PHOSPHOENOLPYRUVATE CARBOXYKINASE FROM HIGHER-PLANTS - PURIFICATION FROM CUCUMBER AND EVIDENCE OF RAPID PROTEOLYTIC CLEAVAGE IN EXTRACTS FROM A RANGE OF PLANT-TISSUES

Phosphoenolpyruvate carboxykinase (PEPCK) was purified 600-fold to hom ogeneity from the cotyledons of cucumber (Cucumis sativus L.) and a po lyclonal antiserum raised. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the purified preparation contained a s ingle polypeptide of 62 kDa, consistent with previous studies of this enzyme in C-4 grasses. Immunoblots of crude extracts showed that a for m of PEPCK of approximately this molecular mass predominated in cucumb er cotyledons and in a range of plant tissues (cotyledons of fat-stori ng seedlings, leaves of C-4 and Crassulacean acid metabolism plants). However, when these tissues were extracted in the presence of SDS and the extracts analysed by immunoblotting, a larger polypeptide of 68-77 kDa was detected. Thus the enzyme generally measured in crude extract s is a smaller form which arises by rapid proteolysis. This phenomenon means that the native form of PEPCK has never been purified from plan ts nor its properties determined.