Regulation of phospholipase activity in potato leaves by calmodulin and protein phosphorylation-dephosphorylation

Abstract

High levels of phospholipase activity were measured in potato ( Solanum tuberosum L. cv. Kennebec or Russett Burbank) leaf extracts using a new fluorometric phospholipase assay based on 1-acyl-2-[6-[(7-nitro-2, 1, 3 benzoxadiazol-4-yl)amino]-caproyl] phosphatidylcholine (C 6-NBD-PC). Time-course studies revealed that phospholipase activity could be stimulated for a brief time by the addition of calmodulin or the catalytic subunit of cyclic AMP-dependent protein kinase. The short-lived calmodulin stimulation or protein kinase stimulation of phospholipase activity could be prolonged by either conducting the time-course reactions in the cold (5°C) or adding sodium fluoride (a phosphatase inhibitor) to the reaction mixtures. Centrifugation studies revealed that calmodulin-stimulated or protein kinase-stimulated phospholipase activities were soluble and not associated with membranes. When potatp leaves were homogenized in the presence of either of two phosphatase inhibitors, the levels of phospholipase activity in the corresponding high-speed supernatant fractions were 36–47% higher than in controls. These experiments suggest a possible protein phosphorylation-dephosphorylation mechanism for the regulation of phospholipase activity in potato leaves.