Involvement of distinct populations of phosphatidylglycerol and phosphatidylcholine molecules in photosynthetic electron-flow activities

When spinach thylakoid membranes were treated with pancreatic phospholipase A₂, phospholipids were degraded and the uncoupled non-cyclic electron-flow activity (from H₂O to NADP⁺) was progressively inhibited. To discriminate between the relative contributions of the hydrolysis products (free fatty acids and lysophospholipids) and of the phospholipid depletion per se to inhibit the activity, we made use of the known property of bovine serum albumin to remove such hydrolysis products from membranes. Using careful washings and adequate lipid extraction procedures, we could ascertain that all hydrolysis products generated by phospholipase A₂ were effectively removed from the thylakoid membrane by bovine serum albumin treatment. When bovine serum albumin was added to thylakoid membranes after various incubation times with the phospholipase A₂, the electron-flow activity was rapidly, but not completely restored. However, when phospholipid hydrolysis exceeded a certain extent (70–85%), the activity was totally inhibited and its restoration by albumin was no longer possible. Addition of EGTA to the phospholipase A₂-treated membranes blocked both the enzyme action and the progress of electron-flow inhibition. Under these conditions, the amplitude of the albumin-induced restoration of electron-flow rate did not depend on the time span between EGTA block and albumin addition. We show that phospholipid depletion of thylakoid membranes is entirely responsible for the irreversible (albumin-insensitive) inhibition of the electron flow from H₂O to NADP⁺ by phospholipase A₂. Plotting the extent (%) of this inhibition vs. the extent (%) of phospholipid depletion allowed us to distinguish three populations of both phosphatidylglycerol and phosphatidylcholine. The first one, which was easily accessible to the enzyme, did not support greatly the electron-flow activity (around 40% of each phospholipid destroyed vs. only 10% or less inhibition). On the other hand, the electron-flow activity strongly depended on the second, less accessible population of phospholipids (around 40% of each phospholipid destroyed vs. 90% inhibition). Finally, the third population of phospholipids was not involved in the uncoupled non-cyclic electron flow activity.