bluuebunny/crossref_metadata_2025 huggingface dataset:
Uridylate Trapping Induced by the C‐2‐Modified d‐Glucose Analogs Glucosone, Fluoroglucose, and Glucosamine
Authors: HOLSTEGE Axel , SCHULZ‐HOLSTEGE Christa , HENNINGER Hanspeter , REIFFEN Karl A. , SCHNEIDER Friedhelm , KEPPLER Dietrich O. R.
Published: March 2005

The interference of nucleotide metabolism induced by d‐arabino‐hexos‐2‐ulose (glucosone), 2‐deoxy‐2‐fluoro‐d‐glucose (fluoroglucose) and, for comparison, 2‐amino‐2‐deoxy‐d‐glucose (glucosamine) was studied in suspension cultures of TA3 mammary tumor cells. High‐performance liquid chromatography, [14C]uridine labeling and enzymatic analyses revealed that glucosone is metabolized to the UDP derivative leading to a diversion of [14C]uridylate from UTP into UDP‐glucosone. The accumulation of UDP‐glucosone, that was measured in TA3 as well as AS‐30D hepatoma cells, was associated with a depletion of UTP pools to less than 15% of control and a reduction of CTP contents to one third. UTP and CTP deficiency could be rapidly rcversed by addition of uridine to the cell suspension.

Fluoroglucose acted both as a uridylate‐trapping and as a guanylate‐trapping hexose analog lowering the cellular contcnts of UTP and GTP to 5% and 27% of control, respectively. The lowering of GTP was accompanied by an increase in total acid‐soluble guanine nucleotides and a diversion of 84% of the [14C]guanylate into GDP‐fluoroglucose.

As compared to fluoroglucose and glucosone, glucosamine was less effective in TA3 cells in decreasing the content of UTP. The uridylate‐trapping effect of glucosamine was in part compensated by the most active expansion of the acid‐soluble uridylate pool; this reflects an enhanced rate of dc novo pyrimidine synthesis that could be suppressed by the inhibitor 6‐azauridine.

As a side effect probably resulting from phosphate trapping, glucosone, fluoroglucose, and glucosamine depressed the levels of ATP and of total adenine 5′‐nucleotides to a similar though moderate extent.

The depletion of UTP, induced by these C‐2‐modified d‐glucose analogs, and additionally of GTP in the case of fluoroglucose, represent a metabolic lesion that may explain their growth inhibitory and cytotoxic effects.
Biosynthesis of Pairs of Peptides Related to Melanotropin, Corticotropin and Endorphin in the Pars Intermedia of the Amphibian Pituitary Gland
Authors: MARTENS Gerard J. M. , JENKS Bruce G. , VAN OVERBEEKE A. P.
Published: March 2005

This study concerns the biosynthesis of a number of peptides in the neurointermediate lobe of the pituitary gland of the aquatic toad, Xenopus laevis. Using pulse‐chase incubations in vitro and high‐performknce liquid chromatographic analysis, it could be shown that these peptides are synthesized through processing of a pro‐hormone, pro‐opiomelanocortin; all peptides were released into the incubation medium. On the basis of electro‐phoretic analysis, selective amino acid incorporation and immunoprecipitation, as well as peptide mapping by high‐performance liquid chromatography, the peptides were classified into three distinct groups: two related to melanocyte‐stimulating hormone (melanotropin), two related to adrenocorticotropic hormone (corticotropin) and two endorphin‐like peptides. Using tryptic and chymotryptic maps of synthetic α‐melanotropin and des‐AcαN‐α‐melanotropin as references, one of the melanotropin‐like peptides was identified as des‐AcαN‐α‐melanotropin; the other one represents neither α‐melanotropin nor any other known melanotropic peptide. The two peptides that were immunologically related to corticotropin had characteristics consistent with a structures resembling a peptide previously named ‘corticotropin‐like intermediate lobe peptide’, corticotropin‐(18‐39). The two endor‐phin‐like peptides, although highly related, do not have the same primary structure. In view of the apparent structural differences between the two peptides in each group, the possible occurrence of two prohormones is discussed.
Cloning of Xenopus laevis Nuclear Poly(A)‐Rich RNA Sequences
Authors: KNÖCHEL Walter , JOHN Maliyakal E.
Published: March 2005

cDNA/RNA hybridization experiments of polysomal and nuclear poly(A)‐rich RNA from early tadpole stages of Xenopus laevis revealed that part of the nuclear poly(A)‐rich RNA sequences are not present within the polysomal polyadenylated RNA. For a more detailed analysis of these sequences we have cloned double‐stranded cDNA derived from tadpole nuclear poly(A)‐rich RNA in the PstI cleavage site of pBR 322. By colony screening with 32P‐labelled cDNA from polysomal and nuclear poly(A)‐rich RNA of the tadpole stage we could identify and isolate some of the cloned sequences, which are present only within the nuclear RNA. However, hybridization with cDNA from polysomal poly(A)‐rich RNA of the gastrula stage indicated that at least one of those sequences which are confined to the nucleus at tadpole stage may serve as mRNA at gastrula stage. We present evidence tliat nuclear and polysomal poly(A)‐rich RNA molecules containing the same nucleotide sequence differ in size and that size reduction at the level of processing precedes and may enable cytoplasmic export. We conclude that, besides stage‐specific regulation of transcription, post‐transcriptional control mechanisms are also involved in gene expression during embryonic development.
The Organization of the Chloroplast DNA in Wheat and Maize in the Region Containing the LS Gene
Authors: KOLLER Barbara , DELIUS Hajo , DYER Tristan A.
Published: March 2005

