bluuebunny/crossref_metadata_2025_split huggingface dataset:
The photochemical reaction center of Chloroflexus aurantiacus is composed of two structurally similar polypeptides
Authors: SHIOZAWA Judith A. , LOTTSPEICH Friedrich , FEICK Reiner 
Published: March 2005

A method has been devised which allowed the isolation of highly purified reaction center from the thermophilic green bacterium, Chloroflexus aurantiacus. The procedure consisted of three chromatography steps. The final step was fast protein liquid chromatography on Mono Q in the presence of nonanoyl‐N‐methylglucamide (Mega‐9). The purified reaction center complex was photochemically active and had an A280/A813 of 1.4 or less. Under non‐denaturing conditions, a pigmented protein band having a Mr of 52000–55000 was observed in sodium dodecyl sulfate gels. When the isolated complex was heat‐dissociated in the presence of sodium dodecyl sulfate, just two polypeptides having very similar Mr (24000 and 24500) were observed. Two protein bands were also observed in two‐dimensional isoelectric focusing/sodium‐dodecyl‐sulfate polyacrylamide gel electropheresis; the PI values of the two polypeptides were 6.5 and 6.7. Partial peptide mapping of the two isolated subunits, using both enzymatic and chemical cleavage techniques, yielded almost identical patterns which indicated a high degree of sequence homology between the two polypeptides.The N‐terminal amino acid sequences of the two polypeptides were identical and did not exhibit any homology to reaction center subunits of purple sulfur bacteria.The Chloroflexus reaction center is believed to be composed of one molecule of each polypeptide, the photoactive bacteriochlorophyll a dimer and, as accessory pigments, an additional bacteriochlorophyll a and three bacteriopheophytins. Hence, it appears to be the smallest photochemically active reaction center isolated to date.
A structural and dynamic model for the nicotinic acetylcholine receptor
Authors: KOSOWER Edward M. 
Published: March 2005

Folding of the five polypeptide subunits (α2βγδ), of the nicotinic acetylcholine receptor (AChR) into a functional structural model is described. The principles used to arrange the sequences into a structure include: (1) hydrophobicity → membrane‐crossing segments; (2) amphipathic character → ion‐carrying segments (ion channel with single group rotations); (3) molecular shape (elongated, pentagonal cylinder) → folding dimensions of exobilayer portion; (4) choice of acetylcholine binding sites → specific folding of exobilayer segments; (5) location of reducible disulfides (near agonist binding site) → additional specification of exobilayer arrangement; (6) genetic homology → consistency of functional group choices; (7) noncompetitive antagonist labeling → arrangement of bilayer helices. The AChR model is divided into three parts: (a) exobilayer consisting of 11 antiparallel β‐strands from each subunit; (b) bilayer consisting of four hydrophobic and one amphiphilic α‐helix from each subunit; (c) cytoplasmic consisting of one (folded) loop from each subunit.The exobilayer strands can form a closed ‘flower’ (the ‘resting state’) which is opened (‘activated’) by agonists bound perpendicular to the strands. Rearrangement of the agonists to a strand‐parallel position and partial closing of the ‘flower’ leads to a desensitized receptor. The actions of acetylcholine and succinoyl and suberoyl bis‐cholines are clarified by the model. The opening and closing of the exobilayer ‘flower’ controls access to the ion channel which is composed of the five amphiphilic bilayer helices. A molecular mechanism for ion flow in the channel is given. Openings interrupted by short duration closings (50 μs) depend upon channel group motions. The unusual photolabeling of intrabilayer serines in α, β and δ subunits but not in γ subunits near the binding site for non‐competitive antagonists is explained along with a mechanism for the action of these antagonists such as phencyclidine. The unusual α 192Cys–193Cys disulfide may have a special peptide arrangement, such as a cis‐peptide bond to a following proline (G.A. Petsko and E.M. Kosower, unpublished results). The position of phosphorylatable sites and proline‐rich segments are noted for the cytoplasmic loops.The dynamic behavior of the AChR channel and many different experimental results can be interpreted in terms of the model. An example is the lowering of ionic conductivity on substitution of bovine for Torpedoδ M2 segment. The model represents a useful construct for the design of experiments onAChR.
Characterization of a soluble Mr‐30000 catalytic fragment of the neuronal calmodulin‐dependent protein kinase II
Authors: LEVINE Harry , SAHYOUN Naji E. 
Published: March 2005

