bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Properties of the Bifunctional Proline Dehydrogenase/1‐Pyrroline‐5‐Carboxylate Dehydrogenase fromPseudomonas aeruginosa
Authors: MEILE Leo , LEISINGER Thomas
Published: March 2005

Proline dehydrogenase/1‐pyrroline‐5‐carboxylate dehydrogenase (Pro/P5C dehydrogenase), a bifunctional enzyme catalyzing the two consecutive reactions of the oxidation of proline to glutamic acid, was purified fromPseudomonas aeruginosastrain PAO1. Pro/P5C dehydrogenase oxidized L‐proline in an FAD‐dependent reaction to L‐Δ1‐pyrroline‐5‐carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L‐glutamic acid. The purification procedure involved DEAE‐cellulose chromatography, affinity chromatography on Matrex gel red A and gel filtration on Sephadex G‐200. It resulted, after 40‐fold purification with 11% yield, in a homogeneous preparation (< 98% pure).

The molecular weight of the single subunit was determined as 119000. Gel filtration of purified Pro/P5C dehydrogenase yielded a molecular weight of 242000 while polyacrylamide gel electrophoresis under native conditions led to the appearance of two catalytically active forms of the enzyme with molecular weights of 241000 and 470000. Manual Edman degradation revealed proline, alanine and aspartic acid as theN‐terminal amino acid sequence.

Pro/P5C dehydrogenase was highly specific for the L‐forms of proline and Δ1‐pyrroline‐5‐carboxylic acid. Its apparentKmvalues were 45 mM for L‐proline, 0.03 mM for NAD and 0.17 mM for NADP. The saturation function for Δ1‐pyrroline‐5‐carboxylic acid was non‐hyperbolic.
Quantitative Recovery, Selective Removal and One‐Step Purification of Human Parotid and Leukemic Lysozymes by Immunoadsorption
Authors: MacKAY Bruce J. , IACONO Vincent J. , ZUCKERMAN Joan M. , OSSERMAN Elliot F. , POLLOCK Jerry J.
Published: March 2005

Immunoadsorption affinity chromatography was used to isolate and purify human lysozyme. The immunoadsorbent was prepared by coupling sheep anti‐(human leukemic lysozyme) IgG to epoxy‐activited Sepharose 6B. Lyophilized parotid saliva (21) was resupended in distilled water (325 ml, 50 mg/ml, w/v) and applied to a column which had a capacity to bind 4.25 mg human enzyme. Non‐adsorbed material did not contain lysozyme, as determined by enzymatic and immunological analyses. All lysozyme activity present in the applied sample (1.97 mg) bound to and was desorbed from the column by elution with 0.2 M sodium acetate HCl buffer, pH 1.8. The isolated material was homogeneous as determined by cationic and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, ultracentrifugation, amino acid and amino‐terminal analyses, and immunoelectrophoretic analysis. The one‐step purification procedure yielded a 1370‐fold increase in specific activity. Human lysozyme was also selectively purified by this method from an ammonium sulfate precipitate of the urine of a patient with chronic monocytic leukemia. Amino acid and polyacrylamide gel electrophoretic analyses indicated that the purified enzyme was identical to human lysozyme isolated from leukemic urine by classical biochemical techniques.
Isolation and Characterization of Streptomyces aureofaciens Protein‐Synthesis Elongation Factor Tu in an Aggregated State
Authors: WEISER Jaroslav , MIKULÍK Karel , ŽIŽKA Zdeněk , ŠŤASTNÁ Jana , JANDA Ivan , JIRÁŇOVÁ Anna
Published: March 2005

The ability of EF‐Tu to aggregate spontaneously was employed for the purification of homogeneous EF‐Tu · GDP from Streptomyces aureofaciens. The formation of filamentous structures in the aggregated EF‐Tu was demonstrated in a light microscope. The purified factor, with a specific activity of 19 100 ± 1000 units/mg in [3H]GDP exchange, was shown to be active in the translation of poly(U).

Aggregated EF‐Tu · GDP exhibited almost eight‐times lower GDP‐exchange capacity at 2°C than at 30°C. This suggests that GDP‐binding sites are not freely accessible at lower temperatures in the aggregated factor, in contrast to Escherichia coli polymerized EF‐Tu. Turbidimetric assays revealed that the solubilization of diluted aggregated S. aureofaciens EF‐Tu is strongly dependent on temperature and causes an increase in the number of accessible GDP‐binding sites.
Isolation and Structural Properties of a High‐Molecular‐Weight Actin‐Binding Protein (Filamin‐Like Protein) in Hog Thyroid Gland
Authors: ROUSTAN Claude , BOYER Mireille , FATTOUM Abdellatif , JEANNEAU René , BENYAMIN Yves , ROGER Michel , PRADEL Louise‐Anne
Published: March 2005

A high‐molecular‐weight protein has been isolated from hog thyroid gland. This protein, with a molecular weight of 475000 determined by ultracentrifugation and gel filtration, is a complex of two polypeptides with apparent molecular weights of 250000 and 240000. It may be related to filamin‐like proteins by its physicochemical properties and its immunogenic cross‐reactivity towards gizzard filamin antibodies. Furthermore it interacts with F‐actin in a stoichiometry of 1 mol of high‐molecular‐weight protein/∼ 12–14 mol actin monomer allowing microfilament association, as shown by electron microscopy.
The Preferred Conformation of Oligosaccharides Derived from the Complex‐Type Carbohydrate Portions of Glycoproteins
Authors: BOCK Klaus , ARNARP Jan , LÖNNGREN Jörgen
Published: March 2005

The preferred conformation in aqueous solution of oligosaccharides related to the complex‐type carbohydrate portion of glycoproteins has been inferred from high‐resolution nuclear magnetic resonance data (both 1H and 13C) combined with hard‐sphere exo‐anomeric calculations. The results indicate that the N‐acetyl‐lactosamine units are arranged in such a way that they are widely separated in space. Thus, these carbohydrates can cover large surface areas of the attached protein and provide good targets for recognition by other proteins.
Phosphorylation of Myosin Light Chain by a Protease‐Activated Kinase from Rabbit Skeletal Muscle
Authors: TUAZON Polygena T. , STULL James T. , TRAUGH Jolinda A.
Published: March 2005