The DNAs of the plasmids pTac39 and pZmBIB, which contain the wheat chloroplast DNA BunHI fragment 2 and the maize chloroplast DNA BumHI fragment 9 respectively, were analysed by electron microscopy and by Northern blotting. Both plasmids contain the gene for the large subunit of ribulose bisphosphate carboxylase (LS gene). Comparison of thc DNAs of the two species by partial denaturation and heteroduplex analysis shows extended homologies interspersed with a fine pattern of non‐homology. On analysis of wheat‐RNA. maize‐DNA hybrids a region of non‐homology at the 3′ end of the LS mRNA of about 200 bases was found. Analysis of wheat DNA. RNA hybrids showed that the wheat LS mRNA contains 1700±50 bascs and another hybrid, containing 2400 bases was also shown to occur in a position adjacent to that formed by the LS mRNA. Binding of Escherichia coli RNA polymerase and the start of transcription in vitro occurred at well‐ defined sites, which are located between or close to the positions where mRNA . DNA hybrids are found. A modified technique for the preparation and fractionation by electrophoresis of glyoxdlated RNA is described.
Characterisation of the Chick Brain High Molecular Weight Multiply Phosphorylated Microtubule Associated Protein
Authors: BURNS Roy G. , ISLAM Khalid
Published: March 2005

A high‐molecular‐weight microtubule‐associated protein from chick brain, which has previously been shown to be phosphorylated in vitro to the extent of 10‐12 mol.mol−1, is shown to have the same properties as reported for the microtubule‐associated protein MAP2 from calf and porcine brain in terms of its molecular weight, sensitivity to trypsin and α‐chymotrypsin, the molecular weights of the primary proteolytic degradation products, and the failure of certain of the latter components to bind to assembled microtubules. The primary 260000‐Mr tryptic product can be phosphorylated to at least 4 mol. mol−1, in contrast to an earlier‐ report with calf brain MAP2 which noted a single phosphorylation site.
Purification and Properties of Tryptophan 5‐Monooxygenase from Rat Brain‐Stem
Authors: NAKATA Hiroyasu , FUJISAWA Hitoshi
Published: March 2005

Tryptophan 5‐monooxygenase was purified approximately 5500‐fold, to apparent homogeneity with a specific activity of 374 nmol min‐−1 mg−1 at 30°C, from rat brain‐stem using Sepharose CL‐6B, DEAE‐Sepharose CL‐6B and pteridine‐agarose chromatography. Two distinct active forms were separable by DEAE‐Sepharose CL‐6B and designated as form I and form I1 based on their order of elution from the gel column. The apparent molecular weight of form I was determined to be 300000 by gel filtration on Ultrogel AcA 34 and 288000 by gradient polyacrylamide gel electrophoresis. The enzyme gave a single band on sodium dodecylsulfate/poly‐ acrylamide gel electrophoresis, the molecular weight of which was estimated to be 59000, indicating that the enzyme might be composed of four identical subunits. The tetrameric structure of the enzyme was further suggested by cross‐linking studies using dimethyl suberimidate as a bifunctional reagent. The enzyme activity was stimulated approximately 3.5‐fold by the addition of Fe2+. Kinetic studies revealed that this activation was associated with an increase of V value. The purified enzyme had an activity of phenylakdnine hydroxylation but not an activity of tyrosine hydroxylation.
Fragmentation and Modification of r‐Carrageenan and Characterisation of the Polysaccharide Order‐Disorder Transition in Solution
Authors: REES David A. , WILLIAMSON Frank B. , FRANGOU Sarah A. , MORRIS Edwin R.
Published: March 2005

A procedure is described in detail by which the algal polysaccharide, r‐carrageenan, can be modified and fragmented to products of more regular structure and shorter chain length. Since most chains of this product can form uninterrupted helices they undergo the disorder‐order transition without the gelation that is a feature of the behaviour of the natural polymer.

The disorder‐order transition becomes much sharper and occurs at higher temperaturcs with increasing chain length. The sign and magnitude of the accompanying shift in optical rotation can be estimated satisfactorily from the solid‐state geometry of the helix using our semi‐empirical method for correlating optical rotation with the conformations at glycosidic linkages. In solutions of the helical form, but not the coil form, cationic dyes such as methylene blue or acridine orange show induced optical activity and a changed visible spectrum which we attribute to binding and stacking on the helix. This also causcs a shift in the temperature of the helix‐coil transition. We discuss the possibility that the tendency of dyes to stack could drive polysaccharides into conformations that they would not otherwise adopt, with implications for their use as ‘conformational probes’.
Intracellular Compartment ation and Control of Alanine Metabolism in Rat Liver Parenchymal Cells
Authors: GROEN Albert K. , SIPS Herman J. , VERVOORN Richard C. , TAGER Joseph M.
Published: March 2005

The catabolism of alanine in isolated rat liver cells was studied using a perifusion system in order to titrate the cells with different concentrations of alanine.

The Concentrations of the components of the alanine aminotransferase reaction in the cytosolic and mitochondrial compartments were measured after digitonin fractionation of samples of the cell suspension taken during each steady state. Comparison of the mass‐action ratios with the equilibrium constant indicated that the cytosolic enzyme is poised towards pyruvate formation at all concentrations of alanine used, whereas the mitochondrial enzyme is always poised towards alanine formation. The calculated flux through cytosolic alanine aminotransferase, using literature data for the kinetic parameters of the enzyme and experimental data for the concentration of metabolites, was in close agreement with the measured carbon flux. It is concluded that the transamination step in alanine catabolism occurs in the cytosol.