Chymotryptic digestion of postsynaptic densities releases a soluble, catalytically active fragment of the α (Mr 50000) subunit of the neuronal cytoskeletal calmodulin‐dependent protein kinase II. The purified soluble form of the kinase likewise yields the fragment. Denaturation of the enzyme results in more extensive proteolytic degradation. 125I‐Iodopeptide maps of the isolated catalytic portions of both forms of the enzyme are similar and are contained within the map of the isolated α subunit. Catalytic fragments of both forms of the enzyme comigrate on two‐dimensional SDS‐PAGE/isoelectric focusing with pI 6.7–7.2.The fragment phosphorylates microtubule‐associated protein (MAP‐2) but is not activated by Ca+2/calmodulin nor is it inhibited by trifluoperazine. Km values for MAP‐2 and ATP are indistinguishable from those of the holoenzyme, while the Vmax is similar to that of the holoenzyme activated with Ca+2/calmodulin. Overlays of Western blots of fragment with 125I‐calmodulin shows a loss of calmodulin binding. Both the number of phosphorylation sites and the ability to autophosphorylate are markedly reduced in the catalytic fragment.Evaluation of the hydrodynamic parameters of the purified fragment yielded Mr value of 25600 with a frictional ratio (f/fo) of 1.12; the Mr value determined by SDS‐PAGE was 30000. Thus, the catalytic fragment appears to represent an activated form of the kinase with a monomeric, globular structure unlike the native enzyme which exhibits oligomerization and cytoskeletal association. These results are consistent with a tertiary structure for the calmodulin‐dependent protein kinase that contains distinct domains responsible for catalytic activity, regulation by calmodulin, cytoskeletal association and the multimeric organization of enzyme subunits.
Activation and transfer of novel synthetic 9‐substituted sialic acids
Authors: GROSS Hans Jürgen , BÜNSCH Almuth , PAULSON James C. , BROSSMER Reinhard 
Published: March 2005

In this report several NeuAc analogues differently modified at position C‐9 were tested as substrates for CMPsialic acid synthase from bovine brain: the hydroxy group at C‐9 was replaced by an amino, acetamido, benzamido, hexanoylamido and azido group.The synthase was partially purified by chromatography on CDP‐hexanolamine –Sepharose. CMP‐glycosides synthesized were measured by analytical HPLC at 275 nm.Each NeuAc analogue was activated to the respective CMP‐glycoside: Km‐values varied from 0.8 mM to 4.6 mM, the Km for NeuAc was 1.4 mM. Thus affinity of the enzyme was influenced only moderately by chemical modification at C‐9.CMP‐glycosides were synthesized on a preparative scale with good yield and characterized by analytical HPLC. In addition, 500‐MHz 1H‐NMR data of CMP‐9‐amino‐NeuAc and CMP‐9‐acetamido‐NeuAc were obtained.Each CMP‐activated NeuAc analogue was a suitable donor substrate for Galβ1‐4GlcNAc α2,6‐sialyl‐transferase from rat liver. Transfer was determined by the thiobarbituric acid method and by analytical HPLC at 200 nm. The results demonstrate that synthetic, not naturally occurring, non‐labelled NeuAc analogues can be incorporated into glycoprotein with high yield.
Cultured Ito cells of rat liver express the α2‐macroglobulin gene
Authors: ANDUS Tilo , RAMADORI Giuliano , HEINRICH Peter C. , KNITTEL Thomas , MEYER ZUM BÜSCHENFELDE Karl‐Hermann 
Published: March 2005

Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize α2‐macroglobulin was analyzed at different times after isolation and by pulse‐chase experiments. Newly synthesized α2‐macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. α2‐Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5–11 days after the isolation of the cells, increasing amounts of α2‐macroglobulin were synthesized. The results of pulse‐chase experiments performed on day 7 showed that radioactively labeled α2‐macroglobulin decreased in the intracellular compartment and increased in the culture medium. α2‐Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non‐reducing conditions. Furthermore, when unlabeled α2‐macroglobulin was added during the immunoprecipitation a competition was observed. Incubation of pancreatic elastase with culture medium of rat Ito cells or rat hepatocytes led to the same cleavage products as found with α2‐macroglobulin. α2‐Macroglobulin‐specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells.Under the conditions where α2‐macroglobulin was synthesized in Ito cells, no synthesis of α1‐macroglobulin, α1‐inhibitor 3, α1‐proteinase inhibitor, α1‐acid glycoprotein, α1‐acute‐phase globulin (T‐kininogen) and albumin could be demonstrated. It is concluded that α2‐macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases.
Chemical modification of phosphorylase b by tetranitromethane
Authors: CARUSO Carla , DI PRISCO Guido , CACACE Marcello G. 
Published: March 2005

Tetranitromethane, C(NO2)4, a reagent for tyrosyl residues, was found to inactivate irreversibly rabbit skeletal muscle glycogen phosphorylase b. Under the chosen conditions seven tyrosyl residues, namely Tyr‐75, 203, 262, 280, 403, 552 and 647, were found to be nitrated. Inactivation was prevented by the presence of the allosteric activator 5′‐AMP during nitration. Under these latter conditions one of the reactive tyrosyl residues was not modified by C(NO2)4; thus, this residue appeared to be essential for either catalytic activity or allosteric activation.Tryptic digests of phosphorylase b, reacted with C(NO2)4 in the absence and presence of 5′AMP, were fractionated by gel filtration. The peptide mixtures were further purified by reverse‐phase HPLC. One of the peptides contained the tyrosyl residue which was modified by C(NO2)4 only in the absence of 5′AMP. The sequence of this peptide was determined. The amino acid residue which is responsible for the loss of activity upon reaction with C(NO2)4 was identified in the amino acid sequence of phosphorylase b as tyrosine‐75.Of the other residues modified in the presence and in the absence of C(NO2)4, tyrosine‐403 contributes to the glycogen‐storage site whereas Tyr‐280 is close to the α‐d‐glucose‐binding site. These residues, exposed to the solvent both in the presence and in the absence of 5′ AMP, are not essential for catalytic activity.
Amino‐acid sequence of human α2‐antiplasmin
Authors: LIJNEN H. Roger , HOLMES William E. , VAN HOEF Berthe , WIMAN Björn , RODRIGUEZ Henri , COLLEN Désiré 
Published: March 2005

The amino‐acid sequence of human α2‐antiplasmin was determined by Edman degradation of peptides purified from CNBr, tryptic and chymotryptic digests. Of the total sequence of 452 amino acids of mature α2‐antiplasmin, as deduced from the cDNA sequence [Holmes et al. (1987) J. Biol. Chem. 262, 1659–1664], 444 residues were identified by amino‐acid sequencing. Two differences were found between the peptide and cDNA analyses (Gly instead of Leu at position 10 and Gly instead of Ser at position 369).α2‐Antiplasmin contains two disulfide bridges (Cys64‐Cys104 and Cys31‐Cys113) and four glucosamine‐based carbohydrate chains attached to Asn87, Asn256, Asn270 and Asn277. α2‐Antiplasmin is homologous with 12 other proteins belonging to the serine protease inhibitor (serpin) superfamily.
Effects of various metabolic conditions and of the trivalent arsenical melarsen oxide on the intracellular levels of fructose 2,6‐bisphosphate and of glycolytic intermediates in Trypanosoma brucei
Authors: VAN SCHAFTINGEN Emile , OPPERDOES Fred R. , HERS Henry‐Géry 
Published: March 2005

