ISOLATION AND CHARACTERIZATION OF THE CYTOSOLIC AND CHLOROPLASTIC 3-PHOSPHOGLYCERATE KINASE FROM SPINACH LEAVES

The cytosol and chloroplast 3-phosphoglycerate kinases (3-PGK) from spinach (Spinacia oleracea L.) were purified to apparent homogeneity. The procedure included a conventional anion-exchange chromatography on DEAE-cellulose and mainly a series of HPLC columns. The charge differences of the two isoenzymes were so small that separation was only successful by anion-exchange chromatography on a HPLC SynChropak AX 300 column. The portion of the two isoenzmyes in leaf tissue was estimated as 5% and 95%. The major 3-PGK was associated with isolated chloroplasts while the other 3-PGK was only found in the soluble cell fraction. The specific activity of the purified enzymes were in the order of 800 units (per milligram of protein). The molecular weight for the two 3-PGKs under nondenaturing (size exclusion chromatography) and denaturing (SOS-PAGE) condi-tions were in the order of 40 kilodaltons, with the cytosolic 3-PGK being slightly smaller than the chloroplastic 3-PGK. An antiserum against the chloroplastic 3-PGK showed only 4.6% cross-reaction of the chloroplastic 3-PGK with the cytosolic 3-PGK. The kinetics for glycerate-3-phosphate and MgATP2- were biphasic. The pres-ence of Na2SO4 changed the MgATP2- dependence to linearity but not the glycerate-3-phosphate dependence.