No Light Activation and High Malate Sensitivity of Phosphoenolpyruvate Carboxylase in Guard Cell Protoplasts of Commelina communis L.

Guard cell protoplasts (GCP) were prepared from leaves of Commelinacommunis L. and phosphoenolpyruvate carboxylase (PEPc) activityrecorded after injection of the protoplasts directly into theassay medium. The GCP were lysed immediately by the presenceof Triton X-100 and a lowered osmotic concentration in the assaycuvette enabling PEPc activity to be measured with ‘nascent’enzyme. There was no light activation of the enzyme with K_mPEP (about 3.7 mol m^–3) and Vmax being similar in light-ordark-treated protoplasts. Illumination of the GCP in the presenceof CO₂-free air and KCI, a treatment which is known to swellGCP, did not change the kinetics. PEPc activity at saturating PEP was very sensitive to malateinhibition, 20 mmol m^–3 (the I₅₀ value) inhibiting activityby about 50%. Inhibition was similar in light- or dark-treatedprotoplasts. Malate inhibition was, however, much less (I₅₀= 500 mmol m^–3) if the enzyme source was a protoplastextract kept in the absence of glycerol. Inclusion of 20% glycerolin the extraction medium maintained the enzyme in the malate-sensitiveform as occurred in the in vivo assays. The high apparent K_mPEP and the high sensitivity to malate inhibition of GCP PEPcare features unlike those observed with PEPc from leaf tissuesof C₄ and CAM plants and from GCP extracts. PEPc activity increased slightly in the presence of KCI in theassay medium up to about 10 mol m^–3 and thereafter activityslowly declined as KCI concentrations increased further. Key words: Guard cell protoplasts, phosphoenolpyruvate carboxylase