The concentration of the components of the glutamate dehydrogenase and 3‐hydroxybutyrate dehydrogenase reactions in the mitochondrial compartment during each steady state were also measured. The ΔG of the combined reactions did not change appreciably on increasing the extracellular alanine concentration to 0.6 mM. At higher alanine concentrations ΔG became slightly more negative. As the extracellular Concentration of alanine was increased, the concentration of glutamate in both the cytosolic and the mitochondrial compartments increased. However, the cytosolic alanine concentration was always lower than that in the extracellular compartment even at the highest concentration of alanine (6 mM) infused. When increasing concentrations of pyruvate were infused together with a fixed concentration of alanine (0.6 mM), the cytosolic concentration of alanine increased to levels even higher than that in the perifusate. However, there was no significant effect in the rate of formation of nitrogenous products.

It is concluded that the transport of alanine across the plasma membrane is the only rate‐controlling step in alanine catabolism under the conditions used.
Purification and Characterization of a Mannose/Glucose‐Specific Lectin from Vicia cracca
Authors: BAUMANN Christian M. , STROSBERG A. Donny , RÜDIGER Harold
Published: March 2005

The seeds of Vicia cracca are known to contain two lectins: an N‐acetylgalactosamine‐binding lectin, which reacts specifically with human erythrocytes of blood group A, and a mannose/glucose‐binding lectin unspecific towards human blood groups. The second lectin is the object of the present work. Purification was achieved by affinity chromatography on Sephadex G‐100. The native lectin has a molecular weight of 44000. It is composed of two small (Mr 5700) and two large subunits (Mr 17500). Binding of sugars to the lectin was assayed by equilibrium dialysis and spectrophotometrically; the association constants for glucose are 1.38×103 l/mol and 2.5×102 l/mol respectively.

The primary structure of the small subunit was determined by analyses of the whole chain, of tryptic and CNBr peptides. It was shown to consist of 53 amino acid residues. The mannose/glucose‐binding lectin from Vicia cracca is highly homologous to the lectins from Vicia sativa, Vicia faba, Pisum sativum and Lens culinaris. There is, however, only limited homology to the N‐acetylgalactosamine‐specific lectin from Vicia cracca.
Studies on Prophospholipase A2 and Its Enzyme from Human Pancreatic Juice
Authors: GRATAROLI Renerq;e , DIJKMAN Ruuid , DUTILH Christian E. , VAN DER OUDERAA Frans , DE HAAS Gerard H. , FIGARELLA Catherine
Published: March 2005

Upon tryptic activation of pure human prophospholipase A2, a heptapeptide is released from the N‐terminal part of the protein yielding active phospholipase A2 (EC 3.1.1.4). Both the kinetics of the activation process and the amino acid sequence of the activation peptide strongly resemble those of pancreatic zymogens of other mammalian sources.

The kinetic properties of human phospholipase A2 and its zymogen are compared with those of the corresponding porcine enzyme using substrates present as micelles, molecular dispersed solutions or as monomolecular surface films. The most obvious difference between the human and porcine phospholipase A2 is the low enzyme activity of the former protein at pH 8.0 as compared to pH 6.0, both against micellar and monomeric substrates. Neither the Ca2+ binding properties nor the inhibition kinetics of the human enzyme using haloketones can easily explain this different pH optimum.

The sequence analysis of the N‐terminal region of the first 40 residues is reported.
Incorporation of N‐Ethylmaleimide into the Membrane‐Bound ADP/ATP Translocator
Authors: AQUILA Heinrich , EIERMANN Wolfgang , KLINGENBERG Martin
Published: March 2005

The incorporation of N‐ethylmaleimide into the 30000‐Mr component of beef‐heart mitochondria has been studied as a function of various ligands to the ADP/ATP carrier and the isolation of the N‐ethylmaleimide‐labeled protein is reported.

The incorporation of N‐ethylmaleimide into the 30000‐Mr component is specifically stimulated by ADP and ATP. Thus by differential incorporation of N‐ethylmaleimide, the 30000‐Mr component is preferentially labeled.

Addition of carboxyatractylate inhibits, whereas bongkrekate tolerates, the incorporation of N‐ethyl‐maleimide.

After solubilization by Triton the purification of N‐ethylnialeimide‐labeled protein is facilitated in the presence of bongkrekate but not of carboxyatractylate, in agreement with the postulated existence of only a bongkrekate‐ N‐ethylmaleimideprotein complex. The labeled protein was purified to homogeneity on hydroxyapatite in Triton and subsequently, after denaturation in dodecylsulfate, on Sepharose 6B.

The identity of the isolated labeled protein with the formerly isolated bongkrekate‐protein or carboxy‐ atractylate‐protein complexes is confirmed by the isoelectric point and amino acid composition.

Two moles of N‐ethylmaleimide must be incorporated into the 30000‐Mr component in order to inhibit fully the binding of one mole carboxyatractylate. This corresponds to one ‐SH group per unit.
The Reactivity of ‐SH Groups in the ADP/ATP Carrier Isolated from Beef Heart Mitochondria
Authors: AQUILA Heinrich , KLINGENBERG Martin
Published: March 2005

The emergence of the reactivity of ‐SH groups associated with conformational changes has been studied on the ADP/ATP carrier, as isolated in three different inhibitor‐protein complexes.

The bongkrekate‐protein complex incorporates approximately one molecule more of N‐ethylmaleimide than the carboxyatractylate‐protein complex. After extensive denaturation by dodecylsulfate in urea, both inhibitor complexes exhibit four reactive ‐SH groups per subunit. Thus one out of four ‐SH groups per subunit has been unmasked in the bongkrekate‐protein complex.

The interconversion from the bongkrekate‐protein complex to the carboxyatractylate‐protein complex is inhibited after the ‐SH groups have been blocked.

The protein complex, isolated with the more easily dissociable atractylate, is used to demonstrate, by the emergence of the ‐SH groups, the transition into the m‐state. This transition is specifically catalyzed by ADP and ATP.

