LIPID BREAKDOWN IN SMOOTH MICROSOMAL-MEMBRANES FROM BEAN COTYLEDONS ALTERS MEMBRANE-PROTEINS AND INDUCES PROTEOLYSIS

The effects of lipid degradation on proteins of smooth microsomal membranes isolated from young bean cotyledons have been examined by three techniques, viz. fluorescence energy transfer from tryptophan to cis-parinaric acid; protein spin-labelling with 3-maleimido PROXYL; and SDS-PAGE. Lipid degradation was induced in isolated membranes by activating phospholipase D and phosphatidic acid phosphatase through the addition of Ca2+, by treatment with exogenous phospholipase C to simulate the concerted actions of phospholipase D and phosphatidic acid phosphatase or by treatment with exogenous phospholipase A2 to generate endogenous substrate for lipoxygenase. All of the treatments induced time-dependent changes in lipid-protein interaction and in protein conformation, and the treatment with phospholipase A2 also engendered proteolysis. The effects of the Ca2+ and phospholipase C treatments on lipid-protein interaction and protein conformation can presumably be partly attributed to an accumulation of diacylglycerol in the membrane, whereas the induction of proteolysis by phospholipase A2 appears to be due to activated oxygen derived from the lipoxygenase reaction and ensuing lipid peroxidation. Lipid degradation induced by these treatments simulates that which occurs during natural senescence of the cotyledons and hence these observations suggest that loss of protein function and proteolysis in senescing membranes is faciliated by lipolytic and peroxidative activity within the lipid bilayer.