Cloning, biochemical characterisation, tissue localisation and possible post-translational regulatory mechanism of the cytosolic phosphoglucose isomerase from developing sunflower seeds.
Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of glucose-6-phosphate. This glycolytic intermediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phosphoglucose isomerase (cPGI, EC 5.3.1.9). In this report, we describe the cloning of a full-length cDNA encoding cPGI from developing sunflower seeds. The sequence was predicted to code for a protein of 566 residues characterised by the presence of two sugar isomerase domains. This cDNA was heterologously expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified using immobilised metal ion affinity chromatography and biochemically characterised. The enzyme had a specific activity of 1,436 micromol min(-1) mg(-1) and 1,011 micromol min(-1) mg(-1) protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respectively. Activity was not affected by erythrose-4-phosphate, but was inhibited by 6-P gluconate and glyceraldehyde-3-phosphate. A polyclonal immune serum was raised against the purified enzyme, allowing the study of protein levels during the period of active lipid synthesis in seeds. These results were compared with PGI activity profiles and mRNA expression levels obtained from Q-PCR studies. Our results point to the existence of a possible post-translational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring.
Troncoso-Ponce
M A
MA
Instituto de la Grasa, CSIC, Seville, Spain.
Rivoal
J
J
Cejudo
F J
FJ
Dorion
S
S
Garcés
R
R
Martínez-Force
E
E
D000595:Amino Acid Sequence; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D019298:Glucose-6-Phosphate / Q000378:metabolism; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D006368:Helianthus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012639:Seeds / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D010940:Plant Proteins; D012333:RNA, Messenger; D005986:Glyceraldehyde 3-Phosphate; D019298:Glucose-6-Phosphate; D005956:Glucose-6-Phosphate Isomerase