COMPARED PROPERTIES OF PHOSPHOENOLPYRUVATE CARBOXYLASE FROM DARK-ADAPTED AND LIGHT-ADAPTED SORGHUM LEAVES - USE OF A RAPID PURIFICATION TECHNIQUE BY IMMUNOCHROMATOGRAPHY

Two C4-type phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPc) forms of Sorghum leaves, differing by their phosphorylation state (the phosphorylated form being predominant during the day), have' been purified from dark- and light-adapted leaves by using an immunoaffinity chromatography column. The phosphorylation state of these purified PEPc forms was assessed by incubation with either the endogenous PEPc protein-serine kinase, or the catalytic subunit of the cAMP-dependent protein kinase and alkaline phosphatase on the dark and light enzyme, respectively. HPLC gel filtration studies showed that both forms are tetrameric at concentrations used throughout the work. Comparison of the kinetic properties at pH 7.0, 7.3 and 8.0, indicated a cooperativity for phosphoenolpyruvate (PEP) at all pHs considered, with a Hill number of 2 for both forms and higher V(m) (2- to 4-fold) and K(m) for the light form. Malate inhibition mainly affected the V(m) of the dark form, whereas it was an allosteric inhibitor for the light enzyme. The results established that phosphorylation is the molecular basis which mediates the functional properties of the enzyme. In a medium mimicking an illuminated mesophyll cytosol (2.5 mM PEP, 5 mM glucose-6-P, 20 mM malate and 10 mM MgCl2 (pH 7.3), the activity of the enzyme from light-adapted leaves was twice that of the dark one. This increased activity due to phosphorylation may be of importance for the regulation of C4 photosynthesis.