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  • Life Sciences (General)  (1)
  • cDNA clone (encoding sucrose-phosphate synthase)  (1)
  • 1
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    Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation (1999)
    In:  Other Sources
    Details
    Publication Date: 2011-08-24
    Description: Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.
    Keywords: Life Sciences (General)
    Type: The Plant journal : for cell and molecular biology (ISSN 0960-7412); Volume 17; 4; 407-13
    Format: text
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  • 2
    Electronic Resource
    Electronic Resource
    Purification, cloning and expression of spinach leaf sucrose-phosphate synthase in Escherichia coli (1993)
    Springer
    Planta 189 (1993), S. 174-181 
    Details
    ISSN: 1432-2048
    Keywords: cDNA clone (encoding sucrose-phosphate synthase) ; Expression (spinach sucrose-phosphate synthase in E. coli) ; Nucleotide sequence (sucrosephosphate synthase) ; Spinacea ; Sucrose-phosphate synthase (purification, cloning, expression)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Sucrose-phosphate synthase (SPS) from leaves of spinach (Spinacia oleracea L.) has been purified to homogeneity by a procedure involving precipitation with polyethylenglycol and chromatography over diethylaminoethylcellulose, Ω-aminohexylagarose, Mono Q and Blue Affinity columns. The purification factor was 838 and the final specific activity was 1.3 nkat · (mg protein)−1. On denaturing gels the major polypeptide was 120 kDa but there was also a variable amount of smaller polypeptides in the range of 90 to 110 kDa. A new activity stain was developed to allow visualization of SPS in gels. The holoenzyme had a molecular weight of about 240 and 480 kDa in native gels and Sepharose, respectively. A high-titre polyclonal antibody was obtained which reacted with SPS from other species including wheat, potato, banana and maize. Screening of a spinach-leaf cDNA-expression library with the antibody allowed the isolation of a full-length clone. Sequencing revealed a predicted molecular weight of 117649 Da, and considerable homology with the recently published sequence for maize leaf (Worrell et al. 1991, Plant Cell 3, 1121–1130). Expression of the spinach-leaf SPS gene in Escherichia coli resulted in biological activity, revealed by the presence of SPS activity in extracts and the accumulation of sucrose-6-phosphate and sucrose in the bacteria.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00195074
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    Paper (German National Licenses)
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