Upon differential centrifugation of cell‐free extracts of Trypanosoma brucei, 6‐phosphofructo‐2‐kinase and fructose‐2,6‐bisphosphatase behaved as cytosolic enzymes. The two activities could be separated from each other by chromatography on both blue Sepharose and anion exchangers. 6‐phosphofructo‐2‐kinase had a Km for both its substrates in the millimolar range. Its activity was dependent on the presence of inorganic phosphate and was inhibited by phosphoenolpyruvate but not by citrate or glycèrol 3‐phosphate. The Km of fructose‐2,6‐bisphosphatase was 7 μM; this enzyme was inhibited by fructose 1,6‐bisphosphate (Ki= 10 μM) and, less potently, by fructose 6‐phosphate, phosphoenolpyruvate and glycerol 3‐phosphate. Melarsen oxide inhibited 6‐phosphofructo‐2‐kinase (Ki < 1 μM) and fructose‐2,6‐bisphosphatase (Ki= 2 μM) much more potently than pyruvate kinase (Ki > 100 μM).The intracellular concentrations of fructose 2,6‐bisphosphate and hexose 6‐phosphate were highest with glucose, intermediate with fructose and lowest with glycerol and dihydroxyacetone as glycolytic substrates. When added with glucose, salicylhydroxamic acid caused a decrease in the concentration of fructose 2,6‐bisphosphate, ATP, hexose 6‐phosphate and fructose 1,6‐bisphosphate. These studies indicate that the concentration of fructose 2,6‐bisphosphate is mainly controlled by the concentration of the substrates of 6‐phosphofructo‐2‐kinase. The changes in the concentration of phosphoenolpyruvate were in agreement with the stimulatory effect of fructose 2,6‐bisphosphate on pyruvate kinase.At micromolar concentrations, melarsen oxide blocked almost completely the the formation of fructose 2,6‐bisphosphate induced by glucose, without changing the intracellular concentrations of ATP and of hexose 6‐phosphates. At higher concentrations (3–10 μM), this drug caused cell lysis, a proportional decrease in the glycolytic flux, as well as an increase in the phosphoenolpyruvate concentrations which was restricted to the extracellular compartment. Similar changes were induced by digitonin. It is concluded that the lytic effect of melarsen oxide on the bloodstream form of T. brucei is not the result of an inhibition of pyruvate kinase.
Molecular cloning and characterization of the gene encoding cholinephosphate cytidylyltransferase in Saccharomyces cerevisiae
Authors: TSUKAGOSHI Yuko , NIKAWA Jun‐ichi , YAMASHITA Satoshi 
Published: March 2005

