bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced stability of RNA coding for yolk polypeptide 3 in Drosophila melanogaster ovary
Authors: WILLIAMS John Lewis , BOWNES Mary
Published: March 2005

In Drosophila three yolk polypeptides (YP1, YP2 and YP3) are synthesised at two sites in the adult female: in the fat body tissue, from which they are transported via the haemolymph to the ovary, and in the ovarian follicle cells which surround the developing oocytes. All three yolk polypeptides are synthesised at equal levels in the fat body. In this paper we show that the steady‐state level of YP3 RNA is significantly reduced in the ovary in comparison with the fat body, and that none of the yolk protein genes is amplified either in the fat body or the follicle cells.

In order to determine the basis of the reduced level of YP3 RNA in the ovary, which could result from a lower rate of transcription or through a decreased stability of the RNA, we have devised an in vivo method of determining relative rates of gene transcription. In both the fat body and the ovary all three yolk proteins are transcribed at similar rates. Thus we infer that YP3 RNA is destablished in the ovary, accounting for the reduction in its steadystate level.
A (re)initiation‐dependent cell‐free protein‐synthesis system from mouse erythroleukemia cells
Authors: BADER Michael , SARRE Thomas F.
Published: March 2005

Cultured mouse erythroleukemia cells (MEL cells) can be induced in vivo to erythroid differentiation which is marked by the onset of globin mRNA and haemoglobin synthesis. When these cells are briefly exposed to hypertonic growth medium prior to lysis, the resulting post‐mitochondrial supernatants show a high in vitro protein‐synthesis activity. Amino acid incorporation is linear up to 60 min; more than 80% of this is due to (re)initiation, as shown by the inhibition with edeine. Extracts from induced cells reach only a third of overall incorporation as compared to extracts from uninduced cells. This reduction of the protein‐synthesizing capacity is also observed in vivo. Polyacrylamide gel electrophoresis shows that extracts from uninduced cells faithfully translate their endogenous mRNA, whereas in extracts from induced cells, non‐globin protein synthesis is reduced and globin is preferentially synthesized. Haemin (40 μM) as well as purified eukaryotic initiation factor 2 (eIF‐2) from rabbit reticulocytes enhance amino acid incorporation in both kinds of extracts, which suggests that both uninduced and induced MEL cells contain a haemin‐controlled eIF‐2α kinase. This system should be useful for studying the mechanisms controlling protein synthesis in a nucleated differentiating cell.
Structural relationships in the adenylate kinase family
Authors: SCHULZ Georg E. , SCHILTZ Emile , TOMASSELLI Alfredo G. , FRANK Rainer , BRUNE Martin , WITTINGHOFER Alfred , SCHIRMER R. Heiner
Published: March 2005

The sequences of five distantly related adenylate kinases have been aligned. The local conservation of amino acids is discussed in the light of the known three‐dimensional structure of one of the enzymes, the cytosolic isoenzyme 1 (AK1) from porcine muscle. The similarity profile outlines clearly the active site in the cleft of the spatial structure of AK1. The alignment reveals further that the enzyme family can be subdivided into small and large variants according to the presence or absence of a particular segment of about 30 residues in the middle of the chain. The extra segments of the large variants are strongly conserved.
Catalysis by enzymes entrapped into hydrated surfactant aggregates having lamellar or cylindrical (hexagonal) or ball‐shaped (cubic) structure in organic solvents
Authors: KLYACHKO Natalya L. , LEVASHOV Andrey V. , PSHEZHETSKY Alexey V. , BOGDANOVA Natalya G. , BEREZIN Iliya V. , MARTINEK Karel
Published: March 2005

Instead of aqueous solutions, universally recognized in enzymology, ternary systems of the water/organic solvent/surfactant type are suggested as liquid‐crystalline media for enzymatic reactions. Two systems, water/octane/Aerosol OT and water/cyclohexane/Brij 96, have been used to solubilize acid and alkaline phosphatases and peroxidase. The enzymes under study do function in liquid‐crystalline mesophase having lamellar, cylindrical (reversed hexagonal) and ball‐shaped (reversed cubic) packing of the surfactant molecules. A significant result is that the phase transition from one liquid‐crystalline structure to another entails, as a rule, a reversible change in the catalytic activity of the solubilized enzyme.
ADP‐ribosylation of platelet actin by botulinum C2 toxin
Authors: AKTORIES Klaus , ANKENBAUER Thomas , SCHERING Beate , JAKOBS Karl H.
Published: March 2005

Botulinum C2 toxin is a microbial toxin which possesses ADP‐ribosyltransferase activity. In human platelet cytosol a 43‐kDa protein was ADP‐ribosylated by botulinum C2 toxin. Labelling of the 43‐kDa protein using [32P]NAD as substrate was reduced by unlabelled NAD and nicotinamide. The label was removed by treatment with snake venom phosphodiesterase. Half‐maximal and maximal ADP‐ribosylation occurred at 0.1 μg/ml and 3 μg/ml botulinum C2 toxin, respectively. The Km value of the ADP‐ribosylation reaction for NAD was about 1 μM. The peptide map of the ADP‐ribosylated 43‐kDa protein was almost identical with platelet actin. The ADP‐ribosylated 43‐kDa substrate protein bound to and was eluted from immobilized DNase I in a manner similar to G‐actin. Trypsin treatment of platelet cytosol decreased subsequent ADP‐ribosylation of the 43‐kDa protein without occurrence of smaller labelled polypeptides. Purified platelet actin was also ADP‐ribosylated by botulinum C2 toxin with similar characteristics found with actin in platelet cytosol. Phalloidin decreased the ADP‐ribosylation of actin in platelet cytosol and of isolated platelet actin. Half‐maximal and maximal, about 90%, reduction of actin ADP‐ribosylation was observed at 0.4 μM and 10μM phalloidin, respectively. ADP‐ribosylation of purified actin, induced by botulinum C2I toxin, abolished the formation of the typical microfilament network. The data indicate that platelet G‐actin but not F‐actin is a substrate of botulinum C2 toxin and that this covalent modification largely affects the functional properties of actin.
Substrate specificity of protein kinase C
Authors: WOODGETT James R. , GOULD Kathleen L. , HUNTER Tony
Published: March 2005