A protease‐activated protein kinase that phosphorylates the P light chain of myosin in the absence of Ca2+ and calmodulin has been isolated from rabbit skeletal muscle. The enzyme has properties similar to protease‐activated kinase I from rabbit reticulocytes [S. M. Tahara and J. A. Traugh (1981) J. Biol. Chem. 256, 11588–11564], which has been shown to phosphorylate the P light chain of myosin [P. T. Tuazon, J. T.Stull, and J. A. Traugh (1982) Biochem. Biophys. Res. Commun. 108, 910–917]. The protease‐activated kinase from skeletal muscle has been partially purified by chromatography on DEAE‐cellulose, phosphocellulose and hydroxyapatite. The enzyme phosphorylates histone as well as the P light chain of myosin following activation by proteolysis. Stoichiometric phosphorylation of myosin light chain was observed with the protease‐activated kinase and myosin light chain kinase. The sites phosphorylated by the protease‐activated kinase and myosin light chain kinase were examined by two‐dimensional peptide mapping following chymotryptic digestion. The phosphopeptides observed with the protease‐activated kinase were different from those obtained with the Ca2+ ‐dependent myosin light chain kinase, indicating that the two enzymes phosphorylated different sites on the P light chain of skeletal muscle myosin. When actomyosin from skeletal muscle was examined as substrate, the P light chain was phosphorylated following activation of the protease‐activated kinase by limited proteolysis.
Mapping of Distinct Structural Domains on Microtubule‐Associated Protein 2 by Monoclonal Antibodies
Authors: SCHERSON Talma , GEIGER Benjamin , ESHHAR Zelig , LITTAUER Uriel Z.
Published: March 2005

Monoclonal antibodes against microtubule‐associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by α‐chymotrypsin to generate a series of high‐molecular‐mass fragments ranging between 270 and 140 kDa. The precursor‐product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270‐kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215‐kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270‐kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule‐binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60‐kDa domain(s) distal to the microtubule‐binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.
Thermodynamics of Stacking and of Self‐Association of the Dinucleoside Monophosphate m62A‐U from Proton NMR Chemical Shifts:
Authors: HARTEL Arnold J. , LANKHORST Peter P. , ALTONA Cornelis
Published: March 2005

Chemical shifts of base and sugar protons of the modified ribodinucleoside monophosphate N6‐dimethyl‐adenylyl(3′‐5′)uridine (m62A‐U) were measured at 100, 360 and 400 MHz in aqueous solution. Seven different samples were used with concentrations ranging from 0.28 mM to 32.7 mM. The temperature was varied from –5 °C to 105 °C. An internal temperature calibration was used. The effects of intermolecular self‐association and of intramolecular stacking on the chemical shifts were quantitatively separated by means of a new approach: differential concentration/temperature profiles (DCTP). Several computational models were tested and the analysis allowed deeper insight into the behaviour of m62A‐U at the molecular level. The simple two‐state approach for both self‐association and stacking already afforded a significant improvement over models in which the association is entirely neglected. A computer least‐squares analysis of the chemical shift behaviour of each individual proton yielded thermodynamic parameters for self‐association and stacking. However, the two‐state model did not suffice to reproduce accurately all of the observations. A satisfactory fit required two additional assumptions: (a) the aromatic protons experience different association shifts in stacked and in unstacked molecules; (b) a temperature‐dependent conformational equilibrium exists between sets of unstacked microstates. The stacked state is taken to represent a single conformational species. The implementation of this extended model in the leastsquares optimization allowed the reproduction of over one thousand chemical shift observations within experimental error. Thermodynamic equilibrium parameters deduced for intramolecular stacking are: ΔHox=–28.8 kJ mol−1, ΔSox=–93 J mol−1 K−1. These numbers agree well with those obtained earlier by us from circular dichroism spectra. The equilibrium enthalpy and entropy values deduced for the association process are: ΔHoA=–35 kJ mol−1 and ΔSoA=–95 J mol−1 K−1.
Induction of Sporulation in Bacillus brevis
Authors: PSCHORN Wolfgang , PAULUS Henry , HANSEN Jutta , RISTOW Hansjürgen
Published: March 2005

This paper presents evidence that the two peptide antibiotics tyrocidine and linear gramicidin, produced by Bacillus brevis ATCC 8185, are required for the induction of sporulation in the producer organism. When tyrocidine synthesis was specifically blocked with 2‐amino‐3‐hydroxy‐3‐phenylpropanoic acid [Mach, B., Reich, E., and Tatum, E. L. (1963) Proc. Natl Acad. Sci. USA, 50, 175–181], sporulation and gramicidin synthesis were inhibited, but both processes could be restored by the addition of tyrocidine. Certain other amino acids such as L‐tyrosine inhibited both sporulation and peptide antibiotic synthesis in nitrogen‐limited cultures. When either tyrocidine or linear gramicidin was added together with L‐tyrosine, neither sporulation nor peptide antibiotic synthesis was restored. On the other hand, the addition of both tyrocidine and linear gramicidin effectively reversed the inhibition of sporulation by L‐tyrosine. These experiments demonstrate that sporulation of B. brevis depends on either the endogenous synthesis or the addition of both tyrocidine and linear gramicidin. The fact that endogenous as well as exogenous peptides could effect sporulation argues against the involvement of artifacts, such as the depletion of intracellular nucleotide pools caused by the surfactant properties of added peptide antibiotics.
DNA‐Dependent RNA Polymerase III from the Fungus Podospora comata
Authors: BARREAU Christian , BEGUERET Joel
Published: March 2005

