Factors affecting reaction of cucumber root lipoxygenase in phospholipid vesicle dispersions

Abstract Reactions of cucumber root lipoxygenase (CRLOX) were investigated using l -α-phosphatidylcholine dilinolenoyl (PCdiLNA) vesicles as substrates at pH 5.5. The progress of the enzyme reaction was followed by measuring oxygen consumption using an oxygen electrode. The CRLOX activity was drastically changed according to the time of sonication treatment for the preparation of substrate vesicles. Dynamic light scattering (DLS) data suggested that a large unilamellar vesicle was most susceptible to the reaction of CRLOX. The addition of free fatty acid (FFA) such as oleic acid to PCdiLNA vesicles enhanced the enzymatic activity effectively, whereas the addition of phosphatidic acid (PA) slightly suppressed the activity. In order to reveal the factors affecting the enzymatic activity, measurements of DLS, ζ -potential, 31 P-NMR and electron microscopy observations were made for PCdiLNA vesicle dispersions, which contained FFA and PA. The particle size and ζ -potential of the vesicles were found to be closely related to the enzymatic reaction. The electron microscopy observations and 31 P-NMR spectra indicated that FFA and PA differently modified the structure of PCdiLNA vesicles, thereby modulating the enzymatic activity. These results suggest that CRLOX strictly can recognize the physical states of a PCdiLNA assemblage when catalyzing oxygenation of the phospholipids.