecular organizations of the outer membrane in the various strains are discussed.
Property and Activity of Mycoloyl Esters of Methyl Glucoside and Trehalose
Authors: DURAND Elisabeth , GILLOIS Monique , TOCANNE Jean‐François , LANEELLE Gilbert
Published: March 2005

Methyl α‐D‐glucoside and α,α′‐D‐trehalose acylated on their primary hydroxyl group by α‐alkyl‐β‐hydroxy acids (mycolic acids) with 18, 32 and more than 80 carbon atoms were studied. These cord factors and analogues when dispersed in water gave hydrated and organized phases which bore resemblances and differences to phospholipid liposomes. Phase transition temperature was determined by fluorescence for the C32 derivatives and by differential scanning calorimetry for the C80 derivatives. A narrow temperature interval between the gel and the fluid states was observed, indicating that this transition was strongly cooperative phenomenon.

The activity on mitochondria of these diacyl trehaloses was studied with succinate as substrate. The fluid suspensions were more active than the rigid ones. There was no influence of the relative configuration of the mycoloyl residue. Acyl chains with 18, 32 and more than 80 carbon atoms had rather similar activities on phosphorylation, but differences appeared when considering effects on respiration. Electron microscopy suggested that cord factor weakened the peripheral part of the inner membrane, but cristae looked unchanged. These observations, together with the results of our previous monolayer studies of these glycolipids, led us to propose a model for interaction of cord factor with mitochondria.
Kinetic Analysis of the Interaction of the Phosphatidylcholine Exchange Protein with Unilamellar Vesicles and Multilamellar Liposomes
Authors: WIRTZ Karel W. A. , VRIEND Gerrit , WESTERMAN Jan
Published: March 2005

The mode of action of the phosphatidycholine exchange protein from bovine liver has been studied by using unilamellar vesicles and multilamellar liposomes both of which membranes contain phosphatidylcholine and phosphatidic acid. The protein‐mediated exchange of phosphatidylcholine between vesicles and liposomes fit the kinetic model presented in a previous study [V. D. Besselaar et al. (1975) Biochemistry, 14, 1852]. Kinetic analysis of the rates of exchange indicate that the apparent dissociation constant of the exchange protein‐vesicle complex decreases with an increasing phosphatidic acid content of the vesicles. Both vesicles and liposomes of 10 mol% phosphatidic acid show the same dissociation constant; on the other hand, both the formation and the disruption of the protein‐membrane complex was 50–100‐times higher for the vesicles than for the liposomes. This implies that the exchange protein can discriminate between vesicles and liposomes. Equilibrium gel chromatography on a column of Bio Gel A‐5m confirmed that the exchange protein binds more strongly to vesicles of an increased phosphatidic acid content. The protein‐mediated exchange of phosphatidylcholine in the vesicle‐liposome system demonstrates a pH optimum at 4.0 to 5.5. The Kinetic analysis at pH 5.0 as compared to pH 7.4 indicates that the enhanced exchange at pH 5.0 can solely be accounted for by an altered interaction of the exchange protein with the liposomes.
Retinol‐Binding Protein from Human Urine and Its Interaction with Retinol and Prealbumin
Authors: FEX Göran , HANSSON Barbro
Published: March 2005

In freshly collected urine from a patient with glomerulotubular proteinuria there were two bands which contained retinol‐binding proteins. The cathodal band showed fluorescence in the ultraviolet. After extraction with organic solvents only the anodal non‐fluorescent band remained. After addition of an excess retinol only one band remained which by mobility corresponded to the cathodal band. The anodal of the two bands was therefore probably the apo form and the cathodal the holo form of the same retinol‐binding protein. Their proportions, determined by densitometric scanning were approximately 4/1 (anodal/cathodal band).

More than 85% of the retinol‐binding protein in the urine bound to prealbumin‐Sepharose.

The apo retinol‐binding protein from urine had the same electrophoretic mobility on agarose gel electrophoresis and the same pattern on isoelectric focusing as an retinol‐binding protein prepared from serum.