Although the Ca2+/phospholipid‐dependent protein kinase, protein kinase C, has a broad substrate specificity in vitro, the enzyme appears considerably less promiscuous in vivo. To date only a handful of proteins have been identified as physiological substrates for this protein kinase. In order to determine the basis for this selectivity for substrates in intact cells, we have probed the subsrate primary sequence requirements of protein kinase C using synthetic peptides corresponding to sites of phosphorylation from four of the known physiological substrates. We have also identified the acetylated N‐terminal serine of chick muscle lactate dehydrogenase as an in vitro, site of phosphyorylation for this protein kinase. These comparative studies have demonstrated that, in vivo, the enzyme exhibits a preference for one basic residue C‐terminal to the phosphorylatable residue, as in the sequence: Ser/Thr‐Xaa‐Lys/Arg, where Xaa is usually an uncharged residue. Additional basic residues, both N and C‐terminal to the target amino acid, enhance the Vmax and Km parameters of phosphorylation. None of the peptides based on physiological phosphrylation sites of protein kinase C was an efficient substrate of cAMP‐dependent protein kinase, emphasizing the distinct site‐recognition selectivities of these two pleiotropic protein kinases. The favorable kinetic parameters of several of the synthetic peptides, coupled with their selectivity for phosphorylation by protein kinase C, will facilitate the assay of this enzyme in the presence of other protein kinases in tissue and cell extracts.
Comparative binding of wheat germ agglutinin and its succinylated form on lymphocytes
Authors: MOULLIER Philippe , DAVELOOSE Denis , LETERRIER François , HOEBEKE Johan
Published: March 2005

We have compared the binding parameters of native wheat germ agglutinin (WGA) and its succinylated form (SWGA) to rat lymphocytes. Scatchard plots were obtained with the fluoresceinated lectins in a concentration range of 10 nM to 0.1 mM. Association and dissociation rate parameters were also measured. The following differences were observed: at low concentration of WGA, binding is positively cooperative with a Hill coefficient of 1.75, whereas binding of SWGA is not. The numbers of high‐affinity sites are respectively (2.5 ± 0.8) × 106 and (6.4 ± 1.3) × 105 for WGA and SWGA. Association constants were found to be (4.7 ± 1.7) × 106 l mol−1 for WGA and (1.42 ± 0.36) × 107 l mol−1 for SWGA, which is 35 times higher than for native WGA. Neuraminidase treatment decreases the Hill coefficient as well as the number of sites involved in the cooperative binding of native WGA. Equilibrium data were obtained at three temperatures to determine the thermodynamic parameters (ΔH° and ΔS°). These results are indicative of an oligomerization process dynamically formed at the membrane level before tight binding of the lectin to its receptors could occur.
Metabolism of 25‐hydroxycholecalciferol in human promyelocytic leukemia cells (HL‐60)
Authors: ISHIZUKA Seiichi , MATSUI Toshimitsu , NAKAO Yoshinobu , FUJITA Takuo , OKABE Tetsuro , FUJISAWA Michio , WATANABE Junichi , TAKAKU Fumimaro , BISHOP June E. , REICHEL Helmut , NORMAN Anthony W.
Published: March 2005

Human promyelocytic leukemia cells incubated with 25‐hydroxy[26,27‐methyl‐3H] cholecalciferol (1 μCi) or non‐radioactive 25‐hydroxycholecalciferol (550 μg) produced significant quantities of two vitamin D3 metabolites. The two metabolites were isolated and purified by methanol chloroform extraction and a series of chromatographic procedures. The metabolite purification and elution positions on these columns were followed by radioactivity and their ultraviolet absorption at 310 nm. The two metabolites have been unequivocally identified as (5Z)‐ and (5E)‐19‐nor‐10‐oxo‐25‐hydroxycholecalciferol by ultraviolet absorption spectrophotometry, mass spectrometry, Fourier‐transform infrared spectrophotometry and co‐chromatography with synthetic compounds on a high‐performance liquid chromatograph. (5E)‐ but not (5Z)‐19‐nor‐10‐oxo‐25‐hydroxycholecalciferol was able to induce HL‐60 cell phenotypic and functional differentiation. However, these two metabolites of 25‐hydroxycholecalciferol did not bind specifically to the chick intestinal 3.7 S. receptor protein for 1α,25‐dihydroxycholecalciferol, The precise biological role of these metabolites is as yet unclear.
Core region of Citrobacter lipopolysaccharide from strain PCM 1487
Authors: ROMANOWSKA Elzbieta , GAMIAN Andrzej , DABROWSKI Janusz
Published: March 2005

The core structure of Citrobacter PCM 1487 lipopolysaccharide has been established using methylation analysis/mass spectrometry, chemical degradations and one‐ and two‐dimensional 1H‐NMR spectroscopy at 500 MHz. 1H‐NMR assignments are given for all sugar components of the core oligosaccharide. In the formula shown below, the alternative locations of branch terminal heptose (LD Hep) and diphosphorylethanolamine (PPEtN) residues are marked by dashed lines; dOclA stands for 3‐deoxy‐D‐manno‐octulosonic acid.

