Phospholipase D of rice bran. I. Purification and characterization

Abstract

Phospholipase D has been purified 75-fold from the crude extract of rice bran in an overall yield of 18%. The purification procedures involved acetone precipitation, fractionation with ammonium sulfate, gel filtration on Sephadex G-200 and CM-cellulose chromatography. Purity was established by electrophoresis on polyacrylamide gels and by gel filtration. The optimum reaction pH was found to be at 5.8–6.0. The purified enzyme was unstable under various conditions, but the activity of the enzyme could be stabilized by the inclusion of 1 mM each of Mg 2+ and Ca 2+ in the eluting buffer at the final step of purification. The molecular weight of the enzyme was found to be about 75 000 by gel filtration. The amino acid composition of the enzyme indicated the presence of high content of aspartate and/or asparagine, glutamate and/or glutamine, alanine, glycine, lysine and leucine, and low content of histidine and methionine. The K m of the enzyme was 1.67 mM for egg yolk L-α-lecithin. Calcium ion and sodium dodecyl sulfate (SDS) remarkably stimulated the activity of the enzyme. But the stimulatory effect of either calcium ion or SDS required the concomitant presence of both calcium ion and SDS in the reaction mixture. The optimum concentrations for enzyme stimulation were 100 and 7 mM for calcium ion and SDS, respectively. p-Chloromercuribenzoate at a concentration of 10 −5 M completely abolished the enzyme activity. EDTA and spermine strongly inhibited the enzyme activity. The overall characteristics of the enzyme were found to be very similar to those of the phospholipase D from cabbage.