DNA‐dependent RNA polymerase III has been purified to homogeneity from the filamentous fungus Podospora comata. The enzyme was extracted at low ionic strength, separated from the polymerases I and II by DEAF‐Sephadex chromatography and purified by heparin‐Sepharose and phosphocellulose chromatography; 0.1–0.2 mg highly purified homogenous enzyme with a specific activity of 220 units/mg could be obtained from 9 kg wet mycelium. The subunit composition of the enzyme was determined after sodium dodecyl sulphate/polyacrylamide gel electrophoresis; thirteen putative subunits of molecular weight 174000 (a), 129000 (b), 87000 (c), 50000 (d), 39000 (e), 23500 (f), 21000 (g), 19000 (h), 17000 (i), 16500 (j), 13500 (k), 11000 (l) and 10000 (m) were identified. All of the polypeptïde components of the enzyme are present in about integral stoichiometric amounts as judged by dyebinding. The presence of subunit M, = 87000 in a molar ratio 1:1 is necessary to obtain very active enzyme. Thirteen homologous subunits were observed in a preparation of RNA polymerase from Podospora anserina, which is a related species. Only subunit i is different in the two species.
Properties of the Charge‐Transfer Transition Observed in Glyceraldehyde‐3‐phosphate Dehydrogenase from Sturgeon Muscle Alkylated by 3‐Chloroacetylpyridine — adenine Dinucleotide
Authors: BRANLANT Guy , TRITSCH Denis , EILER Brigitte , WALLEN Leif , BIELLMANN Jean‐Francois
Published: March 2005

An absorption band at 340 nm is shown to be formed concomitantly with the covalent bond between the affinity label 3‐chloroacetylpyridine‐adenine dinucleotide (clac3PdAD+) and glyceraldehyde‐3‐phosphate om sturgeon. This band corresponds to a charge‐transfer transition. Its intensity depends upon the pH and the ionic strength but is almost independent of the nature of the anions present in the medium. The pH dependence shows an inflexion point at pH 7.1. This result suggests the participation of a residue with a pKa of 7.1 within the active site of the enzyme in the formation of this transition. Using various techniques, the amino acid alkylated by clac3PdAD+ is s OW be the essential Cys‐149, thus excluding the participation of this residue in the formation of the charge‐transfer transition. On the other hand, the modification of Cys‐153 seems not to affect this charge‐transfer band. Other possible donors are proposed, such as the invariant His‐176 or Tyr‐317 residues. These amino acids might be implicated in the formation of the Racker band.
The Antibiotics Kirromycin and Pulvomycin Bind to Different Sites on the Elongation Factor Tu from Esclzerichia coli
Authors: PINGOUD Alfred , BLOCK Wolfgang , URHANKF Claus , WOIF Heinz
Published: March 2005

Pulvomycin and kirromycin, two antibiotics which inhibit protein biosynthesis in Escherichiu coli by complex formation with the elongation factor Tu (EF‐Tu), bind to different sites on the protein. While only one molecule of kirromycin can be bound to one molecule of EF‐Tu, more than one molecule of pulvomycin interacts with a molecule of EF‐Tu. This has been deduced from experiments in which the aminoacyl‐tRNA binding and the GTPase activity of EF‐Tu were measured in the presence of varying amounts of both antibiotics. These experi‐ ments are interpreted to mean that pulvomycin but not kirromycin can replace the other antibiotic in its respective site. Our conclusions are supported by circular dichroism spectroscopy.
Cell‐Associated Glycosaiminoglycans of Human Teratocarcinoma‐Derived Cells of Line PA 1
Authors: RASILO Maija‐Liisa , RENKONEN Ossi
Published: March 2005

Human teratocarcinoma‐derived cells of line PA 1, which are capable of differentiating in vitro [Zeuthen, J. et al. (1980) Int. J. Cancer, 25, 19–32], incorporate label from radioactive sulfate and/or glucosamine into several large‐sized glycosaminoglycans including hyaluronate, chondroitin sulfate/dermatan sulfate co‐polymers, heparan sulfate and keratan‐sulfate‐like molecules. All these polysaccharide fractions were identified by specific degradation methods. The labeled hyaluronate was degraded into a mixture of unsaturated octa‐, hexa‐ and tetra‐ saccharides by a treatment with Strrptomyces hyaluronidase (EC 4.2.2.1). The chondroitin sulfate/dermatan sulfate co‐polymers were cleaved with chondroitin AC lyase (EC 4.2.2.5) into unsaturated disaccharides and a series of unsaturated oligosaccharides; the latter were degraded by a treatment with chondroitin ABC lyase (EC 4.2.2.4) into unsaturated disaccharides. Heparan sulfate was degraded with nitrous acid into free inorganic [35S]sulfate and a series of [35S]sulfate‐labeled oligosaccharides and/or glycopeptides. The keratan‐sulfate‐like molecules were hydrolyzed by a treatment with endo‐β‐galactosidase from Escherichiafieundii into a series of distinct [35S]sulfate‐labeled oligosaccharides; small oligosaccharides were liberated also from [3H]galactose‐labeled molecules. The smallest one of the liberated oligosaccharides was tentatively identified as a sulfated disaccharide.
Organization of Nucleotide Sequences in the Chicken Genome
Authors: OLOFSSON Birgitta , BERNARDI Giorgio
Published: March 2005

The four major components of chicken DNA were prepared by density gradient centrifugation and characterized in several basic properties: relative amounts, dG + dC content, buoyant densities, compositional heterogeneity, and reassociation kinetics. While the relative amounts and the compositions of the major components of chicken DNA were similar to those found in mammalian genomes, their compositional heterogeneities were found to be narrower. The relative amounts of interspersed repeated and unique sequences were strikingly different in different components and also different from those found in the corresponding major components of mouse and human DNAs. If one takes into consideration that major DNA components (a) account for practically all of main‐band DNA and (b) derive by preparative breakage from vey long DNA segments of fairly homogeneous composition, the isochores, our findings indicate that the distribution of interspersed repeats is different chromosomal regions and is species‐specific.
Inhibitory Effect of 1‐β‐d‐Arabinofuranosylcytosine on the Synthesis of Phosphatidyl‐dCMP
Authors: IUJVIDIN Sonia , FELEDI Catalina , MEDRANO Estela E. , MORDOH José
Published: March 2005