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The sample of the core oligosaccharide showed some microheterogeneity due to a slightly incomplete substitution by terminal N‐acetylgalactosamine and a partial splitting of diphosphorylethanolamine residues.
Hexokinase PII from Saccharomyces cerevisiae is regulated by changes in the cytosolic Mg2+‐free ATP concentration
Authors: MORENO Fernando , FERNANDEZ Teresa , FERNANDEZ Rosa , HERRERO Pilar
Published: March 2005

Hexokinase PII is not inhibited by high Mg‐ATP concentrations if the Mg2+‐free ATP is kept at low levels (0.01 mM) in the assay mixture. Hexokinase PI activity is not affected either by Mg2+‐free ATP nor by free Mg2+ in the assay mixture. Thus, hexokinase PI and PII activities appear not to be regulated by substrate inhibition as proposed previously [Kopetzki, E. & Entian, K. D. (1985) Eur. J. Biochem. 146, 657–662]. However, the level of Mg2+‐free ATP in the hexokinase PII assay mixture strongly affects the enzyme activity by decreasing the Vmax and increasing the Km value for Mg‐ATP from 0.15 mM to 5.0 mM.

The physiological role of this inhibition, which has not been described previously, was investigated by determining the cytosolic ATP and Mg2+ concentrations in yeast cells grown under derepressing and repressing conditions. Derepression is accompanied by an important loss of Mg2+ from the cells, maintaining the ATP concentration constant. This produces an increase of Mg2+‐free ATP in the cytosol from 0.01 mM to 0.1 mM. This free ATP concentration would lead to a maximal inhibition of hexokinase PII.
Erabutoxin b
Authors: LOW Barbara W. , CORFIELD Peter W. R.
Published: March 2005

A study has been made, following high‐resolution refinement at 0.14 nm, of the structure of erabutoxin b, prototype postsynaptic neurotoxin from snake venom. The detailed patterns of intramolecular van der Waal's interactions have been determined. From information, hitherto unavailable, about atomic temperature parameters, the relative mobilities in different regions of the molecule have been estimated. A detailed model of structure/function relationships in these neurotoxins, which bind to the acetylcholine receptor, has thus been established: the probable dynamic mode of toxin‐receptor binding is described. The model identifies, and the binding mode depends on a unique structural feature of these protein toxins: the hydrophobic ‘Trp’ cleft. Chargecharge interactions are implicated in initial toxin orientation on the receptor surface. Possible reactive‐site extension in short‐chain toxins is described. Modifications in binding mode of long‐chain toxins are considered. The relative mobilities of antigenic site residues are discussed.
Stereoselective photoaffinity labelling of the purified 1,4‐dihydropyridine receptor of the voltage‐dependent calcium channel
Authors: STRIESSNIG Jörg , MOOSBURGER Kurt , GOLL Alexandra , FERRY David R. , GLOSSMANN Hartmut
Published: March 2005

The voltage‐dependent calcium channel from guinea‐pig skeletal muscle T‐tubules has been isolated with a rapid, two‐step purification procedure. Reversible postlabelling of the channel‐linked 1,4‐dihydropyridine receptor and stereoselective photolabelling as a novel approach were employed to assess purity. A 135‐fold purification to a specific activity of 1311 ± 194 pmol/mg protein (determined by reversible equilibrium binding with (+)‐[3H]PN200‐110) was achieved. Three polypeptides of 155 kDa, 65 kDa and 32 kDa were identified in the purified preparation. The 155‐kDa band is a glycoprotein. The arylazide photoaffinity probe (−)‐[3H]azidopine bound with high affinity to solubilized membranes (Kd= 0.7 ± 0.2 nM) and highly purified fractions (Kd= 3.1 ± 2 nM), whereas the optical antipode (+)‐azidopine was of much lower affinity. Irradiation of (−)‐[3H]azidopine and (+)‐[3H]azidopine receptor complexes with ultraviolet light led to preferential incorporation of the (−) enantiomer into the 155‐kDa polypeptide in crude solubilized and purified preparations. The pharmacological profile of irreversible labelling of the 155‐kDa glycoprotein by (−)‐[3H]azidopine is identical to that found in reversible binding experiments. Specific photolabelling of the 155‐kDa band by (−)‐[3H]azidopine per milligram of protein increases 150‐fold upon purification, whereas incorporation into non‐specific bands in the crude solubilized material is identical for both, (−) and (+)‐[3H]azidopine.
Isolation and characterization of basement membrane and cell proteoheparan sulphates from HR9 cells
Authors: KELLER Ruprecht , FURTHMAYR Heinz
Published: March 2005

The mouse teratocarcinoma cell line HR9 was investigated for proteoheparan sulphate production. Four species of proteoheparan sulphate molecules were isolated and purified to homogeneity. The proteoheparan sulphate isolated from the tissue‐culture medium contains four heparan sulphate side‐chains of 25 kDa each, and its core protein has an approximate molecular mass of 50 kDa. The proteoheparan sulphates associated with the cells were separated into three individual species: cell proteoheparan sulphate I exhibits structural chracteristics which are very similar to the proteoheparan sulphate isolated from the tissue culture medium; cell proteoheparan sulphates II and III contain one heparan sulphate chain of 25 kDa and 20 kDa, and core proteins of approximately 30 kDa and 25 kDa respectively. Antisera, raised against the medium form, react specifically with basement membranes in various tissues by immunofluorescence. This staining pattern was compared to the pattern observed with an antiserum which we have obtained to a proteoheparan sulphate species isolated from the plasma membrane of bovine aortic endothelial cells. The structural and immunological data suggest that basement membrane and plasma membrane proteoheparan sulphates are different biosynthetic products and are not directly related to each other.
Calmodulin‐dependent multifunctional protein kinase
Authors: SHENOLIKAR Shirish , LICKTEIG Ron , HARDIE D. Grahame , SODERLING Thomas R. , HANLEY Rochelle M. , KELLY Paul T.
Published: March 2005