Using 2,2′‐dinitro‐5,5′‐dithiodibenzoate, the appearance of the ‐ SH groups on transition from the c‐state to the m‐state can be followed spectrophotometrically. The specificity for the catalyzing nucleotides is identical with that for transport. The Km for ADP and ATP is in the range of 1 μM.

In conclusion, the thiol groups of the isolated ADP/ATP carrier behave as in the mitochondria1 membrane. The unmasking of ‐SH groups is in full accordance with the concept of two conformational states (c and m)
Heterogeneity of Binding Sites for Adsorptive Pinocytosis of Simple Proteins by Rat Yolk Sacs
Authors: LIVESEY Geoffrey , WILLIAMS Kenneth E.
Published: March 2005

When rat yolk sacs were incubated in serum‐free medium 199, the 251I‐labelled forms of both formaldehyde‐treated bovine serum albumin and ribonuclease were captured far more rapidly than 125I‐labelled polyvinylpyrrolidone. Extensive adsorption of these proteins to the plasma membrane is the main cause of this effect.

Quantitative analysis of the adsorptive‐phase pinocytosis of these two proteins showed curved Hofstee plots, suggesting either the presence of multiple classes of binding site on the surface of pinocytically‐active yolk‐sac cells or a single class of binding site that exhibits negative cooperativity in the binding of these proteins. The values of Km were similar, ranging over 0.6‐ 11.8 μM for 125I‐labelled ribonuclease and over 0.31 ‐4.7 μM for formaldehyde‐treated 125I‐labelled albumin.

Competitive uptake studies, in which tracer amounts of each of the 125I‐labelled proteins were ingested from serum‐free medium containing a higher concentration of one of a series of unlabelled proteins, revealed marked differences between the two radiolabelled proteins. These findings suggest that formaldehyde‐denatured albumin is captured by binding to hydrophobic binding sites on the plasma membrane whereas ribonuclease is captured by binding to negatively charged sites.
Isolation from Thyroid Cells of Purified Plasma Membranes with Associated Actin Microfilaments
Authors: REGNOUF Francoise , DELOBBE André , GABRION Jacqueline , MESNIER Danielle , PRADEL Louise‐Anne
Published: March 2005

Plasma membranes of thyroid cells were purified from hog thyroid glands following two procedures. Their homogeneity was tested by electron microscopy and by measurements of the activity of membrane‐bound enzyme markers. According to the procedure used the membrane fractions obtained present some differences in their morphological features as well as in the repartition of the activities of the membrane‐bound enzyme markers. However, whatever the composition of the membrane fraction examined (membrane vesicles, single membrane sheets with junctional complexes), decoration with heavy meromyosin clearly shows the presence of actin filaments attached to these fragments. Analysis of proteins by polyacrylamide gel electrophoresis indicates the presence of about twelve major components with actin. Treatment of membranes with Triton X‐100 results in an insoluble core which contains all the actin and most of the major proteins. The selective extraction of these components by buffers differing in their ionic strength, pH, or the presence or absence of ATP. Mg has been used to characterize some of the proteins associated to actin; among them are filamin, myosin, α‐actinin, tropomyosin.
Picrate as an Inhibitor of Photosystem II in Photosynthetic Electron Transport
Authors: OETTMEIER Walter , MASSON Klaus
Published: March 2005

2,4,6‐Trinitrophenol (picric acid) is an effective inhibitor of photosynthetic electron transport (pI50 value 6.82). By means of artificial donor and acceptor systems its site of inhibition was located at the reducing side of photo‐system II. In addition, picric acid also uncouples photophosphorylation but a much higher concentration is required than for inhibition of electron transport (pI50 value 4.40). By use of radioactivity labelled [U‐14C]picnic acid (spec. act. 2.1 Ci/mol) a binding constant of 0.25 μM and a number of binding sites of 2.84 nmol/mg chloro‐ phyll was measured. This corresponds to one molecule of picric acid per 394 molecules of chlorophyll or one molecule per electron transport chain. Picric acid is competitively displaced from the thylakoid membrane by 3‐(3,4‐dichlorophenyl)‐1,1 ‐dimethylurea (and functionally related compounds) and phenolic inhibitors as well. In this property picric acid, despite its phenol character, is distinct from other phenolic inhibitors.
Preparation of a mRNA‐Dependent Cell‐Free Translation System from Whole Cells of Saccharomyces cerevisiae
Authors: HOFBAUER Reinhold , FESSL Friederike , HAMILTON Barbara , RUIS Helmut
Published: March 2005

A cell‐free protein‐synthesizing system has been prepared from Saccharomyces cerevisiae by mechanical breakage of cells, isolation of a 30000xg supernatant fraction and removal of endogenous mRNA by treatment with micrococcal nuclease. The system thus isolated is dependent on added mRNA and translates yeast mRNA to discrete products, many of them identical with yeast proteins synthesized in vivo. Activity and properties of this system are comparable to those of other eukaryotic cell‐free translation systems. It offers the following advantages compared to yeast translation systems described previously. (a) Its isolation is simple and fast. (b) Since it is not isolated from spheroplasts there is no danger of its inactivation by contaminants in enzymes used for spheroplast preparation. (c) Isolation appears to be less strain‐dependent and can be carried out starting from cells in various physiological states.
Changes of the Quaternary Structure of Microvillus Aminopeptidase in the Membrane
Authors: SJÖSTRÖM Hans , NORÉN Ove
Published: March 2005

