Differential regulation of three catalytic activities of platelet-activating factor (PAF)-dependent transacetylase

We have previously established that PAF-dependent transacetylase (TA) purified to apparent homogeneity from rat kidney membranes and cytosol contains three separate catalytic activities, namely PAF lysophospholipid transacetylase (TA(1)), PAF sphingosine transacetylase (TA(s)), and PAF acetylhydrolase (AH), In the present investigation, we studied the biochemical factors and mechanism(s) that differentially regulate these three TA activities of the purified enzymes. We found that only the TA(s) activity of the TA purified from the membranes was stimulated by phosphatidylserine (PS) with optimal concentration of activation occurring at 25 muM, Other acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylinositol I-phosphate (PIP), are partially effective, while diacylglycerol and free fatty acids had no effect on the TA(s) activity. PS exerted its effect on the TA(s) activity through the increases of both K-m and V-max. In addition, N-ethylmalimide (NEM) and dithiobis-(2-nitro-5-thiobenzoic acid) (DTNB) strongly inhibited the TA(L) activity and partially decreased the TA(L) and AH activities of the purified membrane enzyme in a dose-dependent manner. The addition of PS, but not by its substrate, sphingosine, could prevented the inhibition by NEM on the basal level of TA(s). On the other hand, the inhibition of TAL by NEM and DTNB were partially protected by the substrate, lysoplasmalogens, Furthermore, PAF fully protects the inhibition of AH, partially protects the inhibition of TA(L), and does not protect the inhibition of TA(s) by NEM, These results suggested that the three individual catalytic activities of TA have different dependencies on the thiol-containing residue(s) of the enzyme, i.e., cysteine. Furthermore, the nonresponsiveness of the purified cytosolic TA(s) to PS activation is consistent with our previous notions that membrane and cytosolic TA are posttranslationally distinct. (C) 2001 Academic Press.