The structural gene for cholinephosphate cytidylyltransferase (CCT) was isolated from a Saccharomyces cerevisiae genomic library by means of complementation in a mutant of the yeast defective in the enzyme. The cloned DNA restored both the growth and cholinephosphate cytidylyltransferase activity of the mutant. Whereas the enzyme of the mutant was thermolabile, the enzyme produced by the transformant was indistinguishable in heat stability from that produced by the wild type.
Strains carrying a multicopy recombinant plasmid overproduced cholinephosphate cytidylyltransferase. The overproduction of the enzyme brought about an increase in the synthesis of CDPcholine in the transformant, but there was no increase in the overall rate of phosphatidylcholine synthesis.
The cloned DNA was subcloned into a 2.5‐kb DNA fragment. The nucleotide sequence which contained CCT was determined by the dideoxy chain‐termination method. The sequence contained an open reading frame capable of encoding a protein of 424 amino acid residues with a calculated relative molecular mass of 49379.31. Northern blot analysis showed that this DNA segment is transcribed in yeast cells and the length of the transcript is consistent with the putative translation product.
Hydropathy analysis according to Kyte and Doolittle indicated that the primary translation product contains extended hydrophilic stretches in its N‐ and C‐terminal regions.
The primary translation product contains a region showing local sequence homology with nucleotidyl‐transfer enzymes such as DNA polymerase (Escherichia coli), CDPdiacylglycerol pyrophosphatase (E. coli), 3‐deoxy‐manno‐octulosonate cytidylyltransferase (E. coli) and DNA ligase (T4 phage), suggesting that these five enzymes are evolutionarily related. Statistically significant sequence homology was also noted between the human c‐fos gene product and the enzyme.
A new type of prosome‐like particle, composed of small cytoplasmic RNA and multimers of a 21‐kDa protein, inhibits protein synthesisin vitro
Authors: AKHAYAT Omar , INFANTE Anthony A. , INFANTE Dzintra , MARTINS de SA Cezar , GROSSI de SA M.‐Fatima , SCHERRER Klaus 
Published: March 2005

A large fraction of the translationally repressed non‐globin messenger RNA in duck erythroblasts is present in non‐polyribosomal free mRNP structures which sediment in the 30–40‐S range (‘35 S’). In 0.5 M KCl, they form core complexes which show a pronounced peak at about 32 S containing mRNA and a discrete spherical RNP particle with a diameter of about 12 nm and the typical morphology of a prosome [H.‐P. Schmid et al. (1984)EMBO J. 3, 29–34]. Buoyant density measurements and chromatography on oligo(dT)‐cellulose indicate that this particle is bound to mRNA; it can be released from the mRNA by treatment of the free mRNP fraction with SDS.This prosome‐like particle inhibits the translation of mRNAin vitro.It is composed primarily of multimers of a single 21‐kDa protein and at least one species of RNA of about 80–100 nucleotides. It is resistant to dissociation by 2 M Cs2SO4and 1% SDS; the 21‐kDa protein is not attacked by proteinase K unless the particle is extracted with phenol prior to treatment with the protease. The small RNA moiety of the particle hybridizes to the poly(A)‐rich mRNA derived from the free mRNPs, as well as to polyribosomal mRNA.These data indicate that prosomes may serve to regulate mRNA translation; they show furthermore that prosome‐like particles (about 600 kDa mass) may be built of up to 25 molecules of a single specific protein, rather than of the entire set of about 20 prosomal proteins previously indentified.
Isolation and characterization of three different carbohydrate chains from melanoma tissue plasminogen activator
Authors: POHL Gunnar , KENNE Lennart , NILSSON Bo , EINARSSON Monica 
Published: March 2005

Electrophoretic analysis of endoglycosidase‐treated tissue plasminogen activator obtained from human melanoma cells showed that the heterogeneity observed for the protein in these preparations is caused by an N‐glycosidically linked N‐acetyllactosamine type of carbohydrate chain which is present in about 50% of the molecules. An oligomannose type and an N‐acetyllactosamine type of glycan is present in all molecules.Three glycopeptides were isolated and characterized by 1H‐NMR, sugar determination, methylation analysis and amino acid determination. The exact attachment site for each of the three glycans could be deduced from the amino acid compositions of the glycopeptides. Asn‐117 carries the oligomannose type of glycan, the structure of which was completely determined. Asn‐184 is the site where the presence or absence of a biantennary N‐acetyllactosamine type of glycan causes the size heterogeneity. The third N‐glycosylation site, Asn‐448, was found to carry a triantennary or tetraantennary N‐acetyllactosamine type of carbohydrate chain.
An NMR study of polymorphous behaviour of the mismatched DNA octamer d(m5C‐G‐m5C‐G‐A‐G‐m5C‐G) in solution
Authors: ORBONS Leonard P. M. , van der MAREL Gijs A. , van BOOM Jacques H. , ALTONA Cornelis 
Published: March 2005