Calcium/calmodulin‐dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross‐reactivity with affinity‐purified polyclonal antibodies to the 50‐kDa catalytic subunit of the brain calmodulin‐dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One‐dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases with S. aureus V8 protease, and two‐dimensional fingerprints of the 125I‐labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56‐kDa and/or 58‐kDa polypeptides of the skeletal muscle calmodulin‐dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50‐kDa and 60‐kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two‐dimensional peptide fingerprints, indicated considerable structural homology among the 50‐kDa and 60‐kDa subunits of the brain calmodulin‐dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51‐kDa subunit, skeletal muscle 56‐kDa, and the brain 50‐kDa and 60‐kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin‐dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3–5 mol phosphate/mol protein. The two‐dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of 32P‐labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin‐dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin‐dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin‐dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.
Phosphorylation of the glycogen‐binding subunit of protein phosphatase‐1G by cyclic‐AMP‐dependent protein kinase promotes translocation of the phosphatase from glycogen to cytosol in rabbit skeletal muscle
Authors: HIRAGA Akira , COHEN Philip
Published: March 2005

The glycogen‐bound form of protein phosphatase‐1 (termed protein phosphatase‐1G) is composed of the catalytic (C) subunit complexed to a glycogen‐binding (G) subunit that anchors the enzyme to glycogen [Strålfors et al. (1985) Eur. J. Biochem. 149, 295–303]. Incubation of purified protein phosphatase‐1G with cyclic‐AMP‐dependent protein kinase and MgATP, which leads to stoichiometric phosphorylation of the G‐subunit [Caudwell et al. (1986) FEBS Lett. 194, 85–90], was found to promote the release of the phosphatase from glycogen; similar observations were made using glycogen‐protein particle preparations. An intravenous injection of adrenaline decreased protein phosphatase‐1 activity associated with the glycogen‐protein particles by 50% with a corresponding increase in the amount present in the cytosol. By contrast, adrenaline did not affect the distribution of glycogen synthase or glycogen phosphorylase which remained entirely bound to glycogen in these experiments. The specific release of protein phosphatase‐1 from glycogen may facilitate its inactivation by inhibitor‐1 in the cytosol, thereby preventing dephosphorylation of the glycogen metabolising enzymes. Translocation of protein phosphatase‐1 may represent a novel mechanism for the activation of glycogenolysis and inhibition of glycogen synthesis by adrenaline.
Effects of inverse changes in dietary lipid and carbohydrate on the synthesis of some pancreatic secretory proteins
Authors: WICKER Catherine , PUIGERVER Antoine
Published: March 2005

The effect of ingesting isocaloric and isonitrogenous diets with increasing amounts of lipid (0–30%) and consequently decreasing amounts of carbohydrates (68.7–1.25%) on the exocrine pancreas was studied in adult male Wistar rats. Pancreatic contents of chymotrypsin, lipase and colipase activity, as well as synthesis of amylase, lipase, procarboxypeptidases and individual serine proteases were examined. Lipid‐free diets and diets containing 1% lipid were found to have little effect on pancreatic proteins as compared with lipid‐rich diets where two distinct patterns of response were observed. Ingestion of diets containing 3–20% lipid resulted in a progressive increase in the activity of lipase, colipase and chymotrypsin up to 2‐fold in the first case and 1.6‐fold in the two other cases when animals were fed the 20% fat diet. Under the latter conditions, the relative synthesis of secretory proteins, as expressed as percentage of the radioactivity incorporated into individual proteins compared to that incorporated into the total mixture of exocrine proteins, was unchanged for procarboxypeptidases, whereas it was stimulated for lipase (2‐fold) and serine proteases (1.6‐fold). Amylase relative synthesis progressively decreased as the lipid content of diets increased. Consumption of hyperlipidic diets containing 25% and 30% fat resulted in a further enhancement in the activity of lipase and colipase in the gland in contrast with chymotrypsin activity which was unchanged as compared to the control diet (3% lipid). As far as biosynthesis was concerned, a plateau in the relative synthesis of lipase and serine protease was reached. Amylase relative synthesis further decreased down to 2.2‐fold when rats were fed the 30% fat‐rich diet whereas that of procarboxypeptidases was markedly increased (about 1.7‐fold). Absolute rates of synthesis of total pancreatic secretory proteins, as expressed with regard to the DNA content of the tissue, indicated that biosynthesis of all secretory pancreatic proteins was stimulated by hyperlipidic diets (at least 2‐fold with the 30% lipid diet). Consequently, when such an increase was stimulated by hyperlipidic diets (at least 2‐fold with the 30% lipid diet). Consequently, when such an increase was taken into consideration, the absolute synthesis of amylase was found to be unchanged throughout the dietary manipulations, whereas that of lipase, procarboxypeptidases and serine proteases were stimulated by 4.0‐fold, 3.4‐fold and 3.2‐fold, respectively.
Purification and characterization of Desulfovibrio vulgaris (Hildenborough) hydrogenase expressed in Escherichia coli
Authors: VOORDOUW Gerrit , HAGEN Wilfred R. , KRÜSE‐WOLTERS K. Margreet , van BERKEL‐ARTS Anita , VEEGER Cees
Published: March 2005