The synthesis of phosphatidyl‐dCMP in mouse thymocytes is inhibited by the antineoplastic agent 1‐β‐d‐arabinofuranosyl cytosine (AraCyt)50% inhibition (ID50) being reached at an AraCyt concentration of 0.18 mM. IN the same cells, ID50 for DNA synthesis is 0.03 mM. This inhibition is probably mediated by the phosphorylated derivative of AraCyt (aCTP) since the synthesis of phosphatidyl‐dCMP from dCTP using permeabilized thymocytes is inhibited by a aCTP (ID50= 0.11 mM). The incorporatioin of [3H]AraCyt inbto the organic phase could also be detected, suggesting that this drug may act as a substrate for the enzyme that catalyzes the transfer of dCTP into phosphatidic acid.
Demonstration and Characterisation of a Testicular Receptor for 1,25‐Dihydroxycholecalciferol in the Rat
Authors: MERKE Jürgen , KREUSSER Wilhelm , BIER Bert , RITZ Eberhard
Published: March 2005

Defective luteinizing‐hormone‐mediated cAMP generation in rat testis was previously demonstrated by us in acute and chronic uremia. This defect was abolished by administration of 1,25‐dihydroxycholecalciferol (1,25‐dihydroxycalciol). In the present study, we furnish evidence for a cytosolic 1,25‐dihydroxycalciol receptor in the rat testis.

Testes of non‐rachitic, rachitic or acutely uremic male Sprague‐Dawley rats were homogenized in 0.3 M KCl, 10 mM Tris/HCl, 1.5 mM EDTA, 1 mM dithiothreitol, pH 7.4. Fresh purified cytosol was incubated with 1,25‐[3H]‐dihydroxycalciol (with or without 200‐fold molar excess of unlabeled 1,25‐dihydroxycalciol, subjected to 5–20% sucrose density‐gradient separation (centrifugation with 255000xg, 21 h, 4°C) and analysed with the hydroxyapatite assay. The data document the presence of a macromolecule in testicular cytosol with the properties of the intestinal 1,25‐dihydroxycalciol receptor. The binding macromolecule migrated at 3.5 S and could clearly be distinguished from the 6‐S tissue‐binding site for 25‐hydroxycholecalciferol. The 1,25‐dihydroxycalciol receptor was present in non‐rachitic, rachitic and acutely uremic rats.

Scatchard analysis of the receptor demonstrates high affinity and selectivity (1,25‐dihydroxycalciol ≫ 25‐hydroxycholecalciferol > 1 α‐hydroxycholecalciferol > 24(R),25‐dihydroxycholecalciferol), low capacity (Nmax= 82 fmol/mg protein) and an equilibrium constant of Kd= 2.6 × 10−10 M. The receptor is a protein according to enzyme degradation studies (trypsin, pronase, RNAase, DNAase). Competition studies with and without the alkylating reagent N‐ethylmaleimide demonstrate a cysteine residue in or close to the binding site for 1,25‐dihydroxycalciol. Binding of 1,25‐dihydroxycalciol protects the receptor against inactivation with N‐ethylmaleimide.
The Structure of 11‐S Globulins from Sunflower and Rape Seed
Authors: PLIETZ Paul , DAMASCHUN Gregor , MÜLLER Jürgen Joachim , SCHWENKE Klaus‐Dieter
Published: March 2005

The 11‐S globulin from sunflower seed has a radius of gyration Rs of 3.95 nm and a maximum dimension L of 11.0 nm. For the 11‐S globulin from rape seed Rs and L amount to 4.1 nm and 11.2 nm, respectively. Both molecules have an almost spherical shape with slightly differing dimensions. Both molecules consist of six subunits arranged as a trigonal antiprism with dihedral point group symmetry 32.
NAD‐Linked l(+)‐Lactate Dehydrogenase from the Strict Aerobe Alcaligenes eutrophus
Authors: STEINBÜCHEL Alexander , SCHLEGEL Hans G.
Published: March 2005

The l(+)‐lactate dehydrogenase (EC 1.1.1.27) of Alcaligenes eutrophus catalyzes the NADH‐dependent reduction of pyruvate and a few other 2‐oxoacids. The Km values for NADH, NAD, pyruvate and l(+)‐lactate are 0.075 mM, 0.130 mM, 0.820 mM and 7.10 mM, respectively. The reaction follows a rapid equilibrium ordered bi‐bi mechanism and involves the formation of a dead‐end EBQ complex. The competitive inhibition of pyruvate reduction caused by NAD (with respect to NADH) is regarded to be of physiological importance.

The enzyme is strongly inhibited by oxaloacetate, oxalate and to a less extent by oxamate. Oxaloacetate was found to be the most powerful inhibitor of the enzyme and exerts an almost complete inhibition of the reduction of pyruvate and some 2‐oxoacids at concentrations of 1 μM and less. At 0.1 μM oxaloacetate the inhibition of pyruvate reduction is about 90%.

The kinetics of pyruvate reduction in the presence of oxaloacetate is characterized by a burst phase followed by a decreased steady‐state velocity. During the burst phase, which lasts from several seconds to some minutes, the enzyme undergoes transition to a less active enzyme form. The inhibition studies revealed the lactate dehydrogenase to be a hysteretic enzyme, due to its slow response to the ligand. The characteristics of the transient were examined.

