Phosphoenolpyruvate carboxykinase from higher plants: Purification from cucumber and evidence of rapid proteolytic cleavage in extracts from a range of plant tissues

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) was purified 600-fold to homogeneity from the cotyledons of cucumber (Cucumis sativus L.) and a polyclonal antiserum raised. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the purified preparation contained a single polypeptide of 62 kDa, consistent with previous studies of this enzyme in C4 grasses. Immunoblots of crude extracts showed that a form of PEPCK of approximately this molecular mass predominated in cucumber cotyledons and in a range of plant tissues (cotyledons of fat-storing seedlings, leaves of C4 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 extracts is a smaller form which arises by rapid proteolysis. This phenomenon means that the native form of PEPCK has never been purified from plants nor its properties determined.

Inducibility of crassulacean acid metabolism (CAM) in Clusia species; physiological/biochemical characterisation and intercellular localization of carboxylation and decarboxylation processes in three species which exhibit different degrees of CAM

“…The carboxylation reaction of PEPCK was assayed as described by Walker et al 1995. The assay contained, in 1 ml: 80 mM 2-(N-morpholino)ethanesulfonic acidNaOH (pH 6.7), 0.35 mM NADH, 5 mM DTT, 2 mM MnCl 2 , 2 mM PEP, 2 mM ADP, 50 mM KHCO 3 , 6 units malate dehydrogenase.…”

“…The supernatant was discarded and pellets were re-suspended in SDS-PAGE sample buer and boiled for 2 min. Both SDS-PAGE and immunoblotting were performed as described by Walker and Leegood (1996). The antibody to PEPCK was raised against the puri®ed enzyme from Cucumis sativus (Walker et al 1995) and the PEPCase antibody (raised against the puri®ed enzyme from Panicum miliaceum) was a gift from Prof. H Bohnert (Department of Molecular and Cellular Biology, University of Arizona, Tucson, USA).…”

“…Both SDS-PAGE and immunoblotting were performed as described by Walker and Leegood (1996). The antibody to PEPCK was raised against the puri®ed enzyme from Cucumis sativus (Walker et al 1995) and the PEPCase antibody (raised against the puri®ed enzyme from Panicum miliaceum) was a gift from Prof. H Bohnert (Department of Molecular and Cellular Biology, University of Arizona, Tucson, USA). The Rubisco antibody was raised against the puri®ed enzyme from Brassica napa (Te csi et al 1994) whilst a polyclonal antiserum to the P-protein component of the GDC complex puri®ed from oat leaves was kindly provided by Jennifer Lorang and Thomas Wolpert (University of Oregon, Corvallis, USA).…”

Characteristics of C4 photosynthesis in stems and petioles of C3 flowering plants

“…Plants were left for 30 min,¯ash frozen, and heated in 70% ethanol to remove soluble sugars; sections were then exposed to X-ray ®lm. We conducted enzyme assays on either leaf lamina of tobacco or the veins of tobacco petioles separated from the cortex manually with a razor blade; results are expressed per unit chlorophyll 25,30 . .…”

Changes in germination and glyoxylate and respiratory enzymes ofPinuspineaseeds under various abiotic stresses

“…(1 cm (1 ) (Walker et al 1995). Malate synthase (MS EC 4.1.3.2), assay contained 140 mM K-phosphate (pH 7.5), 5 mM MgCl 2, 3.6 mM DTNB, 1 mM acetil-CoA, 3.3 mM Na-glyoxylate, 50 ml extract The activity was measured spectrophotometrically at A 405, (o 013,6 mM (1 cm (1 ) (Bajaracharya & Schopfer 1979).…”