The carboxy‐terminal amino acid sequence of the retinol‐binding protein from freshly collected urine that bound to prealbumin‐Sepharose, was ‐Arg‐Leu. The amino‐terminal sequence was Glu‐Arg‐Asp‐Cys‐Arg‐Val‐Ser‐X‐Phe‐Arg‐Val‐Lys‐Glu‐Asn‐Phe‐Asp‐Lys‐Ala‐ Arg‐Phe‐X‐Gly‐Thr‐Trp‐Tyr‐. This sequence and the amino acid composition are compatible with the view that the retinol‐binding protein in urine is the same as in plasma.
The Inhibition of Photosynthetic Electron Transport in Spinach Chloroplasts by Low Osmolarity
Authors: LOCKAU Wolfgang
Published: March 2005

The reversible inhibition, by low osmolarity, of the rate of electron transport through photosystem 1 has been investigated in spinach chloroplasts. By use of different electron donor systems to photosystem 1, inhibitors of plastocyanin, and by measurement of the extent of photooxidation of the photosystem 1 reaction center P700, the inhibition site has been localized on the electron donor side of this photosystem. From comparison of the influence of impermeant and permeant salts on the electron transport rate, and from the effect of ionic strength on the oxidation of externally added plastocyanin by subchloroplast preparations, it is concluded that low ionic strength within the thylakoids inhibits the photooxidation of endogenous plastocyanin by P700. The results are taken as evidence that plastocyanin is oxidized by P700 at the internal (lumen) side of the osmotic barrier in the thylakoid membrane.
Respiratory Control in Escherichia coli K 12
Authors: BURSTEIN Claude , TIANKOVA Lina , KEPES Adam
Published: March 2005

Contrary to the generl view point that respiratory control does not exist in Escherichia coli, we have been able to demonstrate this phenomenon in this micro‐organism. E. coli cells growing aerobically on an oxidizable carbon source respire at a high rate, which cannot be increased by the addition of uncouplers of oxidative phosphorylation. Upon removal of the carbon source, the rate of respiration decreases progressively to reach, after 10 min, less that 1/20 of its previous value. Under these conditions the addition of an uncoupler increases the residual respiration by a factor of 3 to 9. This increase is due to the release of respiratory control. Release of respiratory control cannot be observed upon addition of ADP which does not penetrate across the membrane. Respiratory control can also be shown in the presence of non‐limiting respiratory substrate provided that it is not a carbon source. This was shown by measuring the rate of oxidation of ascorbate and phenazine methosulfate.

NADH oxidase activity of membrane vesicles obtained by high pressure disintegration was increased by a factor of about 1.5 upon the addition of uncouplers and also upon addition of ADP plus phosphate. In contrast, membrane vesicles depleted from BF1 ATPase (a soluble bacterial coupling factor presenting ATPase activity) did not exhibit respiratory control. It could be restored either by rebinding of BF1 ATPase or by addition of dicyclohexylcarbodiimide, an ATPase inhibitor.
Diglyceride Kinase from Escherichia coli
Authors: BOHNENBERGER Elisabeth , SANDERMANN Heinrich
Published: March 2005

The diglyceride kinase activity of membranes from Escherichia coli was extracted into acidic butan‐1‐ol. The enzyme was purified in organic solvent by precipitation at − 20°C, chromatography on DEAE‐cellulose and repeated chromatography on Sephadex LH‐60. The final 1460‐fold purified enzyme preparation gave a single protein band upon isoelectric focusing in the presence of Triton X‐100 (pI, 4.0) and upon polyacrylamide‐gel electrophoresis in the presence of sodium dodecylsulphate. The latter method as well as gel chromatography on Sephadex LH‐60 indicated a molecular weight of about 15400.

The purified enzyme was devoid of lipid, and it required re‐addition of lipid for activity. sn‐1,2‐Dipalmitate and ceramide were phosphorylated, whereas the C55‐isoprenoid alcohol, ficaprenol, did not serve as a substrate under the same conditions. Conversely, the butanol‐soluble C55‐isoprenoid‐alcohol kinase from Staphylococcus aureus did not phosphorylate sn‐1,2‐dipalmitate.
The Physiological Role of Pyruvate Carboxylation in Hamster Brown Adipose Tissue
Authors: CANNON Barbara , NEDERGAARD Jan
Published: March 2005

Pyruvate carboxylase is present in brown adipose tissue mitochondria.

In isolated mitochondria, pyruvate, bicarbonate and ATP, the substrates for pyruvate carboxylase, are able to replace added malate in supplying a condensing partner for acetyl‐CoA formed from β‐oxidation of fatty acids.