Microvillus aminopeptidase (EC 3.4.11.2) is an enzyme with a molecular weight around 300000. Normal preparations contain three different subunits (subunit A, Mr 162000; subunit B, Mr 123000; subunit C, Mr 61000). The relationship between the three subunits was studied by immunoelectrophoresis using specific antibodies against individual denatured subunits and by densitometric scanning of polyacrylamide gels after separation of the three subunits. The results suggest that microvillus aminopeptidase initially appears in the membrane as a symmetric molecule built up to two identical A subunits. These subunits are then split into equimolar amounts of subunit B and subunit C by trypsin. Subunit B cannot generate subunit C but may be further degraded. The reaction sequence described is one which occurs in vivo. Treatment of purified aminopeptidase with trypsin increases the specific activity twofold. This phenomenon does not seem to be correlated to the generation of subunit B and subunit C or to the transformation of amphiphilic form into hydrophilic form.
Transport of l‐Aspartate and l‐Glutamate in Plasma‐Membrane Vesicles from Rat Liver
Authors: SIPS Herman J. , DE GRAAF Peter A. , VAN DAM Karel
Published: March 2005

An Na + concentration gradient (outside > inside) drives the uphill transport of l‐aspartate and l‐glutamate in rat‐liver plasma membrane vesicles. Competition and exchange‐diffusion experiments indicate the presence of a transport system specific for l‐aspartate and l‐glutamate with a high substrate affinity: an apparent Km of 5.4 μM and 7.4 μM was found for l‐aspartate and l‐glutamate, respectively. The uphill transport is Na +‐specific, but also requires the presence of K + for optimal functioning. The possible physiological relevance of such a transport system is discussed.
Ribitol Dehydrogenase Messenger RNA from an Enzyme Superproducer Strain of Klebsiella aerogenes
Authors: BAHRAMIAN Mohammad B. , HARTLEY Brian S. , LOVINY T.
Published: March 2005

Ribitol dehydrogenase messenger RNA, from a strain of Klebsiella aerogenes that had been evolved to superproduce this enzyme, has been purified in a single step by labelling extracted polysomes with rabbit anti‐(ribitol dehydrogenase) and immunoprecipitating with sheep anti‐(rabbit IgG).

The extracted mRNA is stable in a protein synthesis system in vitro and directs synthesis 35–40‐times more efficiently than RNA from coliphages MS2 or Qβ, to give ribitol dehydrogenase as sole major product.

Its size distribution shows a major band of 1500 nucleotides plus fragments 400–1400 nucleotides, with only traces of size 2400–3000 nucleotides. Only the latter could encode both proteins of the operon: ribitol dehydrogenase and D‐ribulokinase.

Ribitol dehydrogenase mRNA represents 24% of total mRNA in cells harvested just after a ‘switch’ point in mid‐exponential phase. About half of the polysomes containing this mRNA are unattached to DNA, whereas only 3% of other mRNAs are unattached to DNA.

This mRNA is not outstandingly stable in vivo, though there are indications that it may be more stable than average. Hence the high level of synthesis of ribitol dehydrogenase (up to 30% of total protein in an extract) seems to be due to very efficient transcription and translation from multiple copies of a constitutive rbtD gene.
Photoaffinity Labeling of Chromatin
Authors: NIELSEN Peter Eigil
Published: March 2005

The syntheses and properties of potential photoaffinity labels for the proteins in chromatin studies are described. One compound, N, N'‐bis‐(9‐acridinyl)‐4‐aza‐4‐(4‐azidobenzoyl)‐1,7‐diaminoheptane dihydrochloride, has been studied more closely. This photolabel shows high affinity towards DNA (Ka∼ 3 × 105 M−1) and photoreacts with histones at wavelengths in the range 260–450 nm. The photoreaction was monitored fluori‐ metrically, and labeling of histones H1, H2A/H2B and H3 was observed. When chromatin was photolabeled, labeling of H1 was exclusively quenched by NaCl at concentrations which are known to cause dissociation of this histone from the DNA. It is thus inferred that only DNA‐associated proteins are photolabeled by the reagent.
The Complete Nucleotide Sequence of the Chicken Ovotransferrin mRNA
Authors: JELTSCH Jean‐Marc , CHAMBON Pierre
Published: March 2005

The nucleotide sequence of an almost double‐stranded cDNA copy [Cochet, M., Perrin, F., Gannon, F., Krust, A., Chambon, P., McKnight, G. S., Lee, D. C., Mayo, K. E., and Palmiter, R. D. (1979) Nueleic Acids Res. 6, 2435–2452] of chicken ovotransferrin (conalbumin) mRNA has been determined. Taking into account the previously reported 5′‐end sequence [Cochet, M., Gannon, F., Hen, R., Maroteaux, L., Perrin, F., and Chambon, P. (1979) Nature (Lond.) 282, 567–574] we present the complete nucleotide sequence of the ovotransferrin mRNA from which the amino acid sequence of the protein is inferred. A computer and statistical analysis of the nucelotide sequence reveals a pattern of internal homology which confirms that the present‐day chicken ovotransferrin gene (and by extrapolation the transferrin genes of other species) has evolved by duplication and gives some support to the quadruplication hypothesis of transferrin evolution.
The Primary Structure of Hen Ovotransferrin
Authors: WILLIAMS John , ELLEMAN Thomas C. , KINGSTON I. Barry , WILKINS Adrian G. , KUHN Katherine A.
Published: March 2005

Peptide sequences obtained from hen ovotransferrin are compared with the complete amino acid sequence of the protein deduced from a cDNA sequence (Jeltsch and Chambon, preceding paper). Of the 705 positions of the whole protein 605 can be matched by the peptide sequences. Some possible discrepancies between the two methods are pointed out. The two halves of the chain show marked similarities in their sequences with 37% identical residues. The positions of the 15 disulphide bridges are shown; there are 6 homologous bridges in each half of the molecule and 3 extra bridges which occur only in the C‐terminal half. The terminal residues of the half‐ molecule fragments obtained by limited proteolysis are identified. The two domains are joined by a 9‐residue connecting peptide. Sequence variability has been found at 9 positions. The sequence of hen ovotransferrin IS compared with the partial sequence available for human transferrin. From this some tentative conclusions about the identities of the metal‐binding residues and about the evolution of transferrin are reached.
Studies on the Transmembrane Orientation of Cytochrome c Oxidase in Phospholipid Vesicles
Authors: CASEY Robert P. , ARIANO Beatrice H. , AZZI Angelo
Published: March 2005

