Separation, purification, and comparative properties of chloroplast and cytoplasmic phosphoglycerate kinase from barley leaves

The chloroplast and cytoplasmic isoenzymes of phosphoglycerate kinase (PGK) (EC. 2.7.2.3) from Hordeum vulgare leaves have been separated and purified for the first time to apparent homogeneity. The method for purifying the isoenzymes is described here and consists of DEAE Sephacel chromatography followed by affinity chromatography on ATP Sepharose. This consistently provided a 500- to 900-fold purification of each isoenzyme. Most of the total PGK in green barley leaves was found to be in the chloroplasts with only 10% in the cytoplasm. The immunological properties of the two isoenzymes were compared. The antisera raised to the separate isoenzymes showed cross-reactivity, although there is evidence that each isoenzyme possesses some distinct epitopes. The isoenzymes differ in overall charge with isoelectric points at 5.2 and 5.4 for the chloroplast and cytoplasmic isoenzymes, respectively. Molecular mass estimations by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis provided similar values of approximately 38 kilodaltons for each isoenzyme, some 4 to 5 kilodaltons less than the values calculated from the cDNA sequences of the wheat isoenzymes. The isoenzymes have broadly similar pH optima of pH 7 to 8. The cytoplasmic isoenzyme is more thermally stable than the chloroplast isoenzyme. Further studies are now in progress to compare both the regulatory properties of the isoenzymes and also their three-dimensional structures as compared with the yeast enzyme