In brown adipocytes, pyruvate and CO2 increase the rate of norepinephrine‐stimulated respiration synergistically.

The norepinephrine‐stimulated respiration in brown adipocytes is diminished when pyruvate transport into the mitochondria is inhibited.

Pyruvate carboxylation increases the intramitochondrial level of citric acid cycle intermediates, as shown by titrations of malonate inhibition of respiration.

Pyruvate carboxylation can continuously supply the mitochondria with citric acid cycle intermediates, as evidenced by its ability to maintain respiration when oxoglutarate conversion to glutamate is stimulated.

Pyruvate carboxylation is necessary for maximal oxygen consumption even when drainage of the citric acid cycle for amino acid synthesis is eliminated.

Pyruvate carboxylation explains observed effects of CO2 on respiration in brown adipocytes, and may also explain the increased glucose uptake by brown adipose tissue during thermogenesis in vivo.
Replication of M13 Duplex DNA in Soluble Extracts of Escherichia coli
Authors: VAN DORP Bob , SCHNECK Peter K. , STAUDENBAUER Walter L.
Published: March 2005

Soluble extracts of M13‐am5‐infected Escherichia coli cells can carry out multiple rounds of M13 duplex DNA replication when supplemented with helix‐destabilising protein of E. coli. Similarly addition of the helix‐destabilising M13 gene 5 protein in low concentrations (up to 30 μg/ml) stimulates the replication of double‐stranded M13 DNA. In contrast, higher concentrations of gene 5 protein (but not of E. coli helix‐destabilising protein) cause a preferential inhibition of complementary strand synthesis resulting in a switch from double‐strand replication to single‐strand synthesis. Depending on the addition of the appropriate amounts of these two helix‐destabilising proteins either stage of M13 DNA replication can now be studied with cell‐free preparations.
The Formate Dehydrogenase Involved in Electron Transport from Formate to Fumarate in Vibrio succinogenes
Authors: KRÖGER Achim , WINKLER Edith , INNERHOFER Anna , HACKENBERG Heinz , SCHÄGGER Hermann
Published: March 2005

The formate dehydrogenase of Vibrio succinogenes, which is involved in electron transport with fumarate as terminal acceptor, was solubilized with Triton X‐100 and purified some 200‐fold by means of chromatography on hydroxypatite, sucrose‐density‐gradient centrifugation and chromatography on DEAE‐Sephadex. Gel filtration failed to increase the specific activity of the enzyme while gel electrophoresis in the presence of dodecylsulfate revealed that 73% of the protein of the preparation consisted of a polypeptide of Mr 110000. The Mr of the functional enzyme was found to be 263000 on the basis of the Stokes radius (5.8 nm) and the sedimentation coefficient (11.3 S).

The preparation contained 9 μmol molybdenum/g protein and about 170 μmol iron‐sulfur/g protein. The contents of b and c cytochromes varied and were lower than that of molybdenum. The low‐potential cytochrome b [Kröger, A. and Innerhofer, A. (1976) Eur. J. Biochem. 69, 497–506] present in the preparation was reduced by formate.

The preparation catalyzed the reduction of a variety of dyes by formate, but not of NAD, FMN, ferrdoxin or oxygen. The reduction of CO2 or bicarbonate by reduced methyl viologen was not catalyzed. The reaction with benzyl viologen obeyed the rate law consistent with a ping‐pong mechanism. The Km for formate was 1.5 mM at infinite concentration of benzyl viologen while that for benzyl viologen was 0.53 mM at infinite formate concentration. Enzymic activity was inhibited by azide, KCN and HgCl2, but not by 4‐chloromercuriphenylsulfonate or 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide, both of which inhibit overall electron transport. The inhibition by azide was competitive with formate; the Ki was 45 μM.

The midpoint potential of the low‐potential cytochrome b of the membrane fraction was shifted – 40 mV by the presence of 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide.

