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 purifled 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 (SDS-PAGE) conditions 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 MgATP(2-) were biphasic. The presence of Na(2)SO(4) changed the MgATP(2-) dependence to linearity but not the glycerate-3-phosphate dependence.
Köpke-Secundo
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E
Institut für Pflanzenphysiologie und Mikrobiologie, Freie Universität Berlin, Königin-Luise-Strasse 12-16a, D-1000 Berlin 33 (West), Federal Republic of Germany.
Molnar
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I
Schnarrenberger
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C