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.
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Abbreviations
- Ω-AHA:
-
Ω-aminohexylagarose
- cDNA:
-
copy DNA
- DEAE:
-
diethylaminoethyl
- Fru6P:
-
fructose-6-phosphate
- Glc6P:
-
glucose-6-phosphate
- PEG:
-
polyethylene glycol
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- SPS:
-
sucrose-phosphate synthase
- UDPGlc:
-
uridine 5′-diphosphoglucose
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This work was supported by the Deutsche Forschungsgemeinschaft (SFB TP B8, W.P. Quick) and the Bundesminister für Forschung und Technologie (U. Sonnewald, K.-P. Krause). We are grateful to T. Voelker for technical advice during the course of this work. We thank Regina Feil for typing the manuscript. We also thank Astrid Basner (IGF) for excellent technical assistance during the sequencing.
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Sonnewald, U., Quick, W.P., MacRae, E. et al. Purification, cloning and expression of spinach leaf sucrose-phosphate synthase in Escherichia coli . Planta 189, 174–181 (1993). https://doi.org/10.1007/BF00195074
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DOI: https://doi.org/10.1007/BF00195074