Hydrogenase from Desulfovibrio vulgaris (Hildenborough) is a heterologous dimer of molecular mass 46 + 13.5 kDa. Its two structural genes have been cloned on a 4664‐base‐pair fragment of known sequence in the vector pUC9. Expression of hydrogenase polypeptides in Escherichia coli transformed with this plasmid is poor (∼0.1% w/w of total protein). Deletion of up to 1.9 kb of insert DNA brings the gene encoding for the large subunit in close proximity to the lac promotor of pUC9 and results in a 50‐fold increased expression of hydrogenase polypeptides in E. coli. The protein formed is inactive and was purified in order to delineate its defect. Complete purification was achieved with a procedure similar to that used for the isolation of active hydrogenase from D. vulgaris H. The derived protein is also an αβ dimer and electron‐paramagnetic resonance studies indicate the presence of the electron‐transferring ferredoxin‐type iron‐sulfur clusters. In contrast to the native protein from D. vulgaris H, these can only be reduced with dithionite, not with hydrogen, indicating that the hydrogen‐binding active centre which also contains an iron‐sulfur cluster is missing.
Nucleotide sequence of cloned cDNA specific for rat ribosomal protein L31
Authors: TANAKA Tatsuo , KUWANO Yuh , KUZUMAKI Takejiro , ISHIKAWA Kiichi , OGATA Kikuo
Published: March 2005

A cDNA clone, specific for rat ribosomal protein L31, was isolated by hybrid‐selected translation from a cDNA library made for 8–9‐S poly(A)‐rich RNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. It consists of 25 base pairs from the 5′ leading sequence, the entire coding sequence of 378 base pairs and the 3′ trailing sequence of 36 base pairs besides the poly(A) tail. The primary structure of protein L31 was deduced from the nucleotide sequence. It consists of 124 amino acids with a relative molecular mass of 14331. The calculated amino acid composition is consistent with the reported composition determined for the hydrolysate of L31. The amino acid sequence showed marked homology with that of yeast ribosomal protein L34.
Chemical characterization of enterobacterial common antigen isolated from Plesiomonas shigelloides ATCC 14029
Authors: BASU Sumanta , KUHN Hella‐M. , NESZMELYI András , HIMMELSPACH Karl , MAYER Hubert
Published: March 2005

Serologically characterized samples of enterobacterial common antigen (ECA) from Plesiomonas shigelloides, Salmonella montevideo and Shigella sonnei were investigated by chemical methods including methylation and NMR techniques. All showed the same sugar composition and contained a lipid moiety with palmitic acid as main fatty acid and with a phosphodiester group. Additional enzymatic studies, reported in the preceding paper, provided evidence that the lipid moiety is an L‐glycerophosphatidyl residue attached via a phosphodiester linkage to C‐1 of GlcNAc as the reducing end of the ECA sugar chain.

ECA of P. shigelloides showed the best‐resolved 13C‐NMR spectra, especially after the removal of non‐stoichiometric O‐acetyl groups at C‐6 of GlcNAc of the ECA repeating unit and of the lipid moiety by mild acid hydrolysis (0.01 M HCl, 100°C, 10 min). Subsequent 13C‐NMR studies were therefore carried out with the mild‐acid‐treated ECA of P. shigelloides which allowed a tentative assignment of all resonances of the ECA repeating unit. 13C‐NMR spectra of Salmonella and Shigella ECA were essentially the same as those obtained with Plesiomonas ECA.

The same trisaccharide repeating unit was encountered as demonstrated previously in the cyclic form of ECA isolated from S. sonnei by Dell et al. [Carbohydr. Res. 133, 95–104 (1984)]. Methylation analysis, however, afforded small amounts of terminal GlcNAc thus proving, in combination with the demonstration of the attached lipid moiety, an acyclic nature of ECA from P. shigelloides and from the two enterobacterial species. The question of whether the cyclic form co‐exists in S. sonnei phase I and possibly in other enterobacterial species or, whether it had been formed during extraction as an artifact, has not yet been answered. The way in which ECA was isolated in our studies would preclude the presence of a non‐amphiphilic (cyclic) polysaccharide.

The finding that the sugar chain of ECA is attached to an L‐glycerophosphatidyl residue is in full corroboration with serological, enzymatical and gel electrophoretic studies shown in the preceding paper and with the character of ECA as a surface antigen being anchored by hydrophobic interactions in the outer membrane of Entero‐bacteriaceae and P. shigelloides.
Ribosomal protein phosphorylation in vivo and in vitro by vaccinia virus
Authors: BUENDIA Brigitte , PERSON‐FERNANDEZ Anne , BEAUD Georges , MADJAR Jean‐Jacques
Published: March 2005

Ribosomal protein phosphorylation was investigated in Ehrlich ascites tumor cells infected with vaccinia virus (Copenhagen strain). After 90 min of simultaneous infection and 32P‐labelling, ribosomal proteins Sa, S2 and S13 appear specifically phosphorylated as well as Sb/La, P1 and S6, which are also phosphorylated in control cells. Sa is an acidic protein, whose phosphorylation has not been observed previously. A kinetic study showed that S2 is phosphorylated very rapidly within 10 min after the beginning of infection and it is complete 1 h later. The phosphorylation of S13 begins after a lag time of about 1 h and is completed after about 2.5 h of infection. Moreover only one phosphate is incorporated into S13 on a serine residue while up to four phosphates are incorporated into S2, the first on a serine and the three following on thereonine residues. In vivo experiments, carried out in the presence of cycloheximide and cordycepin, suggest a viral origin for the kinase involved in the phosphorylation of S2 and S13. Moreover, in vitro experiments demonstrated that the kinase associated with the viral cores is capable of phosphorylating S2 on a serine residue only. In our cell/virus system, no significant difference in S6 phosphorylation was detected, when compared to uninfected cells. It is concluded that the specific and efficient phosphorylation of three ribosomal proteins from the 40S ribosomal subunit correlate well with possible translational mechanisms ensuring the efficient expression of early and late genes of vaccinia virus. In the light of these and previous results [Person, A. and Beaud, G. (1986) J. Biol. Chem. 261, 8283–8289], a mechanism is proposed for the shut‐off of host protein synthesis and the selective translation of mRNAs of viral origin.
Interaction of non‐histone proteins HMG1 and HMG2 with core histones in nucleosomes and core particles revealed by chemical cross‐linking
Authors: ŠTROS Michal , KOLÍBALOVÁ Anna
Published: March 2005