By means of one‐ and two‐dimensional NMR spectroscopy the solution structures of the partly self‐complementary octamer d(m5C‐G‐m5C‐G‐A‐G‐m5C‐G) were investigated. It is shown that this DNA fragment, under conditions of high DNA concentration (8 mM DNA) and/or high ionic strength prefers to adopt a duplex structure. At low DNA concentration (0.4 mM DNA), the duplex exists in a 1:1 slow equilibrium with a monomeric hairpin form. Addition of salt destabilizes the hairpin structure in favour of the dimer. At high temperatures the hairpin form, as well as the dimer structure, exist in a fast equilibrium with the random‐coil form. For the hairpin/random‐coil equilibrium a Tm of 329 K and a ΔH° of ‐121 kJ · mol−1 were deduced. These thermodynamic parameters are independent of the DNA concentration, as is expected for a monomeric structure. For the dimer to coil transition a Tm of 359 K(1 M DNA) and a ΔH° of ‐285 kJ · mol duplex−1 were derived. The thermodynamic data of the hairpin‐coil transition mutually agree with those recently reported for the hairpin to random coil equilibrium of the DNA octamer d(m5C‐G‐m5C‐G‐T‐G‐m5C‐G) [Orbons, L. P. M., van der Marel, G. A., van Boom, J. H. & Altona, C. (1987) J. Biomol. Struct. Dyns. 4, 939–963].It is demonstrated that the dimer structure exhibits B‐DNA characteristics, as is witnessed by the NOESY experiments and the analysis of the proton‐proton coupling data. It is shown that the base‐pair formation of the G × A mismatches is anti‐anti. A comparison of 1H and 31P chemical‐shift data of the title compound with those of a well‐characterized B‐DNA structure reveals large differences in the dm5C(3)‐dG(4)‐dA(5) part of the mismatched dimer structure. These differences apparently indicate some major local structural changes due to the incorporation of the G × A mismatches.Under the most extreme conditions used (i.e. up to 3 M NaCl or 75% CH3OH in the presence of 10 mM MgCl2) no Z‐DNA structure was observed.It is shown that the structural features of the hairpin form of the title compound mimic those of the hairpin structure of d(m5C‐G‐m5C‐G‐T‐G‐m5C‐G). An energy‐minimized model of the hairpin form is given.
Characterization and partial purification of a novel mannosylphosphodolichol phosphodiesterase from chicken liver microsomes
Authors: TOMITA Yoshio , MOTOKAWA Yutaro 
Published: March 2005

Mannosylphosphodolichol phosphodiesterase, which catalyzes the release of mannose from mannosylphosphodolichol, was solubilized from chicken liver microsomes by treatment with the non‐ionic detergent, Emulgen 909. The enzyme was partially purified using ammonium sulfate precipitation, DEAE‐cellulose chromatography, and gel filtration on Sepharose 6B. The enzyme showed absolute requirement for sulfhydryl reducing agents. The enzyme activity was stimulated by the addition of CaCl2 and Emulgen 909 and exhibited a pH optimum around 5.3. The Km value for mannosylphosphodolichol was found to be 0.43 μM. The activity was competitively inhibited by dolichyl phosphate and dolichol and the Ki value for dolichyl phosphate was estimated to be 12.5 μM. The purified preparation had no activity toward N‐acetylglucosaminyldiphosphodolichol, glucosylphosphodolichol, mannose 1‐phosphate, or artificial substrates for mannosidases, glucosidases, acid phosphatase, and acid phosphodiesterase.A heat‐stable factor which stabilizes the mannosylphosphodolichol phosphodiesterase was separated from the enzyme by DEAE‐cellulose chromatography. It was precipitated by trichloroacetic acid and not extracted into lipid solvents. The separation resulted in the complete loss of the enzyme activity and the restoration of the activity was not observed when the factor was added back to the enzyme solution.