Instituto de la Grasa, CSIC, Avda. Padre Garcia Tejero 4, 41012 Seville, Spain.
Phytochemistry. 2012 Jul;79:27-38. doi: 10.1016/j.phytochem.2012.04.001. Epub 2012 May 1.
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.
从发育中的向日葵（Helianthus annuus L.）种子中克隆并鉴定了三个编码不同磷酸甘油酸激酶（PGK，EC 2.7.2.3）同工型的 cDNA，两个细胞质（HacPGK1 和 HacPGK2）和一个质体（HapPGK）。这些基因的表达谱在异养组织中存在差异，例如发育中的种子和根，其中 HacPGK1 占优势，而 HapPGK 在光合组织中高度表达。使用固定化金属离子亲和层析将 cDNA 在大肠杆菌中表达，并将相应的蛋白质纯化至电泳纯，然后进行生物化学特性分析。尽管序列之间具有高度的同一性，但 HacPGK1 同工型在比活、热稳定性和 pH 敏感性方面与 HacPGK2 和 HapPGK 表现出强烈的差异。针对纯化的 HacPGK1 同工型制备了多克隆免疫血清，该血清与其他 PGK 同工型表现出交叉免疫反应性。该血清允许在胚胎组织中定位 PGK 同工型的高表达水平。