Chemical cross‐linking was used to study the interaction of the non‐histone chromosomal proteins HMG1 and HMG2 with core histones in H1,H5‐depleted nucleosomes or core particles. Cross‐linking with a ‘zero‐length’ cross‐linker 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide and with a longer (cleavable) cross‐linker dimethyl‐3,3′‐dithiobispropionimidate revealed an interaction of HMG1 and HMG2 with (or proximity to) core histones in both types of particles. These results indicated that the presence of the 40–50‐base‐pairs‐long segment of the ‘linker’ DNA in nucleosomes was not necessary for the establishment of mutual contacts of HMG1 and HMG2 proteins with core histones. Possible implications of the interaction of HMG1 and HMG2 proteins with histones for the structure and functioning of chromatin are discussed.
Sequence of carbonic anhydrase II cDNA from chick retina
Authors: ROGERS John H.
Published: March 2005

Sequences of three cDNA clones for carbonic anhydrase II (CA‐II) from chick retina are presented. The longest cDNA clone encodes all but the first three amino acids of CA‐II, and the encoded sequence generally agrees with published fragments of CA‐II sequence from chick red blood cells. It is 70% identical to human CA‐II; the active‐site residues are conserved, but the chick protein has six extra cysteines. There is a long 3′‐untranslated region which contains a second open reading frame, but this is not conserved. There appears to be a single CA‐II gene in the chick. Some anomalies in cDNA synthesis and in Bal31 deletion are noted.
On the mechanism of action of bovine intestinal mucosa 5′‐nucleotide phosphodiesterase
Authors: CUMMINS Jane H. , POTTER Barry V. L.
Published: March 2005

The diastereoisomers of adenosine 5′‐O‐phosphorothioate O‐methyl ester have been synthesised. Only the Sp diastereoisomer is a substrate for the 5′‐nucleotide phosphodiesterase from bovine intestinal mucosa. The previously unidentified enantiomer of 4‐nitrophenyl phenyl phosphonothioate hydrolysed by the enzyme is shown to have the Sp configuration. Digestion of the Sp diastereoisomer of adenosine 5′‐O‐phosphorothioate O‐methyl ester by the enzyme in 18O‐labelled water gave 18O‐labelled adenosine 5′‐O‐phosphorothioate which was stereochemically analysed by methylation and subsequent 31P‐NMR spectroscopy and shown to posses the Sp configuration. Thus the enzyme‐catalysed cleavage proceeded with retention of configuration at phosphorus, presumably via a double‐displacement mechanism. This provides strong evidence for the existence of a nucleotidyl‐enzyme intermediate on the reaction pathway.
The Ca2+‐dependent actions of the α‐adrenergic agonist phenylephrine on hepatic glycogenolysis differ from those of vasopressin and angiotensin
Authors: KLEINEKE Jochen , SÖLING Hans‐Dieter
Published: March 2005

The stimulation of hepatic glycogenolysis by the Ca2+‐dependent hormones phenylephrine, vasopressin and angiotensin II was studied as a function of intracellular and extracellular Ca2+.

In the isolated perfused rat liver the decline in glucose formation was monophasic (‘half‐life’∼ 3 min) with vasopressin (1 nM) or angiotensin II (0.05 μM), but biphasic (half‐life of 4.8 min and 17.6 min) in the presence of the α‐agonist phenylephrine (0.01 mM), indicating either a different mode of mobilization or the mobilization of additional intracellular calcium stores.

Under comparable conditions an elevated [Ca2+] level was maintained in the cytosol of hepatocytes for at least 10 min in the presence of phenylephrine, but not vasopressin.

Titration experiments performed in the isolated perfused liver to restore cellular calcium revealed differences in the hormone‐mediated uptake of Ca2+. The onset in glucose formation above that seen in the absence of exogenous calcium occurred at approximately 30 μM or 70–80 μM Ca2+ in the presence of phenylephrine or vasopressin respectively. The shape of the response curve was sigmoidal for vasopressin and angiotensin II, but showed a distinct plateau between 0.09 mM and 0.18 mM in the presence ofphenylephrine. The plateau was also observed at phenylephrine concentrations as low as 0.5 μM. The formation of plateaus observed after treatment of the liver with A 23187, but not after EGTA, is taken as an indication that intracellular calcium stores are replenished.

A participation of the mitochondrial compartment could be excluded by pretreatment of the liver with the uncoupler 2,4‐dinitrophenol.

Differences in the Ca2+ dependence of the glycogenolytic effects of these hormones were also revealed by kinetic analysis.

It is concluded that phenylephrine differs from vasopressin and angiotensin II in that, in addition to a more common, non‐mitochondrial pool, which is also responsive to the vasoactive peptides, the agonist mobilizes Ca2+ from a second, non‐mitochondrial pool.