The inhibition of lactate dehydrogenase from A. eutrophus by oxaloacetate is considered to be of great physiological importance, allowing its function only at a low oxaloacetate concentration and consequently at high NADH/NAD ratios.
Determination of Protonation Sites in Thermospermine and in Some Other Polyamines by 15N and 13C Nuclear Magnetic Resonance Spectroscopy
Authors: TAKEDA Yasuko , SAMEJIMA Keijiro , NAGANO Kozo , WATANABE Mayumi , SUGETA Hiromu , KYOGOKU Yoshimasa
Published: March 2005

The pK values of thermospermine, a novel asymmetric tetraamine (4,8‐diazadodecane‐1.12‐diamine), have been determined for the first time by the conventional titration method with subsequent data analyses. It was found to be the most basic polyamine among naturally occurring tetraamines. The 15N NMR titration method was applied to determine its protonation site. Natural‐abundance 15N magnetic resonance spectra of the polyamine have been recorded as a function of pH from its free to the protonated form. Its apparent pK values, as well as the intrinsic chemical shifts for each stage of protonation, have been calculated from the NMR titration curve by using a nonlinear least‐squares method. The four protonation sites of thermospermine have been successfully determined to be N12, N1, N‐8, and N‐4 in that order from the free to the completely protonated form. In a similar manner the protonation sites of spermidine and two symmetric tetraamines, thermine and spermine, have been established. 13C NMR experiments for the four polyamines were almost consistent with those of 15N NMR. Owing to the much higher concentration of the polyamines furnished for NMR titration experiments, the pK values obtained by NMR were larger than those derived by the conventional titration methods.
Nucleosomal Particles Open as the Histone Core Becomes Hyperacetylated
Authors: BODE Jürgen , GÓMEZ‐LIRA M. Macarena , SCHRÖTER Hennrik
Published: March 2005

Lymphoblastoid cells grown in the presence of the deacetylase inhibitor butyrate were used to isolate nucleosomal particles in a hyperacetylated state. During a non‐denaturing gel electrophoresis these particles revealed a heterogeneity which is only in part due to the presence of nonhistone proteins. Monomers that are free from histone H1 and high‐mobility‐group (HMG) proteins 14 and 17 yield a subfractionation according to the degree of core histone acetylation beyond a limiting value of 10 acetyl groups/particle. It is shown that hyperacetylation provides particles with low mobilities and a considerable conformational freedom in contrast to HMG protein 14 which locks them in a conformation that has a similar electrophoretic behaviour but is more defined.
Amino‐Acid Sequences of the Active‐Site Sulfhydryl Peptide and Other Thiol Peptides from the Cysteine Proteinase α‐Clostripain
Authors: GILLES Anne‐Marie , DE WOLF André , KEIL Borivoj
Published: March 2005

One free‐SH group in the heavy chain of α‐clostripain reacts rapidly with N‐tosyllysine chloromethyl ketone which inactivates the enzyme. Iodoacetic acid also reacts with the thiol group required for enzyme activity but more slowly. A tryptic peptide containing the reactive sulfhydryl group labelled with iodo[1‐14C]acetic acid was isolated and determined to be Gln‐Ser‐Val‐Asp‐Leu‐Leu‐Ala‐Phe‐Asp‐Ala‐Cys‐Met. All other cysteine peptides were isolated from the trypsin hydrolysate of the [14C]carboxymethylated enzyme. Moreover N‐terminal and C‐terminal sequences of both chains of α‐clostripain were determined.

The sequences representing 20% of the primary structure of α‐clostripain are not homologus with either other cysteine proteinases or with any other protein structure known to date.
Desulfovibrio Gigas Hydrogenase: Redox Properties of the Nickel and Iron‐Sulfur Centers
Authors: TEIXEIRA Miguel , MOURA Isabel , XAVIER António V. , DERVARTANIAN Daniel V. , LEGALL Jean , PECK Harry D. , HUYNH Boi Hanh , MOURA José J. G.
Published: March 2005

Below 30 K, oxidized Desulfovibrio gigas hydrogenase presents an intense electron paramagnetic resonance (EPR) signal centered at g= 2.02, typical of an iron‐sulfur center. In addition a rhombic EPR signal, attributed to Ni(III) species, is also observed [LeGall, J., Ljungdahl, P., Moura, I., Peck, H. D., Jr, Xavier, A. V., Moura, J. J. G., Teixeira, M., Huynh, B. H., and DerVartanian, D. V. (1982) Biochem. Biophys. Res. Commun. 106, 610–616; and Cammack, R., Patil, D., Aguirre, R., and Hatchikian, E. C., (1982) FEBS Lett. 142, 289–292]. At higher temperatures (77 K) the iron‐sulfur EPR signal is broader and all the EPR features of the rhombic nickel signal can easily be observed. We have now obtained additional information concerning the redox properties of these EPR active centers, using an EPR redox titration method in the presence of dye mediators at pH = 8.5. The mid‐point potential was determined to be ‐ 70 mV for the Fe, S cluster and ‐ 220 mV for the Ni center. Intermediate oxidation states were obtained upon partial reduction with either dithionite or hydrogen. Although upon dithionite reduction the centers are reduced in the order of decreasing mid‐point reduction potentials, under a hydrogen atmosphere the nickel center reduces preferentially. This suggests a catalytic involvement of the nickel redox center in the binding of hydrogen. Preliminary Mössbauer studies on Desulfovibrio gigas hydrogenase reveal the presence of a paramagnetic 3 Fe center and two 4 Fe centers. The 3 Fe center is responsible for the g= 2.02 EPR signal but the two 4 Fe centers have been so far undetectable by EPR.
Gangliosides of Human Chronic Lymphocytic Leukemia and Hairy Cells
Authors: GOFF Barbara A. , LEE William M. F. , WESTRICK Mary Alice , MACHER Bruce A.
Published: March 2005

Gangliosides were purified from the cells of two patients with hairy cell leukemia and one patient with chronic lymphocytic leukemia. Quantification of these componds showed that these cells contain only 5–15% of the amount of lipid‐bound sialic acid (gangliosides) per cell as normal lymphocytes. Structural charaterization by gasliquid chromatography, glycosidase treatment and high‐performance liquid chromatography demonstrated that the major gangliosides of these leukemia cells were of the lactosyl type. Hairy cells contained monosialyl‐lactosylceramide (II3NeuAc‐LacCer), whereas chronic lymphocytic leukemia cells contained both monosialyl and disialyl lactosylceramide [II3 (NeuAc)2‐LacCer]. Chronic lymphocytic leukemia cells contained lesser amounts of three other gangliosides of the neolacto or lacto series as determined by endo‐β‐galactosidase treatment. None of these leukemia cells contained detectable quantities of NeuAc‐LcOse3Cer, a ganglioside found in normal leukocytes.
Hydroxylamine Mutagenesis: Observation of Inverted Watson‐Crick Base‐Pairing Between N4‐Methoxycytosine and Adenine with the Aid of Natural‐Abundance High‐Resolution 15N NMR Spectroscopy
Authors: KIERDASZUK Borys , STOLARSKI Ryszard , SHUGAR David
Published: March 2005