We report investigations into the direction of orientation of cytochrome c oxidase in reconstituted vesicles and the factors determining this. Measurement of the enzyme orientation employed two independent techniques: monitoring of the level of haem reduction by membrane‐permeant and membrane‐impermeant reagents and a kinetic analysis of the reduction of a spin label covalently bound to the oxidase surface. The method of preparation of the oxidase vesicles had a pronounced effect on the enzyme orientation and the two measurement techniques agreed in indicating that the proportion of mitochondrially oriented enzyme was approximately 85% and 50% for vesicles prepared by cholate dialysis and sonication respectively. Our results show that the membrane orientation of the oxidase is determined by interactions between the phospholipid bilayer and the portion of the enzyme embedded therein, as opposed to gross physical constraints. In particular, we demonstrate that the orientation of the oxidase is affected by the fluidity and surface charge of the membrane.
The Central Tryptic Fragment of Histones H1 and H5 Is a Fully Compacted Domain and Is the Only Folded Region in the Polypeptide Chain
Authors: TIKTOPULO Elizaveta I. , PRIVALOV Peter L. , ODINTSOVA Tatyana I. , ERMOKHINA Tatyana M. , KRASHENINNIKOV Igor A. , AVILES Francesc X. , CARY Peter D. , CRANE‐ROBINSON Colyn
Published: March 2005

Scanning microcalorimetry has been used to show that the globular structure in histones H1 and H5 melts reversibly over a relatively narrow temperature range as a single domain with enthalpy of melting very close to the van't Hoff enthalpy. These proteins therefore exhibit macroscopic cooperativity like that of many well‐studied small proteins. For histone H5 the observed melting temperature and melting enthalpy coincide with those previously obtained [Eur. J. Biochem. 67, 379‐388 (1976)] using intrinsic Cotton effects in the circular dichroism spectrum, but not with those from extrinsic Cotton effects or NMR chemical shifts. Calorimetric measurements on a central tryptic fragment from histones H1 and H5 show these fragments to have the same molar melting enthalpy as the parent histones. The folded structures of H1 and H5 are therefore located only within these fragments. For histone H1 this conclusion is supported by the finding that the C‐terminal fragment (residues 122‐213) shows no peak in the specific heat curve.
Carp Insulin: Amino Acid Sequence, Biological Activity and Structural Properties
Authors: MAKOWER Alexander , DETTMER Rudolf , RAPOPORT Tom A. , KNOSPE Siegfried , BEHLKE Joachim , PREHN Siegfried , FRANKE Peter , ETZOLD Gerhard , ROSENTHAL Sinaida
Published: March 2005

The amino acid sequence of insulin of carp (Cyprinus carpio) has been determined and correlated with its biological activity in a fat‐cell test and its structural properties as measured by circular dichroism and sedimentation analysis.

The amino acid sequence of carp insulin displays some unusual features: the B chain is longer at the N ter‐ minus by two residues as compared with mammalian insulins and there are substitutions of the charged residues, found in most insulins at positions B21 and B22, by proline and threonine respectively. On the other hand, all amino acid residues essential for biological activity and for the association of insulin monomers are the same in carp insulin. Accordingly, the half‐maximal response in a fat‐cell test is reached with carp insulin at concentrations which are only three times higher than with porcine insulin and the maximal response is the same. The circular dichroism spectrum of carp insulin resembles greatly that of bovine insulin indicating that it has a similar spatial structure. Despite amino acid substitutions in the dimer‐dimer contact region, carp insulin is able to form hexamers.
Citrate Synthesis in Intact Rat‐Liver Mitochondria Is Irreversible
Authors: GREKSAK Miloslav , LOPES‐CARDOZO Matthijs , VAN DEN BERGH Simon G.
Published: March 2005

Rat‐liver mitochondria were incubated with [1,5‐14C]citrate in the presence of fluorocitrate to block its oxidation in the Krebs cycle. The reaction products were analysed enzymatically and by anion‐exchange chromatography. Incorporation of 14C into acetyl‐l‐carnitine or ketone bodies via a backward action of citrate synthase was not observed. The optimal rate of citrate synthesis from pyruvate and malate in the presence of fluorocitrate was 15 nmol. mg−1. min−1. In the absence of fluorocitrate, but in the presence of malonate, citrate was oxidized to succinate at a rate of 4 nmol . mg−1 . min−1.

We conclude that the synthesis of citrate by intact rat liver mitochondria is an irreversible process. The possible mechanism underlying this phenomenon and the consequence for metabolic regulation are discussed.
Generation of Prothrombin‐Converting Activity and the Exposure of Phosphatidylserine at the Outer Surface of Platelets
Authors: BEVERS Edouard M. , COMFURIUS Paul , VAN RIJN Jan L. M. L. , HEMKER H. Coenraad , Robert F. A ZWAAL
Published: March 2005

In the present paper we compare prothrombin‐converting activities of platelets non‐activated, or activated by collagen, thrombin or collagen plus thrombin in the absence and presence of added factor Va. In all experiments described, the rate of thrombin formation for platelets activated by the combined action of collagen and thrombin is greater than that of platelets stimulated by collagen or thrombin alone. The presence of added factor Va enhanced the rate of thrombin formation in all cases, but the higher activity observed with platelets stimulated by collagen plus thrombin remains. When platelets are activated by collagen plus thrombin in the presence factor Xa and prothrombin, a lag period of approximately 10 min is observed before the rate of thrombin formation reaches a steady state. Addition of an excess of factor Va in this experiment reduces the lag time to 3 min. This lag period is interpreted as the time required to generate extra binding sites for the prothrombinase complex at the platelet surface. These extra sites explain the difference in thrombin formation rate between these platelets and platelets activated by either collagen or thrombin only.

