title: Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds.
abstract: Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues.
authors.affiliation: Instituto de la Grasa, CSIC, Avda. Padre Garcia Tejero 4, 41012 Seville, Spain.
authors.forename: M A
authors.initials: MA
authors.lastname: Troncoso-Ponce
authors.forename: J
authors.initials: J
authors.lastname: Rivoal
authors.forename: M
authors.initials: M
authors.lastname: Venegas-Calerón
authors.forename: S
authors.initials: S
authors.lastname: Dorion
authors.forename: R
authors.initials: R
authors.lastname: Sánchez
authors.forename: F J
authors.initials: FJ
authors.lastname: Cejudo
authors.forename: R
authors.initials: R
authors.lastname: Garcés
authors.forename: E
authors.initials: E
authors.lastname: Martínez-Force
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D019143:Evolution, Molecular; D018506:Gene Expression Regulation, Plant; D006368:Helianthus / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D018087:Plastids / Q000201:enzymology; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012639:Seeds / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D010735:Phosphoglycerate Kinase