The results are consistent with the proposal that Ca2+ transport across subcellular membranes may be subject to different hormonal control.
Fluctuation domains in myoglobin
Authors: ALBANI Jihad , ALPERT Bernard
Published: March 2005

The dynamics of two domains in the myoglobin molecule, close to the heme and inside the protein medium including the surface, are investigated through the study of the fluorescence oxygen quenching of two probes imbedded in the heme pocket: zinc protoporphyrin IX (with a fluorescence lifetime of 2.1 ns) and metal‐free protoporphyrin IX (with a fluorescence lifetime of 17.8 ns).
Isolation and characterization of a DNA‐uptake‐stimulating protein from the culture medium of Neurospora crassa slime strain
Authors: TÓTH György , BÉKÉSI István , SCHABLIK Marcella , SZABÓ Gábor
Published: March 2005

A protein fraction was purified to homogeneity from the culture medium of the wall‐less (slime) strain of Neurospora crassa (FGSC 1118), which proved to be identical with DNA‐uptake‐stimulating factor (designated DUSF), which has been described earlier [Schablik, M. and Szabó, G. (1981) FEMS Microbiol Lett. 10, 395–397]. The quantitiy of DUSF is measured by the amount of [3H]DNA uptake by Neurospora cells at standard conditions. Its relative molecular mass was 230000. It has an isoelectric point of pH 5.5. This protein consists of two identical subunits, relative molecular mass 110000.
Biosynthesis of 1‐alkenes in higher plants
Authors: NEY Peter , BOLAND Wilhelm
Published: March 2005

Uneven numbered 1‐alkenes, such as 1‐pentadecene, 1‐heptadecene, 1,8‐heptadecadiene, 1,8,11‐heptadecatriene and 1,8,11,14‐heptadecatetraene are the predominant hydrocarbons in germinating safflower (Carthamus tinctorius L.). According to their chain length and positions of double bonds they are derived from palmitic, stearic, oleic, linoleic or linolenic acid, respectively. This has been proved by administration of synthetic α‐, β‐, or γ‐deuterated 12‐phenyldodecanoic acids to germinating safflower. Mass spectrometric analysis of the resulting deuterium‐labelled 11‐phenyl‐1‐undecenes shows that only a single hydrogen from C‐3 and carbon dioxide from C‐1 of the precursor fatty acid is lost during vinyl group formation. A strong isotope effect (kH/kD≥ 10) during the conversion of racemic 12‐phenyl[3‐2H]dodecanoic acid to 11‐phenyl‐1‐[2‐2H]undecene is consistent with an initial enzymatic attack on a non‐activated hydrogen at C‐3 accompanied by simultaneous fragmentation into the respective 1‐alkene and carbon dioxide. Mechanisms, based on ionic or radical abstraction of a hydrogen from C‐3 of the fatty acid, followed by decarboxylation are discussed. The results are applicable to the formation of various vinylic substituents in natural products such as porphyrins, acetylenic hydrocarbons, pheromones from marine brown algae and terpenoids.
Large‐scale purification of hnRNP proteins from HeLa cells by affinity chromatography on ssDNA‐cellulose
Authors: PANDOLFO Massimo , VALENTINI Omar , BIAMONTI Giuseppe , ROSSI Paola , RIVA Silvano
Published: March 2005

A purification procedure for proteins which bind heterogeneous nuclear RNA (hnRNP proteins) is described. The procedure, which entails standard chromatographic fractionations (single‐stranded DNA cellulose, hydroxyapatite) and detection with specific antibodies, allows a large‐scale preparation of these proteins and the partial separation of different polypeptides. By this method, polypeptides of higher molecular mass (53–55 kDa) can be purified, which are structurally and antigenically related to the ‘canonical’ hnRNP core proteins (34–43 kDa) that constitute the 40S hnRNP complexes. We also show that HeLa cells contain a protease that cleaves hnRNP core proteins to discrete smaller polypeptides of 22–28 kDa. Such protease, which has been partially purified, appears to copurify extensively with some of the hnRNP proteins.
Protein synthesis in Drosophila melanogaster embryos
Authors: MATEU Mauricio G. , VICENTE Oscar , SIERRA José M.
Published: March 2005

Eukaryotic initiation factor 2 (eIF‐2) was purified from the high‐salt wash fraction of Drosophila melanogaster embryos. This factor, with a molecular mass of about 90 kDa, consists of two subunits of 47 kDa and 39 kDa on dodecylsulfate/polyacrylamide gel electrophoresis. The 39‐kDa subunit is phosphorylated by the hemin‐controlled inhibitor of rabbit reticulocytes in a terminal fragment which can be cleaved by mild treatment with trypsin. Drosophila eIF‐2 is not a substrate for protein kinases capable of phosphorylating the β subunit of eIF‐2 from rabbit reticulocytes. It is also shown that Drosophila eIF‐2 can form a ternary complex with GTP and Met‐tRNAi, which can be efficiently transferred to 40S ribosomes in the presence of AUG and Mg2+. This factor is able to form a binary complex with GDP. Furthermore, purified eIF‐2 contains about 0.3 mol bound GDP/mol suggesting a high affinity of the factor for this nucleotide. Data supporting the notion that this affinity is increased in the presence of Mg2+, which impairs the GDP/GTP exchange on eIF‐2, are presented. The properties of Drosophila eIF‐2 suggest that this factor may be susceptible to regulation by a mechanism like that operating on rabbit reticulocyte eIF‐2.
The phosphoglycerate kinases from Trypanosoma brucei
Authors: MISSET Onno , OPPERDOES Fred R.
Published: March 2005

Trypanosoma brucei has two phosphoglycerate kinase (PGK) isoenzymes, one is particle‐bound and localized in glycosomes while the other is present in the cytosol. The cytosolic isoenzyme (cPGK) was 900‐fold purified from cultured procyclic trypanosomes by hydrophobic interaction chromatography on phenyl‐Sepharose followed by affinity chromatography on 2′,3′‐ATP‐Sepharose and had a specific activity of 275 units/mg protein. cPGK was compared with the purified glycosomal isoenzyme (gPGK) from bloodstream‐form trypanosomes as well as with the commercially available PGKs from yeast, rabbit muscle and Spirulina platensis, a blue‐green alga.

