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S-Adenosylmethionine synthetase from Escherichia coli 总被引:16,自引:0,他引:16
G D Markham E W Hafner C W Tabor H Tabor 《The Journal of biological chemistry》1980,255(19):9082-9092
Adenosylmethionine (AdoMet) synthetase has been purified to homogeneity from Escherichia coli. For this purification, a strain of E. coli which was derepressed for AdoMet synthetase and which harbors a plasmid containing the structural gene for AdoMet synthetase was constructed. This strain produces 80-fold more AdoMet synthetase than a wild type E. coli. AdoMet synthetase has a molecular weight of 180,000 and is composed of four identical subunits. In addition to the synthetase reaction, the purified enzyme catalyzes a tripolyphosphatase reaction that is stimulated by AdoMet. Both enzymatic activities require a divalent metal ion and are markedly stimulated by certain monovalent cations. AdoMet synthesis also takes place if adenyl-5'yl imidodiphosphate (AMP-PNP) is substituted for ATP. The imidotriphosphate (PPNP) formed is not hydrolyzed, permitting dissociation of AdoMet formation from tripolyphosphate cleavage. An enzyme complex is formed which contains one equivalent (per subunit) of adenosylmethionine, monovalent cation, imidotriphosphate, and presumably divalent cation(s). The rate of product dissociation from this complex is 3 orders of magnitude slower than the rate of AdoMet formation from ATP. Studies with the phosphorothioate derivatives of ATP (ATP alpha S and ATP beta S) in the presence of Mg2+, Mn2+, or Co2+ indicate that a divalent ion is bound to the nucleotide during the reaction and provide information on the stereochemistry of the metal-nucleotide binding site. 相似文献
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Precise excision of Tn10 occurs at significantly elevated frequencies in cultures of the polA7 mutant strain of Salmonella typhimurium, is further increased in polA7 dam-1 and polA7 mutB strains and decreased in a polA7 mutH background. The numbers of precise excision events occurring in polA7 strains are also significantly increased when methionine (20 micrograms/ml or less) is present in the medium but decreased when ethionine (again, 20 micrograms/ml or less) is present. When both amino present, the outcome is about a 2-fold increase in precise excision events. The involvement of mismatch repair and methylation patterns in precise excision events is discussed. 相似文献
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A 'top-down' approach to the determination of control coefficients in metabolic control theory 总被引:2,自引:0,他引:2
A new approach to the determination of flux and concentration control coefficients in metabolic pathways is outlined. Linear pathways are conceptually divided in two around an intermediate metabolite (or group or metabolites) and the control coefficients of the two parts are derived from the elasticity coefficients of the two parts to the intermediate. Branched pathways are treated similarly, the control coefficients of the branches being derived either from the elasticities of the branches to their common intermediate or from the relative flux changes of the branches. Repeating this analysis around other intermediates in the pathway allows the control coefficients of smaller and smaller groups of enzymes to be determined. In complex systems this approach to describing control may have several advantages over determining the control coefficients of individual enzymes and is a potentially useful complementary approach. 相似文献
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Merima Bublin Maria Kostadinova Julian E. Fuchs Daniela Ackerbauer Adolfo H. Moraes Fabio C. L. Almeida Nina Lengger Christine Hafner Christof Ebner Christian Radauer Klaus R. Liedl Ana Paula Valente Heimo Breiteneder 《PloS one》2015,10(11)
Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients’ sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes. 相似文献
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RP Tucker K Drabikowski JF Hess J Ferralli R Chiquet-Ehrismann JC Adams 《BMC evolutionary biology》2006,6(1):60-17
Background
Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes. 相似文献10.
Jožica Gričar Martin de Luis Polona Hafner Tom Levanič 《Trees - Structure and Function》2013,27(6):1669-1680