It is concluded that the formate dehydrogenase of V. succinogenes is isolated as a dimer consisting of two identical subunits of Mr 110000, each of which carries one atom of molybdenum and iron‐sulfur groups. The low‐potential cytochrome b is the direct acceptor for the electrons of formate dehydrogenase in the electron transport of formate‐fumarate reduction of V. succinogenes. Inhibition of electron transport of the membrane fraction between formate dehydrogenase and menaquinone by 2‐(n‐nonyl)‐4‐hydroxyquinoline‐N‐oxide [Kröger, A. and Innerhofer, A. (1976) Eur. J. Biochem. 69, 487–495] is caused by the inhibitor binding to the low‐potential cytochrome b.
Glucosamine Itself Mediates Reversible Inhibition of Protein Glycosylation
Authors: KOCH H. Uwe , SCHWARZ Ralph T. , SCHOLTISSEK Christoph
Published: March 2005

The metabolism of glucosamine in chick embryo fibroblasts was studied at different concentrations of the amino sugar added to the culture medium.

In glucose‐containing medium the well‐known metabolites, UDP‐N‐acetylglucosamine,N‐acetylglucosamine 6‐phosphate andN‐acetylglucosamine, are detectable after inhibition of glycosylation resulting from glucosamine treatment. Especially when the cells were infected with influenza virus, high intracellular concentrations of non‐metabolized glucosamine are demonstrable in addition.

Removal of the inhibitor from the medium results in release of the block of influenza virus glycoprotein glycosylation within 10 min. The onset of glycosylation is paralleled by a rapid reduction of intracellular levels of glucosamine without significant changes in the concentrations of its metabolites. Furthermore, concentrations of GDP‐mannose, UDP‐glucose, and UDP‐galactose remain constant for at least 30 min after reversal of the block.

It is concluded that glucosamine as such exerts its effects on glycosylation, rather than one of its metabolites being responsible for this effect.
Biosynthesis of Peptidoglycan in Pseudomonas aeruginosa
Authors: MIRELMAN David , NUCHAMOWITZ Yael
Published: March 2005

Ether‐treated cells of pseudomonas aeruginosa catalyze the formation of crosslinked peptidoglycan from the two nucleotide precursors uridinediphospho‐N‐acetylglucosamine and uridine‐diphospho‐N‐acetylmuramyl‐l‐alanyl‐d‐γ‐glutamyl‐meso‐ diaminopimelyl‐d‐alanyl‐d‐alanine. The main enzymatic reactions of biosynthesis were similar to those found in Escherichia coli. Part of the reaction products were soluble in 4% sodium dodecylsulfate whereas the other part was covalently bound to the preexisting cell wall peptidoglycan sacculus. The incorporation into cell wall is carried out by a transpeptidation reaction in which the nascent peptidoglycan functions mainly as the donor and the preexisting one as acceptor. The detergent‐soluble peptidoglycan is composed of partially crosslinked peptidoglycan strands as well as low‐molecular‐weight peptidoglycan fragments. Pulse‐chase biosynthesis experiments show that the detergent‐soluble peptidoglycan is an intermediate that eventually becomes covalently bound to the wall. The dd‐carboxypeptidase activity of P. aeruginosa is membrane‐bound and does not hydrolyse C‐terminal d‐alanine residues from the l‐lysine‐containing nucleotide‐precursor analogue. An ld‐carboxypeptidase was also detected in P. aeruginosa.
Biosynthesis of Peptidoglycan in Pseudomonas aeruginosa
Authors: MIRELMAN David , NUCHAMOWITZ Yael
Published: March 2005

The intrinsic effect of various β‐lactam antibiotics on the biosynthesis of peptidoglycan of Pseudomonas aeruginosa X‐48 was investigated. Most of the cephalosporins and penicillins tested already at 0.5 μg/ml strongly inhibited (a) the incorporation of nascent peptidoglycan into the detergent‐insoluble fraction (> 75%), (b) the formation of peptide crosslinkages (> 60%) and (c) the activity of the DD‐carboxypeptidase and partially that of the transpeptidase (∼ 90% and ∼ 40% respectively). Another group of β‐lactam drugs did not inhibit incorporation into the material insoluble in sodium dodecylsulfate, the formation of peptide crosslinkages nor transpeptidase activity. They only partially inhibited the activity of the DD‐carboxypeptidase–endopeptidase system (40–50% at 0.5 μg/ml). The results obtained differ from those of Presslitz and Ray [Antimicrob. Agents Chemother. 7, 578–581 (1975)] and show some resemblance to the effects of β‐lactams on the biosynthesis of Escherichia coli peptidoglycan.