Like all other PGKs, cPGK was a monomeric protein with a molecular mass of approximately 45 kDa similar to that of the PGKs from other organisms but 2 kDa smaller than that of gPGK. Despite this difference in length and a great difference in isoelectric point, the two trypanosome isoenzymes strongly resembled each other in several respects. (a) The kinetic parameters did not differ significantly from each other or from the PGKs of other organisms. (b) Both trypanosome enzymes resembled the enzyme from S. platensis in that they had an almost absolute requirement for ATP, contrary to the enzymes from yeast and rabbit muscle, which were capable of utilizing GTP and ITP also. This difference in substrate specificity may be related to the amino acid substitutions, Trp 308→His and Ala 306→Glu in the adenine‐binding site, which are only found in the two Trypanosoma isoenzymes. Kinetic analysis showed that these substitutions do not prevent binding of the ATP analogoues, but probably prevent phosphoryl‐group transfer. (c) Both isoenzymes displayed an activity optimum at pH 6.0—9.0 similar to that for the enzyme of yeast.

Both gPGK and cPGK were inhibited by the trypanocidal drug Suramin. This inhibition could be described as competitive both with ATP and 3‐phosphoglycerate with two inhibitor molecules binding to one molecule of enzyme. The gPGK, however, was much more sensitive (Kappi= 8.0 μM) to Suramin than either the cPGK (Kapp.i= 20 μM) or the enzymes from rabbit muscle (Kapp.i= 55 μM), yeast (Kapp.i= 167 μM) or S. platensis (Kapp.i= 250 μM). It is suggested that positive charges on the enzyme's surface may play an important role in the potentiation of the binding of the negatively charged Suramin molecule.
Characterization of the cytochrome system of a nitrogen‐fixing strain of a sulfate‐reducing bacterium: Desulfovibrio desulfuricans strain Berre‐Eau
Authors: MOURA Isabel , FAUQUE Guy , LeGALL Jean , XAVIER Antonio V. , J. G. MOURA José
Published: March 2005

Two c‐type cytochromes were purified and characterized by electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopic techniques, from the sulfate‐reducer nitrogen‐fixing organism, Desulfovibrio desulfuricans strain Berre‐Eau (NCIB 8387). The purification procedures included several chromatographic steps on alumina, carboxymethylcellulose and gel filtration. A tetrahaem and a monohaem cytochrome were identified. The multihaem cytochrome has visible, EPR and NMR spectra with general properties similar to other low‐potential bis‐histidinyl axially bound haem proteins, belonging to the class of tetrahaem cytochrome c3 isolated from other Desulfovibrio species. The monohaem cytochrome c553 is ascorbate‐reducible and its EPR and NMR data are characteristic of a cytochrome with methionine‐histidine ligation. Their properties are compared with other homologous proteins isolated from sulfate‐reducing bacteria.
Reversible inhibition of sarcoplasmic reticulum Ca‐ATPase by altered neuromuscular activity in rabbit fast‐twitch muscle
Authors: LEBERER Ekkehard , HÄRTNER Karl‐Thomas , PETTE Dirk
Published: March 2005

A 50% decrease in both the initial rate and the total capacity of Ca2+ uptake by the sarcoplasmic reticulum (SR) occurred 2 days after the onset of chronic (10 Hz) nerve stimulation in rabbit fast‐twitch muscle. Prolonged stimulation (up to 28 days) did not lead to further decreases. This reduction, which was detected in muscle homogenates using a Ca2+‐sensitive electrode, was reversible after 6 days cessation of stimulation and was not accompanied by changes in the immunochemically (ELISA) determined tissue level or isozyme characteristics of the SR Ca2+‐ATPase protein. However, as measured in isolated SR, it correlated with a reduced specific activity of the Ca2+‐ATPase. Kinetic analyses demonstrated that affinities of the SR Ca2+‐ATPase towards Ca2+ and ATP were unaltered. Positive cooperativity for Ca2+ binding (h= 1.5) was maintained. However, a 50% decrease in Ca2+‐dependent phosphoprotein formation indicated the presence of inactive forms of Ca2+‐ATPase in stimulated muscle. The reduced phosphorylation of the enzyme was accompanied by an approximately 50% lowered binding of fluorescein isothiocyanate, a competitor at the ATP‐binding site. In view of the unaltered affinity for ATP, this finding suggests that active Ca2+‐ATPase molecules coexist in stimulated muscle with inactive enzyme molecules, the latter displaying altered properties at the nucleotide‐binding site.
α‐Glucan synthesis on a protein primer
Authors: MORENO Silvia , CARDINI Carlos E. , TANDECARZ Juana S.
Published: March 2005

Reconstitution experiments with the DEAE‐cellulose‐treated enzymes, engaged in a two‐step mechanism of synthesis of α‐glucan bound to protein, are performed. Urea/sodium dodecyl sulfate/polyacrylamide gel electrophoretic analysis of the radioactive products synthesized by the reconstituted system shows highly glucosylated, labeled bands, whose apparent molecular masses change with the acrylamide concentration in the gels. The long carbohydrate chains synthesized during the second step arise from the sequential addition of glucosyl moieties to the glucoprotein formed during the first step. A deglucosylation experiment confirms that the product of the reconstituted system originates from the 38‐kDa glucosylated component of the reaction 1 product by the addition of β‐amylase‐sensitive glucosyl moieties. Our data suggest that specific phosphorylases and starch synthetases are found in potato tuber, which are capable of utilizing reaction 1 product as primer for the synthesis of protein‐bound glucan.