Natural‐abundance high‐resolution 15N NMR spectra have been recorded for 1‐methyl‐N4‐methoxycytosine (the product of reaction of 1‐methylcytosine with the mutagen methoxyamine), 2′,3′,5′‐tri‐O‐methyladenosine, and a 1:1 mixture of the two, all in chloroform solution. The spectra were obtained by application of a sequence of impulses of the type referred to as insensitive nuclear enhancement by polarization transfer (INEPT). Assignments of all the nitrogen resonances were based on nitrogen‐proton coupling constants and literature data. Analysis of the 15N chemical shifts demonstrated the formation of base pairs with hydrogen bonds involving the ring N(1) and an N6‐H of adenine, and the N(3)‐H and C(2)=O of N4‐methoxycytosine, viz. N(1)·H‐N(3) and N6‐H·O2=C. The hydrogen‐bond‐stabilized complex formed thus involves an inverse type of Watson‐Crick base‐Pairing. The data excluded formation of Hoogsteen‐type base‐pairing. The results are considered in relation to hydroxylamine and methoxyamine mutagenesis, to types of base‐pairing at the monomer and polymer levels, and to interpretations of 15N NMR spectra of heterocyclic rings in general.
Occurrence of an Inhibitory Guanine Nucleotide‐Binding Regulatory Component of the Adenylate Cyclase System in cyc− Variants of S49 Lymphoma Cells
Authors: JAKOBS Karl H. , GEHRING Ulrich , GAUGLER Bernhard , PFEUFFER Thomas , SCHULTZ Günter
Published: March 2005

In membranes of S49 lymphoma cyc− variants, which lack a functional guanine nucleotide‐binding component (Ns protein) mediating adenylate cyclase stimulation by hormones, guanine nucleotides decreased the forskolin‐stimulated adenylate cyclase activity by maximally 40–60%. The potency order of the guanine nucleotides studied was guanosine 5′‐[γ‐thio]triphosphate(GTP[S])> guanosine 5′[β,γ‐imido]triphosphate ≫ GTP > guanosine 5′‐[β‐thio]diphosphate (GDP[S]). GTP and GDP[S] acted as partial inhibitors; they competitively antagonized the GTP[S]‐induced inhibition, which was half‐maximal and maximal at about 3 nM and 100 nM GTP[S], respectively. Cholera toxin did not enhance the inhibitory action of GTP. The cyc− adenylate cyclase inhibition by GTP[S] occurred after a short lag period and persisted after washing the membranes. The inhibition was not affected by the pH (6.5–9.0) of the incubation medium and was largely independent of the concentrations of MgATP (up to 200 μM) and Mg2+ (up to 10 mM). In contrast, Mn2+ potently reduced the GTP[S]‐induced cyc− adenylate cyclase inhibition. Similarly as observed with forskolin, GTP[S] decreased the cyc− adenylate cyclase activity stimulated by purified, preactivated Ns protein. Apart from Mn2+, inhibition of the cyc− adenylate cyclase by GTP[S] was prevented or reversed by various treatments, which have been shown to obliterate hormone‐induced adenylate cyclase inhibition in other cell types, such as by N‐ethylmaleimide, by limited proteolysis with trypsin and by a factor extracted from bovine sperm particles. The data indicate that in membranes of cyc− variants, which lack a functional Ns protein, a guanine nucleotide‐binding component (Ni) is present, which mediates adenylate cyclase inhibition by guanine nucleotides. Such a component apparently also mediates adenylate cyclase inhibition by hormones in other cell types. Comparison of adenylate cyclase stimulation and inhibition by guanine nucleotides suggests that Ni is activated and inactivated by similar but not identical mechanisms as the Ns protein and that the relative activity states of these coupling components determine the activity of the adenylate cyclase.
Activation of the Enzymic Activity of Hepatic Lipase by Apolipoprotein A‐II
Authors: JAHN Claus E. , OSBORNE James C. , SCHAEFER Ernst J. , BREWER H. Bryan
Published: March 2005

Human post‐heparin plasma contains two major lipases, lipoprotein lipase and hepatic lipase. Lipoprotein lipase has been investigated extensively and in the presence of a specific activator, apolipoprotein C‐II, is of major importance in modulating the hydrolysis of triacylglycerol rich lipoprotein particles in plasma. The metabolic function and regulation of hepatic lipase is less well established. We have reported previously that apolipoprotein A‐II(apoA‐II)in vitro increased the activity of hepatic lipase by 3.6‐fold [Jahn et al. (1981) FEBS Lett. 131, 366–368]. We have extended these studies and report here the effects of normal plasma, very low density, low density and high density lipoproteins (VLDL, LDL and HDL) and apolipoproteins A‐I, A‐II, C‐I, C‐II and C‐III on hepatic lipase enzymic activity. Normal plasma and HDL activated hepatic lipase by 255 ± 53% (n = 9) and 288 ± 55% (n = 7) respectively, whereas VLDL inhibited hepatic lipase to 25% and LDL did not affect hepatic lipase enzymic activity (n = 7). Isolated apoA‐I, apoC‐I, apoC‐II, and apoC‐III inhibited hepatic lipase enzymic activity to 25 ± 8% (n = 5), 25 ± 15% (n = 5), 23 ± 7% (n = 5) and 13 ± 10% (n = 5) of the basal level.

