LIGHT REGULATION OF PHOSPHOENOLPYRUVATE CARBOXYLASE IN BARLEY MESOPHYLL PROTOPLASTS IS MODULATED BY PROTEIN-SYNTHESIS AND CALCIUM, AND NOT NECESSARILY CORRELATED WITH PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE-ACTIVITY

The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1. 31) and PEPCase kinase was investigated using barley (Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resu lted in a reduction in the sensitivity of PEPCase to the inhibitor L-m alate; PEPCase from protoplasts incubated in the light for 1 h was inh ibited 48 +/- 2% by 2 mM malate, whereas the enzyme from protoplasts i ncubated for Ih in the dark was inhibited by 67 +/- 2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cyclohe ximide (CHM), indicating the involvement of protein synthesis. The PEP Case kinase in protoplasts increased with time after isolation in dark ness, and increased still further following light treatment. The incre ase in kinase activity in the light was sensitive to CHM. When protopl asts were illuminated in the presence of EGTA and the calcium ionophor e A23187 to reduce intracellular Ca2+, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase acti vity was detected than in protoplasts illuminated in the absence of EG TA and A23187. Incubation with 3-(3',4'-dichlorophenyl)-1,1-dimethylur ea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kina se. These results indicate that there is a constitutive PEPCase kinase activity in C-3 leaf tissue, that there is another kinase which is li ght-activated in a CHM-sensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase ki nase actvity, and that PEPCase and PEPCase kinase are regulated in a d ifferent manner in C-3 protoplasts than in C-4 protoplasts or leaf tis sue.