Inhibition of membrane damage due to phospholipase activity during fractionation of wheat aleurone tissue

Fractionation of [Me-14C]choline-labelled wheat aleurone tissue in the absence of phospholipase inhibitors resulted in a 60% loss of label from phosphatidylcholine and even larger losses of NADH-cytochrome c reductase activity from microsomal fractions. Several putative inhibitors of phospholipase D were tested for their ability to protect the membranes during fractionation. The addition of choline and O-phosphorylcholine, together with glycerol-1-phosphate to inhibit any phosphatidic acid phosphatase activity, proved to be the best protectants. In tissue from freshly imbibed seeds, however, the addition of p-chloromercuribenzoate to a cocktail of inhibitors was necessary for the best recovery of radiolabelled membranes. Effects of the inhibitors on phospholipase D activity in cell free extracts were studied in an attempt to confirm the enzyme as the cause of membrane damage.