The exposure of phospholipids at the platelet outer surface was studied with phospholipases after various activations of the platelets. It is demonstrated that activation by collagen plus thrombin is accompanied by increased susceptibility of platelet phospholipids towards phospholipase A2. Among these degradable phospho‐ lipids are 25% of the phosphatidylserine and 30% of the phosphatidylethanolamine. On the other hand, little no phosphatidylserine is exposed at the membrane exterior of thrombin‐treated or control platelets. We propose that the exposure of phosphatidylserine at the outer surface of platelets activated with thrombin plus collagen essential for the rate enhancement of thrombin formation observed under these conditions. The possibility of transbilayer movement of phospholipids in the platelet membrane as a result of the activation process will be discussed.
Inhibition of Mouse Fibroblast Interferon by Gangliosides
Authors: KRISHNAMURTI Chitra , BESANÇON Françoise , JUSTESEN Just , POULSEN Kenneth , ANKEL Helmut
Published: March 2005

Gangliosides are potent inhibitors of the antiviral activity of mouse fibroblast and other β‐interferons.We have compared the effects of gangliosides on antiviral and antigrowth activities of mouse fibroblast interferon and on the induction of (2′‐5′)oligoadenylate synthetase, one of the enzymes implicated in the antiviral state induced by interferon. Whereas both biological effects appear to be inhibited by gangliosides in an analogous fashion, inhibition of induction of (2′‐5′)oligoadenylate synthetase does not correlate with inhibition of vesicular stomatitis virus replication. Ganglioside concentrations that inhibit the interferon‐induced (2′‐5′)oligoadenylate synthetase to levels close to those of uninduced cells, still allow for a 100–1000‐fold reduction of viral yield.Significantly higher ganglioside concentrations are required to prevent completely the antiviral effect. This biphasic relationship between (2′‐5′)oligoadenylate synthetase levels and inhibition of viral yield suggests that no or very small increases in synthetase levels are involved in inhibition of virus by between two and three orders of magnitude.
The Complete Nucleotide Sequence of a 23‐S rRNA Gene from Tobacco Chloroplasts
Authors: TAKAIWA Furnio , SUGIURA Masahiro
Published: March 2005

The nucleotide sequence of a tobacco chloroplast 23‐S rRNA gene, including the spacer between it and the 4.5‐S rRNA gene, has been determined. The 23‐S rRNA coding region is 2804‐base‐pairs long. A comparison with the 23‐S rRNA sequence of Escherichia coli reveals strong homology and further shows a similarity between the chloroplast 4.5‐S rRNA and the 3′‐terminal region of E. coli 23‐S rRNA. However, the 101‐base‐pair spacer sequence between the 23‐S and 4.5‐S rRNA genes has little homology with E. coli 23‐S rRNA.
Spin‐Label Characterisation of the Lamellar‐to‐Hexagonal (HII) Phase Transition in Egg Phosphatidylethanolamine Aqueous Dispersions
Authors: HARDMAN Paul D.
Published: March 2005

The thermotropic behaviour of egg yolk phosphatidylethanolamine dispersions in excess aqueous phase has been investigated by spin label electron spin resonance spectroscopy and differential thermal analysis. Phosphatidylethanolamine isomers spin‐labelled at six different positions along the.acyl chain, and steroid spin labels, indicate both gel‐fluid lamellar and lamellar‐reverse hexagonal(HII) phase transitions, in agreement with complementary calorimetric studies. Analysis of spin label data shows that the transition to the HII phase is accompanied by an increase in conformational freedom of the acyl chain, more pronounced towards the methyl terminus, and representing an increase in the population of gauche isomers which can only be accommodated by a transition to the non‐bilayer phase. Raising the bulk pH to, and above, pH 8.5 results in stabilisation of the bilayer phase and no transition to the HII phase is observed. The phosphatidylethanolamine spin labels also indicate a polarity profile which is characteristic of each phase.
The Structure of the EF‐Tu · GDP · Me2+ Complex
Authors: WITTINGHOFER Alfred , GOODY Roger S. , ROESCH Paul , KALBITZER Hans Robert
Published: March 2005

The structure of the MgGDP complex at the active site of elongation factor (EF‐Tu) has been investigated by using phosphorothioate analogs of GDP in the absence and presence of various metal ions, electron para‐magnetic resonance (EPR) and nuclear magnetic resonance (NMR) measurements. The high stereoselectivity of EF‐Tu for the diastereomers of guanosine 5′‐0‐(1‐thiodiphosphate) (GDP[αS]) is independent of the nature of the metal ion and is caused by the interaction of the protein with the α‐phosphate of GDP. By using GDP analogs where the oxygens at either thea‐phosphate or thep‐phosphate have been selectively labelled with 17O and measuring their effect on the EPR spectrum of EF‐Tu‐bound manganese we are able to show that only the β‐phosphate of GDP is coordinated to the metal ion in the EF‐Tu. Me2+. GDPcomplex. 31P‐NMR studies on GDP and guanosine 5′‐0‐(2‐thiodiphosphate) (GDP[βS]) bound to EF‐Tu indicate that in the EF‐Tu Me2+ GDP complex Mg2+ interacts more strongly with the β‐phosphate than with the α‐phosphate. Together with binding studies using GDP[βS] our NMR results also indicate that the protein is complexed to the β‐phosphorous of GDP via two oxygens.
Processing of a Chloroplast‐Translated Membrane Protein in vivo
Authors: REISFELD Avi , MATTOO Autar K. , EDELMAN Marvin
Published: March 2005

