STUDY OF FATTY-ACID SPECIFICITY OF SUNFLOWER PHOSPHOLIPASE-D USING DETERGENT PHOSPHOLIPID MICELLES/

The fatty acid specificity of phospholipase D purified from germinatin g sunflower seeds was studied using mixed micelles with variable deter gent/phospholipid ratios. The main advantage of this approach is that since the substrate is integrated in the detergent micelles, compariso ns can be made between the kinetic constants of a wide range of phosph atidylcholine (PtdCho) compounds with various fatty acid contents, Pho spholipase D is subject to interfacial activation as it is most active on water-insoluble substrates. It is not active on sphingomyelin and only slightly on lysophosphatidylcholine. By fitting the curves based on the experimental kinetic data, the interfacial dissociation constan t of phospholipase B, the maximum hydrolysis rate V-m and the kinetic constant K-m(B) were determined with the micellar substrate. The speci ficity of various substrates was examined by comparing the V-m/K-m(B) values, and it was noted that sunflower phospholipase D is most active on medium-chain fatty PtdCho compounds, With long-chain natural phosp holipids, the specificity of phospholipase D was slightly dependent an the: level of fatty acid unsaturation. The purr: enzyme was able to h ydrolyse the sunflower phospholipids present in mixed detergent micell es but not the phospholipids integrated in the natural sunflower oil b ody structure. We concluded, however, that during the germination of s unflower seeds, phospholipase D might he involved in the degradation o f oil bodies, since other factors present in crude seed extracts may m ake phospholipids accessible to the enzyme.