ApoA‐II recombined with HDL lipids activated by 273 ± 42% (n = 5) whereas apoC‐III recombined with HDL lipids inhibited hepatic lipase to 30% of basal activity. Activation of hepatic lipase by apoA‐II was dependent on temperature and pH, with maximal activation (5.75‐fold over basal levels) occurring at pH 7.5 and 37°C. The addition of lipid‐free apoA‐II 10 min after the hydrolysis of triacylglycerol by hepatic lipase had begun resulted in an increase in rate from 1.9 nmol min−1 ml−1 to 7.0 nmol min−1 ml−1. The rate observed by the addition of apoA‐II before the start of the reaction was 9.5 nmol min−1 ml−1. These results are consistent with the concept that apoA‐II modulates the enzymic activity of hepatic lipase, and may play a role in vivo in the regulation of hepatic lipase activity and HDL lipoprotein metabolism.
Serum‐Induced Stimulation of Nucleoplasmic and Nucleolar Transcription in Mouse 3T3 Fibroblasts Revisited
Authors: TIERCY Jean‐Marie , WEIL Roger
Published: March 2005

Transition of 3T3 mouse fibroblasts from a quiescent to a growing state was induced by the addition of 10% fresh bovine serum to the culture medium. The number of DNA‐synthesizing cells began to increase 10‐11 h after the addition of serum and reached a maximum of 70‐80% around 24 h. In quiescent cells, maintained in 0.5% serum, residual RNA synthesis represented manly nucleoplasmic transcription (hn RNA, 5‐S RNA and tRNA). Synthesis of tRNA was 2‐3‐fold increased by 1‐2 h after addition of serum; however, a significant stimulation of hnRNA, 5‐S RNA and 45‐S pre‐rRNA synthesis could only be observed around 4 h. The experimental data also revealed a close temporal relationship between the oneset of serum‐stimulated hnRNA and overall protein synthesis. Determined colorimetrically, the cellular RNA and protein content began to increase by 4–5 h and had doubled by 24 h. Virtually the same results on RNA and protein synthesis were obtained when the experiments were performed in the presence of cytosine arabinoside, and inhibitor of DNA synthesis. From our results we concluded that serum‐stimulated overall RNA and protein synthesis preceded by several hours serum‐induced S phase and was independent of DNA replication.
Maturatio of 5‐S rRNA: Ribonuclease E Cleavages and Their Dependence on Precursor Sequences
Authors: Roy Monoj K. , SINGH Balraj , RAY Bimal K. , APIRION David
Published: March 2005

9‐S RNA is a processing intermediate that accumulates in an RNase E− strain of Escherichia coli. It spans from the RNase III cleavage site, after 23‐S rRNA, to the 3′ end of the transcript and is derived from rRNA genes which do not contain tRNAs distal to 5‐S rRNA. Here, we have studied the processing of 9‐S RNA with ribonuclease E. RNase E cleaves 9‐S RNA in two sites: one of these is three nucleotides upstream from the 5′ end of 5‐S rRNA, the other downstream from its 3′ end. Both cleavages are probably introduced by the same enzyme, since both cleavages are thermolabile when an extract of a temperature‐sensitive RNase E mutant was used for processing in vitro.

In order to asses the role of 5′ and 3′ end precursor‐specific sequences in the RNase E reaction, we isolated the molecules lacking nucleotides at the 5′ or 3′ end. Molecules having the 5′ end of 9‐S RNA but missing nucleotides from the 3′ end (called 8‐S RNA) were as good a substrate for RNase E as 9‐S, RNA itself. However, molecules having the 3′ end of 9‐S RNA but the 5′ end of p5 (called 7‐S RNA), were less efficient substrates for RNase E. Finally, the removal of as little as seven nucleotides from the 5′ end of 8‐S RNA rendered it almost completely unstuitable as a substrate for RNase E.
Sequence Analysis of the Cloned mRNA Coding for Glyceraldehyde‐3‐Phosphate Dehydrogenase from Chicken Heart Muscle
Authors: DOMDEY Horst , WIEBAUER Karin , KLAPTHOR Heidi , ARNOLD Hans Henning
Published: March 2005

Using a cloned cDNA (pGAP30) the nucleotide sequence for chicken glyceraldehyde‐3‐phosphate dehydrogenase mRNA has been determined. The cDNA insert contains 1051 nucleotides representing the amino acid coding sequence, with the exception of 49 NH2‐terminal amino acids, and includes the entier 3′‐noncoding region. Sequence information on the missing 5′ terminus of the mRNA, not represented in the clone pGAP30, was obtained by extension of the cDNA using an 85‐nucleotide‐long internal fragment as a primer. Thus the sequence of 310 amino acids of chicken glyceraldehyde‐3‐phosphate dehydrogenase representing 93% of the complete primary structure could be derived. The coding portion exhibits non‐random utilization utilization of synonymous codons with a strong bias for codons with G or C at the third position. The non‐coding region contains several octanucleotides which are repeated and shows a potentially stable stem‐and‐loop structure located towards the end of the mRNA. Hypothetical functional implications of the putative secondary structure are discussed.
Immunological Evidence for Apoproteins of the Light‐Harvesting Chlorophyll‐Protein Complex in a Mutant of Barley Lacking Chlorophyllb
Authors: RYRIE Ivan J.
Published: March 2005

A barley mutant lacking chlorohyllband the pigmented light‐harvesting chlorophyll‐protein of photo‐system 2 is shown by several criteria to contain functional apoproteins of the light‐harvesting complex.

Electrophoretic comparison of thylakoid polypeptide Patterns, and the effects of trypsin treatment on these, seuggests that the mutant contains several polypeptides equivalent in mobility to those of the wild‐type complex.

An antibody monospecific for the light‐harvesting complex agglutinated both wild‐type and mutant thylakoids.

‘Western blot’ immunoelectrophoretic analysis indicated that of four distinct subunits of the light‐harvesting complex in the wild‐type thylakoids, three are detectable in the mutant.