The 32000‐dalton (Da) shield protein regulating electron transport in Spirodelu oligorrhiza is an integral chloroplast membrane polypeptide. It is rapidly synthesized, constituting a major chloroplast‐translation product in vivo. Following in vitro translation of spirodela chloroplast RNA in a wheat germ system, a 33500‐Da polypeptide is produced. Synthesis of a 33500‐Da protein, associated with the chloroplast membrane. is also seen in vivo, within 2 min of pulse‐labeling spirodela with radioactive amino acids. Comparative analyses among these polypeptides reveal: (a) all three are deficient in lysine residues; (b) the two 33500‐Da species have indistinguishable partial proteolytic digestion patterns while that for the 32000‐Da protein differs only slightly from them; (c) radio‐activity from the 33500‐Da polypeptide is rapidly chased in vivo into the 32000‐Da protein, even in the presence of protein synthesis inhibitors. These results show the 33500‐Da proteins synthesized in vitro and in vivo to be the precursor form of the 32000‐Da shield protein in spirodela, with processing commencing only alter completion of the precursor polypeptide chain and insertion into the membrane.
Fructose‐2,6‐bisphosphatase from Rat Liver
Authors: VAN SCHAFTINGEN Emile , DAVIES Dewi R. , HERS Henri‐Gèry
Published: March 2005

An enzyme that catalyzes the stoichiometric conversion of fructose 2,6‐bisphosphate into fructose 6‐phosphate and inorganic phosphate has been purified from rat liver. This fructose 2,6‐bisphosphatase copurified with phosphofructokinase 2 (ATP: D‐fructose 6‐phosphate 2‐phosphotransferase) in the several separation procedures used. The enzyme was active in the absence of Mg2+ and was stimulated by triphosphonucleotides in the presence of Mg2+ and also by glycerol 3‐phosphate, glycerol 2‐phosphate and dihqdroxyacetone phosphate. It was strongly inhibited by fructose 6‐phosphate at physiological concentrations and this inhibition was partially relieved by glycerol phosphate and dihydroxyacetone phosphate. The activity of fructose 2,6‐bisphosphatase was increased severalfold upon incubation in the presence of cyclic‐AMP‐ dependent protein kinase and cyclic AMP. The activation resulted from an increase in V (rate at infinite concentration of substrate) and from a greater sensitivity to the stimulatory action of ATP and of glycerol phosphate at neutral pH. The activity of fructose 2,6‐bisphosphatase could also be measured in crude liver preparations and in extracts of hepatocytes. It was then increased severalfold by treatment of the cells with glucagon, when measured in the presence of triphosphonucleotides.
pH and Temperature Dependences of Thermolysin Catalysis
Authors: KUNUGI Shigeru , HIROHARA Hideo , ISE Norio
Published: March 2005

The pH dependences of kCat/Km, kcat and Km of thermolysin‐catalyzed hydrolysis of N‐furylacryloylglycyl‐l‐leucinamide (Fua‐Gly‐LeuNH2) and N‐furylacryloylglycyl‐l‐phenylalaninamide (Fua‐Gly‐PheNHz) were in‐ vestigated. Taking the buffer dependences into account, the kcat/Km profile was explained by a simple bell‐shaped curve with pKa1= 5.0 and pKa2= 8.25, at 25°C. Both kcat and Km increased with pH at lower pH and took larger values for Fua‐Gly‐LeuNHz than for Fua‐Gly‐PheNHz at 25°C. The pH dependence of inhibitory actions by amino acids and dipeptides, such as carbobenzyloxy‐L‐phenylalanine and L‐phenylalanyl‐I–leucinamide, showed characteristic features depending on their charge states: anionic or neutral ones inhibited the enzyme more strongly at lower pH while cationic ones did so at neutral pH.

Temperature dependences of kcat/Km, Ki and the two pKa values in the kcat/Km profile were measured. The kcat/Km showed non‐linear dependence while Ki increased linearly with temperature on a logarithmic scale. The calculated AH values of deprotonation for pKal and pKa1 were 33.4 kJ/mol and 35.1 kJ/mol, respectively. The value for pKal is too large to be assigned to the carboxylic group of Glu‐143, in contrast to the generally accepted view.

A mechanism for thermolysin catalysis is presented with particular emphasis on the binding specificity and the catalytic role of zinc‐coordinated water
Studies on the Alkali Light Chains of Vertebrate Skeletal Muscle Myosin
Authors: BURKE Morris , WANG Hong Lyieuh
Published: March 2005

The structures of the alkali light chain subunits A1 and A2 have been studied by examining the effect of the conformationally sensitive reagent tetranitromethane, which reacts specifically with tyrosyl residues. Whereas reaction in the presence of 6 M guanidine hydrochloride results in modification of the three tyrosyl residues of both these light chains, only two tyrosyl residues are exposed to the reagent in the native conformations of these proteins. By gel chromatography of the CNBr‐cleaved chains it was demonstrated that the two reactive tyrosyls are those located in the CB‐1 and CB‐3 segments and that these tyrosyl residues are modified simultaneously and not sequentially. The unreactive tyrosyl residue is in the CB‐6 segment and is separated by two residues from the single cysteinyl residue of these chains. It is found that the modified light chains cannot be made to reassociate with the heavy chains by the NH4Cl hybridization procedure of Wagner and Weeds [J. Mol. Biol. 109,455–470 (1977)] or by the thermal hybridization procedure [Burke and Sivaramakrishnan (1981) Biochemistry 20, 5908–59131. Furthermore, reduction of the nitrotyrosyl groups to aminotyrosyl residues by sodium dithionite does not restore this effect. The data suggest that regions of the light chains at CB‐1 and CB‐3 are involved in the association to the heavy chains.