As in wild‐type lamellae at least one of the light‐harvesting complex polypeptides is phosphorylated by the endogenous protein kinase. The results are considered in terms of a general role for the light‐harvesting complex polypeptides in membrane appression and the regulation of excitation energy distribution within thylakoids.
Experimental Evidence and Theoretical Discussion for Long‐Term Oscillations of Phosphofructokinase in a Compartmentalized System
Authors: HERVAGAULT Jean‐François , THOMAS Daniel
Published: March 2005

The activity of rabbit muscle phosphofructokinase (EC 2.7.1.11) has been followed as a function of time under conditions where the enzyme is separated from the bulk solution by an inert membrane.

An enzymatic coupling assay allows continuous measurement of the variations in NADH concentration, which is directly related to the enzyme catalytic activity. For given concentrations of substrates (ATP and Fru6P) in the outside reservoir and a given ratio between diffusion coefficients of both substrates, the activity of phosphofructo‐kinase exhibits an oscillatory behavior during a period of about 5 h. The phenomenon is explained in terms of coupling between diffusion of metabolites and non‐linear enzyme reaction.
Faithful Transcription of Ribosomal 5‐S RNA in vitro Depends on the Presence of Several Factors
Authors: SHI Xiao‐Ping , WINGENDER Edgar , BÖTTRICH Johannes , SEIFART Klaus H.
Published: March 2005

Cytoplasmic extracts from HeLa cells, capable of transcribing the cloned genes for ribosomal 5‐S RNA, were employed to study the factors involved in this process. Two factors can be isolated, by gel filtration through Sephadex G‐100, which are devoid of RNA polymerase activity. They significantly enhance the extent and specificity of the transcription of 5‐S rRNA. Both proteins can jointly be purified by affinity chromatography on immobilized DNA containing the genes for ribosomal 5‐S RNA from Xenopus borealis. Besides a protein of approximately 45 kDa, possibly corresponding to TF IIIA isolated from Xenopus oocytes, a second protein with a molecular mass of 22 ± 1 kDa stimulates the formation of 5‐S RNA. This protein is contained in the breakthrough of DEAE‐cellulose; it binds to and is eluted from phosphocellulose with 0.6 M KCl.

In addition, it was found that the exclusion volume obtained after gel filtration on Sephadex G‐100 contains functional complexes, which are capable of transcribing the cloned 5‐S genes and hence contain all the required factors. Direct evidence is presented that the protein of 22 kDa described above is contained in and can be isolated from such complexes. It is postulated from indirect evidence that an additional factor with a molecular mass in excess of 100 kDa is required which can be removed from functional polymerase complexes by gel filtration through Bio‐Gel A5m.
Isolation of a 3‐Deoxy‐d‐mannooctulosonic Acid Disaccharide from Salmonella minnesota Rough‐Form Lipopolysaccharides
Authors: BRADE Helmut , GALANOS Chris , LÜDERITZ Otto
Published: March 2005

Lipopolysaccharides of Salmonella minnesota rough mutants were treated with 20 mM acetate buffer pH 4.4 at 70°C/3 h. After dialysis of the hydrolysates about one third of the total 3‐deoxy‐d‐mannooctulosonic acid (dOclA) content but no neutral sugars were found in the dialysate. By high‐voltage paper electrophoresis, a compound with the mobility of 1.2 relative to dOclA could be isolated from the dialysate. It was identified as a dOclA disaccharide by hydrolysis without or after reduction with sodium borohydride and by analysis with the thiobarbituric acid assay under different conditions. The ketosidic linkage in the disaccharide is assumed to be 2.4 or 2.5.
Activity Patterns of Aminoacyl‐tRNA Synthetases, tRNA Methylases, Arginyltransferase and Tubulin: Tyrosine Ligase during Development and Ageing of Caenorhabditis elegans
Authors: GABIUS Hans‐Joachim , GRAUPNER Gerhart , CRAMER Friedrich
Published: March 2005

As a step in the characterization of development and ageing in the nematode Caenorhabditis elegans, the activities of different groups of enzymes that supposedly exert modulating functions in and after protein synthesis have been determined. From embryonic (E), the four juvenile larval stages (L1–L4) and the gravid adult (A, A+), the selection of defined developmental stages extends to two different preparations of aged nematodes (S10, S12). Some aminoacyl‐tRNA synthetase activities remain nearly unchanged in all stages up to the adult, some increase continuously during the larval stages and the remaining activities show stage‐specific alterations. Upon ageing all activities except the one for tryptophan decrease sharply. tRNA methylase activities increase from E to L4, decrease from L4 to adult and to aged nematodes with only qualitative alterations in substrate specificity. The activity of tubulin:tyrosine ligase exhibits a parallel pattern, while arginyltransferase activity has a plateau between L2 and L4. The results are consistent with the idea of a modulation of protein synthesis and other cellular processes by quantitative activity changes during development and ageing.
Comparison of the Physical Maps and Redundant Ends of thre Chromosomes of Phages 2C, SP01, SP82 and φe
Authors: HOET Philippe , COENE Marc , COCITO Carlo
Published: March 2005

The physical map of 2C DNA (cf. following paper in thi9s journbal) was compared to the maps of SP01, SP82 and φe (Three other Bacillus Subtilis phages containing hydroxymethluracil in place of th ymine in their DNA). The overall organizatio9n of the four genomes was remarkab le similar, as indicated by the topology of Hae III and Sal I cleavage sements. The proof was gatheredfor ther pressence in the four phage DNAs of large redundant ends carerying a single Hae III recognition site. The location of the latter proved identical for 2C and SP01, but to two shifted in the DNAs of SP82 and φe. Since the redundant end components of these hydroxymethluracil genomes are colinear, as shown by cross‐hbridization studies, the shifting of the Hae III cleavage site is presumabley due to tow base substitutions, suppressing an endonulease recognition site and establishing a new site elsewhere. Relateddness between the genomes of this family of viruses was evaluated from the fraction of conserved restriction fragments. According to these calculation, 6% base substitutions have occurred within the four viral DNA, as shown by crosshybridization with restroiction fragments. These data indicate the occurrence of deletions, in addition to base subsitutions, as evolutionary mechanisms prevailing in the genomes of this family fo phages.